PhD thesis - ku Wohlfahrt Rasmussen.pdf · PhD thesis Magnus Wohlfahrt Rasmussen ... THE MRNA DECAY FACTOR PAT1 FUNCTIONS IN A PATHWAY INCLUDING MAP ... 54 SDS-PAGE and

U N I V E R S I T Y O F C O P E N H A G E N

F A C U L T Y O F S C I E N C E

PhD thesis

Magnus Wohlfahrt Rasmussen

MAP Kinase 4 substrates and plant innate immunity

Supervisor: Morten Petersen

Handed in: 31/12/2015

Table of Contents

PREFACE ............................................................................................................................ 5

Aknowlegements ............................................................................................................................................................... 5

ABSTRACT ......................................................................................................................... 6

SAMMENDRAG .................................................................................................................. 7

LIST OF ABBREVIATIONS ................................................................................................ 8

PLANT INNATE IMMUNITY .............................................................................................. 11

Pathogen associated molecular patterns (PAMPs) ...................................................................................................... 11

Pathogenic effector molecules and host resistance (R) proteins ................................................................................. 12

Autoimmunity ................................................................................................................................................................. 14

References........................................................................................................................................................................ 15

MAP KINASES IN ARABIDOPSIS INNATE IMMUNITY................................................... 21

Abstract ........................................................................................................................................................................... 21

Introduction .................................................................................................................................................................... 21

MAPK cascades in PAMP triggered immunity (PTI) ................................................................................................. 21

MAPK cascades in effector triggered immunity (ETI) ............................................................................................... 22

WRKY transcription factors ......................................................................................................................................... 23

MAPK in general stress signaling ................................................................................................................................. 24

References........................................................................................................................................................................ 24

MRNA DECAY IN PLANT IMMUNITY .............................................................................. 27

Abstract ........................................................................................................................................................................... 27

Introduction .................................................................................................................................................................... 27

Plant innate immunity .................................................................................................................................................... 28

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mRNA decay and autoimmunity ................................................................................................................................... 29

Conclusion ....................................................................................................................................................................... 35

References........................................................................................................................................................................ 35

THE MRNA DECAY FACTOR PAT1 FUNCTIONS IN A PATHWAY INCLUDING MAP KINASE 4 AND IMMUNE RECEPTOR SUMM2 ............................................................... 42

Abstract ........................................................................................................................................................................... 42

Introduction .................................................................................................................................................................... 42

Results .............................................................................................................................................................................. 43

AtPAT1 is an ScPAT1 orthologue and interacts with MPK4 in planta ....................................................................... 43

PAT1 is required for decapping of selected mRNAs ................................................................................................... 45

PAT1 is an MPK4 substrate ......................................................................................................................................... 45

pat1 mutants exhibit autoimmunity similar to mpk4 .................................................................................................... 46

PAT1 detection in P-bodies is induced by PAMPs ...................................................................................................... 47

The MPK4 suppressor summ2 also suppresses the pat1 resistance phenotype ............................................................ 47

Discussion ........................................................................................................................................................................ 48

Materials and Methods ................................................................................................................................................... 51

Plant materials and growth conditions ......................................................................................................................... 51

Cloning and transgenic lines ........................................................................................................................................ 52

Mutagenesis of His6-PAT1 .......................................................................................................................................... 52

PAT1 protein purification and in vitro kinase assays................................................................................................... 52

Yeast transformation .................................................................................................................................................... 52

Flg22 kinetics ............................................................................................................................................................... 52

Semi-quantitative and qRT-PCR ................................................................................................................................. 53

RNA extraction and RACE PCR ................................................................................................................................. 53

Quantification of capped versus uncapped transcripts ................................................................................................. 53

Confocal microscopy ................................................................................................................................................... 53

Infection assays ............................................................................................................................................................ 53

Transient expression in N. benthamiana ...................................................................................................................... 53

Protein extraction and immunoprecipitation in N. benthamiana .................................................................................. 53

Arabidopsis protein extraction and immunoprecipitation for mass spectrometry analysis .......................................... 54

SDS-PAGE and immunoblotting ................................................................................................................................. 54

Antibodies .................................................................................................................................................................... 54

In-gel digestion, TiO2 chromatography and mass spectrometry .................................................................................. 54

Acknowledgements ......................................................................................................................................................... 54

2

Author contributions ...................................................................................................................................................... 55

Conflict of Interest .......................................................................................................................................................... 55

References........................................................................................................................................................................ 55

Supplemental figures ...................................................................................................................................................... 58

AOC3 IS AN MPK4 SUBSTRATE AND ITS OVEREXPRESSION INDUCE IMMUNITY AGAINST PST DC3000 ..................................................................................................... 66

Introduction .................................................................................................................................................................... 66

Results .............................................................................................................................................................................. 70

AOC3 Interacts with MPK4 in planta. ......................................................................................................................... 70

Over-expression of AOC3 leads to increased resistance .............................................................................................. 72

AOC3 is an MPK4 substrate ........................................................................................................................................ 72

MPK4 associates with chloroplasts. ............................................................................................................................. 75

Discussion ........................................................................................................................................................................ 77

Materials and methods ................................................................................................................................................... 78

References........................................................................................................................................................................ 79

EIF4E-THR6 IS SPECIFICALLY PHOSPHORYLATED IN VITRO BY MPK4 AND FORM A COMPLEX IN THE NUCLEUS IN VIVO. ....................................................................... 83

Introduction .................................................................................................................................................................... 83

Results .............................................................................................................................................................................. 87

eIF4E is an MPK4 substrate......................................................................................................................................... 87

eIF4E interacts with MPK4 in planta ........................................................................................................................... 88

eif4e mutants does not exhibit autoimmunity similar to mpk4 ..................................................................................... 88

ClYVV susceptibility is not dependent on eIF4E phosphorylation ............................................................................. 90

MPK4 and eIF4E interact in the nucleus. .................................................................................................................... 92

Discussion ........................................................................................................................................................................ 92

Materials and methods ................................................................................................................................................... 94

Plants growth conditions .............................................................................................................................................. 94

SDS-PAGE and immunoblotting ................................................................................................................................. 95

Antibodies .................................................................................................................................................................... 95

Bacterial infection assays ............................................................................................................................................. 96

Transient expression in Nicotiana benthamiana ........................................................................................................... 96

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Protein extraction and immunoprecipitation in Nicotiana benthamiana and Arabidopsis ........................................... 96

Purification of X7 polymerase ..................................................................................................................................... 97

Genotyping................................................................................................................................................................... 97

Cloning and transgenic lines ........................................................................................................................................ 97

Recombinant protein purification and in vitro kinase assays ....................................................................................... 98

Virus inoculation and detection by ELISA .................................................................................................................. 98

References........................................................................................................................................................................ 99

CONCLUDING REMARKS ............................................................................................. 104

References...................................................................................................................................................................... 105

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Preface

This thesis concludes my PhD work at the department of Biology, University of Copenhagen. My

main research focus has been on substrates of the immune regulating MAP kinase 4 and their

involvement in immunity. The thesis consist of:

(i) A general introduction to plant innate immunity.

(ii) A review article “MAP Kinases in Arabidopsis innate immunity” published in “Frontiers

in Plant Science”. This article serves as an introduction, giving an overview on the

function of MAP kinases immune signaling in Arabidopsis.

(iii) An invited review article “mRNA decay in plant immunity” under revision for

publication in “Cellular and Molecular Life Science”. This article gives an overview on

our current understanding of the involvement of mRNA decay in plant immunity and

serves as a more comprehensive introduction to (iv).

(iv) A research article “The mRNA decay factor PAT1 functions in a pathway including

MAP kinase 4 and immune receptor SUMM2” published in ”The EMBO Journal”.

(v) Two draft manuscripts regarding novel MPK4 substrates and their putative function in

defense.

Aknowlegements

An era has ended and I will finally leave the PMB lab! I am sure that I will be missed and trust me;

I will miss all of you. I would like to thank my main supervisor Morten Petersen and co-supervisor

John Mundy for their support and guidance during my research and the opportunity to work in your

lab. I will also take the opportunity to thank all the current and previous lab members for making

my stay in the lab such a wonderful time. I would in particular like to thank my work wife no.1

Milena for all the interesting chitchat and for being my personal scientific oracle and my work wife

no.2 Àngels (even though you left me for a real job!) for simply putting up with me and my crazy

ideas. Lastly, thank you Tine for bear with me the numerous times when I’ve told you that that I’m

almost on my way home and end up staying two hours extra in the lab.

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Abstract

Multi-layered defense responses are activated in plants upon recognition of invading pathogens.

Transmembrane receptors recognize conserved pathogen-associated molecular patterns and activate

MAP kinase cascades, which regulate changes in gene expression to produce appropriate immune

responses. For example, Arabidopsis MPK4 regulates the expression of a subset of defense genes

via at least one WRKY transcription factor. We report here that MPK4 is found in complexes in

vivo with (i) PAT1, component of the mRNA decapping machinery, (ii) AOC3, a component in the

biosynthesis pathway of JA and (iii) eIF4E, a component in the translational initiation protein

complex. For PAT1 and eIF4E we show that MPK4 phosphorylates specific Ser and Thr residues in

vitro, and that MPK4 also phosphorylates AOC3 at an unmapped residue. Specific in vivo

phosphorylation for PAT1 is shown in response to pathogen recognition, which also induce its

localization to cytoplasmic processing bodies. All three proteins; PAT1, AOC3 and eIF4E also

interacts with MPK4 in vivo although the functional outcome of these interactions are still elusive.

The thesis comprise a general introduction to plant innate immunity followed by two review articles

“MAP kinase cascades in Arabidopsis innate immunity” published in Frontiers in Plant Science

and “mRNA decay in plant immunity” under revision for Cellular and Molecular Life Science.

Together these sections gives a comprehensive overview of Arabidopsis defense signaling. The

results are presents as one manuscript “The mRNA decay factor PAT1 functions in a pathway

including MAP kinase 4 and immune receptor SUMM2” published in The EMBO Journal and two

draft manuscripts summarizing our data on regarding AOC3 and eIF4E in respect to MPK4.

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Sammendrag

Flere forsvarslag aktiveres i planter ved genkendelse af invaderende patogener. Transmembrane

receptorer genkender konserverede patogen-associeret molekylære mønstre og aktivere MAP-

kinase kaskader, som regulerer ændringer i gen-ekspression for at producere passende

immunresponser. For eksempel, Arabidopsis MPK4 regulerer ekspressionen af en undergruppe af

forsvarsgener via mindst en WRKY transkriptionsfaktor. Vi rapporterer her, at MPK4 findes i

komplekser in vivo med (i) PAT1, en komponent i ”mRNA decapping”, (ii) AOC3, en komponent i

biosyntesevejen af JA og (iii) eIF4E, en komponent i translationsinitierings komplekset. For PAT1

og eIF4E viser vi, at MPK4 phosphorylerer specifikke Ser og Thr aminosyrerestre in vitro, og at

MPK4 også phosphorylerer en ukendt aminosyreester i AOC3. Specifik in vivo phosphorylering af

PAT1 sker som reaktion på patogen genkendelse, som derudover også inducerer dens lokalisering

til cytoplasmiske ”procesing bodies”. Alle tre proteiner; PAT1, AOC3 og eIF4E interagerer med

MPK4 in vivo, selvom det funktionelle resultat af disse interaktioner stadig er ukendte.

Afhandlingen omfatter en generel introduktion til at planters medfødte immunforsvar efterfulgt af to

review-artikler " MAP kinase cascades in Arabidopsis innate immunity " offentliggjort i Frontiers

in Plant Science og "mRNA decay in plant immunity" under revision for Cellular and Molecular

Life Science. Disse afsnit giver tilsammen et samlet overblik over Arabidopsis forsvar signalering.

Resultaterne er præsenteret som et manuskript " The mRNA decay factor PAT1 functions in a

pathway including MAP kinase 4 and immune receptor SUMM2" offentliggjort i The EMBO

Journal og to udkast til manuskripter som opsummerer vores viden vedrørende AOC3 og eIF4E i

forhold til MPK4.

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List of Abbreviations

4E-BP eIF4E BINDING PROTEIN

ACD ACCELERATED CELL DEATH

AGO ARGONAUTE

AOC ALLENE OXIDE CYCLASE

AOS ALENE OXIDE SYNTHASE

ARF AUXIN RESPONSE FACTOR

At Arabidopsis thaliana

BAK1 BRI1-ASSOCIATED RECEPTOR KINASE1

BIK1 BOTRYTIS-INDUCED KINASE1

CC Coiled-coiled

CDPK Ca2+ dependent kinase

ClYVV Clover Yellow Vein Virus

Col Columbia

CSD COPPER/ZINC SUPEROXIDE DISMUTASE

CTR Constitutively Triple Response

DCL1 DICER-LIKE 1

DCP1 DECAPPING 1

EBF EIN3 binding F-box protein

EDS1 ENHANCED DISEASE SUSCEPTIBILITY1

EFR EF-TU RECEPTOR

eIF eukaryotic INITIATION FACTOR

EIN Ethylene Insensitive

ERF Ethylene Response Factor

ET Ethylene

ETI Effector Triggered Immunity

FLS2 FLAGELLIN-SENSITIVE2

HR Hypersensitivity response

JA Jasmonic acid

LAZ LAZARUS

Ler Landsberg

LMM Lesion Mimic Mutant

LOX LIPOXYGENASE

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MBP MYELIN BASIC PROTEIN

MKS1 MAP KINASE 4 SUBSTRATE 1

MNK MAPK-INTERACTING KINASE

MAPK MAP Kinase

MAP2K MAP Kinase Kinase

MAP3K MAP Kinase Kinase Kinase

MPK4 MAP KINASE 4

NADPH Nicotinamide adenine dinucleotide hosphate

NDR1 NONRACE-SPECIFIC DISEASE RESISTANCE1

NMD Non-sense Meadiated Decay

NPR NON-EXPRESSOR OF PR-GENES

OPDA Oxo-phytodienoic acid

OPR OPDA REDUCTASE

PAD PHYTOALEXIN DEFICIENT

PAMP Pathogen Associated Molecular Pattern

PABP poly(A) binding protein

PAT PROTEIN ASOIATED WITH TOPOISOMERASE-II

PBs Processing Bodies

PCD Programmed cell death

PR Pathogenesis-Related

PRR Pattern Recognizing Receptor

pst Pseudomonas syringae pv. tomato

PTI PAMP Triggered Immunity

PTC Premature termination codon

PTGS Post Transcriptionally Gene Silencing

RBOHD RESPIRATORY BURST OXIDASE HOMOLOG D

R-gene Resistance gene

R-protein Resistance Protein

RIN4 RPM1-INTERACTING PROTEIN4

RIPK RPM1-INDUCED PROTEIN KINASE

RISC RNA-induced silencing complex

ROS Reactive oxygen species

RPM1 RESISTANCE TO PSEUDOMONAS SYRINGAE

RPS2 RESISTANCE TO PSEUDOMONAS SYRINGAE2

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SA Salicylic acid

SAR Systemic Acquired Resistance

SID2 ISOCHORISMATE SYNTHASE1

siRNA Small interfering RNA

SP Serine-Proline

SUMM2 SUPPRESSOR of MKK1 MKK2 2

TAL Transcription Activator-Like

TF Transcription factor

TIR Toll and Interleukin-1 Receptor homology

TP Threonine-Proline

VCS VARICOSE

WT Wild type

Xoo Xanthomonas oryzae pathovar oryzae

XRN Exoribonuclease

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Plant innate immunity

Plants convert sunlight and atmospheric carbon dioxide into chemical energy in the form of

carbohydrates (Lodish et al., 2000). Thus, plants are a source of nutrient for numerous organisms. It

is logical to assume that plants defend themselves against pathogens or herbivores to avoid

depletion of their nutrients. Plants have therefore developed various defense mechanisms to avoid

hosting malevolent organisms (Katagiri and Tsuda, 2010). All biological processes, including

defense responses, are energy consuming, thus a tight regulation of defense can avoid superfluous

use of scarce resources. Defense responses are spatial and temporal regulated, in most cases by

direct or indirect recognition of pathogens. For example, plants can instigate defense by direct

recognition of bacterial flagellin or indirectly by recognizing host tissue damaged by pathogens or

herbivores (Gómez-Gómez and Boller, 2000; Krol et al., 2010). Plants have adapted both a

mechanical defense system in the form of structural fortification of the cell wall and active defense

system in form of both transmembrane and cytoplasmic immune receptors that upon activation

boosts defense responses (Boller and He, 2009; Malinovsky et al., 2014). The role of the rigid cell

wall and regulation of its components in response to pathogens are reviewed by Malinovsky et al

(2014) and will not be covered here whereas a general introduction to immune receptors and their

activation are described below.

Pathogen associated molecular patterns (PAMPs)

PAMP is a collective term used to describe conserved molecular patters found in microorganisms

that on their own is enough to induce defense responses. PAMPs are recognized by transmembrane

immune receptors located in the plasma membrane. The recognition of bacterial flagellin by the

pattern recognition receptor (PRR) FLAGELLIN-SENSING 2 (FLS2) in Arabidopsis is one of the

best characterized PAMP recognition systems in plants (Boller and Felix, 2009; Gómez-Gómez and

Boller, 2000). Another example is the perception of bacterial elongation factor-Tu by the EF-Tu

(EFR1) receptor (Kunze et al., 2004; Zipfel et al., 2006). Whereas FLS2 is widely conserved across

plant species, EFR belongs only to the Brassicaceae family (Zipfel et al., 2006). Transgenic

expression of Arabidopsis EFR in species outside the Brassicaceae family make plants sensitive to

EF-Tu induced immunity (Lacombe et al., 2010). Thus, signaling downstream of PRRs is likely

well conserved across species.

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Both EFR and FLS2 hetero dimerizes with the co-receptor BRI1-ASSOCIATED KINASE 1

(BAK1) in a ligand induced interaction (Chinchilla et al., 2007, -; Roux et al., 2011; Schulze et al.,

2010). Phosphorylation of BOTRTYTIS-INDUCED KINASE 1 (BIK1) is induced upon activation

of both FLS2 and EFR (Lu et al., 2010; Zhang et al., 2010). In Arabidopsis BIK1 resides in a

complex with both BAK1 and FLS2 or EFR and upon PAMP perception BIK1 is rapidly

phosphorylated which trigger BIK1 to activate downstream immune responses (Lu et al., 2010;

Zhang et al., 2010).

One of the early effects of PAMP triggered immunity (PTI) is the generation of reactive oxygen

species (ROS) in an oxidative burst producing apoplastic superoxide (O2-), which can be converted

into hydrogen peroxide (H2O2) (Torres et al., 2006). The transmembrane RESPIRATORY BURST

OXIDASE HOMOLOG D (ROBHD) is a key enzyme in producing superoxide, it oxidize

intracellular NADPH and transports the electron to the apoplastic space where it reacts with

molecular oxygen producing superoxide (Torres et al., 2005, 2006). Production of ROS, like

hydrogen peroxide is directly toxic to pathogens and it induce cell wall strengthening while also

functioning as a systemic signaling molecules (Kadota et al., 2015; Miller et al., 2009). Recent

studies have shown that ROBHD sits in a complex with FLS2/EFR, BAK1 and BIK1, when PTI is

induced, BIK1 directly phosphorylates resides in RBOHD. When hindering this phosphorylation

RBOHD is not activated properly resulting in increased susceptibility against non-adapted

pathogens (Kadota et al., 2014; Li et al., 2014).

In general, PTI is relatively mild and mostly effective against non-adapted pathogens that do not

employ effector molecules evolved to circumvent PTI signaling. In addition to ROS signaling other

canonical signaling pathways include mitogen activated protein kinases (MAPKs) that for example

can adjust transcript accumulation of defense related genes. The involvement of MAPKs in PTI is

reviewed in more details in “MAP Kinases in Arabidopsis innate immunity”.

Pathogenic effector molecules and host resistance (R) proteins

Successful pathogens have evolved effector proteins or molecules that can modify the host and

support growth of the pathogen. Bacterial pathogens deliver these effectors by types of secretion

systems that can effectively inject effector proteins directly into the host cell. Mutations that disrupt

the type-three secretion system in gram-negative bacterial pathogens are often accompanied by

weakened virulence and are inadequate of overcoming PTI (Cunnac et al., 2009).

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Effector proteins function in numerous ways. One example is Transcription Activator-Like (TAL)

effectors functioning as transcription factors, which bind the promoter of specific host genes and

induce their transcription. For effective infection in rice the pathogen Xanthomonas oryzae

pathovar oryzae (Xoo) deploy the TAL effector pthXo1 (Yang et al., 2006). PthXo1 binds the

promoter and induce the induction of OsSWEET11, which is important for Xoo infection (Chen et

al., 2010; Yang et al., 2006; Yuan et al., 2010). OsSWEET11 is presumably a sugar transporter that

induce sugar efflux in favor of the pathogen (Chen et al., 2010), however it has also been suggested

that OsSWEET11 functions as a cupper transporter that reduce the cupper concentration in the

xylem sap to facilitate infection of the cupper sensitive Xoo (Yuan et al., 2010). In Arabidopsis

infection with Pseudomonas (Pst) DC3000 induce accumulation of transcripts from 7 SWEET

genes, whereas infections with the Pst DC3000 HrcU mutant which cannot inject type three-

effector proteins into the host did not induce accumulation of three of these transcript (Chen et al.,

2010). Plants resistant to pathogens deploying specific TAL effectors have evolved defense genes

that are under control of promoters mimicking that of the TAL effector targets, which thereby are

transcribed in presence of the TAL effectors inducing a defense response (Hutin et al., 2015).

Effector proteins can also modify host proteins to quench host defense responses. RPM1

INTERACTING PROTEIN 4 (RIN4) is one of the best characterized targets of bacterial effectors.

RIN4 is anchored to the plasma membrane and resides in complex with or are in close proximity to

FLS2 and activation of FLS2 induces phosphorylation of RIN4 important for full activation of PTI

(Chung et al., 2014; Qi et al., 2011). Although the molecular function of RIN4 is not fully

understood, its role in immunity is emphasized by being a target of no less than four bacterial

effectors comprising AvrRpm1, AvrB, AvrRpt2 and HopF2 while being a guardee of the two R

proteins RPM1 and RPS2 in Arabidopsis (Axtell and Staskawicz, 2003, 2; Mackey et al., 2002,

2003; Selote and Kachroo, 2010; Wang et al., 2010; Wilton et al., 2010). These effector proteins

and conjugant R proteins function by different mechanisms. AvrRpt2 functions as a protease and

when introduced into the host it degrades RIN4, the lack of RIN4 in turns activate RPS2 causing

effector triggered immunity (ETI) (Axtell and Staskawicz, 2003; Mackey et al., 2003, 4). PTI and

ETI signaling is somewhat similar although ETI responses are more prolonged and pronounced and

can include localized cell death (Cui et al., 2014). Arabidopsis rps2 loss-of-function mutants are

more susceptible to AvrRpt2 expressing pathogens, thus it is likely that resistant plants have

evolved RPS2 in response AvrRpt2 mediated bacterial virulence to restore resistance (Afzal et al.,

2011).

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AvrB and AvrRpm1 on the other hand mediate RIN4 phosphorylation through RPM1-INDUCED

PROTEIN KINASE (RIPK). FLS2 and RIPK mediated RIN4 phosphorylation occurs on different

residues and whereas FLS2 facilitates PTI through RIN4, RIPK mediated phosphorylation dampens

PTI. In Arabidopsis the R protein RPM1 sense RIPK mediated phosphorylation of RIN4 and

instigate ETI (Chung et al., 2011, 2014; Liu et al., 2011).

The fourth effector protein HopF2 inhibits accumulation of PAMP induced phosphorylated RIN4,

dampening PTI (Chung et al., 2014). Besides its interaction with RIN4, HopF2 also targets the

FLS2 co-receptor BAK1 and BIK1, thus HopF2 reduced accumulation of PAMP induced

phosphorylation of RIN4 is possible to occur at multiple levels (Chung et al., 2014; Wang et al.,

2010; Zhou et al., 2014). Expression of HopF2 in Arabidopsis dampens PTI and is not associated

with activation of ETI, therefore HopF2 represents a functional effector protein apparently not

detected by Arabidopsis R proteins.

Detection of AvrB, AvrRpm1 and AvrRpt2 modified RIN4 by RPM1 and RPS2 are examples of

indirect recognition of effector proteins. In this model host proteins are guarded by R proteins that

detect modification of their guardees (Dangl and Jones, 2001; Van Der Biezen and Jones, 1998).

Some R proteins directly recognize effector proteins and trigger ETI, this is true for Arabidopsis R

protein RRS1 which directly recognize the Ralstonia solanacearum effector protein AvrPopP2

(Deslandes et al., 2003).

A third class of R proteins has evolved as decoys, mimicking potential host effector targets. This

hypothesis was first suggested by Hoorn and Kamoun (2008). A direct example of this is the

Arabidopsis R protein RRS1 that contain a WRKY domain normally associated with transcription

factors. The R. solanacearum effector protein PopP2 directly acetylates a group of defense related

WRKY transcription factors inhibiting their DNA binding capabilities. PopP2 also bind the decoy

RRS1, which is paired with another R protein, RPS4 and when PopP2 bind the RRS1 decoy the

RRS1/RPS4 complex activate ETI (Le Roux et al., 2015; Sarris et al., 2015).

Autoimmunity

HR is often associated with ETI and is typically characterized by rapid, localized programmed cell

death at the site of pathogen invasion (Mur et al., 2008). Evidence linking ETI to HR include; (i)

presence of paired effectors and R proteins (ii) over-expression of R proteins and (iii) expression of

auto-active R proteins are all sufficient to in duce HR (Gao et al., 2011; Grant et al., 2000; Peart et

al., 2005; Stokes et al., 2002). Programmed cell death during HR seems to rely on light dependent

14

ROS production in the chloroplasts as inhibition or induction of chloroplast ROS production

respectively delay or increase cell death when applied with virulent pathogens (Zurbriggen et al.,

2010). Pathogen induced HR also appear to depend on autophagy as autophagy deficient mutants do

not display full HR (Hofius et al., 2009; Munch et al., 2015). It is however still unclear whether ETI

triggered HR is cause or a consequence of disease resistance. Some evidence support HR as a cause

of ETI against strictly biotrophic pathogens (Wang et al., 2011). Whereas other findings support

that HR can be uncoupled from ETI (Bendahmane et al., 1999; Bulgarelli et al., 2010; Coll et al.,

2010; Heidrich et al., 2011; Munch et al., 2015).

Autoimmune mutants display constitutive ETI phenotypes. We have previously characterized the

autoimmune mutants accelerated cell death 11 (acd11) and map kinase 4 (mpk4) (Brodersen et al.,

2002; Petersen et al., 2000). ACD11 is a putative sphingolipid transfer protein, but its precise role

during these processes is still unknown. acd11 is a so called lesion mimic mutants as it exhibits

constitutive defense responses and cell death without pathogen perception (Brodersen et al., 2002;

Palma et al., 2010). Activation of the R protein LAZARUS 5 (LAZ5) is responsible for the acd11

phenotype. LAZ5 is activated in absence ACD11, although the mechanism of this detection remains

unknown, but laz5 loss-of-function mutants completely rescue the lesion mimic phenotype (Palma

et al., 2010). The mpk4 mutant similarly exhibits autoimmunity caused at least by activation of the

R protein SUPRESSOR OF MKK1 MKK2 2 (SUMM2) (Petersen et al., 2000; Zhang et al., 2012).

The function of MPK4 is discussed in detail in “MAP Kinases in Arabidopsis innate immunity”. It

is clear that Arabidopsis loss-of-function mutants shadowed by autoimmune phenotypes are often

the result of inappropriate activation or R-proteins. However, since these mutants constitutively

exhibits immune responses they are often categorized as negative regulators of defense due to

pleiotropic effects of ETI (Rodriguez et al., 2015). It is therefore important to elucidate this issue

when investigating the true in vivo function of these proteins.

References

Afzal, A. J., Cunha, L. da, and Mackey, D. (2011). Separable Fragments and Membrane Tethering of Arabidopsis RIN4 Regulate Its Suppression of PAMP-Triggered Immunity. Plant Cell 23, 3798–3811. doi:10.1105/tpc.111.088708.

Axtell, M. J., and Staskawicz, B. J. (2003). Initiation of RPS2-specified disease resistance in Arabidopsis is coupled to the AvrRpt2-directed elimination of RIN4. Cell 112, 369–377.

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Bendahmane, A., Kanyuka, K., and Baulcombe, D. C. (1999). The Rx Gene from Potato Controls Separate Virus Resistance and Cell Death Responses. Plant Cell 11, 781–791. doi:10.1105/tpc.11.5.781.

Boller, T., and Felix, G. (2009). A renaissance of elicitors: perception of microbe-associated molecular patterns and danger signals by pattern-recognition receptors. Annu. Rev. Plant Biol. 60, 379–406.

Boller, T., and He, S. Y. (2009). Innate Immunity in Plants: An Arms Race Between Pattern Recognition Receptors in Plants and Effectors in Microbial Pathogens. Science 324, 742–744. doi:10.1126/science.1171647.

Brodersen, P., Petersen, M., Pike, H. M., Olszak, B., Skov, S., Ødum, N., et al. (2002). Knockout of Arabidopsis ACCELERATED-CELL-DEATH11 encoding a sphingosine transfer protein causes activation of programmed cell death and defense. Genes Dev. 16, 490–502. doi:10.1101/gad.218202.

Bulgarelli, D., Biselli, C., Collins, N. C., Consonni, G., Stanca, A. M., Schulze-Lefert, P., et al. (2010). The CC-NB-LRR-Type Rdg2a Resistance Gene Confers Immunity to the Seed-Borne Barley Leaf Stripe Pathogen in the Absence of Hypersensitive Cell Death. PLoS ONE 5, e12599. doi:10.1371/journal.pone.0012599.

Chen, L.-Q., Hou, B.-H., Lalonde, S., Takanaga, H., Hartung, M. L., Qu, X.-Q., et al. (2010). Sugar transporters for intercellular exchange and nutrition of pathogens. Nature 468, 527–532. doi:10.1038/nature09606.

Chinchilla, D., Zipfel, C., Robatzek, S., Kemmerling, B., Nurnberger, T., Jones, J. D. G., et al. (2007). A flagellin-induced complex of the receptor FLS2 and BAK1 initiates plant defence. Nature 448, 497–500. doi:10.1038/nature05999.

Chung, E.-H., da Cunha, L., Wu, A.-J., Gao, Z., Cherkis, K., Afzal, A. J., et al. (2011). Specific Threonine Phosphorylation of a Host Target by Two Unrelated Type III Effectors Activates a Host Innate Immune Receptor in Plants. Cell Host Microbe 9, 125–136. doi:16/j.chom.2011.01.009.

Chung, E.-H., El-Kasmi, F., He, Y., Loehr, A., and Dangl, J. L. (2014). A Plant Phosphoswitch Platform Repeatedly Targeted by Type III Effector Proteins Regulates the Output of Both Tiers of Plant Immune Receptors. Cell Host Microbe 16, 484–494. doi:10.1016/j.chom.2014.09.004.

Coll, N. S., Vercammen, D., Smidler, A., Clover, C., van Breusegem, F., Dangl, J. L., et al. (2010). Arabidopsis Type I Metacaspases Control Cell Death. Science. doi:10.1126/science.1194980.

Cui, H., Tsuda, K., and Parker, J. E. (2014). Effector-Triggered Immunity: From Pathogen Perception to Robust Defense. Annu. Rev. Plant Biol. doi:10.1146/annurev-arplant-050213-040012.

Cunnac, S., Lindeberg, M., and Collmer, A. (2009). Pseudomonas syringae type III secretion system effectors: repertoires in search of functions. Curr. Opin. Microbiol. 12, 53–60. doi:10.1016/j.mib.2008.12.003.

16

Dangl, J. L., and Jones, J. D. G. (2001). Plant pathogens and integrated defence responses to infection. Nature 411, 826–833. doi:10.1038/35081161.

Deslandes, L., Olivier, J., Peeters, N., Feng, D. X., Khounlotham, M., Boucher, C., et al. (2003). Physical interaction between RRS1-R, a protein conferring resistance to bacterial wilt, and PopP2, a type III effector targeted to the plant nucleus. Proc. Natl. Acad. Sci. 100, 8024–8029. doi:10.1073/pnas.1230660100.

Gao, Z., Chung, E.-H., Eitas, T. K., and Dangl, J. L. (2011). Plant intracellular innate immune receptor Resistance to Pseudomonas syringae pv. maculicola 1 (RPM1) is activated at, and functions on, the plasma membrane. Proc. Natl. Acad. Sci. 108, 7619–7624. doi:10.1073/pnas.1104410108.

Gómez-Gómez, L., and Boller, T. (2000). FLS2: An LRR Receptor-like Kinase Involved in the Perception of the Bacterial Elicitor Flagellin in Arabidopsis. Mol. Cell 5, 1003–1011. doi:10.1016/S1097-2765(00)80265-8.

Grant, M., Brown, I., Adams, S., Knight, M., Ainslie, A., and Mansfield, J. (2000). The RPM1 plant disease resistance gene facilitates a rapid and sustained increase in cytosolic calcium that is necessary for the oxidative burst and hypersensitive cell death. Plant J. 23, 441–450. doi:10.1046/j.1365-313x.2000.00804.x.

Heidrich, K., Wirthmueller, L., Tasset, C., Pouzet, C., Deslandes, L., and Parker, J. E. (2011). Arabidopsis EDS1 Connects Pathogen Effector Recognition to Cell Compartment–Specific Immune Responses. Science 334, 1401–1404. doi:10.1126/science.1211641.

Hofius, D., Schultz-Larsen, T., Joensen, J., Tsitsigiannis, D. I., Petersen, N. H. T., Mattsson, O., et al. (2009). Autophagic Components Contribute to Hypersensitive Cell Death in Arabidopsis. Cell 137, 773–783. doi:10.1016/j.cell.2009.02.036.

Hoorn, R. A. L. van der, and Kamoun, S. (2008). From Guard to Decoy: A New Model for Perception of Plant Pathogen Effectors. Plant Cell 20, 2009–2017. doi:10.1105/tpc.108.060194.

Hutin, M., Pérez-Quintero, A. L., Lopez, C., and Szurek, B. (2015). MorTAL Kombat: the story of defense against TAL effectors through loss-of-susceptibility. Plant Biot. Interact., 535. doi:10.3389/fpls.2015.00535.

Kadota, Y., Shirasu, K., and Zipfel, C. (2015). Regulation of the NADPH Oxidase RBOHD During Plant Immunity. Plant Cell Physiol. 56, 1472–1480. doi:10.1093/pcp/pcv063.

Kadota, Y., Sklenar, J., Derbyshire, P., Stransfeld, L., Asai, S., Ntoukakis, V., et al. (2014). Direct Regulation of the NADPH Oxidase RBOHD by the PRR-Associated Kinase BIK1 during Plant Immunity. Mol. Cell 54, 43–55. doi:10.1016/j.molcel.2014.02.021.

Katagiri, F., and Tsuda, K. (2010). Understanding the Plant Immune System. Mol. Plant. Microbe Interact. 23, 1531–1536. doi:10.1094/MPMI-04-10-0099.

Krol, E., Mentzel, T., Chinchilla, D., Boller, T., Felix, G., Kemmerling, B., et al. (2010). Perception of the Arabidopsis Danger Signal Peptide 1 Involves the Pattern Recognition Receptor

17

AtPEPR1 and Its Close Homologue AtPEPR2. J. Biol. Chem. 285, 13471–13479. doi:10.1074/jbc.M109.097394.

Kunze, G., Zipfel, C., Robatzek, S., Niehaus, K., Boller, T., and Felix, G. (2004). The N Terminus of Bacterial Elongation Factor Tu Elicits Innate Immunity in Arabidopsis Plants. Plant Cell 16, 3496–3507. doi:10.1105/tpc.104.026765.

Lacombe, S., Rougon-Cardoso, A., Sherwood, E., Peeters, N., Dahlbeck, D., van Esse, H. P., et al. (2010). Interfamily transfer of a plant pattern-recognition receptor confers broad-spectrum bacterial resistance. Nat. Biotechnol. 28, 365–369. doi:10.1038/nbt.1613.

Le Roux, C., Huet, G., Jauneau, A., Camborde, L., Trémousaygue, D., Kraut, A., et al. (2015). A Receptor Pair with an Integrated Decoy Converts Pathogen Disabling of Transcription Factors to Immunity. Cell 161, 1074–1088. doi:10.1016/j.cell.2015.04.025.

Li, L., Li, M., Yu, L., Zhou, Z., Liang, X., Liu, Z., et al. (2014). The FLS2-Associated Kinase BIK1 Directly Phosphorylates the NADPH Oxidase RbohD to Control Plant Immunity. Cell Host Microbe 15, 329–338. doi:10.1016/j.chom.2014.02.009.

Liu, J., Elmore, J. M., Lin, Z.-J. D., and Coaker, G. (2011). A Receptor-like Cytoplasmic Kinase Phosphorylates the Host Target RIN4, Leading to the Activation of a Plant Innate Immune Receptor. Cell Host Microbe 9, 137–146. doi:16/j.chom.2011.01.010.

Lodish, H., Berk, A., Zipursky, S. L., Matsudaira, P., Baltimore, D., and Darnell, J. (2000). CO2 Metabolism during Photosynthesis. Available at: http://www.ncbi.nlm.nih.gov/books/NBK21472/ [Accessed December 23, 2015].

Lu, D., Wu, S., Gao, X., Zhang, Y., Shan, L., and He, P. (2010). A receptor-like cytoplasmic kinase, BIK1, associates with a flagellin receptor complex to initiate plant innate immunity. Proc. Natl. Acad. Sci. 107, 496–501. doi:10.1073/pnas.0909705107.

Mackey, D., Belkhadir, Y., Alonso, J. M., Ecker, J. R., and Dangl, J. L. (2003). Arabidopsis RIN4 is a target of the type III virulence effector AvrRpt2 and modulates RPS2-mediated resistance. Cell 112, 379–389.

Mackey, D., Holt III, B. F., Wiig, A., and Dangl, J. L. (2002). RIN4 interacts with Pseudomonas syringae type III effector molecules and is required for RPM1-mediated resistance in Arabidopsis. Cell 108, 743–754.

Malinovsky, F. G., Fangel, J. U., and Willats, W. G. T. (2014). The role of the cell wall in plant immunity. Plant Biot. Interact. 5, 178. doi:10.3389/fpls.2014.00178.

Miller, G., Schlauch, K., Tam, R., Cortes, D., Torres, M. A., Shulaev, V., et al. (2009). The Plant NADPH Oxidase RBOHD Mediates Rapid Systemic Signaling in Response to Diverse Stimuli. Sci Signal 2, ra45–ra45. doi:10.1126/scisignal.2000448.

Munch, D., Teh, O.-K., Malinovsky, F. G., Liu, Q., Vetukuri, R. R., Kasmi, F. E., et al. (2015). Retromer Contributes to Immunity-Associated Cell Death in Arabidopsis. Plant Cell 27, 463–479. doi:10.1105/tpc.114.132043.

18

Mur, L. A. J., Kenton, P., Lloyd, A. J., Ougham, H., and Prats, E. (2008). The hypersensitive response; the centenary is upon us but how much do we know? J. Exp. Bot. 59, 501–520. doi:10.1093/jxb/erm239.

Palma, K., Thorgrimsen, S., Malinovsky, F. G., Fiil, B. K., Nielsen, H. B., Brodersen, P., et al. (2010). Autoimmunity in Arabidopsis acd11 Is Mediated by Epigenetic Regulation of an Immune Receptor. PLoS Pathog 6, e1001137. doi:10.1371/journal.ppat.1001137.

Peart, J. R., Mestre, P., Lu, R., Malcuit, I., and Baulcombe, D. C. (2005). NRG1, a CC-NB-LRR Protein, together with N, a TIR-NB-LRR Protein, Mediates Resistance against Tobacco Mosaic Virus. Curr. Biol. 15, 968–973. doi:10.1016/j.cub.2005.04.053.

Petersen, M., Brodersen, P., Naested, H., Andreasson, E., Lindhart, U., Johansen, B., et al. (2000). Arabidopsis MAP Kinase 4 Negatively Regulates Systemic Acquired Resistance. Cell 103, 1111–1120. doi:10.1016/S0092-8674(00)00213-0.

Qi, Y., Tsuda, K., Glazebrook, J., and Katagiri, F. (2011). Physical association of pattern-triggered immunity (PTI) and effector-triggered immunity (ETI) immune receptors in Arabidopsis. Mol. Plant Pathol. 12, 702–708. doi:10.1111/j.1364-3703.2010.00704.x.

Rodriguez, E., el Ghoul, H., Mundy, J., and Petersen, M. (2015). Making sense of plant autoimmunity and “negative regulators.” FEBS J., n/a–n/a. doi:10.1111/febs.13613.

Roux, M., Schwessinger, B., Albrecht, C., Chinchilla, D., Jones, A., Holton, N., et al. (2011). The Arabidopsis Leucine-Rich Repeat Receptor–Like Kinases BAK1/SERK3 and BKK1/SERK4 Are Required for Innate Immunity to Hemibiotrophic and Biotrophic Pathogens. Plant Cell Online 23, 2440–2455. doi:10.1105/tpc.111.084301.

Sarris, P. F., Duxbury, Z., Huh, S. U., Ma, Y., Segonzac, C., Sklenar, J., et al. (2015). A Plant Immune Receptor Detects Pathogen Effectors that Target WRKY Transcription Factors. Cell 161, 1089–1100. doi:10.1016/j.cell.2015.04.024.

Schulze, B., Mentzel, T., Jehle, A. K., Mueller, K., Beeler, S., Boller, T., et al. (2010). Rapid Heteromerization and Phosphorylation of Ligand-activated Plant Transmembrane Receptors and Their Associated Kinase BAK1. J. Biol. Chem. 285, 9444–9451. doi:10.1074/jbc.M109.096842.

Selote, D., and Kachroo, A. (2010). RIN4-like proteins mediate resistance protein-derived soybean defense against Pseudomonas syringae. Plant Signal. Behav. 5, 1453–1456. doi:10.4161/psb.5.11.13462.

Stokes, T. L., Kunkel, B. N., and Richards, E. J. (2002). Epigenetic variation in Arabidopsis disease resistance. Genes Dev. 16, 171–182. doi:10.1101/gad.952102.

Torres, M. A., Jones, J. D. G., and Dangl, J. L. (2005). Pathogen-induced, NADPH oxidase–derived reactive oxygen intermediates suppress spread of cell death in Arabidopsis thaliana. Nat. Genet. 37, 1130–1134. doi:10.1038/ng1639.

Torres, M. A., Jones, J. D. G., and Dangl, J. L. (2006). Reactive Oxygen Species Signaling in Response to Pathogens. Plant Physiol. 141, 373–378. doi:10.1104/pp.106.079467.

19

Van Der Biezen, E. A., and Jones, J. D. G. (1998). Plant disease-resistance proteins and the gene-for-gene concept. Trends Biochem. Sci. 23, 454–456. doi:10.1016/S0968-0004(98)01311-5.

Wang, W., Barnaby, J. Y., Tada, Y., Li, H., Tör, M., Caldelari, D., et al. (2011). Timing of plant immune responses by a central circadian regulator. Nature 470, 110–114. doi:10.1038/nature09766.

Wang, Y., Li, J., Hou, S., Wang, X., Li, Y., Ren, D., et al. (2010). A Pseudomonas syringae ADP-Ribosyltransferase Inhibits Arabidopsis Mitogen-Activated Protein Kinase Kinases. Plant Cell 22, 2033–2044. doi:10.1105/tpc.110.075697.

Wilton, M., Subramaniam, R., Elmore, J., Felsensteiner, C., Coaker, G., and Desveaux, D. (2010). The type III effector HopF2 Pto targets Arabidopsis RIN4 protein to promote Pseudomonas syringae virulence. Proc. Natl. Acad. Sci. 107, 2349–2354. doi:10.1073/pnas.0904739107.

Yang, B., Sugio, A., and White, F. F. (2006). Os8N3 is a host disease-susceptibility gene for bacterial blight of rice. Proc. Natl. Acad. Sci. 103, 10503–10508. doi:10.1073/pnas.0604088103.

Yuan, M., Chu, Z., Li, X., Xu, C., and Wang, S. (2010). The Bacterial Pathogen Xanthomonas oryzae Overcomes Rice Defenses by Regulating Host Copper Redistribution. Plant Cell 22, 3164–3176. doi:10.1105/tpc.110.078022.

Zhang, J., Li, W., Xiang, T., Liu, Z., Laluk, K., Ding, X., et al. (2010). Receptor-like Cytoplasmic Kinases Integrate Signaling from Multiple Plant Immune Receptors and Are Targeted by a Pseudomonas syringae Effector. Cell Host Microbe 7, 290–301. doi:10.1016/j.chom.2010.03.007.

Zhang, Z., Wu, Y., Gao, M., Zhang, J., Kong, Q., Liu, Y., et al. (2012). Disruption of PAMP-Induced MAP Kinase Cascade by a Pseudomonas syringae Effector Activates Plant Immunity Mediated by the NB-LRR Protein SUMM2. Cell Host Microbe 11, 253–263. doi:10.1016/j.chom.2012.01.015.

Zhou, J., Wu, S., Chen, X., Liu, C., Sheen, J., Shan, L., et al. (2014). The Pseudomonas syringae effector HopF2 suppresses Arabidopsis immunity by targeting BAK1. Plant J. 77, 235–245. doi:10.1111/tpj.12381.

Zipfel, C., Kunze, G., Chinchilla, D., Caniard, A., Jones, J. D. G., Boller, T., et al. (2006). Perception of the Bacterial PAMP EF-Tu by the Receptor EFR Restricts Agrobacterium-Mediated Transformation. Cell 125, 749–760. doi:10.1016/j.cell.2006.03.037.

Zurbriggen, M. D., Carrillo, N., and Hajirezaei, M.-R. (2010). ROS signaling in the hypersensitive response. Plant Signal. Behav. 5, 393–396.

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MINI REVIEW ARTICLEpublished: 24 July 2012

doi: 10.3389/fpls.2012.00169

MAP kinase cascades in Arabidopsis innate immunityMagnus W. Rasmussen, Milena Roux, Morten Petersen and John Mundy*

Department of Biology, University of Copenhagen, Copenhagen, Denmark

Edited by:

Alex Jones, The Sainsbury Laboratory,UK

Reviewed by:

Birgit Kersten, Johann Heinrich vonThünen Institute, GermanyJesus V. Jorrin Novo, University ofCordoba, Spain

*Correspondence:

John Mundy, Department ofBiology, University of Copenhagen,Ole Maaløes Vej 5, 2200Copenhagen, Denmark.e-mail: [email protected]

Plant mitogen-activated protein kinase (MAPK) cascades generally transduce extracellularstimuli into cellular responses.These stimuli include the perception of pathogen-associatedmolecular patterns (PAMPs) by host transmembrane pattern recognition receptors whichtrigger MAPK-dependent innate immune responses. In the model Arabidopsis, moleculargenetic evidence implicates a number of MAPK cascade components in PAMP signaling,and in responses to immunity-related phytohormones such as ethylene, jasmonate, and sal-icylate. In a few cases, cascade components have been directly linked to the transcriptionof target genes or to the regulation of phytohormone synthesis. Thus MAPKs are obvioustargets for bacterial effector proteins and are likely guardees of resistance proteins, whichmediate defense signaling in response to the action of effectors, or effector-triggered immu-nity.This mini-review discusses recent progress in this field with a focus on the ArabidopsisMAPKs MPK3, MPK4, MPK6, and MPK11 in their apparent pathways.

Keywords: calcium signaling, hypersensitive response, MAP kinase cascade, MAP kinase substrates, pathogen

effectors, pattern recognition receptors, reactive oxygen species, resistance proteins

INTRODUCTIONPlants have evolved an effective basal defense system to detectand limit the growth of pathogens. Pathogens may be recognizedby the host via the perception of conserved microbial structurestermed pathogen-associated molecular patterns (PAMPs). PAMPsare recognized via transmembrane pattern recognition recep-tors (PRRs) that bind specific PAMPs and initiate intracellularimmune responses (Zipfel, 2008). These PAMP-triggered immu-nity (PTI) responses include the generation of reactive oxygenspecies (ROS), extracellular alkalinization, and protein phospho-rylation with associated gene regulation that ultimately restrictsthe growth of the microbial intruder (Gimenez-Ibanez andRathjen, 2010).

Mitogen-activated protein kinase (MAPK) signaling plays cen-tral roles in such intracellular immunity pathways. In general,MAP kinase signaling is initiated by the stimulus-triggered activa-tion of a MAP kinase kinase kinase (MAP3K; also called MEKK).MAP3K activation, which may be directly or indirectly effectedby a PRR, in turn leads to the phosphorylation and activationof downstream MAP kinase kinases (MAP2K; also called MKK orMEK). Subsequently, the MAP2K phosphorylates the downstreamMAPK sequentially leading to changes in its subcellular localiza-tion and/or phosphorylation of downstream substrates includingtranscription factors which alter patterns of gene expression (seeFigure 1). General functions of MAPK cascades in plant biologyhave recently been reviewed elsewhere (Fiil et al., 2009; Rodriguezet al., 2010; Komis et al., 2011).

MAPK CASCADES IN PTIA few PRRs have been documented to stimulate MAPK signalingupon perception of PAMPs. These include the flagellin receptorFLS2 (Felix et al., 1999; Gómez-Gómez and Boller, 2000), the bac-terial elongation factor EF-Tu receptor EFR (Zipfel et al., 2006),and the chitin receptor CERK1 (Miya et al., 2007).

The Arabidopsis genome encodes 60 MAP3Ks, 10 MAP2Ks,and 20 MAPKs (Ichimura et al., 2002). This indicates that MAPKcascades may not simply consist of single MAP3Ks, MAP2Ks,and MAPKs connected together. Instead, it suggests that thereis some level of redundancy, and that the spatial and temporalactivities of different components may be strictly regulated tominimize wanton cross-talk. The three MAPKs MPK3, MPK4,and MPK6 are the most intensively studied plant MAPKs, and allthree were implicated in defense signaling a decade ago (Petersenet al., 2000; Asai et al., 2002). MPK11, a close homolog to MPK4,has also recently been shown to be activated by PAMP treatment(Bethke et al., 2012).

MPK3, MPK4, and MPK6 are all activated by PAMPs suchas flg22 (a conserved 22 amino acid flagellin peptide) and elf18(elongation factor-Tu peptide; Felix et al., 1999; Zipfel et al.,2006). However, these three MAPK cascades are differently reg-ulated already at the PRR level. For example, the two receptorkinases BAK1 and BKK1 genetically regulate PAMP signalingthrough their interactions with cognate PRRs (Roux et al., 2011;Schwessinger et al., 2011). The BAK1 mutant allele bak1-5 car-ries a Cys408Tyr substitution adjacent to its kinase catalytic loop.This impairs its flg22-regulated kinase activity and inhibits phos-phorylation of MPK4. However, the catalytic complex formedbetween mutant BAK1 in bak1-5 and FLS2 is still able to inducephosphorylation of MPK3/MPK6 (Roux et al., 2011; Schwessingeret al., 2011). Interestingly, MPK3/MPK6 phosphorylation wasimpaired in only the double bak1-5 bkk1 background and notin the individual bak1-5 and bkk1 lines (Roux et al., 2011).

Asai et al. (2002) developed an elegant protoplast expressionsystem in an attempt to identify signaling components down-stream of FLS2. With this system they were able to show acomplete MAPK cascade downstream of FLS2 consisting of theMAP3K MEKK1, two MAP2Ks (MKK4 and MKK5), and theMAPKs MPK3/MPK6. However, genetic evidence later showed

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Rasmussen et al. MAP kinase cascades in immunity

FIGURE 1 | (A) MAPK signaling cascades are attractive targets forbacterial effectors. The P. syringae HopAI1 effector irreversibly inactivatesMPK4 to prevent immune responses. The R protein SUMM2 may guardprocesses downstream of MPK4 independent from MKS1, and triggersa hypersensitive response in the event of loss or inactivation of MPK4.(B) PAMP perception by PRRs instigates a signaling cascade, often via

co-receptors, which causes activation of MAP3K MEKK1 and twoMAP2Ks MKK1 and MKK2. These phosphorylate and activate MPK4which then phosphorylates its substrate MKS1, releasing MKS1 incomplex with WRKY33. MPK3/MPK6 sequentially phosphorylateWRKY33 allowing it to promote PAD3 transcription, thus activatingplant defense.

that MEKK1 kinase activity was dispensable for MPK3/MPK6activation, although mekk1 plants were impaired in MPK4 acti-vation (Rodriguez et al., 2007). Interestingly, expressing a kinasedead version of MEKK1 in mekk1 plants completely restored theactivation of MPK4 upon treatment with flg22, suggesting thatMEKK1 may “simply” act as a scaffold protein (Rodriguez et al.,2007). Biochemical and genetic studies further revealed that thetwo MAP2Ks MKK1 and MKK2 interact with both MEKK1 andwith MPK4, and that flg22-induced MPK4 activation is impairedin the double mkk1 mkk2 mutant. This indicates that MKK1 andMKK2 are partially redundant in MPK4 mediated downstreamsignaling (Gao et al., 2008; Qiu et al., 2008b).

MPK4 was originally reported as a negative regulator of plantimmunity because the mpk4 mutant accumulates high levels ofsalicylic acid, constitutively expresses pathogenesis-related (PR)genes, and has a severely dwarfed growth phenotype (Petersenet al., 2000). This phenotype is very similar to that of the mekk1single and mkk1 mkk2 double mutants, further supporting theirfunctional relationships (Rodriguez et al., 2007; Gao et al., 2008;Qiu et al., 2008b).

MAPK CASCADES IN EFFECTOR-TRIGGERED IMMUNITYIn addition to PTI, plants also employ resistance (R) proteins as cytoplasmic receptors to directly or indirectly recognize specific pathogenic effector proteins injected into host cells as virulence

factors. Effector-triggered immunity (ETI) and PTI share a num-ber of responses, although ETI also includes varying levels of rapid,localized cell death in what is called the hypersensitive response. Rprotein-dependent recognition initiates immune responses in ETI.R proteins may recognize effector proteins either directly or indi-rectly by monitoring changes in the effector’s host target(s). Thislatter case gave rise to the guard hypothesis in which R proteinsguard host guardees that are manipulated by pathogen effectors(Van Der Biezen and Jones, 1998).

The genetic characterization of the MEKK1/MKK1–MKK2/MPK4 cascade as a negative regulatory pathway of defenseresponses was at odds with the activation of the pathway byPAMPs. Instead, it was possible that the severe phenotypes ofthe kinase knockout mutants were caused by activation of one ormore R protein(s) guarding this kinase pathway. Indeed, in anelegant screen for suppressors of the mkk1 mkk2 double mutant,Zhang et al. (2012) identified the R protein SUMM2 (suppressorof mkk1 mkk2). The T-DNA insertion line summ2-8 completelysuppressed the severe mkk1 mkk2 phenotype in respect to mor-phology, cell death, ROS levels and PR gene expression (Zhanget al., 2012). The analogous knockout phenotype of the upstreamMAP3K mekk1 is also completely suppressed in the summ2-8 back-ground. Interestingly, although the mpk4 mutant shares a similarphenotype with the knockouts of its upstream kinase partners,the mpk4 phenotype is not fully suppressed by the summ2-8

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mutation, as double mpk4 summ2-8 mutants still retain resid-ual cell death and low levels of ROS. This suggests that MPK4is involved in other pathways independent of SUMM2, and thatMPK4 may be guarded by additional R proteins (Zhang et al., 2012;Figure 1A).

The importance of MAPK signaling in immunity is empha-sized by studies reporting bacterial effector proteins targetingMAPK cascades for downregulation (Zhang et al., 2007a,b, 2012;Cui et al., 2010). For example, the Pseudomonas syringae effectorprotein HopAI1 targets and irreversibly inactivates MPK3, MPK4,and MPK6, thereby suppressing immune responses which wouldotherwise inhibit bacterial growth (Zhang et al., 2007a, 2012). Inaddition, the P. syringae effector protein AvrB has been shown tointeract with and induce the phosphorylation of MPK4, althoughit has not been shown if this phosphorylation occurs as a directeffect of AvrB action or via recognition of AvrB by the plantimmune system (Cui et al., 2010).

In plants carrying functional SUMM2 alleles, immuneresponses are activated by bacterial effector proteins targeting theMPK4 pathway (Figure 1A). For example, inducible expressionof the bacterial HopAI1 effector in wild-type plants gives rise toa defense phenotype similar to that seen in mekk1, mkk1 mkk2,and mpk4 mutants including elevated levels of ROS, PR geneexpression, and cell death (Zhang et al., 2012). SUMM2 appar-ently does not interact directly with the kinase components of theMEKK1/MKK1–MKK2/MPK4 signaling cascade, suggesting thatSUMM2 most likely guards a downstream target of MPK4 activity(Zhang et al., 2012). At present, the best studied in vivo substrateof MPK4 activity is MPK4 substrate 1 (MKS1) which forms anuclear complex with MPK4 and the WRKY33 transcription fac-tor (Andreasson et al., 2005; Qiu et al., 2008a). Phosphorylationof MKS1 follows MPK4 activation by flg22 perception and, oncephosphorylated, MKS1 is released from complexes with MPK4,thereby releasing the WRKY33 transcription factor to bind to itscognate target genes (Qiu et al., 2008a). It has therefore been pro-posed that MPK4 and MKS1 sequester WRKY33 in the absence ofpathogens, and free WRKY33 to induce resistance upon pathogenperception (Figure 1B, left).

As MKS1 is the only known direct target of MPK4, Zhanget al. (2012) tested whether MKS1 interacted with the R pro-tein SUMM2 that seemingly guards MPK4 activity. However, nointeraction between SUMM2 and MKS1 was detected. Since mks1mutants have a wild-type growth phenotype, and the mpk4 phe-notype is strongly suppressed in the mks1 background, SUMM2may guard a process downstream of MPK4 that is independent ofMKS1 (Petersen et al., 2010).

WRKY TRANSCRIPTION FACTORSThe plant-specific WRKY family is a large group of transcrip-tion factors which bind a conserved W-box sequence in thepromoters of numerous genes including those encoding PRproteins. WRKY33 was found to induce the transcription of PHY-TOALEXIN DEFICIENT 3 (PAD3) which encodes the cytochromeP450 monooxygenase 71B15 required for synthesis of the antimi-crobial compound camalexin (Zhou et al., 1999; Qiu et al., 2008a;Figure 1B). The wrky33 mutant exhibits enhanced susceptibil-ity toward necrotrophic pathogens such as Botrytis cinerea, while

WRKY33 overexpression results in increased resistance due toenhanced PAD3 expression (Zheng et al., 2006).

MPK3 and MPK6 activities also induce the production ofcamalexin. Transient overexpression of the constitutively active,phospho-mimic mutant forms of MKK4/MKK5 (MKK4DD andMKK5DD), which are the upstream MAP2Ks of MPK3/MPK6,has been reported to induce transcription of both PAD2, whichencodes γ-glutamylcysteine synthetase functioning in glutathionebiosynthesis, and PAD3. Both PAD2 and PAD3 are necessaryfor camalexin production (Parisy et al., 2007; Ren et al., 2008).Pathogen-induced camalexin accumulation is partially comprisedin mpk3 but not notably in mpk6 mutants, yet camalexin accu-mulation in mpk3 mpk6 double mutants is almost completelyabolished (Ren et al., 2008). While this implicates MPK3/MPK6in camalexin synthesis, caution should be applied in evaluat-ing results obtained from the mpk3 mpk6 double mutant as itis arrested at the cotyledon stage and is unable to initiate trueleaves (Wang et al., 2007). Upstream of MPK3/MPK6 in camalexininduction, MKK4 and MKK5 are activated by the MAP3KsMEKK1 and MAPKKKα in response to fungal pathogens (Renet al., 2008). Yet another MAP2K, MKK9, whose upstreamMAP3K(s) remains unidentified, is also involved in MPK3/MPK6activation, as plants expressing phospho-mimic MKK9DD pro-duce even more camalexin than plants expressing MKK4DD orMKK5DD (Xu et al., 2008).

To delineate the link between MPK3/MPK6 activation andcamalexin accumulation, Mao et al. (2011) elegantly introducedthe phospho-mimic mutant NtMEK2DD, an MKK4 and/or MKK5ortholog from Nicotiana tabacum, into an array of different wrkymutants in a search for essential transcription factors involvedin MPK3/MPK6 mediated camalexin induction. Interestingly,NtMEKK2DD was able to induce camalexin accumulation in alltested mutant lines except wrky33. In addition, WRKY33 provedto be a substrate of MKP3/MPK6 activity, and overexpression ofnon-phosphorylatable forms of WRKY33 could not fully com-plement the inability of wrky33 mutants to express PAD3 andaccumulate camalexin (Mao et al., 2011; Figure 1B, right).

WRKY33-induced PAD3 expression therefore appears toinvolve both MPK4- and MPK3/MPK6-mediated signaling(Andreasson et al., 2005; Qiu et al., 2008a; Mao et al., 2011). Maoet al. (2011) proposed a model in which PAD3-mediated camalexininduction occurs differentially depending on the type of pathogencausing the immune response. In this model, bacterial pathogensinduce an MPK4 mediated response while fungal pathogens ini-tiate an MPK3/MPK6 mediated response. This hypothesis isbased on overexpression of the constitutively active MKK4/MKK5ortholog NtMEKK2DD, rendering MPK3/MPK6 hyperactive andable to induce PAD3 expression (Mao et al., 2011). In support ofthis hypothesis, the mpk3 mpk6 double mutant is comprised in B.cinerea-induced PAD3 induction (Ren et al., 2008). Nonetheless,and as noted above, some care should be taken with experimentsbased on mpk3 mpk6 double mutants given their developmentallethality (Wang et al., 2007).

An alternative model may therefore be proposed which com-bines the MPK4 and MPK3/MPK6 pathways into a dual control ofPAD3 regulation in response to pathogen perception (Figure 1B).In such a model, WRKY33 is sequestered in a nuclear complex

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comprising at least MPK4 and MKS1 in unchallenged plants,and is released following PAMP perception (Qiu et al., 2008a).Phosphorylation is dispensable for WRKY33 to bind its cognateW-box cis-elements, although it does promote transcriptionalactivation (Mao et al., 2011). This is illustrated by the fact thatPAD3 expression is induced in mpk4 plants (Qiu et al., 2008a),perhaps due to the basal activity of free non-phosphorylatedWRKY33 or by free WRKY33 activated by basal MPK3 and/orMPK6 activity. In this scenario, once WRKY33 is released fromits nuclear complex with MPK4 and MKS1, it is phosphorylatedand hence activated by MPK3/MPK6, thereby inducing camalexinlevels through PAD3 expression. The elevated PAD3 expressioninduced from NtMEKK2DD hyper-activated MPK3/MPK6 (Maoet al., 2011) is not in conflict with this model, as it is likely thathyperactive MPK3/MPK6 are able to phosphorylate residual freeWRKY33, thus bypassing other possible feedback mechanisms inPAD3 expression.

In this model, MPK4 and MPK3/MPK6 function togetheras a binary switch conferring dual level regulation. Clarifica-tion of the mode of action in which MPK4 and MPK3/MPK6function clearly needs further elucidation and should includeexperiments using catalytically inactive and/or inactivatableMPK4 (Petersen et al., 2000; Brodersen et al., 2006). Applica-tion of fungal PAMPs to plants expressing catalytically inactiveMPK4 might indicate whether phosphorylation of free WRKY33by endogenous MPK3/MPK6 is enough to induce expressionof PAD3.

MAPK IN GENERAL STRESS SIGNALINGThe refined work of Popescu et al. (2009) identified a MAP2K–MAPK phosphorylation network covering 570 MAPK substratesby combinatorially pairing active MAP2Ks with MAPKs, and thensubjecting them to a protein microarray phosphorylation assay.Interestingly, the substrates identified were enriched for tran-scription factors involved in stress responses. Notably, MPK6phosphorylated 32% of the identified targets, of which 40%overlapped with MPK3 targets (Popescu et al., 2009). This isin agreement with earlier data, similarly obtained from a pro-tein microarray study (Feilner et al., 2005). Equally noteworthyis the finding that MPK3 also shared 50% of its targets withMPK4, revealing intensive synergy in MAPK signaling (Popescuet al., 2009).

In addition to MAPK cascades, ROS also play a pivotal role instress signaling (Rodriguez et al., 2010). OXI1, a serine/threoninekinase induced by general ROS-generating stimuli, is required forfull activation of MPK3/MPK6 after treatment with H2O2 (Rentelet al., 2004). Although OXI1 is characterized as an upstream regu-lator of MPK3/MPK6 activation, MPK3/MPK6 have been shownto phosphorylate OXI1 in vitro. This suggests that there is a

feedback loop, but in vivo data supporting such a loop has notbeen shown (Forzani et al., 2011).

In addition to MAPK cascade signaling, PAMP perception alsoinduces Ca2+ dependent kinases (CDPKs) by regulating Ca2+influx channels (Ma et al., 2009; Kwaaitaal et al., 2011). Recentfindings indicate that Ca2+ ATPases regulate Ca2+ efflux and func-tion to regulate innate immune defenses (Zhu et al., 2010). Ofparticular interest is the Ca2+ ATPase ACA8 which was shown tointeract with FLS2, and which may well regulate CDPK signalingthrough flg22 perception (Frei dit Frey et al., 2012).

MPK8 activity has been shown to negatively regulate the expres-sion of OXI1 in order to maintain ROS homeostasis. Remarkably,activation of MPK8 is not limited to the upstream MAP2K MKK3,as the Ca2+ binding protein calmodulin (CaM) is able to bind andactivate MPK8 in an Ca2+-dependent manner (Takahashi et al.,2011). CaM-mediated MPK8 activation is interesting because itbypasses the traditional, sequential activation of MAPKs and alsounequivocally links MAPK activation with the ROS burst andion flux during stress signaling. In addition, CaM also medi-ates MAPK downregulation. MAP kinase phosphatase 1 (MKP1),which interacts with MPK3, MPK4, and MPK6 (Ulm et al., 2002),binds CaM in a Ca2+-dependent manner and stimulates MKP1phosphatase activity (Lee et al., 2008). The associations betweenCDPKs and MAPK cascades have recently been review elsewhere(Wurzinger et al., 2011).

Much progress has been made in understanding how MAPKsignaling functions in plant immunity. In Arabidopsis, 3 of the 60identified MAP3Ks are involved in defense, namely MEKK1 (Asaiet al., 2002), EDR1 (Frye et al., 2001), and MEKKα (del Pozo et al.,2004; Ren et al., 2008). In addition, at least 6 of the 10 identifiedMAP2Ks (MKK1, MKK2, MKK4, MKK5, MKK7, and MKK9) areinvolved in defense signaling (Asai et al., 2002; Djamei et al., 2007;Dóczi et al., 2007; Zhang et al., 2007b; Yoo et al., 2008). This situ-ation requires tight regulation of the spatial and temporal kinaseactivities in order to impose specificity upon downstream signal-ing. To shed light on this regulation, high-throughput methodssuch as those used by Popescu et al. (2009) are particularly valuableand help to outline MAPK signaling cascades. While this progressmay be lauded, further work needs to focus on identifying direct,in vivo kinase substrates and their respective phosphorylation sites.This may bring us closer to bridging the apparent gap betweenPRRs and MAPK cascades, and to understanding how specificityis achieved among MAPK pathways both spatially and temporally.

ACKNOWLEDGMENTSThis work was supported by grants to John Mundy from the Dan-ish Research Council for Technology and Production (23-03-0076) and the Strategic Research Council (09-067148) and to Milena Roux from the Natural Science Council (11-116368).

REFERENCESAndreasson, E., Jenkins, T., Broder-

sen, P., Thorgrimsen, S., Petersen, N.H., Zhu, S., Qiu, J.-L., Micheelsen,P., Rocher, A., Petersen, M., New-man, M. A., Bjørn Nielsen, H., Hirt,H., Somssich, I., Mattsson, O., andMundy, J. (2005). The MAP kinase

substrate MKS1 is a regulator ofplant defense responses. EMBO J. 24,2579–2589.

Asai, T., Tena, G., Plotnikova, J., Will-mann, M. R., Chiu, W. L., Gomez-Gomez, L., Boller, T., Ausubel,F. M., and Sheen, J. (2002).MAP kinase signalling cascade in

Arabidopsis innate immunity. Nature415, 977–983.

Bethke, G., Pecher, P., Eschen-Lippold, L., Tsuda, K., Katagiri,F., Glazebrook, J., Scheel, D.,and Lee, J. (2012). Activation ofthe Arabidopsis thaliana mitogen-activated protein kinase MPK11 by

the flagellin-derived elicitor peptide,flg22. Mol. Plant Microbe Interact. 25,471–480.

Brodersen, P., Petersen, M., Nielsen, H.B., Zhu, S., Newman, M., Shokat, K.M., Rietz, S., Parker, J., and Mundy,J. (2006). Arabidopsis MAP kinase 4regulates salicylic acid- and jasmonic

24



“fpls-03-00169” — 2012/7/22 — 17:06 — page 5 — #5


acid/ethylene-dependent responsesvia EDS1 and PAD4. Plant J. 47,532–546.

Cui, H., Wang, Y., Xue, L., Chu, J., Yan,C., Fu, J., Chen, M., Innes, R. W., andZhou, J.-M. (2010). Pseudomonassyringae effector protein AvrB per-turbs Arabidopsis hormone signalingby activating MAP kinase 4. Cell HostMicrobe 7, 164–175.

del Pozo, O., Pedley, K. F., and Martin,G. B. (2004). MAPKKK[alpha] is apositive regulator of cell death asso-ciated with both plant immunity anddisease. EMBO J. 23, 3072–3082.

Djamei, A., Pitzschke, A., Nak-agami, H., Rajh, I., and Hirt,H. (2007). trojan horse strategy inAgrobacterium transformation: abus-ing MAPK defense signaling. Science318, 453–456.

Dóczi, R., Brader, G., Pettkó-Szandtner,A., Rajh, I., Djamei, A., Pitzschke, A.,Teige, M., and Hirt, H. (2007). TheArabidopsis mitogen-activated pro-tein kinase kinase MKK3 is upstreamof group C mitogen-activated pro-tein kinases and participates inpathogen signaling. Plant Cell 19,3266–3279.

Feilner, T., Hultschig, C., Lee, J.,Meyer, S., Immink, R. G. H., Koenig,A., Possling, A., Seitz, H., Bev-eridge, A., Scheel, D., Cahill, D. J.,Lehrach, H., Kreutzberger, J., andKersten, B. (2005). High throughputidentification of potential Arabidop-sis mitogen-activated protein kinasessubstrates. Mol. Cell. Proteomics 4,1558–1568.

Felix, G., Duran, J. D., Volko, S.,and Boller, T. (1999). Plants have asensitive perception system for themost conserved domain of bacterialflagellin. Plant J. 18, 265–276.

Fiil, B. K., Petersen, K., Petersen, M.,and Mundy, J. (2009). Gene regula-tion by MAP kinase cascades. Curr.Opin. Plant Biol. 12, 615–621.

Forzani, C., Carreri, A., de la Fuentevan Bentem, S., Lecourieux, D.,Lecourieux, F., and Hirt, H. (2011).The Arabidopsis protein kinase Pto-interacting 1-4 is a common target ofthe oxidative signal-inducible 1 andmitogen-activated protein kinases.FEBS J. 278, 1126–1136.

Frei dit Frey, N., Mbengue, M.,Kwaaitaal, M., Nitsch, L., Altenbach,D., Haweker, H., Lozano-Duran, R.,Njo, M. F., Beeckman, T., Huettel,B., Borst, J. W., Panstruga, R., andRobatzek, S. (2012). Plasma mem-brane calcium ATPases are importantcomponents of receptor-mediatedsignaling in plant immune responsesand development. Plant Physiol. 159,798–809.

Frye, C. A., Tang, D., and Innes, R.W. (2001). Negative regulation ofdefense responses in plants by a con-served MAPKK kinase. Proc. Natl.Acad. Sci. U.S.A. 98, 373–378.

Gao, M., Liu, J., Bi, D., Zhang, Z.,Cheng, F., Chen, S., and Zhang,Y. (2008). MEKK1, MKK1/MKK2and MPK4 function together in amitogen-activated protein kinase cas-cade to regulate innate immunity inplants. Cell Res. 18, 1190–1198.

Gimenez-Ibanez, S., and Rathjen, J.P. (2010). The case for the defense:plants versus Pseudomonas syringae.Microbes Infect. 12, 428–437.

Gómez-Gómez, L., and Boller, T.(2000). FLS2: an LRR receptor-likekinase involved in the perception ofthe bacterial elicitor flagellin in Ara-bidopsis. Mol. Cell 5, 1003–1011.

Ichimura, K., Shinozaki, K., Tena, G.,Sheen, J., Henry, Y., Champion, A.,Kreis, M., Zhang, S., Hirt, H., Wil-son, C., Heberle-Bors, E., Ellis, B. E.,Morris, P. C., Innes, R. W., Ecker, J.R., Scheel, D., Klessig, D. F., Machida,Y., Mundy, J., Ohashi, Y., and Walker,J. C. (2002). Mitogen-activated pro-tein kinase cascades in plants: a newnomenclature. Trends Plant Sci. 7,301–308.

Komis, G., Illés, P., Beck, M., andŠamaj, J. (2011). Microtubules andmitogen-activated protein kinase sig-nalling. Curr. Opin. Plant Biol. 14,650–657.

Kwaaitaal, M., Huisman, R., Maintz,J., Reinstädler, A., and Panstruga, R.(2011). Ionotropic glutamate recep-tor (iGluR)-like channels mediateMAMP-induced calcium influx inArabidopsis thaliana. Biochem. J. 440,355–365.

Lee, K., Song, E. H., Kim, H. S., Yoo, J.H., Han, H. J., Jung, M. S., Lee, S. M.,Kim, K. E., Kim, M. C., Cho, M. J.,and Chung, W. S. (2008). Regulationof MAPK phosphatase 1 (AtMKP1)by calmodulin in Arabidopsis. J. Biol.Chem. 283, 23581–23588.

Ma, W., Qi, Z., Smigel, A., Walker, R.K., Verma, R., and Berkowitz, G. A.(2009). Ca2+, cAMP, and transduc-tion of non-self perception duringplant immune responses. Proc. Natl.Acad. Sci. U.S.A. 106, 20995–21000.

Mao, G., Meng, X., Liu, Y., Zheng, Z.,Chen, Z., and Zhang, S. (2011). Phos-phorylation of a WRKY transcriptionfactor by two pathogen-responsiveMAPKs drives phytoalexin biosyn-thesis in Arabidopsis. Plant Cell 23,1639–1653.

Miya, A., Albert, P., Shinya, T.,Desaki, Y., Ichimura, K., Shirasu, K.,Narusaka, Y., Kawakami, N., Kaku,H., and Shibuya, N. (2007). CERK1,

a LysM receptor kinase, is essen-tial for chitin elicitor signaling inArabidopsis. Proc. Natl. Acad. Sci.U.S.A. 104, 19613–19618.

Parisy, V., Poinssot, B., Owsianowski,L., Buchala, A., Glazebrook, J., andMauch, F. (2007). Identification ofPAD2 as a γ-glutamylcysteine syn-thetase highlights the importance ofglutathione in disease resistance ofArabidopsis. Plant J. 49, 159–172.

Petersen, K., Qiu, J.-L., Lütje, J.,Fiil, B. K., Hansen, S., Mundy, J.,and Petersen, M. (2010). ArabidopsisMKS1 is involved in basal immunityand requires an intact N-terminaldomain for proper function. PLoSONE 5, e14364. doi: 10.1371/jour-nal.pone.0014364

Petersen, M., Brodersen, P., Naested,H., Andreasson, E., Lindhart, U.,Johansen, B., Nielsen, H. B., Lacy,M., Austin, M. J., Parker, J. E.,Sharma, S. B., Klessig, D. F., Mar-tienssen, R., Mattsson, O., Jensen, A.B., and Mundy, J. (2000). Arabidop-sis MAP kinase 4 negatively regulatessystemic acquired resistance. Cell 103,1111–1120.

Popescu, S. C., Popescu, G. V., Bachan,S., Zhang, Z., Gerstein, M., Snyder,M., and Dinesh-Kumar, S. P. (2009).MAPK target networks in Arabidop-sis thaliana revealed using functionalprotein microarrays. Genes Dev. 23,80–92.

Qiu, J.-L., Fiil, B. K., Petersen, K.,Nielsen, H. B., Botanga, C. J., Thor-grimsen, S., Palma, K., Suarez-Rodriguez, M. C., Sandbech-Clausen,S., Lichota, J., Brodersen, P., Grasser,K. D., Mattsson, O., Glazebrook, J.,Mundy, J., and Petersen, M. (2008a).Arabidopsis MAP kinase 4 regulatesgene expression through transcrip-tion factor release in the nucleus.EMBO J. 27, 2214–2221.

Qiu, J.-L., Zhou, L., Yun, B.-W., Nielsen,H. B., Fiil, B. K., Petersen, K.,MacKinlay, J., Loake, G. J., Mundy, J.,and Morris, P. C. (2008b). Arabidop-sis mitogen-activated protein kinasekinases MKK1 and MKK2 have over-lapping functions in defense signalingmediated by MEKK1, MPK4, andMKS1. Plant Physiol. 148, 212–222.

Ren, D., Liu, Y., Yang, K.-Y., Han, L.,Mao, G., Glazebrook, J., and Zhang,S. (2008). A fungal-responsive MAPKcascade regulates phytoalexin biosyn-thesis in Arabidopsis. Proc. Natl. Acad.Sci. U.S.A. 105, 5638–5643.

Rentel, M. C., Lecourieux, D., Ouaked,F., Usher, S. L., Petersen, L., Okamoto,H., Knight, H., Peck, S. C., Gri-erson, C. S., Hirt, H., and Knight,M. R. (2004). OXI1 kinase is nec-essary for oxidative burst-mediated

signalling in Arabidopsis. Nature427, 858.

Rodriguez, M. C., Adams-Phillips, L.,Liu, Y., Wang, H., Su, S. H., Jester, P.J., Zhang, S., Bent, A. F., and Krysan,P. J. (2007). MEKK1 is requiredfor flg22-induced MPK4 activationin Arabidopsis plants. Plant Physiol.143, 661.

Rodriguez, M. C., Petersen, M., andMundy, J. (2010). Mitogen-activatedprotein kinase signaling in plants.Annu. Rev. Plant Biol. 61, 621–649.

Roux, M., Schwessinger, B., Albrecht,C., Chinchilla, D., Jones, A., Holton,N., Malinovsky, F. G., Tör, M., DeVries, S., and Zipfel, C. (2011).The Arabidopsis leucine-rich repeatreceptor-like kinases BAK1/SERK3and BKK1/SERK4 are required forinnate immunity to hemibiotrophicand biotrophic pathogens. Plant Cell23, 2440–2455.

Schwessinger, B., Roux, M., Kadota,Y., Ntoukakis, V., Sklenar, J.,Jones, A., and Zipfel, C. (2011).Phosphorylation-dependent differ-ential regulation of plant growth,cell death, and innate immunity bythe regulatory receptor-like kinaseBAK1. PLoS Genet. 7, e1002046. doi:10.1371/journal.pgen.1002046

Takahashi, F., Mizoguchi, T., Yoshida,R., Ichimura, K., and Shinozaki, K.(2011). Calmodulin-dependent acti-vation of MAP kinase for ROS home-ostasis in Arabidopsis. Mol. Cell 41,649–660.

Ulm, R., Ichimura, K., Mizoguchi, T.,Peck, S. C., Zhu, T., Wang, X., Shi-nozaki, K., and Paszkowski, J. (2002).Distinct regulation of salinity andgenotoxic stress responses by Ara-bidopsis MAP kinase phosphatase 1.EMBO J. 21, 6483–6493.

Van Der Biezen, E. A., and Jones, J. D. G.(1998). Plant disease-resistance pro-teins and the gene-for-gene concept.Trends Biochem. Sci. 23, 454–456.

Wang, H., Ngwenyama, N., Liu, Y.,Walker, J. C., and Zhang, S. (2007).Stomatal development and pattern-ing are regulated by environmentallyresponsive mitogen-activated proteinkinases in Arabidopsis. Plant Cell 19,63–73.

Wurzinger, B., Mair, A., Pfister, B.,and Teige, M. (2011). Cross-talkof calcium-dependent protein kinaseand MAP kinase signaling. Plant Sig-nal. Behav. 6, 8–12.

Xu, J., Li, Y., Wang, Y., Liu, H., Lei, L.,Yang, H., Liu, G., and Ren, D. (2008).Activation of MAPK kinase 9 inducesethylene and camalexin biosynthesisand enhances sensitivity to salt stressin Arabidopsis. J. Biol. Chem. 283,26996–27006.

25



“fpls-03-00169” — 2012/7/22 — 17:06 — page 6 — #6


Yoo, S.-D., Cho, Y.-H., Tena, G., Xiong,Y., and Sheen, J. (2008). Dual controlof nuclear EIN3 by bifurcate MAPKcascades in C2H4 signalling. Nature451, 789–795.

Zhang, J., Shao, F., Li, Y., Cui, H., Chen,L., Li, H., Zou, Y., Long, C., Lan,L., Chai, J., Chen, S., Tang, X., andZhou, J. M. (2007a). A Pseudomonassyringae effector inactivates MAPKsto suppress PAMP-induced immu-nity in plants. Cell Host Microbe 1,175–185.

Zhang, X., Dai, Y., Xiong, Y., DeFraia, C.,Li, J., Dong, X., and Mou, Z. (2007b).Overexpression of Arabidopsis MAPkinase kinase 7 leads to activationof plant basal and systemic acquiredresistance. Plant J. 52, 1066–1079.

Zhang, Z., Wu, Y., Gao, M., Zhang, J.,Kong, Q., Liu, Y., Ba, H., Zhou, J.,

and Zhang, Y. (2012). Disruption ofPAMP-induced MAP kinase cascadeby a Pseudomonas syringae effectoractivates plant immunity mediated bythe NB-LRR protein SUMM2. CellHost Microbe 11, 253–263.

Zheng, Z., Qamar, S. A., Chen,Z., and Mengiste, T. (2006). Ara-bidopsis WRKY33 transcription fac-tor is required for resistance tonecrotrophic fungal pathogens. PlantJ. 48, 592–605.

Zhou, N., Tootle, T. L., and Glaze-brook, J. (1999). Arabidopsis PAD3, agene required for camalexin biosyn-thesis, encodes a putative cytochromeP450 monooxygenase. Plant Cell 11,2419–2428.

Zhu, X., Caplan, J., Mamillapalli,P., Czymmek, K., and Dinesh-Kumar, S. P. (2010). Function

of endoplasmic reticulum calciumATPase in innate immunity-mediatedprogrammed cell death. EMBO J. 29,1007–1018.

Zipfel, C. (2008). Pattern-recognitionreceptors in plant innate immunity.Curr. Opin. Immunol. 20, 10–16.

Zipfel, C., Kunze, G., Chinchilla, D.,Caniard, A., Jones, J. D. G., Boller,T., and Felix, G. (2006). Perceptionof the bacterial PAMP EF-Tu by thereceptor EFR restricts Agrobacterium-mediated transformation. Cell 125,749–760.

Conflict of Interest Statement: Theauthors declare that the research wasconducted in the absence of any com-mercial or financial relationships thatcould be construed as a potential con-flict of interest.

Received: 02 May 2012; paper pendingpublished: 24 May 2012; accepted: 09 July2012; published online: 24 July 2012.Citation: Rasmussen MW, Roux M,Petersen M and Mundy J (2012) MAPkinase cascades in Arabidopsis innateimmunity. Front. Plant Sci. 3:169. doi:10.3389/fpls.2012.00169This article was submitted to Frontiers inPlant Proteomics, a specialty of Frontiersin Plant Science.Copyright © 2012 Rasmussen, Roux,Petersen and Mundy. This is an open-access article distributed under the termsof the Creative Commons AttributionLicense, which permits use, distributionand reproduction in other forums, pro-vided the original authors and sourceare credited and subject to any copy-right notices concerning any third-partygraphics etc.

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mRNA decay in plant immunity

Magnus W. Rasmussen, Àngels M. Regué, John Mundy and Morten Petersen*

Review article submitted to Cellular and Molecular Life science

*Corresponding author: [email protected]

Abstract

Strict regulation of mRNA metabolism is required in plant immunity in order to control defense

responses and regulation of immune receptors. Transcription is specific and is typically regulated

through activation and release of transcription factors, equally important is transcript degradation.

The mechanisms of mRNA decay are well studied, but the mechanisms regarding specificity is are

not well understood. Mutants defective in both nonsense mediated decay and decapping display

autoimmune phenotypes implying functions in immune regulation. In this review, we will focus on

the few published examples concerning mRNA decay and immune responses in Arabidopsis.

Introduction

mRNA metabolism is strictly regulated to control the fate of cells in response to developmental and

environmental stimuli. mRNA metabolism refers to any event in the lifecycle of mRNAs including

transcription, processing and degradation. Much is known about changes in transcript levels during

plant development (Becker and Theißen, 2003) and in response to abiotic stress (Akhtar et al., 2012;

Rasmussen et al., 2013) or biotic stresses including microbial perception (Andreasson et al., 2005; Mao

et al., 2011; Qiu et al., 2008). Changes in specific transcript levels are often attributed to changes in

their transcription rates, although there are relatively few reports of global or specific transcription

rates (Sidaway-Lee et al., 2014). While transcription of genes responding to specific stimuli clearly

affects the accumulation of mRNAs (Akhtar et al., 2012), the contribution of mRNA decay is less

studied. A primary example of the importance of mRNA decay is illustrated by Xu and Chua (Xu

and Chua, 2012, 1) who showed that phosphorylation of Decapping 1 (DCP1) by MAP kinase 6

(MPK6) upon dehydration stress promotes decapping, and that plants expressing a non-

phophorylatable version of DCP1 were hypersensitive to osmotic stress.

Eukaryotic mRNAs contain two integral stability determinants, the 5’ 7-methylguanosine

triphosphate cap (m7G) and the 3’ poly(A) tail, both of which are incorporated co-transcriptionally.

These structures interact with cellular proteins: eukaryotic initiation factor 4E (eIF4E) binds directly

27

to the 5’ cap and the poly(A) binding protein (PABP) binds to the 3’ poly(A) tail. The binding of

eIF4E and PABP promotes translational initiation and, at the same time, prevents degradation by

exoribonucleases. Thus, to initiate mRNA decay these determinants must either be removed or the

mRNA must be cleaved by an endonuclease followed by an exoribonuclease attack on the newly

exposed 3’ and 5’ ends. Eukaryotic mRNAs are primarily degraded by removal of the poly(A) tail

with subsequent removal of the 5’ cap and 5’ to 3’ degradation by exoribonucleases, or via 3’ to 5’

degradation by the exosome complex. The components of the Arabidopsis exosome complex are

reviewed by Lange and Gagliardi (Lange and Gagliardi, 2010), and Chiba and Green (Chiba and Green,

2009) provide a comprehensive comparison of yeast and plant mRNA decay machinery. Transcripts

can also be degraded via nonsense mediated decay (NMD), a eukaryotic RNA surveillance

mechanism that controls transcripts with aberrant features and targets them for degradation. These

aberrant features include premature termination codons (PTC), PTC-independent long 3’

untranslated regions, 3’ UTR-positioned introns, and upstream open reading frames (Wachter and

Hartmann, 2014). In addition to controlling mRNA integrity, NMD has also been described in

mammalian cells to target different mRNA isoforms, providing an additional regulation point for

important biological processes (Smith and Baker, 2015). This review mainly focuses on mRNA

degradation in the context of immune signaling in plants. While most published work in this field

concerns microRNA (miRNA) mediated decay of transcripts, we primarily focus here on the few

examples of non-miRNA immune mediated mRNA.

Plant innate immunity

Plants have two layers of immunity (Jones and Dangl, 2006). The first layer is comprised of pattern

recognition receptors (PRRs) in the plasma membrane, which recognize pathogen-associated

molecular patterns (PAMPs). This recognition induces PAMP triggered immunity (PTI) (Greeff et

al., 2012). PAMPs and their cognate PPRs include bacterial flagellin (and its peptide derivative

flg22) recognized by the receptor FLS2 (Felix et al., 1999; Gómez-Gómez and Boller, 2000), bacterial

elongation factor EF-Tu by EFR1 (Kunze et al., 2004; Zipfel et al., 2006) and fungal chitin recognized

by CERK1 (Miya et al., 2007). Immune responses triggered by PAMP recognition include protein

phosphorylation, induction of defense genes, extracellular alkalization and production of reactive

oxygen species (Rasmussen et al., 2012). The second layer of defense employs cytoplasmic immune

receptors known as resistance (R) proteins that can detect pathogenic virulence determinants,

commonly referred to as effectors, which are injected into the host cell. R proteins recognize

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effectors by direct interaction or via effector modification of host proteins and activate effector

triggered immunity (ETI) (Jones and Dangl, 2006). PTI and ETI signaling is somewhat similar

although ETI responses are prolonged and more pronounced and can include localized cell death

(Cui et al., 2014). Most R-proteins are nucleotide-binding domain, leucine-rich repeat (NB-NLR)

proteins that can be further separated into coiled-coil- (CC-) or Toll/interleukin-1 receptor- (TIR-)

NB-LRRs (Caplan et al., 2008). The mechanism(s) by which R-proteins confer resistance against

specific pathogens remain largely unknown, and their signaling mechanisms are probably diverse

(Bonardi and Dangl, 2012). Nonetheless, different genes are required for NB-LRR mediated resistance

as ENHANCED DISEASE SUSCEPTIBILITY1 (EDS1) and PHYTOALEXINDEFICIENT4 (PAD4)

are required for TIR-NB-LRR defenses whereas NO DISEASE RESPONSES1 (NDR1) is required

for most defense responses mediated by CC-NB-LRRs (Century et al., 1997; Parker et al., 1996). R

proteins are tightly regulated as their overexpression may lead to increased immune signaling and

autoimmune phenotypes in non-infected plants (Alcázar and Parker, 2011; Nandety et al., 2013).

Conversely, plants with loss-of-function R genes are more susceptible to specific pathogen attacks.

mRNA decay and autoimmunity

Some mutants implicated in mRNA decay exhibit autoimmune phenotypes. These include protein

associated with topoisomerase protein 1 (pat1) involved in decapping of transcripts, as well as

upframeshift1 (upf1), upf3 and suppressor with morphogenic effect on genitalia7 (smg7)

functioning in non-sense mediated decay (NMD) (Fig. 1A) (Gloggnitzer et al., 2014; Jeong et al., 2011;

Rayson et al., 2012a; Riehs-Kearnan et al., 2012; Roux et al., 2015). The autoimmune phenotypes of these

NMD deficient mutants resemble those of lesion mimic mutants such as mpk4 (Petersen et al., 2000;

Zhang et al., 2012, 2) and accelerated cell death11 (acd11) (Brodersen et al., 2002; Palma et al., 2010,

11) in which autoimmunity is caused by inappropriate activation of ETI. Similar to R protein

mediated ETI, autoimmunity in NMD deficient mutants is also repressed at elevated temperatures

or high humidity (Alcázar and Parker, 2011; Riehs-Kearnan et al., 2012; Zhu et al., 2010). In addition,

inactivation of PAD4 but not NDR1 fully suppresses the retarded growth observed in the smg7 and

upf1 NMD deficient mutants. This indicates activation of ETI by one or more TIR-NB-LRRs in

these mutants (Riehs-Kearnan et al., 2012).

29

Figure 1: Nonsense-mediated mRNA decay

A, The ribosome stalls at a premature termination codon (PTC), giving rise to a long 3’UTR sensed by UPF1 (Hogg and

Goff, 2010). UPF1 then associates with translation release factor eRF3 and subsequently recruits UPF2 and UPF3. A

phosphatidylinositol 3-kinase-related kinase phosphorylates UPF1 which enables translocation of the UPF1/2/3-mRNA

complex to p-bodies by binding SMG7 in a phospho-dependent manner (Kerényi et al., 2013).

B, NMD mutant plants display autoimmune phenotypes reassembling effector triggered immunity and resistance (Jeong et

al., 2011; Rayson et al., 2012b; Riehs-Kearnan et al., 2012). This may arise either from (i) activation of R-proteins

guarding NMD components such as UPF1-3 or SMG7 (ii) hyper-accumulation of R-gene transcripts normally down-

regulated by NMD (Gloggnitzer et al., 2014).

NMD is apparently regulated during immune responses because plants challenged with the virulent

bacteria Pseudomonas syringae pv. tomato (pto) DC3000 have reduced levels of UPF1 and UPF3

transcripts and accumulate canonical NMD target transcripts (Jeong et al., 2011). RNA-seq data have

shown that 39 TIR-NB-LRRs and 11 CC-NB-LRRs are upregulated in the smg7 pad4 double

mutant compared to wild type (Gloggnitzer et al., 2014). Interestingly, 19 of these TIR-NB-LRRs

30

contain putative NMD target features while these features are absent in the 11 upregulated CC-NB-

LRRs. The half-lives of all but one of the upregulated TIR-NB-LRR transcripts are increased in the

smg7 pad4 double mutant, whereas the half-lives of the upregulated CC-NB-LRR transcripts are

unaltered. This indicates that only TIR-NB-LRR transcripts are affected by mutations in SMG7

(Gloggnitzer et al., 2014). Although NMD seems to regulate TIR-NB-LRR transcripts post-

transcriptionally and hence regulate ETI, NMD is down regulated after PAMP treatment with flg22.

This supports a role for NMD in regulating certain PTI responses (Gloggnitzer et al., 2014). This

raises the question of whether the phenotype of NMD deficient mutants is due to an intrinsic

mechanism stabilizing NMD targeted defense transcripts such as those of TIR-NB-LRRs.

Alternatively, since a natural variant of the TIR-NB-LRR RPS6 can suppress the autoimmune smg7

phenotype (Gloggnitzer et al., 2014) this NMD deficient phenotype could also be due to activation of

RPS6 guarding putative effector targeted steps in the NMD pathway mimicked by mutations in

SMG7. Two models explaining autoimmunity in smg7 is illustrated in Figure 2B.

Thus, autoimmunity in mRNA decay mutants may arise from inappropriate activation of R proteins

caused by mutations in favored effector targets. An example of this comes from analysis of the

decapping mutant pat1 (Roux et al., 2015). Decapping or removal of the mRNA 5’ cap structure is

important for exoribonuclease mediated transcript degradation. Mutants impaired in decapping such

as dcp1, dcp2 and varicose (vcs) are all seedling lethal and accumulate high levels of capped

transcripts (Goeres et al., 2007; Xu et al., 2006, 1). Recent studies have shown that transcript

accumulation due to improper decapping or lack of sequential decay by exoribonucleases or the

exosome results in induced endogenous gene silencing through increased siRNA levels

(Martínez de Alba et al., 2015; Zhang et al., 2015). In addition to gene silencing, miRNA-guided

translational repression also seems to be elevated in plant decapping mutants, as also reported for

mammalian systems (Brodersen et al., 2008; Eulalio et al., 2007). mRNA decapping occurs in discrete

cytoplasmic foci known as processing bodies and, upon activation of PTI with flg22, these foci

increase in number (Balagopal and Parker, 2009; Parker and Sheth, 2007; Roux et al., 2015). PAT1

localizes to processing bodies where it functions as a decapping activator (Marnef and Standart,

2010). Arabidopsis PAT1 interacts and is phosphorylated by MPK4, and PAT1 phosphorylation

increases after flg22 treatment (Roux et al., 2015). MPK4 functions in transducing signals from PPRs

(Rodriguez et al., 2010) and plants lacking MPK4 activity display activation of ETI dependent on the

R protein SUMM2 which guards the MPK4 pathway (Petersen et al., 2000, 4; Zhang et al., 2012, 4).

Mutants lacking PAT1 display constitutive defense responses and, similarly to mpk4 mutants, pat1

is immunosuppressed when crossed into a summ2 background. In addition, PAT1 interacts or is in

31

complex with SUMM2. It is therefore possible that PAT1 is an effector target with a specific

function in immunity that is under surveillance by SUMM2 (Roux et al., 2015). Arabidopsis MPK6,

another stress related MAP kinase, phosphorylates DCP1 upon dehydration stress and thereby

represses decapping (Xu and Chua, 2012, 1). Taken together, these observations indicate that both

biotic and abiotic stress signaling can directly influence mRNA decapping.

An example of how mRNA decay may control important steps in a defense pathway includes the

plant hormone ethylene that regulates both developmental processes and responses to biotic and

abiotic stresses (Johnson and Ecker, 1998). Ethylene is perceived in Arabidopsis by a family of 4

receptors that function as negative regulators (Chang abnd Stadler, 2001). In the absence of ethylene,

these receptors activate Constitutively Triple Response (CTR1), a MAPK kinase kinase (MAP3K)

which also functions as a negative regulator of ethylene signaling (Kieber et al., 1993). Ethylene

Insensitive 2 (EIN2) is genetically downstream of CTR1 and is a positive regulator promoting

ethylene responses through the transcription factors EIN3 and EIN3-like proteins (Alonso et al., 1999,

2; Chao et al., 1997; Guo and Ecker, 2003). In the presence of ethylene EIN3 levels increase rapidly,

while in the absence of ethylene EIN3 is down-regulated post-translationally through a

ubiquitin/proteasome pathway mediated by the EIN3 binding F-box proteins 1 and 2 (EBF1&2

(Gagne et al., 2004; Guo and Ecker, 2003, 3; Potuschak et al., 2003; Yanagisawa et al., 2003). Both EBF1

and EBF2 transcripts have been found to be targets of the Exoribonuclease 4 (XRN4) and hence

accumulate in xrn4 mutants which results in reduced levels of EIN3 protein (Olmedo et al., 2006;

Potuschak et al., 2006, 4). Tight regulation of EIN3, likely through EBF1 and EBF2, is essential for

fine-tuning immunity as EIN3 positively regulates transcription of the PRR FLS2 by binding to its

promoter (Boutrot et al., 2010). EIN3 also regulates transcription of Isochorismate Synthase 1 (ICS1

also known as SID2), involved in the biosynthesis of the defense hormone salicylic acid. In contrast

to its effects on FLS2, EIN3 represses transcription of ICS1 and thereby inhibits salicylic acid

accumulation (Chen et al., 2009). How and why EIN3 reciprocally regulates two genes important for

mounting proper immune responses remains a bit of a mystery. Nonetheless, it seems clear that

EIN3 itself is regulated via EBF1 and EBF2 mRNA decay (Olmedo et al., 2006; Potuschak et al.,

2006). Expression of Ethylene Response Factor 1 (ERF1) is directly induced by EIN3 (Solano et al.,

1998). ERF1 links ethylene and jasmonate signaling in Arabidopsis for appropriate expression of

defense responses against pathogens (Lorenzo et al., 2003). As for EBF1 and EBF2, ERF1 transcripts

are also targeted by XRN4 and hence accumulate in xrn4 mutants (Nguyen et al., 2015). XRN4 is

homologous to yeast XRN1 which degrades RNA species with a single phosphate group at the 5’

end (Kastenmayer and Green, 2000; Souret et al., 2004). It is therefore likely that the ERF1 transcripts,

32

as well as those of EBF1 and EBF2, that accumulate in xrn4 mutants are uncapped and that

decapping in fact regulates these transcripts prior to XRN4 mediated decay. This has not yet been

tested experimentally.

In addition to the above examples, miRNAs provide an additional control over mRNA decay and a

direct link to regulation of immunity. Post transcriptional gene silencing (PTGS) is a key regulatory

mechanism in controlling immune responses in plants (Seo et al., 2013). In PTGS, double stranded

RNAs are cleaved into 20-24 nucleotide small RNA species by DICER-like enzymes. One of the

two RNA strands then associates with ARGONAUTE (AGO) proteins which are catalytic

components of the RNA-induced silencing complex (RISC) promoting mRNA cleavage (Meister,

2013). Different classes of small RNA species including micro RNAs (miRNAs) and short

interfering RNAs (siRNAs) are involved in PTGS. miRNAs are transcribed from the genome by

RNA polymerase II and, due to regions of reverse sequence complementarity, primary or pri-

miRNAs form hairpin-like secondary structures (Ha and Kim, 2014). In plants, mature miRNAs are

formed in the nucleus by cleavage of pri-miRNAs by DICER-LIKE1 (DCL1) proteins in complex

with SERRATE and HYL1. Mature miRNAs are then exported to the cytosol where they associate

with AGO1 and promote mRNA cleavage (Ha and Kim, 2014).

miRNAs has been shown to function in plant immunity (Li et al., 2010; Navarro et al., 2006). In

response to PAMP perception, miRNA miR393 accumulates and negatively regulates auxin

signaling by targeting transcripts encoding the auxin receptors TIR1 (transport inhibitor 1), AFB2

(auxin signaling F-box protein 2) and AFB3 (Navarro et al., 2006). Arabidopsis encodes a number

auxin response factors (ARFs) that can promote or repress the transcription of genes containing

auxin responsive elements (Hagen and Guilfoyle, 2002). The auxin-regulated Aux/IAA proteins

sequester ARFs and thereby repress auxin signaling (Liscum and Reed, 2002). TIR1 is part of a

ubiquitin-ligase complex interacting with Aux/IAA proteins and marking them for degradation

(Gray et al., 2001). Therefore, as a consequence of PAMP triggered miR393 accumulation, Aux/IAA

proteins are stabilized resulting in repressed auxin signaling (Navarro et al., 2006). Overexpression of

transgenic miR393 increases resistance to pto DC3000, while overexpression of AFB1 (a paralog to

TIR1 and AFB2/3 that is resistant to miR393 mediated degradation) renders plants more susceptible

(Navarro et al., 2006). This supports a function of miRNA in regulating proper responses during PTI.

Virulent bacterial pathogens have evolved effector proteins that can suppress the miRNA pathway

and thereby repress host immunity. During infection, Pto DC3000 can employ the effector protein

AvrPtoB that represses transcription of miR393a and miR393b which both encode miR393 (Navarro

et al., 2008). In addition, the effector protein AvrPto stabilizes miR393 precursors thereby reducing

33

the levels of mature miR393 and rendering plants more susceptible to infection (Navarro et al., 2008).

Since AvrPto and AvrPtoB both interact with and inhibit PRRs including FLS2 (Dodds and Rathjen,

2010; Göhre et al., 2008; Xiang et al., 2008) it is possible that these effectors do not only target the

miRNA pathway, but also suppress PTI signaling downstream of FLS2 and other PRRs. Other

miRNAs including miR160 and miR167 are also upregulated during PTI, and both target members

of the ARF transcription factor family. This further supports the involvement of miRNA mediated

gene silencing in auxin signaling during PTI (Fahlgren et al., 2007).

Transcripts encoding R protein immune receptors have also been shown to be direct targets of

miRNAs. miR472 negatively regulates several CC-NB-LRRs by targeting conserved regions

encoding ATP binding p-loop domains (Boccara et al., 2014; Lu et al., 2006). It is likely that in the

absence of infection miR472 promotes degradation of CC-NB-LRR transcripts and that activation

of PTI down regulates or pacifies miR472 to permit accumulation of CC-NB-LRR transcripts

resulting in increased resistance (Boccara et al., 2014). Consistent with this, miR472 deficient and

over-expressor plants are respectively more resistant and susceptible to pto DC3000 (Boccara et al.,

2014). Similarly, tomato miR482 targets a group of CC-NB-LRRs and results in the production of

secondary trans-acting siRNAs that target other immune regulators. Such secondary targets include

PEN3 involved in basal immunity and a proteasomal subunit that is also likely to function in

resistance (Shivaprasad et al., 2012). In addition, and as seen for Arabidopsis miR472 and miR393,

tomato miR482 mediated silencing is impaired in infected plants, likely due to a bacterial effector.

Tomato plants infiltrated with an hrcC mutant of Pto. DC3000 which is incapable of delivering

effectors into host plants showed impaired accumulation of miR482 target transcripts compared to

plants infiltrated with virulent Pto. DC3000 (Shivaprasad et al., 2012). This effector mediated up-

regulation of resistance gene expression via the suppression of host RNA silencing might have

evolved as a counter defense against pathogens similar to ETI.

Plant miRNAs represent a fraction of the total pool of small RNAs which includes siRNAs and

secondary siRNAs (Meyers et al., 2008). In contrast to miRNAs, siRNAs usually perform auto

silencing, while trans-acting siRNAs target mRNAs transcribed from other loci. This amplifies the

effects of siRNA and miRNA mediated PTGS (Ghildiyal and Zamore, 2009). For example, the

Arabidopsis RPP5 locus encodes seven TIR-NB-LRR proteins including SNC1 and RPP4. Most of

these R gene paralogs are all repressed by 21-nucleotide siRNAs corresponding to their LRR

regions which are likely generated from antisense transcription from SNC1 (Yi and Richards, 2007).

34

Conclusion

Our current knowledge of immune regulated mRNA decay and its specificity remains limited.

Control of R proteins through miRNA-mediated mRNA decay implies direct roles in regulating

appropriate immune responses. The examples described above also suggest that the general decay

pathways comprising the exosome, exoribonucleases and NMD are important for immune signaling

as well as for responses and adaptations to abiotic stress. It is difficult to identify the target

transcript specificities of these pathways, in contrast to the sequence homology based identification

of miRNA-mediated decay targets. Nonetheless, since mutations in several of the key components

in mRNA decay pathways have autoimmune phenotypes, they are themselves likely targets of

pathogen effectors. This issue needs further investigation to ensure that conclusions regarding the

functions of these decay components are not based on potential pleiotropic phenotypes of their loss-

of-function mutants, but rather reflect their true in vivo functions.

References

Akhtar, M., Jaiswal, A., Taj, G., Jaiswal, J. P., Qureshi, M. I., and Singh, N. K. (2012). DREB1/CBF

transcription factors: their structure, function and role in abiotic stress tolerance in plants. J. Genet.

91, 385–395.

Alcázar, R., and Parker, J. E. (2011). The impact of temperature on balancing immune responsiveness and

growth in Arabidopsis. Trends Plant Sci. 16, 666–675. doi:10.1016/j.tplants.2011.09.001.

Alonso, J. M., Hirayama, T., Roman, G., Nourizadeh, S., and Ecker, J. R. (1999). EIN2, a Bifunctional

Transducer of Ethylene and Stress Responses in Arabidopsis. Science 284, 2148–2152.

doi:10.1126/science.284.5423.2148.

Andreasson, E., Jenkins, T., Brodersen, P., Thorgrimsen, S., Petersen, N. H., Zhu, S., et al. (2005). The MAP

kinase substrate MKS1 is a regulator of plant defense responses. EMBO J 24, 2579–2589.

doi:10.1038/sj.emboj.7600737.

Balagopal, V., and Parker, R. (2009). Polysomes, P bodies and stress granules: states and fates of eukaryotic

mRNAs. Curr. Opin. Cell Biol. 21, 403–408. doi:10.1016/j.ceb.2009.03.005.

Becker, A., and Theißen, G. (2003). The major clades of MADS-box genes and their role in the development

and evolution of flowering plants. Mol. Phylogenet. Evol. 29, 464–489. doi:10.1016/S1055-

7903(03)00207-0.

Boccara, M., Sarazin, A., Thiébeauld, O., Jay, F., Voinnet, O., Navarro, L., et al. (2014). The Arabidopsis

miR472-RDR6 Silencing Pathway Modulates PAMP- and Effector-Triggered Immunity through the

Post-transcriptional Control of Disease Resistance Genes. PLoS Pathog 10, e1003883.

doi:10.1371/journal.ppat.1003883.

Bonardi, V., and Dangl, J. L. (2012). How complex are intracellular immune receptor signaling complexes?

Plant Proteomics 3, 237. doi:10.3389/fpls.2012.00237.

35

Boutrot, F., Segonzac, C., Chang, K. N., Qiao, H., Ecker, J. R., Zipfel, C., et al. (2010). Direct transcriptional

control of the Arabidopsis immune receptor FLS2 by the ethylene-dependent transcription factors

EIN3 and EIL1. Proc. Natl. Acad. Sci. 107, 14502–14507. doi:10.1073/pnas.1003347107.

Brodersen, P., Petersen, M., Pike, H. M., Olszak, B., Skov, S., Ødum, N., et al. (2002). Knockout of

Arabidopsis ACCELERATED-CELL-DEATH11 encoding a sphingosine transfer protein causes

activation of programmed cell death and defense. Genes Dev. 16, 490–502. doi:10.1101/gad.218202.

Brodersen, P., Sakvarelidze-Achard, L., Bruun-Rasmussen, M., Dunoyer, P., Yamamoto, Y. Y., Sieburth, L.,

et al. (2008). Widespread Translational Inhibition by Plant miRNAs and siRNAs. Science 320,

1185–1190. doi:10.1126/science.1159151.

Caplan, J., Padmanabhan, M., and Dinesh-Kumar, S. P. (2008). Plant NB-LRR immune receptors: from

recognition to transcriptional reprogramming. Cell Host Microbe 3, 126–135.

doi:10.1016/j.chom.2008.02.010.

Century, K. S., Shapiro, A. D., Repetti, P. P., Dahlbeck, D., Holub, E., and Staskawicz, B. J. (1997). NDR1,

a Pathogen-Induced Component Required for Arabidopsis Disease Resistance. Science 278, 1963–

1965. doi:10.1126/science.278.5345.1963.

Chang, C., and Stadler, R. (2001). Ethylene hormone receptor action in Arabidopsis. BioEssays 23, 619–627.

doi:10.1002/bies.1087.

Chao, Q., Rothenberg, M., Solano, R., Roman, G., Terzaghi, W., and Ecker†, J. R. (1997). Activation of the

Ethylene Gas Response Pathway in Arabidopsis by the Nuclear Protein ETHYLENE-

INSENSITIVE3 and Related Proteins. Cell 89, 1133–1144. doi:10.1016/S0092-8674(00)80300-1.

Chen, H., Xue, L., Chintamanani, S., Germain, H., Lin, H., Cui, H., et al. (2009). ETHYLENE

INSENSITIVE3 and ETHYLENE INSENSITIVE3-LIKE1 Repress SALICYLIC ACID

INDUCTION DEFICIENT2 Expression to Negatively Regulate Plant Innate Immunity in

Arabidopsis. Plant Cell 21, 2527–2540. doi:10.1105/tpc.108.065193.

Chiba, Y., and Green, P. J. (2009). mRNA Degradation Machinery in Plants. J. Plant Biol. 52, 114–124.

doi:10.1007/s12374-009-9021-2.

Cui, H., Tsuda, K., and Parker, J. E. (2014). Effector-Triggered Immunity: From Pathogen Perception to

Robust Defense. Annu. Rev. Plant Biol. doi:10.1146/annurev-arplant-050213-040012.

Dodds, P. N., and Rathjen, J. P. (2010). Plant immunity: towards an integrated view of plant–pathogen

interactions. Nat. Rev. Genet. 11, 539–548. doi:10.1038/nrg2812.

Eulalio, A., Rehwinkel, J., Stricker, M., Huntzinger, E., Yang, S.-F., Doerks, T., et al. (2007). Target-specific

requirements for enhancers of decapping in miRNA-mediated gene silencing. Genes Dev. 21, 2558–

2570. doi:10.1101/gad.443107.

Fahlgren, N., Howell, M. D., Kasschau, K. D., Chapman, E. J., Sullivan, C. M., Cumbie, J. S., et al. (2007).

High-Throughput Sequencing of Arabidopsis microRNAs: Evidence for Frequent Birth and Death of

MIRNA Genes. PLoS ONE 2, e219. doi:10.1371/journal.pone.0000219.

Felix, G., Duran, J. D., Volko, S., and Boller, T. (1999). Plants have a sensitive perception system for the

most conserved domain of bacterial flagellin. Plant J. 18, 265–276. doi:10.1046/j.1365-

313X.1999.00265.x.

Gagne, J. M., Smalle, J., Gingerich, D. J., Walker, J. M., Yoo, S.-D., Yanagisawa, S., et al. (2004).

Arabidopsis EIN3-binding F-box 1 and 2 form ubiquitin-protein ligases that repress ethylene action

36

and promote growth by directing EIN3 degradation. Proc. Natl. Acad. Sci. U. S. A. 101, 6803–6808.

doi:10.1073/pnas.0401698101.

Ghildiyal, M., and Zamore, P. D. (2009). Small silencing RNAs: an expanding universe. Nat. Rev. Genet. 10,

94–108. doi:10.1038/nrg2504.

Gloggnitzer, J., Akimcheva, S., Srinivasan, A., Kusenda, B., Riehs, N., Stampfl, H., et al. (2014). Nonsense-

Mediated mRNA Decay Modulates Immune Receptor Levels to Regulate Plant Antibacterial

Defense. Cell Host Microbe 16, 376–390. doi:10.1016/j.chom.2014.08.010.

Goeres, D. C., Norman, J. M. V., Zhang, W., Fauver, N. A., Spencer, M. L., and Sieburth, L. E. (2007).

Components of the Arabidopsis mRNA Decapping Complex Are Required for Early Seedling

Development. Plant Cell 19, 1549–1564. doi:10.1105/tpc.106.047621.

Göhre, V., Spallek, T., Häweker, H., Mersmann, S., Mentzel, T., Boller, T., et al. (2008). Plant Pattern-

Recognition Receptor FLS2 Is Directed for Degradation by the Bacterial Ubiquitin Ligase AvrPtoB.

Curr. Biol. 18, 1824–1832. doi:10.1016/j.cub.2008.10.063.

Gómez-Gómez, L., and Boller, T. (2000). FLS2: An LRR Receptor-like Kinase Involved in the Perception of

the Bacterial Elicitor Flagellin in Arabidopsis. Mol. Cell 5, 1003–1011. doi:10.1016/S1097-

2765(00)80265-8.

Gray, W. M., Kepinski, S., Rouse, D., Leyser, O., and Estelle, M. (2001). Auxin regulates SCFTIR1-

dependent degradation of AUX/IAA proteins. Nature 414, 271–276. doi:10.1038/35104500.

Greeff, C., Roux, M., Mundy, J., and Petersen, M. (2012). Receptor-like kinase complexes in plant innate

immunity. Front. Plant Sci. 3. doi:10.3389/fpls.2012.00209.

Guo, H., and Ecker, J. R. (2003). Plant Responses to Ethylene Gas Are Mediated by SCFEBF1/EBF2-

Dependent Proteolysis of EIN3 Transcription Factor. Cell 115, 667–677. doi:10.1016/S0092-

8674(03)00969-3.

Hagen, G., and Guilfoyle, T. (2002). Auxin-responsive gene expression: genes, promoters and regulatory

factors. Plant Mol. Biol. 49, 373–385. doi:10.1023/A:1015207114117.

Ha, M., and Kim, V. N. (2014). Regulation of microRNA biogenesis. Nat. Rev. Mol. Cell Biol. 15, 509–524.

doi:10.1038/nrm3838.

Hogg, J. R., and Goff, S. P. (2010). Upf1 senses 3′UTR length to potentiate mRNA decay. Cell 143, 379–

389. doi:10.1016/j.cell.2010.10.005.

Jeong, H.-J., Kim, Y. J., Kim, S. H., Kim, Y.-H., Lee, I.-J., Kim, Y. K., et al. (2011). Nonsense-Mediated

mRNA Decay Factors, UPF1 and UPF3, Contribute to Plant Defense. Plant Cell Physiol. 52, 2147–

2156. doi:10.1093/pcp/pcr144.

Johnson, P. R., and Ecker, J. R. (1998). THE ETHYLENE GAS SIGNAL TRANSDUCTION PATHWAY:

A Molecular Perspective. Annu. Rev. Genet. 32, 227–254. doi:10.1146/annurev.genet.32.1.227.

Jones, J. D. G., and Dangl, J. L. (2006). The plant immune system. Nature 444, 323–329.

doi:10.1038/nature05286.

Kastenmayer, J. P., and Green, P. J. (2000). Novel features of the XRN-family in Arabidopsis: Evidence that

AtXRN4, one of several orthologs of nuclear Xrn2p/Rat1p, functions in the cytoplasm. Proc. Natl.

Acad. Sci. U. S. A. 97, 13985–13990.

37

Kerényi, F., Wawer, I., Sikorski, P. J., Kufel, J., and Silhavy, D. (2013). Phosphorylation of the N- and C-

terminal UPF1 domains plays a critical role in plant nonsense-mediated mRNA decay. Plant J. 76,

836–848. doi:10.1111/tpj.12346.

Kieber, J. J., Rothenberg, M., Roman, G., Feldmann, K. A., and Ecker, J. R. (1993). CTR1, a negative

regulator of the ethylene response pathway in arabidopsis, encodes a member of the Raf family of

protein kinases. Cell 72, 427–441. doi:10.1016/0092-8674(93)90119-B.

Kunze, G., Zipfel, C., Robatzek, S., Niehaus, K., Boller, T., and Felix, G. (2004). The N Terminus of

Bacterial Elongation Factor Tu Elicits Innate Immunity in Arabidopsis Plants. Plant Cell 16, 3496–

3507. doi:10.1105/tpc.104.026765.

Lange, H., and Gagliardi, D. (2010). “The Exosome and 3′–5′ RNA Degradation in Plants,” in RNA Exosome

Advances in Experimental Medicine and Biology., ed. T. H. Jensen (Springer US), 50–62. Available

at: http://link.springer.com/chapter/10.1007/978-1-4419-7841-7_5 [Accessed April 9, 2015].

Liscum, E., and Reed, J. W. (2002). Genetics of Aux/IAA and ARF action in plant growth and development.

Plant Mol. Biol. 49, 387–400. doi:10.1023/A:1015255030047.

Li, Y., Zhang, Q., Zhang, J., Wu, L., Qi, Y., and Zhou, J.-M. (2010). Identification of MicroRNAs Involved

in Pathogen-Associated Molecular Pattern-Triggered Plant Innate Immunity. Plant Physiol. 152,

2222–2231. doi:10.1104/pp.109.151803.

Lorenzo, O., Piqueras, R., Sánchez-Serrano, J. J., and Solano, R. (2003). ETHYLENE RESPONSE

FACTOR1 Integrates Signals from Ethylene and Jasmonate Pathways in Plant Defense. Plant Cell

15, 165–178. doi:10.1105/tpc.007468.

Lu, C., Kulkarni, K., Souret, F. F., MuthuValliappan, R., Tej, S. S., Poethig, R. S., et al. (2006). MicroRNAs

and other small RNAs enriched in the Arabidopsis RNA-dependent RNA polymerase-2 mutant.

Genome Res. 16, 1276–1288. doi:10.1101/gr.5530106.

Mao, G., Meng, X., Liu, Y., Zheng, Z., Chen, Z., and Zhang, S. (2011). Phosphorylation of a WRKY

Transcription Factor by Two Pathogen-Responsive MAPKs Drives Phytoalexin Biosynthesis in

Arabidopsis. Plant Cell 23, 1639–1653. doi:10.1105/tpc.111.084996.

Marnef, A., and Standart, N. (2010). Pat1 proteins: a life in translation, translation repression and mRNA

decay. Biochem. Soc. Trans. 38, 1602–1607. doi:10.1042/BST0381602.

Martínez de Alba, A. E., Moreno, A. B., Gabriel, M., Mallory, A. C., Christ, A., Bounon, R., et al. (2015). In

plants, decapping prevents RDR6-dependent production of small interfering RNAs from endogenous

mRNAs. Nucleic Acids Res. 43, 2902–2913. doi:10.1093/nar/gkv119.

Meister, G. (2013). Argonaute proteins: functional insights and emerging roles. Nat. Rev. Genet. 14, 447–

459. doi:10.1038/nrg3462.

Meyers, B. C., Axtell, M. J., Bartel, B., Bartel, D. P., Baulcombe, D., Bowman, J. L., et al. (2008). Criteria

for Annotation of Plant MicroRNAs. Plant Cell 20, 3186–3190. doi:10.1105/tpc.108.064311.

Miya, A., Albert, P., Shinya, T., Desaki, Y., Ichimura, K., Shirasu, K., et al. (2007). CERK1, a LysM

receptor kinase, is essential for chitin elicitor signaling in Arabidopsis. Proc. Natl. Acad. Sci. 104,

19613–19618. doi:10.1073/pnas.0705147104.

Nandety, R. S., Caplan, J. L., Cavanaugh, K., Perroud, B., Wroblewski, T., Michelmore, R. W., et al. (2013).

The Role of TIR-NBS and TIR-X Proteins in Plant Basal Defense Responses. Plant Physiol. 162,

1459–1472. doi:10.1104/pp.113.219162.

38

Navarro, L., Dunoyer, P., Jay, F., Arnold, B., Dharmasiri, N., Estelle, M., et al. (2006). A Plant miRNA

Contributes to Antibacterial Resistance by Repressing Auxin Signaling. Science 312, 436–439.

doi:10.1126/science.1126088.

Navarro, L., Jay, F., Nomura, K., He, S. Y., and Voinnet, O. (2008). Suppression of the MicroRNA Pathway

by Bacterial Effector Proteins. Science 321, 964–967. doi:10.1126/science.1159505.

Nguyen, A. H., Matsui, A., Tanaka, M., Mizunashi, K., Nakaminami, K., Hayashi, M., et al. (2015). Loss of

Arabidopsis 5’-3’ exoribonuclease AtXRN4 function enhances heat stress tolerance of plants

subjected to severe heat stress. Plant Cell Physiol., pcv096. doi:10.1093/pcp/pcv096.

Olmedo, G., Guo, H., Gregory, B. D., Nourizadeh, S. D., Aguilar-Henonin, L., Li, H., et al. (2006).

ETHYLENE-INSENSITIVE5 encodes a 5′→3′ exoribonuclease required for regulation of the EIN3-

targeting F-box proteins EBF1/2. Proc. Natl. Acad. Sci. 103, 13286–13293.

doi:10.1073/pnas.0605528103.

Palma, K., Thorgrimsen, S., Malinovsky, F. G., Fiil, B. K., Nielsen, H. B., Brodersen, P., et al. (2010).

Autoimmunity in Arabidopsis acd11 Is Mediated by Epigenetic Regulation of an Immune Receptor.

PLoS Pathog 6, e1001137. doi:10.1371/journal.ppat.1001137.

Parker, J. E., Holub, E. B., Frost, L. N., Falk, A., Gunn, N. D., and Daniels, M. J. (1996). Characterization of

eds1, a mutation in Arabidopsis suppressing resistance to Peronospora parasitica specified by several

different RPP genes. Plant Cell 8, 2033–2046. doi:10.1105/tpc.8.11.2033.

Parker, R., and Sheth, U. (2007). P Bodies and the Control of mRNA Translation and Degradation. Mol. Cell

25, 635–646. doi:10.1016/j.molcel.2007.02.011.

Petersen, M., Brodersen, P., Naested, H., Andreasson, E., Lindhart, U., Johansen, B., et al. (2000).

Arabidopsis MAP Kinase 4 Negatively Regulates Systemic Acquired Resistance. Cell 103, 1111–

1120. doi:10.1016/S0092-8674(00)00213-0.

Potuschak, T., Lechner, E., Parmentier, Y., Yanagisawa, S., Grava, S., Koncz, C., et al. (2003). EIN3-

Dependent Regulation of Plant Ethylene Hormone Signaling by Two Arabidopsis F Box Proteins:

EBF1 and EBF2. Cell 115, 679–689. doi:10.1016/S0092-8674(03)00968-1.

Potuschak, T., Vansiri, A., Binder, B. M., Lechner, E., Vierstra, R. D., and Genschik, P. (2006). The

Exoribonuclease XRN4 Is a Component of the Ethylene Response Pathway in Arabidopsis. Plant

Cell Online 18, 3047–3057. doi:10.1105/tpc.106.046508.

Qiu, J.-L., Zhou, L., Yun, B.-W., Nielsen, H. B., Fiil, B. K., Petersen, K., et al. (2008). Arabidopsis Mitogen-

Activated Protein Kinase Kinases MKK1 and MKK2 Have Overlapping Functions in Defense

Signaling Mediated by MEKK1, MPK4, and MKS1. Plant Physiol. 148, 212–222.

doi:10.1104/pp.108.120006.

Rasmussen, M. W., Roux, M., Petersen, M., and Mundy, J. (2012). MAP kinase cascades in Arabidopsis

innate immunity. Plant Proteomics 3, 169. doi:10.3389/fpls.2012.00169.

Rasmussen, S., Barah, P., Suarez-Rodriguez, M. C., Bressendorff, S., Friis, P., Costantino, P., et al. (2013).

Transcriptome Responses to Combinations of Stresses in Arabidopsis. Plant Physiol. 161, 1783–

1794. doi:10.1104/pp.112.210773.

Rayson, S., Arciga-Reyes, L., Wootton, L., De Torres Zabala, M., Truman, W., Graham, N., et al. (2012a). A

Role for Nonsense-Mediated mRNA Decay in Plants: Pathogen Responses Are Induced in

Arabidopsis thaliana NMD Mutants. PLoS ONE 7, e31917. doi:10.1371/journal.pone.0031917.

39

Rayson, S., Arciga-Reyes, L., Wootton, L., De Torres Zabala, M., Truman, W., Graham, N., et al. (2012b). A

Role for Nonsense-Mediated mRNA Decay in Plants: Pathogen Responses Are Induced in

Arabidopsis thaliana NMD Mutants. PLoS ONE 7, e31917. doi:10.1371/journal.pone.0031917.

Riehs-Kearnan, N., Gloggnitzer, J., Dekrout, B., Jonak, C., and Riha, K. (2012). Aberrant growth and

lethality of Arabidopsis deficient in nonsense-mediated RNA decay factors is caused by

autoimmune-like response. Nucleic Acids Res., gks195. doi:10.1093/nar/gks195.

Rodriguez, M. C. S., Petersen, M., and Mundy, J. (2010). Mitogen-Activated Protein Kinase Signaling in

Plants. Annu. Rev. Plant Biol. 61, 621–649. doi:10.1146/annurev-arplant-042809-112252.

Roux, M. E., Rasmussen, M. W., Palma, K., Lolle, S., Regué, À. M., Bethke, G., et al. (2015). The mRNA

decay factor PAT1 functions in a pathway including MAP kinase 4 and immune receptor SUMM2.

EMBO J., e201488645. doi:10.15252/embj.201488645.

Seo, J.-K., Wu, J., Lii, Y., Li, Y., and Jin, H. (2013). Contribution of Small RNA Pathway Components in

Plant Immunity. Mol. Plant-Microbe Interact. MPMI 26, 617–625. doi:10.1094/MPMI-10-12-0255-

IA.

Shivaprasad, P. V., Chen, H.-M., Patel, K., Bond, D. M., Santos, B. A. C. M., and Baulcombe, D. C. (2012).

A MicroRNA Superfamily Regulates Nucleotide Binding Site–Leucine-Rich Repeats and Other

mRNAs. Plant Cell Online, tpc.111.095380. doi:10.1105/tpc.111.095380.

Sidaway-Lee, K., Costa, M. J., Rand, D. A., Finkenstadt, B., and Penfield, S. (2014). Direct measurement of

transcription rates reveals multiple mechanisms for configuration of the Arabidopsis ambient

temperature response. Genome Biol. 15, R45. doi:10.1186/gb-2014-15-3-r45.

Smith, J. E., and Baker, K. E. (2015). Nonsense-mediated RNA decay – a switch and dial for regulating gene

expression. BioEssays, n/a–n/a. doi:10.1002/bies.201500007.

Solano, R., Stepanova, A., Chao, Q., and Ecker, J. R. (1998). Nuclear events in ethylene signaling: a

transcriptional cascade mediated by ETHYLENE-INSENSITIVE3 and ETHYLENE-RESPONSE-

FACTOR1. Genes Dev. 12, 3703–3714. doi:10.1101/gad.12.23.3703.

Souret, F. F., Kastenmayer, J. P., and Green, P. J. (2004). AtXRN4 Degrades mRNA in Arabidopsis and Its

Substrates Include Selected miRNA Targets. Mol. Cell 15, 173–183.

doi:10.1016/j.molcel.2004.06.006.

Wachter, A., and Hartmann, L. (2014). NMD: Nonsense-Mediated Defense. Cell Host Microbe 16, 273–275.

doi:10.1016/j.chom.2014.08.015.

Xiang, T., Zong, N., Zou, Y., Wu, Y., Zhang, J., Xing, W., et al. (2008). Pseudomonas syringae Effector

AvrPto Blocks Innate Immunity by Targeting Receptor Kinases. Curr. Biol. 18, 74–80.

doi:10.1016/j.cub.2007.12.020.

Xu, J., and Chua, N.-H. (2012). Dehydration stress activates Arabidopsis MPK6 to signal DCP1

phosphorylation. EMBO J. 31, 1975–1984. doi:10.1038/emboj.2012.56.

Xu, J., Yang, J.-Y., Niu, Q.-W., and Chua, N.-H. (2006). Arabidopsis DCP2, DCP1, and VARICOSE Form a

Decapping Complex Required for Postembryonic Development. Plant Cell 18, 3386–3398.

doi:10.1105/tpc.106.047605.

Yanagisawa, S., Yoo, S.-D., and Sheen, J. (2003). Differential regulation of EIN3 stability by glucose and

ethylene signalling in plants. Nature 425, 521–525. doi:10.1038/nature01984.

40

Yi, H., and Richards, E. J. (2007). A Cluster of Disease Resistance Genes in Arabidopsis Is Coordinately

Regulated by Transcriptional Activation and RNA Silencing. Plant Cell 19, 2929–2939.

doi:10.1105/tpc.107.051821.

Zhang, X., Zhu, Y., Liu, X., Hong, X., Xu, Y., Zhu, P., et al. (2015). Suppression of endogenous gene

silencing by bidirectional cytoplasmic RNA decay in Arabidopsis. Science 348, 120–123.

doi:10.1126/science.aaa2618.

Zhang, Z., Wu, Y., Gao, M., Zhang, J., Kong, Q., Liu, Y., et al. (2012). Disruption of PAMP-Induced MAP

Kinase Cascade by a Pseudomonas syringae Effector Activates Plant Immunity Mediated by the NB-

LRR Protein SUMM2. Cell Host Microbe 11, 253–263. doi:10.1016/j.chom.2012.01.015.

Zhu, Y., Qian, W., and Hua, J. (2010). Temperature Modulates Plant Defense Responses through NB-LRR

Proteins. PLoS Pathog 6, e1000844. doi:10.1371/journal.ppat.1000844.

Zipfel, C., Kunze, G., Chinchilla, D., Caniard, A., Jones, J. D. G., Boller, T., et al. (2006). Perception of the

Bacterial PAMP EF-Tu by the Receptor EFR Restricts Agrobacterium-Mediated Transformation.

Cell 125, 749–760. doi:10.1016/j.cell.2006.03.03

41

Article

The mRNA decay factor PAT1 functions in apathway including MAP kinase 4 and immunereceptor SUMM2Milena Edna Roux1,†, Magnus Wohlfahrt Rasmussen1,†, Kristoffer Palma2, Signe Lolle1, Àngels Mateu

Regué1, Gerit Bethke3, Jane Glazebrook3, Weiping Zhang4, Leslie Sieburth4, Martin R Larsen5,

John Mundy1 & Morten Petersen1,*

Abstract

Multi-layered defense responses are activated in plants uponrecognition of invading pathogens. Transmembrane receptorsrecognize conserved pathogen-associated molecular patterns(PAMPs) and activate MAP kinase cascades, which regulatechanges in gene expression to produce appropriate immuneresponses. For example, Arabidopsis MAP kinase 4 (MPK4) regulatesthe expression of a subset of defense genes via at least one WRKYtranscription factor. We report here that MPK4 is found incomplexes in vivo with PAT1, a component of the mRNA decappingmachinery. PAT1 is also phosphorylated by MPK4 and, upon flagel-lin PAMP treatment, PAT1 accumulates and localizes to cytoplas-mic processing (P) bodies which are sites for mRNA decay.Pat1 mutants exhibit dwarfism and de-repressed immunity depen-dent on the immune receptor SUMM2. Since mRNA decapping is acritical step in mRNA turnover, linking MPK4 to mRNA decay viaPAT1 provides another mechanism by which MPK4 may rapidlyinstigate immune responses.

Keywords decapping; immunity; MAP kinases; mRNA decay; phosphorylation

Subject Categories Microbiology, Virology & Host Pathogen Interaction;

Plant Biology; RNA Biology

DOI 10.15252/embj.201488645 | Received 2 April 2014 | Revised 2 December

2014 | Accepted 11 December 2014

Introduction

Plant innate immunity employs multilayered defense responses

comprised of two overlapping mechanisms. In the first layer, plant

pattern recognition receptors detect invading microorganisms by the

presence of conserved pathogen-associated molecular patterns

(PAMPs)(Boller & Felix, 2009). PAMP recognition is exemplified by

the binding of the bacterial flagellin-derived flg22 peptide to the

leucine-rich repeat-receptor-like kinase flagellin sensing 2 (FLS2)

(Gomez-Gomez et al, 2001; Chinchilla et al, 2006). PAMP recognition

initiates downstream signaling, including production of reactive

oxygen species, calcium influx, MAP kinase activation and global

changes in gene expression that induce PAMP-triggered immunity

(PTI) (Chisholm et al, 2006; Zipfel, 2009). Adapted pathogens have

evolved effector proteins that are delivered into host cells to compro-

mise PTI by evading PAMP detection or suppressing defense

responses. In the second layer of immunity, plant resistance (R)

proteins have evolved to directly or indirectly recognize the activities

of pathogen effectors (Jones & Dangl, 2006). In the best-studied exam-

ples, R proteins are found to guard host proteins or complexes (guar-

dees) manipulated by specific pathogen effectors. Pathogen detection

via R proteins leads to induction of strong defenses and to a form of

host programmed cell death known as the hypersensitive response

(HR) to sequester infections. These responses are collectively termed

effector-triggered immunity (ETI) (Jones & Dangl, 2006).

Changes in phosphorylation are important regulatory mechanisms

in cellular signaling. Activation of mitogen-activated protein (MAP)

kinases occurs within 10 min of PAMP application (Asai et al, 2002;

Boller & Felix, 2009) and relies on sequential phosphorylations

between MAPKK-kinases (MEKK), MAPK kinases (MKK) and MAP

kinases (MPK) (Pitzschke et al, 2009; Andreasson & Ellis, 2010;

Rasmussen et al, 2012). In Arabidopsis, several MPKs are activated

by PAMPs. A cascade comprising MEKK1-MKK4/5-MPK3/6 was

initially found to be involved in PTI downstream of FLS2 (Asai et al,

2002; Droillard et al, 2004; Gao et al, 2008). Similarly, flg22 treatment

activates another cascade including MEKK1, MKK1/2 and MPK4

(Petersen et al, 2000; Ichimura et al, 2006; Meszaros et al, 2006;

Suarez-Rodriguez et al, 2007; Gao et al, 2008; Qiu et al, 2008a).

1 Department of Biology, Faculty of Science, University of Copenhagen, Copenhagen, Denmark2 New England Biolabs Ltd, Whitby, ON, Canada3 Department of Plant Biology, University of Minnesota, St. Paul, MN, USA4 Department of Biology, University of Utah, Salt Lake City, UT, USA5 University of Southern Denmark, Institute for Biochemistry and Molecular Biology, Odense, Denmark

*Corresponding author. Tel: +45 353 22127; E-mail: [email protected]†These authors contributed equally to this work

Published online: January 20, 2015

42

Interestingly, Flg22-induced activation of MPK3, MPK4 and MPK6 is

dependent on MKK1, while MPK3 and MPK6 are also activated by

MKK4 (Meszaros et al, 2006). Thus, FLS2 activates two cascades, one

with an unknown MEKK and MKK4/5-MPK3/6, the other with

MEKK1-MKK1/2-MPK4. More recently, the closest homologue of

MPK4, MPK11, was shown to also be activated by PAMPs (Bethke

et al, 2012).

mpk4 mutants were originally found to exhibit autoimmunity,

and MPK4 thus appeared to function genetically as a negative regu-

lator of defense responses (Petersen et al, 2000; Droillard et al,

2004). However, MPK4 is activated in response to pathogens and

PAMP elicitation (Droillard et al, 2004; Teige et al, 2004; Ichimura

et al, 2006; Brader et al, 2007; Qiu et al, 2008a) which is counter-

intuitive for a negative regulator. Subsequently, it was shown that

activated MPK4 interacts with and phosphorylates MAP kinase

substrate 1 (MKS1), bringing about the release of the transcription

factor WRKY33 and induction of the expression of the PHYTO-

ALEXIN DEFICIENT 3 (PAD3) gene required for biosynthesis of the

antimicrobial camalexin (Andreasson et al, 2005; Qiu et al, 2008b).

This illustrates how MPK4 functions as a positive regulator of PTI.

Since it was recently reported that MPK4 is a target for manipula-

tion by pathogen effectors (Zhang et al, 2007, 2012), and as mpk4

mutant phenotypes are partially suppressed by mutations in the R

protein SUMM2 (suppressor or mkk1 mkk2) (Zhang et al, 2012),

one model is that MPK4 is a PTI guardee whose absence triggers

ETI.

Apart from regulation by transcription factors, mRNA translation

and degradation also regulate gene expression, especially when they

are reprogrammed to stabilize bulk mRNAs and favor mRNAs

required for an appropriate stress response (Jiao et al, 2010;

Munchel et al, 2011; Park et al, 2012; Ravet et al, 2012). mRNA is

intrinsically unstable to facilitate rapid changes in mRNA abundance

in response to stimuli. Eukaryotic mRNAs contain stability determi-

nants including the 50 7-methylguanosine triphosphate cap (m7G)

and the 30 poly(A) tail. To achieve fine control of transcript

abundance, mRNA is attacked by a decapping complex that breaks

down RNA in a coordinated manner. mRNA decay is initiated by

deadenylation, the removal of the 30 poly(A) tail, whereafter mRNA

can either be routed to 30–50 degradation by exosomal exonucleases,

or 50–30 by the exoribonuclease activity of the decapping complex

(Garneau et al, 2007). The decapping complex is highly conserved

among eukaryotes (Garneau et al, 2007; Xu & Chua, 2011) and

comprises the decapping enzyme DCP1/2 which removes the 50 m7G

cap, and the exoribonuclease XRN that degrades the monophosph-

orylated mRNA. Deadenylation and decapping are linked in

eukaryotes by the decapping enhancer PAT1 (protein associated

with topoisomerase II) (Hatfield et al, 1996; Wang et al, 1996;

Tharun & Parker, 2001; Ozgur et al, 2010). In yeast, PAT1 promotes

the interaction between mRNA and decapping enzyme, altering

the messenger ribonucleoprotein (mRNP) organization from an

arrangement favoring translation to one promoting decay. mRNA

decay occurs in distinct cytoplasmic foci called processing bodies

(PBs) in eukaryotes (Parker & Sheth, 2007; Balagopal & Parker,

2009), where mRNPs are sequestered for degradation, or from which

they may re-enter polysomal complexes (Brengues, 2005). PBs can

also harbor translationally arrested mRNPs and RNA silencing

machinery (Balagopal & Parker, 2009; Xu & Chua, 2009; Thomas

et al, 2011).

In plants, the decapping machinery includes the decapping

enzyme DCP2 and its activators DCP1, DCP5, VARICOSE (VCS)

(Deyholos et al, 2003; Xu et al, 2006; Goeres et al, 2007; Brodersen

et al, 2008; Xu & Chua, 2009, 2011; Motomura et al, 2012) as well

as the exoribonuclease XRN4 (Olmedo et al, 2006; Potuschak

et al, 2006; Gregory et al, 2008; Rymarquis et al, 2011; Vogel et al,

2011). It is thought that DCP1, DHH1 and DCP5 form mRNPs for

translational repression of target mRNA, which are then subject to

decapping by recruitment of DCP2 and VCS and digestion by XRN4

(Xu & Chua, 2009, 2011). Importantly, although PAT1 functions have

not previously been studied in plants, three homologues are encoded

in the Arabidopsis genome, each of which contains a conserved

C-terminal domain. The decapping activator Sm-like (LSM) proteins,

which interact with PAT1 in eukaryotes (Salgado-Garrido et al, 1999;

Bonnerot et al, 2000; Bouveret et al, 2000; Tharun et al, 2000;

Tharun, 2009), have recently been characterized in Arabidopsis

(Perea-Resa et al, 2012; Golisz et al, 2013). It was found that

LSM1-7 proteins form a complex and lsm1 mutants accumulate

capped mRNA. Furthermore, VCS, DHH1 and PAT1 homologs were

identified in LSM1 immunoprecipitates (Golisz et al, 2013).

The decapping complex plays important roles in eukaryotic

development. In contrast, links between mRNA decapping and

stress signaling are just being uncovered (Jiao et al, 2010; Buchan

et al, 2011; Munchel et al, 2011; Park et al, 2012), and how decap-

ping may be involved in regulating immune systems is largely

unknown. Here, we characterize the Arabidopsis homologue of the

mRNA decay regulator PAT1. We show that PAT1 functions in

decapping of mRNA. Furthermore, PAT1 is phosphorylated in

response to flg22 and localizes to discrete, punctate foci in the cyto-

sol. PAT1 also interacts with MPK4 and with the R protein SUMM2

in planta, and the absence of PAT1 triggers SUMM2 dependent

immunity. This indicates that PAT1 is regulated by MPK4 in a path-

way whose disruption leads to ETI via SUMM2.

Results

AtPAT1 is an ScPAT1 orthologue and interacts withMPK4 in planta

We previously conducted a yeast two-hybrid screen to identify Arab-

idopsis proteins that interact with MPK4 (Andreasson et al, 2005). In

addition to the MPK4 substrate MKS1, we identified two clones encod-

ing PAT1 (At1g79090), an mRNA decapping stimulator involved in

post-transcriptional gene regulation (Coller & Parker, 2005). Two PAT1

homologs are encoded in the Arabidopsis genome (AtPAT1H1

At3g22270; AtPAT1H2 At4g14990, Supplementary Fig S1A). The

steady-state expression level of PAT1 and the homologs compared to

the housekeeping gene ACTIN8 (At1g49240) was analyzed by RT-PCR

(Supplementary Fig S1B). We focused on PAT1 as the other two homo-

logs were not identified by yeast two-hybrids. To confirm the PAT1-

MPK4 interaction in planta, doubly transgenic Arabidopsis lines were

generated in the Ler mpk4-1 background that expressed PAT1 with a

C-terminalMyc tag andHA-taggedMPK4under the control of their own

promoters. Anti-Myc immunoprecipitation from either MPK4-HA or

double transgenic MPK4-HA/Pat1-Myc tissue detected a 50 kDa band

corresponding to MPK4-HA only in double transgenic lines (Fig 1A).

Thus,MPK4 and PAT1 can be found in complex inArabidopsis.

The EMBO Journal mRNA decapping component PAT1 in immunity Milena Edna Roux et al


43

Yeast PAT1 engages with translating mRNPs and is involved in

translational repression and decapping activation (Marnef & Stan-

dart, 2010). Since the function of PAT1 in Arabidopsis was

unknown, we examined whether it functions similarly to yeast

PAT1. To this end, a full-length Arabidopsis PAT1 cDNA was cloned

from Col-0 (Supplementary Fig S1C and D) and transformed into

wild-type yeast (B4742) and a yeast mutant (Y15797) in which yeast

PAT1 was replaced with a G418 resistance cassette (BY4742

(YCR077c) pat1D::KanMX). In contrast to the wild-type, yeast lack-

ing PAT1 (pat1D) display a temperature-sensitive phenotype and

are impaired at 37°C but grow normally at 30°C (Tharun et al,

2005). This phenotype is reverted to wild-type in yeast containing

Arabidopsis PAT1, as growth at 37°C was restored (Fig 1B). As an

additional control, we transformed Arabidopsis PAT1 into wild-type

pat1

B4742

B4742+AtPAT1

pat1 +AtPAT1

1

Dilution

2 3 4 50 1

Dilution

2 3 4 50

Input Myc IP

MPK4-HA

PAT1-Myc

WB: anti-HA

WB: anti-Myc

- - +

++

+

++

kDa

100

50

50

A

B

EIF4A1

EXPL1

UGT87A2

Col-0 pat1-1

D

PAT1-Myc

C

PA

T1-

HA

+LS

M1-

GF

P

LSM

1-G

FP

HA WB

GFP WB

GFP WB

HA WB

Rubisco

GF

P IP

Inp

ut

PA

T1-

HA

Figure 1. MPK4 and PAT1 interact in Arabidopsis.

A MPK4-HA is detected in PAT1-Myc immunoprecipitates from double transgenic Arabidopsis plants. Immunoblots of input and anti-Myc IPs probed with anti-HA andanti-Myc antibodies. Left panel, input; right panel, anti-Myc IP.

B Yeast pat1D mutants are unable to grow at 37°C while wild-type (B4742) strain grows at 30°C (left panel) and 37°C (right). Growth at 37°C is restored in pat1Dexpressing AtPAT1. Serial dilutions of each yeast strain were plated on YPAD agar plates and grown at the indicated temperatures.

C Co-IP between PAT1-HA and LSM1-GFP. Proteins were transiently co-expressed in N. benthamiana and tissue harvested 3 days post-infiltration. Immunoblots of GFPIPs (top panel) and inputs (20 lg each, bottom panel). Immunoblots were cut in half and probed with anti-HA antibodies and anti-GFP antibodies.

D Agarose gel electrophoresis of 50 RACE PCR to detect capped transcripts of EIF4A1, UGT87A2 and EXPL1 in 14-day-old Col-0 and pat1-1.

Source data are available online for this figure.

Milena Edna Roux et al mRNA decapping component PAT1 in immunity The EMBO Journal


44

yeast (B4742/AtPAT1), and this grew similarly to wild-type at 30°C

and almost as well at the wild-type at 37°C (Fig 1B). The expression

of Arabidopsis PAT1 in yeast was confirmed by anti-PAT1 immuno-

blotting of yeast protein extracts (Supplementary Fig S1E). This

provides compelling evidence for the orthologous functions of these

yeast and Arabidopsis PAT1 proteins. As PAT1 is found in complex

with MPK4, these results provide a link between MPK4 and post-

transcriptional regulation of mRNA stability.

We next analyzed the interaction between PAT1 and conserved

components of mRNA decapping. PAT1-LSM1-7 complexes function

in mRNA decapping and deadenylation (Bouveret et al, 2000;

Tharun, 2009; Haas et al, 2010; Ozgur et al, 2010; Totaro et al,

2011). We therefore transiently expressed in Nicotiana benthamiana

LSM1-GFP and PAT1-HA and then immunoprecipitated LSM1 with

GFP Trap beads. PAT1-HA could be detected in LSM1 immunopre-

cipitates but did not adhere to GFP Trap beads in the absence of

LSM1-GFP (Fig 1C). This is consistent with the detection of peptides

corresponding to PAT1 and its homologues in LSM1 immunoprecipi-

tates (Golisz et al, 2013) and supports a role of PAT1 in mRNA

decapping. In other organisms, interactions between PAT1 and

LSM1 are robust, while those between PAT1 and other mRNA decap-

ping proteins, including the DCP1-DCP2 complex and XRN1, are

more transient (Bouveret et al, 2000; Nissan et al, 2010; Ozgur et al,

2010). This is consistent with our difficulty in detecting DCP1 in

complex with PAT1 in Arabidopsis (Supplementary Fig S1F).

PAT1 is required for decapping of selected mRNAs

In order to determine whether PAT1 behaves as an activator of

mRNA decapping, we used 50 RACE to compare the levels of

capped mRNAs in Col-0 and pat1 mutants. To this end, we identi-

fied an allele, pat1-1 (Salk_040660), with a T-DNA insertion in the

last exon of PAT1 (Supplementary Fig S1C). We also generated an

anti-PAT1 antibody against a C-terminal peptide (Supplementary

Fig S1D). Immunoblotting of Col-0 protein extracts with this anti-

body detected a clear band around 90 kDa. In contrast, no protein

could be detected in pat1-1 mutant extracts (Supplementary Fig

S1G). This indicates that pat1-1 harbors either a truncated version

of PAT1, no PAT1 protein, or levels of the protein that are below

detection.

50 RACE was performed on transcripts known to be degraded by the

decapping complex (EXPL1; UGT87A2) (Perea-Resa et al, 2012), as well

as a housekeeping transcript EIF4A1. We found that capped EXPL1 and

UGT87A2 accumulated in pat1-1 mutants, while capped EIF4A1 mRNA

was present in equal amounts in Col-0 and pat1-1 (Fig 1D). This

indicates that PAT1 plays a role in mRNA decay via decapping.

PAT1 is an MPK4 substrate

Since MPK4 and PAT1 are found in complexes in planta, we asked

whether PAT1 is an MPK substrate. PAT1 contains 5 Ser-Pro (SP)

motifs which are commonly phosphorylated by MPKs (Pearson

et al, 2001; Ubersax & Ferrell, 2007) (Supplementary Fig S1D). To

characterize PAT1 phosphorylation in vivo, we identified PAT1

phosphopeptides by mass spectrometry. Since MPK3/4/6 are acti-

vated by flagellin and by virulent strains of Pseudomonas syringae

pv. tomato (Pto) DC3000 (Asai et al, 2002; Brader et al, 2007;

Suarez-Rodriguez et al, 2007; Bethke et al, 2009, 2012; Rasmussen

et al, 2012), PAT1 was immunoprecipitated from extracts of

untreated control and flg22-treated wild-type Col-0 or PAT1-GFP

transgenic lines. Bands corresponding to PAT1-GFP (130 kDa)

were excised from the gel (Supplementary Fig S2), subjected to in-

gel tryptic digestion, and peptides were extracted. Phosphopeptides

were enriched by TiO2 chromatography and analyzed by liquid

chromatography (RP-HPLC) coupled to electrospray ionization

tandem mass spectrometry (LC-ESI-MS/MS). This identified several

phosphopeptides from PAT1-GFP IPs that were not detectable in

the negative control (Col-0) (Table 1; Supplementary Fig S2). The

most abundant phosphopeptide, based on the extracted ion chro-

matogram from the LC-MS/MS analysis, revealed phosphorylation

of Ser208 in an SP motif (Fig 2A). Another peptide was identified

with phosphorylation of Ser343. However, this site is not within

an SP motif, making it a less likely site for phosphorylation by

MPKs. Importantly, both these peptides have previously been

detected in Arabidopsis by mass spectrometry (Phosphat Database,

http://phosphat.mpimp-golm.mpg.de/). It should be noted that the

PAT1 phosphopeptides were detected both whether or not the

sample had been treated with flg22. Thus, under the conditions

used here, PAT1 was phosphorylated and remained so after expo-

sure to flg22.

To determine whether PAT1 is a substrate of a specific MPK, we

carried out in vitro kinase assays using immunoprecipitated, PAMP-

activated MPKs with purified His6-PAT1 protein. Phosphorylated

His6-PAT1 was detectable as a radioactive band around 95 kDa after

incubation with flg22-activated MPK4 (Fig 2B), while MPK6 caused

only low levels of PAT1 phosphorylation and MPK3 did not signifi-

cantly alter PAT1 phosphorylation (Supplementary Fig S3). Each

MPK was also incubated with the generic MPK substrate myelin

basic protein (MBP) to verify their activation (Fig 2B; Supplemen-

tary Fig S3). This confirms that activated MPK4, and to a lesser

extent, MPK6, is responsible for the phosphorylation of PAT1. A

version of His6-PAT1 with Ser208 mutated to alanine (S208A)

was also incubated with MPK4 and MPK6 IPs, and this mutant

Table 1. Phosphopeptides identified in PAT1-GFP IPs by mass spectrometry analysis.

Sequence Phospho-sitesa m/z

Mascot score

PAT1GFP water PAT1GFP flg22

SSFVSYPPPGSISPDQR 1 (S208) 950.93005 71 79

SSFVSYPPPGSISPDQR 2 (S208, S200) 990.91333 46 64

SSSGNYDGMLGFGDLR 1 (S343) 929.87323 70 114

SSSGNYDGMLGFGDLR 2 (S343, S342) 969.85663 39 50

aPhosphosites refers to the number and in brackets the location of the phosphorylation detected by MS analysis. Potential phosphorylation sites are indicated inbold letters. m/z refers to the mass-to-charge ratio of the tabulated peptides.



45

http://phosphat.mpimp-golm.mpg.de/

had significantly lower levels of phosphorylation (Fig 2B). This

supports the identification of S208 as a key phosphorylation site in

PAT1.

pat1 mutants exhibit autoimmunity similar to mpk4

Given that MPK4 is an important immune regulator in Arabidopsis

and that mpk4 mutants have de-repressed defense responses, we

examined whether PAT1 may also be involved in immunity. The

pat1-1 mutant has a distinct leaf serration phenotype and a slightly

smaller rosette than Col-0 (Fig 3). A similar rosette phenotype was

seen in another T-DNA insertion line (pat1-2, WiscDsLox_734_D04

in Supplementary Figs S1C and S4), indicating that this phenotype is

due to loss-of-function of PAT1. Importantly, however, the pheno-

type of pat1-1 is not as extreme as mpk4-2, which is much smaller

and has more pronounced leaf curling and reduced fertility as well

as constitutive defense gene expression (Petersen et al, 2000);

Fig 3).

In order to determine whether pat1-1 is a constitutive defense

mutant similar to mpk4, quantitative reverse-transcription PCR

(qRT-PCR) was used to measure the steady-state level of the

pathogenesis-related PR1 and PR2 genes in adult plants (Fig 4A).

Previous work showed that the enhanced defense response of mpk4

mutants is suppressed by mutations in EDS1 (Brodersen et al, 2006).

Thus, eds1-2 was crossed with pat1-1 to explore the pat1 pheno-

type in the absence of this regulator (Fig 3). Compared to

Col-0, pat1-1 mutants accumulated 1,000-fold more PR1 and 150-fold

more PR2 transcripts (Fig 4A). In contrast, the levels of PR mRNAs

in pat1-1 eds1-2 double mutants were similar to those in wild-type

(Fig 4A). Under the same conditions, mpk4-2 mutants accumulated

4,000-fold more PR1 and 800-fold more PR2 transcripts (Fig 4A).

We found that the elevated PR gene expression in pat1-1 corre-

lated to an enhanced resistance to infection by syringe-infiltrated

Pto DC3000 when compared to Col-0 (Fig 4B). While Pto DC3000

growth reached 5.5 cfu/cm2 in Col-0, pat1-1 mutants supported

tenfold lower accumulation of Pto DC3000. In this experiment,

eds1-2 mutants showed enhanced bacterial growth as expected for

this mutant with compromised defense responses (Fig 4B). pat1

disease resistance is EDS1 dependent, as bacterial growth pat1-1

eds1-2 double mutants was similar to that in eds1-2 (Fig 4B). These

findings indicate that, similar to mpk4, pat1 mutants express

EDS1-dependent autoimmunity in the absence of microbes.

300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900m/z

0

5

10

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Rela

tive

Abun

danc

e

1230

.50

653.

08

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536.

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682.

17

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863.

75

303.

17

1598

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75

1288

.25

1036

.42

1726

.71

1708

.75

y11+P

y12+P Y13+Py15+P

y14+P

b15+Py2y4

y5+P y10+P

y9+P

y8+Py7+P

b6

(M+H)2+ - H3PO4

S Sy15

Fy14

Vy13

Sy12

Yy11

b6

Py10

Py9

Py8

Gy7

S Iy5

Sy4

b13

P Dy2

b15

Q R

A

BHis6-PAT1 His6-PAT1 S208A MBP

4 6- 4 6- 4 6-

Autoradiogram

CBB Gel

y11+P – H3PO4

b13+P – H3PO4

Figure 2. PAT1 is phosphorylated in planta and in vitro.

A Tandem MS spectrum of the phosphopeptide SSFVSYPPPGSISPDQR in which the underlined serine is phosphorylated. Y- and b-ions are indicated that localizephosphorylation to the SP site.

B MPK4 and MPK6 were immunoprecipitated from extracts of Col-0 seedlings treated with 200 nM flg22 for 10 min. For the negative control (�), extracts wereincubated with agarose beads without antibodies. IPs were incubated with His6-PAT1, His6-PAT1 S208A or MBP for 60 min at 37°C before boiling and SDS-PAGE.Autoradiogram (top panel), Coomassie-stained gel for loading control (bottom).




46

PAT1 detection in P-bodies is induced by PAMPs

Processing bodies (PBs) are cytoplasmic granules involved in both

mRNA decay and translational repression pathways (Kulkarni et al,

2010). PAT1 is a conserved PB component in yeast (Rodriguez-Cousino

et al, 1995), C. elegans (Boag et al, 2008), Drosophila (Marnef et al,

2010) and mammals (Scheller et al, 2007) and PBs can be signifi-

cantly induced in Arabidopsis by hypoxia and heat stress (Weber

et al, 2008). To investigate whether PAT1 is found in PBs, we

produced transgenic Arabidopsis lines that express PAT1-GFP from

its native promoter complementing the pat1 phenotype (Supplemen-

tary Fig S5A and B). We detected GFP signal in small numbers of

distinct foci by confocal microscopy in roots of young seedlings

(Fig 5A). Within 20 min of flg22 treatment, a significant increase in

the number of GFP-positive foci could be seen in the root tips

(Fig 5A). To test whether these foci correspond to PBs, we treated

the roots with cycloheximide in DMSO which is known to abrogate

PB formation in plants (Goeres et al, 2007). This revealed that

cycloheximide inhibited flg22-induced foci (Fig 5A). Importantly,

the control DMSO treatment did not reduce the number of foci

(Supplementary Fig S6A and B). As a control, we also tracked the

localization of the known decapping component VCS (Xu et al,

2006). Similar to PAT1-GFP, VCS-GFP was seen in flg22-induced

PBs and absent when treated with cycloheximide (Fig 5B). Interest-

ingly, PAT1 protein, which is hardly detectable under steady-state

conditions, also accumulated in response to flg22 treatment, with a

peak by 60 min, and a return to normal levels after 2 h (Fig 5C).

PAT1-GFP mirrors this effect and was similarly up-regulated in

response to flg22 treatment, as detected by anti-GFP Western blotting

of seedling protein (Fig 5D). Importantly, PAT1 transcript levels were

not highly induced by flg22 treatment in Col-0 seedlings

(Supplementary Fig S6C), suggesting that PAT1 induction occurs

post-transcriptionally. These data indicate that activation of PTI

leads to up-regulation of PAT1 protein levels by an unknown post-

transcriptional mechanism, and this facilitates the detection of PAT1

in PBs. In addition, PTI could induce PB formation as part of cellular

reprogramming, and PAT1 may localize to PBs to engage in this

process.

The MPK4 suppressor summ2 also suppresses the pat1resistance phenotype

Autoimmunity caused by loss of different components of the MPK4

kinase cascade can be suppressed by mutations in the resistance

protein SUMM2 (Zhang et al, 2012). An explanation for this is that

SUMM2 keeps this PAMP responsive pathway under surveillance

and that mutations in its components mimic the effects of microbial

effectors that prevent phosphorylation within or below the cascade

(Zhang et al, 2007). To further probe the connection between MPK4

and PAT1, we generated pat1-1 summ2-8 double mutants. These

mutants retained the leaf serration phenotype of pat1-1 single

mutants (Supplementary Fig S7A). However, double mutants no

longer accumulated excessive PR1 and PR2 transcripts (Fig 6A) and

displayed summ2 levels of susceptibility to syringe-infiltrated Pto

DC3000 (Fig 6B). Importantly, the pat1 growth phenotype was not

caused by overexpression of SUMM2 transcripts, as the level of

SUMM2 in pat1-1 was similar to wild-type (Supplementary Fig S7B).

To test whether the accumulation of 50 capped transcripts in pat1-1

(Fig 1D) was merely an effect of inappropriate activation of

SUMM2, we tested the accumulation of 50 capped UGT87A2 in an

XRN1 sensitivity assay. XRN1 degrades all uncapped RNA leaving 50

capped RNA intact (Blewett & Goldstrohm, 2012). The accumulation

of 50 capped versus uncapped UGT87A2 transcripts was at similar

levels in pat1-1 and pat1-1 summ2-8 (Fig 6C). Col-0 and summ2-8

plants had similarly reduced 50 capped versus uncapped ratios but,

importantly, these were much lower than the levels of pat1-1 and

pat1-1 summ2-8 (Fig 6C). Therefore, the accumulation of capped

transcripts in pat1-1 mutants is not an artifact of defense activation

but reflects a role for PAT1 in mRNA decay. In order to determine

whether the SUMM2-mediated resistance in pat1 mutants is specific

to pathogens of a specific class, we compared susceptibility to

pathogens with different lifestyles. Pto DC300 is a hemi-biotrophic

eds1-2

Col-0 pat1-1 mpk4-2

pat1-1 eds1-2

Figure 3. pat1 mutants have a serrated leaf, semi-dwarf phenotype.Plants photographed at 4 weeks of growth in short day conditions, genotypes as indicated. Col-0 and eds1-2 plants and pat1-1 and pat1-1/eds1-2, respectively, were placed inthe middle of the same pots before the pictures were taken.



47

pathogen, which grows in living tissue during early infection. In

contrast, the necrotrophic fungal pathogen Botrytis cinerea relies on

dying tissue for its propagation in plants (Glazebrook, 2005). Infec-

tion of pat1-1 summ2 double mutants with B. cinerea resulted in

similar growth of the fungus as detected in summ2-8 single mutants

(Supplementary Fig S8).

To more accurately address to what extent PAT1 phosphoryla-

tion is MPK4 dependent and to measure PAT1 phosphorylation

profiles before and after flg22 treatment, we generated PAT1-HA

transgenic lines in the mkk1/2 summ2-8 background. The

suppression of mpk4 by summ2-8 is only partial, but the autoim-

munity phenotype is fully suppressed in mkk1/2 summ2 triple

mutants. We next applied quantitative mass spectrometry (iTRAQ)

to PAT1-HA IPs from Col-0 and mkk1/2-summ2 and this revealed

increased phosphorylation stoichiometry of PAT1 at Ser208 in

untreated Col-0 compared to mkk1/2 summ2 plants (Fig 6D).

Flg22 treatment leads to increased levels of PAT1 Ser208 in both

genotypes, but the levels in Col-0 were higher than what we

found for PAT1 in mkk1/2 summ2 (Fig 6D). Since flg22 treat-

ment augments PAT1 Ser208 phosphorylation in the absence of

MKK1/2, MPK4 might be activated by other upstream kinases or

PAT1 may be phosphorylated by MPK6. Nevertheless, these data

indicate that flg22 treatment leads to increased phosphorylation of

PAT1 at Ser208 and the MKK1/2 MPK4 pathway contributes to

this phosphorylation.

Our data indicate that PAT1 is part of the pathway including

MPK4 and the SUMM2 R protein. SUMM2 and PAT1 might thus

interact in planta. To test this, we transiently expressed and immu-

noprecipitated PAT1-GFP in N. benthamiana also transiently

expressing MPK4-HA or SUMM2-HA. When PAT1-GFP was pulled

down using GFP Trap beads, we could easily detect MPK4-HA and

SUMM2-HA in the immunoprecipitates (Fig 7A). To verify the asso-

ciation of MPK4, SUMM2 and LSM1, we also pulled down PAT1-HA

and detected MPK4-GFP, SUMM2-GFP and LSM1-GFP in the immu-

noprecipitates but not Myc-GFP (Supplementary Fig S9). To further

confirm the association of PAT1 and SUMM2, we used bimolecular

fluorescence complementation (BiFC), which produces a fluorescent

readout upon reconstruction of YFP. The BiFC assay showed detect-

able YFP signal in the cytoplasm when PAT1 and SUMM2 were

co-expressed (Fig 7B). No signals were observed when PAT1 was

expressed with another R protein, At4g12010, implying that the asso-

ciation/reconstruction is specific (Fig 7B, bottom panel). This indi-

cates that PAT1 is in complex with SUMM2 in planta. Thus, MPK4

and PAT1 are both physically and genetically linked to SUMM2.

Discussion

In this study, we identify the Arabidopsis decapping component

PAT1 and show it to be an interactor and substrate of MPK4 in plant

innate immunity. Furthermore, we demonstrate that Arabidopsis

PAT1 complements yeast pat1Δ mutants, indicating that the func-

tion of this conserved protein is maintained in plants. The accumu-

lation of capped mRNAs in pat1 mutants is consistent with a role

for PAT1 in the mRNA decapping pathway.

Pat1 mutants exhibit a distinct leaf serration phenotype (Supple-

mentary Fig S4) resembling those of miRNA-loss-of-function

mutants (Nikovics et al, 2006) such as abh1-8 (Gregory et al, 2008),

serrate (Grigg et al, 2005) and hypomorphic alleles of ago1 (Morel,

2002). This suggests that PAT1 may have a role connected to

microRNA activity. Arabidopsis decapping mutants such as dcp1,

dcp2 and vcs accumulate lower levels of certain miRNAs (Motomura

et al, 2012). Mutants with a pat1-like phenotype, such as vcs, suo6

and amp1, have revealed new components in miRNA-mediated

translational repression (Brodersen et al, 2008; Yang et al, 2012; Li

et al, 2013). In Drosophila, HPat interacts with components of the

miRNA machinery including AGO1 and GW182 (Bari�si�c-Jager et al,

2013). The connection between the mRNA decay activator HPat and

the miRNA effector complex may provide a link to promote the

transition of mRNA from translation to degradation. Although

PAT1 may regulate targets of miRNA-mediated translational

repression, the mechanism by which translational repression occurs

in Arabidopsis is still under investigation.

Col-0 eds1-2 pat1-1 pat1-1eds1-2

4000

3500

3000

2500

2000

1500

1000

500

0Col-0 eds1-2 pat1-1 pat1-1

eds1-2mpk4-2

Fold

cha

nge PR2

PR1

p < 0.0001p < 0.0001

p < 0.0001p < 0.0001

Log(

cfu/

cm2 )

0

1

2

3

4

5

6

7p=0.0243

p=0.0300

p=0.0003 p=0.0006

A

B

Figure 4. pat1 mutants display EDS1-dependent, constitutive defenseresponses.

A PR gene expression is elevated in mpk4-2 and pat1-1 mutants. Four-week-old plants were used for RNA extraction, followed by qRT-PCR.Fold-change in PR1 (gray bars) and PR2 (black) expression is relative toCol-0, normalized to UBQ10. Standard error of the mean is indicated byerrors bars (n = 3). Statistical significance between the mean valueswas determined by ANOVA followed by Fisher’s LSD test, P-values areonly shown for mean values significantly different from Col-0.

B pat1 is more resistant to colonization by P. syringae pv. tomato DC3000.Bacteria were syringe-infiltrated and samples taken 3 days post-infiltration.Data are shown as log10-transformed colony-forming units/cm2 leaf tissue(cfu/cm2). Standard error of the mean is indicated by errors bars (n = 4).Statistical significance between the mean values was determined byANOVA followed by Fisher0s LSD test.



48

We also find here that PAT1 is an MPK4 substrate and that pat1

mutants exhibit autoimmunity as does mpk4. The connection

between MPK4 and PAT1 is further supported by suppression of the

pat1 constitutive defense phenotype by loss-of-function of the

SUMM2 R protein (Fig 6). However, SUMM2 deficiency only

partially rescues mpk4 mutants (Zhang et al, 2012), thus it is

0 5 10 20 60 90 180

Col-0/PAT-GFP

min flg22

anti-GFP

CBB

Col

-0

PAT1

-GFP

PA

T1-G

FP fl

g22

20 m

in+

cycl

ohex

imid

e 60

min

PA

T1-G

FP f

lg22

20

min

GFP

VC

S-G

FP

VC

S-G

FP fl

g22

20 m

in+

cycl

ohex

imid

e 60

min

V

CS

-GFP

flg

22 2

0 m

in

anti-PAT1

CBB

0 5 10 20 60 90 180 H2O

pat1-1

Bright field & GFP Bright field & GFP GFP A B

C D

Figure 5. PAT1-GFP is present in P-bodies after PAMP treatment.

A Confocal microscopy with root elongation zones. Five-day-old Col-0/PAT1-GFP seedlings were treated with 1 lM flg22 on glass slides for 20 min (second panel)followed by treatment with 100 lg/ml cycloheximide for 60 min (bottom panel). The scale bar corresponds to 10 lm.

B Confocal microscopy with 5-day-old VCS-GFP seedlings treated as in (A). The scale bar corresponds to 10 lm.C PAT1 protein is induced by PAMP treatment. Immunoblot detection of equal amounts of protein from Col-0 seedlings at times in minutes as indicated following

vacuum infiltration with 1 lM flg22 or water (180 min after infiltration). Immunoblots were probed with anti-PAT1 antibodies. Negative control pat1-1 was loadedfor comparison. Coomassie brilliant blue protein loading control is indicated by CBB.

D Anti-GFP immunoblotting of proteins extracted from Col-0/PAT1-GFP seedlings treated over a time course with flg22 (top panel) and Coomassie brilliant blue (CBB)-stained PVDF loading control is shown (bottom panel). No PAT1-GFP was detected in negative control Col-0.




49

possible that this partial rescue represents a SUMM2 PAT1 branch in

the MPK4 pathway. Nevertheless, the pat1 constitutive defense

phenotype is suppressed by summ2 such that pat1 summ2 mutants

display a wild-type phenotype in response to biotrophic and necro-

trophic pathogens (Fig 6; Supplementary Fig S8). Since PAT1 and

SUMM2 also interact in planta (Fig 7), PAT1, or a PAT1-containing

complex, is part of a pathway that includes SUMM2 as well as

MPK4. It is therefore possible that PAT1 is under SUMM2 surveil-

lance because it is an effector target with specific functions in immu-

nity. Since pat1 summ2 mutants are not immunosuppressed, PAT1

and its homologues in Arabidopsis may function redundantly during

PTI. An alternative explanation is that we simply have not yet tested

a pathogen whose infection strategy could reveal a role of PAT1 in

immunity.

mRNA decapping is not the only mRNA regulatory pathway char-

acterized by constitutive defense responses. Indeed, mutants of

nonsense-mediated decay including upf3-1, upf1-5 and smg7mutants

display autoimmune phenotypes (Jeong et al, 2011; Rayson et al,

2012a; Riehs-Kearnan et al, 2012; Shi et al, 2012). Their phenotypes

are also suppressed by mutations in immune regulators such as eds1

and pad4 (Rayson et al, 2012b; Riehs-Kearnan et al, 2012), similar

to what we find for pat1. This suggests that these components could

also be under surveillance and may be suppressed by mutations in

specific R genes. Recently, it was suggested that nonsense-mediated

decay controls turnover of R gene mRNAs and cause autoimmunity

in smg7 (Gloggnitzer et al, 2014). However, since autoimmunity in

smg7 depends on a specific allele of the R protein RPS6, it is also

possible that SMG7 is under surveillance. Most significantly, this

suggests that plants have developed complex sensors to monitor the

integrity of these pathways, which is consistent with the importance

of differential gene expression in response to pathogen perception.

We detected PAT1 phosphorylation by PAMP-activated MPK4,

and weakly also by MPK6 in vitro. We also identified PAT1 Ser208

and Ser343 as in planta phosphorylation sites by mass spectrome-

try. Since PAT1 phosphorylation was reduced in vitro when Ser208

was mutated to Ala, this site may be a key target of MPK4 (Fig 2B).

Interestingly, Ser208 is conserved in Physcomitrella patens (moss),

rice, the Arabidopsis PAT1 homologues, and Xenopus PATL2, but

not in human or yeast PAT1 (Supplementary Fig S10). Although

Ser208 corresponds to an SP site in Xenopus PATL1/xPAT1b, PATL1

Col-0 pat1-1 summ2-8 pat1-1summ2-8

0%

20%

40%

60%

80%

100%

0

1

2

3

4

5

6

7

0

500

1000

1500

2000

2500 Fo

ld c

hang

eA B

C SSFVSYPPPGSISPDQR – Ser2081.8

1.6

1.4

1.2

1.0

0.8

0.6

0.4

0.2

0Col-0

PAT1-HA

D

Col-0PAT1-HA

flg22

mkk1/2/summ2PAT1-HA

mkk1/2/summ2PAT1-HA

flg22

Col-0 pat1-1 summ2-8 pat1-1summ2-8

mpk4-2 Col-0 pat1-1 summ2-8 pat1-1summ2-8

PR1 PR2

5’ c

appe

d tr

ansc

ripts

Nor

mal

ized

iTR

AQ

ratio

srel

ativ

eto

Col

PA

T1-H

A

log

(cfu

/cm

2 )

p=0.0012p=0.0010p=0.0071

p=0.0056

Figure 6. SUMM2 is required for the constitutive defense phenotype of pat1 mutants.

A Elevated PR gene expression in pat1 mutants is suppressed by summ2-8. Four-week-old plants were used for RNA extraction, followed by qRT-PCR. Fold-change inPR1 (gray bars) and PR2 (black bars) expression is relative to Col-0, normalized to UBQ10. Standard error of the mean is indicated by errors bars (n = 3).

B pat1 resistance to P. syringae pv. tomato DC3000 is suppressed by summ2-8. Bacteria were syringe-infiltrated and samples taken 3 days post-infiltration. Data arelog10-transformed colony-forming units/cm2 leaf tissue (cfu/cm2). Standard error of the mean is indicated by errors bars (n = 4).

C 14-day-old seedlings grown on ms plates were used for RNA extraction. Next, 1 lg of RNA from each sample was treated with XRN1 (NEB) or mock treated beforeRT-qPCR. Data were normalized to ACT2, and transcript levels were compared between XRN1 and mock-treated RNA for each genotype. Standard error of the mean isindicated by error bars (n = 4). Statistical significance between the mean values was determined by ANOVA followed by Fisher’s LSD test.

D Phosphorylation of the PAT1 peptide SSFVSYPPPGSISPDQR, which include Ser208, in Col-0 and mkk1/1-summ2 before and after flg22 treatment. The phosphorylationstoichiometry is illustrated relative to Col PAT1 without flg22 treatment. The ratios were obtained using quantitative iTRAQ mass spectrometry.



50

is known to be phosphorylated on Ser62 by an unknown kinase

(Marnef et al, 2010). Thus, Ser208 may represent a plant-specific

site or mechanism. While Ser208 is conserved in plant PAT1 ortho-

logs, Ser342/3 is not. As Ser342/3 does not correspond to SP sites,

they may be phosphorylated by kinases other than MPKs. Further-

more, we detected PAT1 phosphorylation in planta irrespective of

PAMP treatment.

PAT1 phosphorylation by MPKs has not been shown in any

system, although several phosphosites have been identified in

human and yeast PAT1 proteins (PhosphoElm, http://phospho.

elm.eu.org/ and Phosida http://www.phosida.com/databases).

However, other decapping complex members are subject to stress-

induced MPK-mediated phosphorylation. For example, human

DCP1a is phosphorylated by c-Jun N-terminal kinase in response to

stress (Rzeczkowski et al, 2011). Similarly, Ste20 phosphorylates

yeast DCP2 upon glucose deprivation (Yoon et al, 2010). In both

cases, phosphorylation seems only to be required for P-body forma-

tion and not for general decapping (Yoon et al, 2010; Rzeczkowski

et al, 2011). Arabidopsis MPK6 was shown to specifically phosphor-

ylate DCP1 in plants during dehydration stress (Xu & Chua, 2012).

Thus, the regulation of mRNA decay machinery by MPKs during

stress responses seems to be a key mechanism in plants and other

organisms, although exactly how this affects mRNA turnover

remains elusive.

Materials and Methods

Plant materials and growth conditions

Arabidopsis thaliana ecotype Columbia (Col-0) was used as a control.

Seeds for T-DNA insertion lines were from NASC (Nottingham, UK).

PAT1

-Nc

& S

UM

M2-

Nn

eYFP Merged

PAT1

-Nc

& S

UM

M2-

Nn

PA

T1-N

c &

At4

g120

10-N

n

150 100

HA WB

HA WB

GFP WB

50

75

100

50

75

100

Inpu

t G

FP IP

SU

MM

2-H

A

MP

K4-

HA

_

PAT1-GFP + A B

kDa

Figure 7. PAT1 is associated with MPK4 and SUMM2 in N. benthamiana.

A Proteins were transiently co-expressed (PAT1-GFP + MPK4-HA or SUMM2-HA) in N. benthamiana and tissue harvested 2 days post-infiltration. Anti-HA immunoblotsof inputs (20 lg each) are shown in the bottom panel. Immunoblots of GFP IPs were probed with anti-HA antibodies, then stripped and probed with anti-GFPantibodies. Molecular weights are shown in kDa on the right.

B Confocal images of yellow fluorescent protein complementation. PAT1-Nc was transiently co-expressed with either SUMM2-Nn or At4g12010 in N. benthamiana.Confocal microscopy on leaf disks was conducted 2 days post-infiltration. Merged pictures show overlay of brightfield, chlorophyll and eYFP. The scale barcorresponds to 25 lm.




51

http://phospho.elm.eu.org/

http://phospho.elm.eu.org/

http://www.phosida.com/data�bas�es

The T-DNA lines for At1g79090 (PAT1), both of which have inser-

tions in the last exon, were Salk_040660 (here named pat1-1) and

WiscDsLox437D04 (pat1-2). The T-DNA insertion in At1g12280

(SUMM2) summ2-8 (SAIL_1152A06), mkk1/2 (Zhang et al, 2012)

and eds1-2 (Parker et al, 1996) have been described. Genotyping

primers for newly described T-DNA lines are provided in

Supplementary Table S1. Arabidopsis plants were grown in 9 × 9 cm

pots at 22°C with a 8-h photoperiod, or on plates containing

Murashige–Skoog (MS) salts medium (Duchefa), 1% sucrose and 1%

agar with a 16-h photoperiod.

Cloning and transgenic lines

The genomic PAT1 (At1g79090) DNA sequence (without stop

codon), plus 2 kb upstream from the start codon, was amplified

from Col-0 genomic DNA and cloned into pENTR-D-TOPO (Invitro-

gen). The entry clone was recombined into pGWB513, pGWB517

and pGWB504 (Nakagawa et al, 2007) to obtain a C-terminal HA,

Myc and GFP tags, respectively. The expression clones were trans-

formed into Agrobacterium tumefaciens strain GV3101 and trans-

formed into Col-0 plants by floral dipping. Transformants were

selected on hygromycin (30 lg/ml) MS agar and survivors tested

for protein expression by Western blotting.

The VCS promoter region was amplified and inserted into pGEM-T

easy and next into the HindIII SalI in pBN:GFP to generate pBNpVCS:

GFP. The VCS coding region was amplified and cloned into pGEM-T

easy next inserted into SalI and KpnI in pBNpVCS:GFP and trans-

formed into Agrobacterium LBA4404 and transformed into Col-0

plants by floral dipping. The cDNA of LSM1 (At3g14080) without

stop codon was cloned into pENTR-D-TOPO (Invitrogen) and recom-

bined into 35S promoter-containing pGWB505 (Nakagawa et al,

2007) to obtain C-terminal GFP-tagged LSM1. Genomic DNA of

MPK4 and cDNA of SUMM2 were cloned into pENTR-D-TOPO

and recombined into pGWB514 (Nakagawa et al, 2007) to obtain

C-terminal HA-tagged constructs. The genomic DNA and promoter of

DCP1 was cloned into pENTR-D-TOPO and recombined into

pGWB513 to obtain C-terminal HA-tagged constructs under the

control of the native promoter. Clones were transformed into

A. tumefaciens strain GV3101 and used for transient expression in

N. benthamiana or floral dipping in Arabidopsis. Genomic PAT1-

GFP was transformed into the Col-0 background. DCP1-HA was

dipped into the Col-0 background and T3 progeny were crossed with

Col-0/PAT1-GFP T4 lines to obtain double transgenic lines. Genomic

PAT1-Myc was transformed into Ler mpk4-1/MPK4-HA transgenic

lines (described in Petersen et al, 2000) to obtain double transgenic

lines.

The entry clones of PAT1 and SUMM2 were further recombined

into pcYGW and pnYGW (Hino et al, 2011), respectively, obtaining

the N- and C-terminal split YFP tags used for BiFC. At4g12010

clones were obtained by the same procedure. Clones were trans-

formed into A. tumefaciens strain GV3101 and used for transient

expression in N. benthamiana.

Mutagenesis of His6-PAT1

Site-directed mutagenesis of Ser208 to Ala was accomplished using

pET15b-PAT1 as a template for PCR mutagenesis. The primers used

were PAT1 S208A F&R (see Supplementary Table S1).

PAT1 protein purification and in vitro kinase assays

For in vitro experiments, PAT1 protein was purified from E. coli.

The PAT1 cDNA was cloned into pET15b (for an N-terminal His

fusion) and transformed into E. coli BL21 (pLysS). Protein expres-

sion was induced by overnight treatment with 0.5 mM IPTG at

18°C, added to cells at OD600 = 0.6. PAT1 protein was insoluble

and thus purified from inclusion bodies using Bugbuster (Nov-

agen). Proteins were solubilized in 6 M urea, 0.7% N-lauroylsarco-

sine, 100 mM Tris-HCl pH 8 and refolded overnight at 4°C in

0.88 M L-arginine, 55 mM Tris-HCl, 2 mM NaCl, 0.88 mM KCl,

protease inhibitors. Protein was then dialyzed against 20 mM Tris-

HCl pH 8, 100 mM NaCl using 3,500 MWCO dialysis tubing. Puri-

fied protein was concentrated using Centriprep 30K spin columns

(Millipore) and then diluted with glycerol to a final concentration

of 0.1 mg/ml.

For kinase assays, Col-0 plants were immersed in 200 nM flg22

for 10 min of to activate MAP kinases. MPK3, 4 and 6 were immu-

noprecipitated from 3 mg total protein extracted from flg22-treated

tissue using 2 lg of each of their specific antibodies (Sigma) and

30 ll EZview protein A agarose beads (Sigma). Four microgram of

purified myelin basic protein (MBP, Sigma) or 20 lg His6-PAT1

protein was incubated with washed MPK immunoprecipitates for

60 min at 37°C with 3 lCi c-ATP in kinase buffer (62.5 lM ATP,

100 mM Tris pH 7.5, 150 mM NaCl, 150 mM MgCl2, 10 mM EGTA,

5 mM DTT, Phosstop inhibitor (Roche)). Kinase reactions were

diluted with 4× SDS buffer and boiled for 5 min before loading on

12% SDS-PAGE gels. Following electrophoresis, gels were stained

with Coomassie Brilliant Blue and incubated with gel drying buffer,

followed by drying on a Bio-Rad gel dryer. Dried gels were exposed

to a phosphor screen overnight.

Yeast transformation

Yeast strains pat1Δ (BY4742 (YCR077c) pat1D::KanMX) and B4742

were obtained from Euroscarf. PAT1 was cloned from A. thaliana

Col-0 cDNA into pENTR-D-TOPO and recombined into yeast expres-

sion vector pVV215 (C-terminal HA tag, -URA selection; (Van

Mullem et al, 2003) by Gateway recombination. Yeast pat1Δ and

B4742 cells were transformed using lithium acetate/polyethylene

glycol according to the Clontech Yeast Protocols handbook.

Transformed yeast was selected on SD-URA agar plates and re-

streaked after 3–4 days onto fresh selection plates. Pat1D and wild-

type-transformed yeast was grown in liquid culture overnight, and

protein was extracted from pelleted cells by vortexing with glass

beads in 1× SDS extraction buffer. Boiled proteins were subjected to

SDS-PAGE and anti-PAT1 immunoblotting. For temperature sensi-

tivity assays, overnight cultures of wild-type, pat1D and PAT1-

expressing transformants were plated on YPAD and grown at 30 and

37°C for 3 days.

Flg22 kinetics

Seedlings were grown on MS agar (Col-0) or MS agar containing

30 lg/ml hygromycin (Col-0/PAT1-GFP) for 5 days before being

transferred to MS liquid medium in 24-well plates. After 10 days,

seedlings were treated by the addition of flg22 to a final concentra-

tion of 100 nM (2 seedlings per well × 3 wells per treatment time



52

point). Seedlings were harvested at the indicated times and immedi-

ately frozen in liquid nitrogen for later RNA or protein extraction.

Semi-quantitative and qRT-PCR

Total RNA was extracted from seedlings with Tri Reagent (Sigma).

RNA samples were treated with DNase Turbo DNA-free (Ambion),

quantified with a NanoDrop spectrophotometer (Thermo Scientific)

and reverse-transcribed into cDNA with SuperScript III reverse

transcriptase (Invitrogen). For semi-quantitative reverse-transcription

PCR (RT-PCR), Col-0 seedling cDNA was used as a template for PCR

with primers specific for ACTIN8, PAT1, PAT1H1 or PAT1H2 using

Sigma Jumpstart REDTaq Readymix. PCR products (20 ll) were

separated on 2% (w/v) agarose gels and visualized with ethidium

bromide. Brilliant II SybrGreen master mix (Agilent) was used for

qPCRs. The UBQ10 (At4g05320) gene was used for normalization.

Gene expression of PR1, PR2, PAT1 and SUMM2 was measured by

qPCR analysis, normalized to UBQ10 expression and plotted relative

to Col-0 expression level. These experiments were repeated in three

independent biological replicates, each with three technical repli-

cates, and representative data are shown. Standard error is repre-

sented by error bars on figures, and statistical significance is

indicated by letters above error bars. These are derived from one-

way ANOVA with Tukey’s multiple comparison test (GraphPad

Prism).

RNA extraction and RACE PCR

RNA extraction used TRI reagent and 10 lg RNA was used for

50 RACE according to instructions (First Choice RACE, Ambion).

PCR was carried out on 1 ll of products from reverse transcription

of capped RNA using DreamTaq polymerase (Fermentas) with 25

cycles. RACE PCR products (10 ll) were separated on 2% (w/v)

agarose gels and visualized with ethidium bromide.

Quantification of capped versus uncapped transcripts

Total RNA was extracted from seedlings with NucleoSpin RNA

columns (Machery-Nagel). To remove RNA with no 50 cap structure,

1 lg of total RNA was incubated with 1 unit XRN1 (New England

Biolabs) or no enzyme at 37°C for 1 h. Next RNA was reverse-tran-

scribed into cDNA with SuperScript III reverse transcriptase (Invitro-

gen). UGT87A2 transcript accumulation was measured by qPCR

using SybrGreen master mix (Agilent) and normalized to ACT2.

Calculating 50 capped versus uncapped transcripts was done by

comparing transcript levels from XRN1 and mock-treated samples

for the individual genotypes.

Confocal microscopy

Col-0/PAT1-GFP plants were grown on MS agar containing 30 lg/ml

hygromycin for 5 days. Seedlings were placed on glass microscope

slides with water, 1% DMSO or 100 nM flg22 for 20 min. For follow-

ing cycloheximide and DMSO treatment, seedlings were removed

from flg22 and placed on new glass microscope slides. Cyclohexi-

mide was used at 100 lg/ml in a 1% DMSO dilution, and the control

was done with 1% DMSO. Imaging was done using a Leica SP5

inverted microscope.

Infection assays

Pseudomonas syringae pv. tomato DC3000 (Pto DC3000) strains

were grown in overnight culture in Kings B medium supplemented

with appropriate antibiotics. Cells were harvested by centrifugation

and pellets resuspended in sterile water to OD600 = 0.002. Bacteria

were infiltrated with a needleless 1-mm syringe into four leaves on

four plants per genotype and maintained in growth chambers for

3 days. Samples were taken using a cork-borer (6.5 mm) to cut leaf

disks from four leaves per plant and four plants per genotype. Leaf

disks were ground in water, serially diluted and plated on Kings B

with appropriate selection. Plates were incubated at 28°C and colo-

nies counted 2–3 days later. These experiments were repeated in

three independent biological replicates, and representative data are

shown.

For drop inoculation of B. cinerea (strain B05.10), 10 ll of

2.5 × 105 spores/ml in Gamborg B5/2% sucrose (pH 5.6) was

placed on the adaxial surface of fully expanded leaves of 4-week-old

plants and sampled by harvesting 10–15 leaf disks per genotype

were at each time point, namely 0, 2 and 3 days. Data are the aver-

age of 3 biological replicates. Disease severity was measured as

accumulation of the B. cinerea CUTINASE A transcript by qPCR rela-

tive to A. thaliana a-SHAGGY KINASE (At5g26751) (primers are

according to Gachon & Saindrenan, 2004).

Transient expression in Nicotiana benthamiana

Agrobacterium tumefaciens strains were grown in LB medium

supplemented with appropriate antibiotics overnight. Cultures were

spun down and resuspended in 10 mM MgCl2 to OD600 = 0.8. A.

tumefaciens strains carrying PAT1-GFP and MPK4-HA or SUMM2-

HA were mixed 1:1 and syringe-infiltrated into 3-week-old N. benth-

amiana leaves. Samples for protein extraction were harvested

2 days post-infiltration (dpi). Agrobacterium tumefaciens strains

carrying either PAT1-HA, LSM1-GFP or PAT1-HA + LSM1-GFP were

mixed 1:1 and syringe-infiltrated into 3-week-old N. benthamiana

leaves. Samples for protein extraction were harvested 3 dpi. For

BiFC, A. tumefaciens strains carrying PAT1 fused to the N-terminal

part of YFP and SUMM2 or At4g12010 fused to the C-terminal part

were mixed 1:1 and syringe-infiltrated into N. benthamiana leaves

at an final OD600 = 0.8. Confocal microscopy on leaf disks was

conducted 2 days post-infiltration under a Leica SP5 inverted micro-

scope.

Protein extraction and immunoprecipitation inNicotiana benthamiana

Leaves were ground in liquid nitrogen and extraction buffer [50 mM

Tris-HCl pH 7.5; 150 mM NaCl; 10% (v/v) glycerol; 10 mM DTT;

10 mM EDTA; 1% (w/v) PVP; protease inhibitor cocktail (Roche);

0.1% (v/v) IGEPAL CA-630 (Sigma); Phosstop (Roche) added at

2 ml/g tissue powder. Samples were clarified by 20 min centrifuga-

tion at 4°C and 13,000 rpm. Supernatants (1.5 ml) were adjusted to

2 mg/ml protein and incubated 2 h at 4°C with 20 ll GFPTrap-Abeads (Chromotek) or anti-HA antibodies (2 lg, Santa Cruz) and

30 ll EZview protein A agarose beads (Sigma). Following incuba-

tion, beads were washed four times with IP buffer, before adding

30 ll 2× SDS and heating at 95°C for 5 min.



53

Arabidopsis protein extraction and immunoprecipitation for massspectrometry analysis

Leaves from adult plants were ground in liquid nitrogen and extrac-

tion buffer [50 mM Tris-HCl pH 7.5; 150 mM NaCl; 10% (v/v) glyc-

erol; 5 mM DTT; 2 mM EDTA; protease inhibitor cocktail (Roche);

0,1% (v/v) IGEPAL CA-630 (Sigma) and Phosstop (Roche) added at

2 ml/g tissue powder. Samples were clarified by 20 min centrifuga-

tion at 4°C 13,000 rpm. Supernatants (45 ml) were adjusted to

3 mg/ml protein and incubated 4 h at 4°C with 50 ll GFPTrap-Abeads (Chromotek) or anti-HA antibodies (4 lg, Santa Cruz) and

100 ll EZview protein A agarose beads (Sigma). Following incuba-

tion, beads were washed four times with IP buffer before adding

2× SDS and heating to 95°C for 5 min.

SDS-PAGE and immunoblotting

SDS-PAGE gels were prepared with either 8, 10 or 12% cross-linking.

Proteins were loaded and gels run at 100–150 V for 1.5 h before elec-

troblotting onto PVDF membrane (GE Healthcare). Membranes were

rinsed in TBS and blocked for 1 h in 5% (w/v) non-fat milk in TBS-

Tween (0.1% (v/v)). Antibodies were diluted in blocking solution to

the following dilutions: anti-GFP (AMS Biotechnology (rabbit)

1:5,000 or Santa Cruz (mouse) 1:1,000), anti-HA 1:1,000 (Santa

Cruz). Membranes were incubated with primary antibodies for 1 h

to overnight. Membranes were washed 3 × 10 min in TBS-T (0.1%)

before 1-h incubation in secondary antibodies, anti-rabbit or anti-

mouse-HRP or anti-rabbit or anti-mouse AP conjugate (Promega; 1:

5,000). Chemiluminescent substrate (ECL Plus, Pierce) was applied

before exposure to film (AGFA CP-BU). For AP-conjugated primary

antibodies, membranes were incubated in NBT/BCIP (Roche) until

bands were visible. For probing immunoblots with multiple antibod-

ies, stripping was carried out using Restore Western Blot Stripping

Buffer (Pierce) for 15–30 min, following by three washes with TBS-

Tween.

Antibodies

Polyclonal anti-PAT1 antibodies were generated by immunizing

rabbits with synthetic peptides derived from the N-terminus

[EQRIPDRTKLYPEPQ] and C-terminus [KRSMLGSQKTEPVLS] of

PAT1. Antibodies (final bleed) were affinity-purified against the

C-terminal peptide (Eurogentec). Antibody specificity was verified

by immunoblotting with plant extracts derived from Col-0 and pat1-1

tissue. Mouse anti-HA and anti-GFP antibodies were obtained from

Santa Cruz. Rabbit anti-GFP antibodies were obtained from AMS

Biotechnology. Anti-MPK3, anti-MPK-4 and anti-MPK-6 antibodies

were obtained from Sigma. Secondary antibodies were obtained

from Promega.

In-gel digestion, TiO2 chromatography and mass spectrometry

Bands excised from SDS-PAGE were chopped into small pieces and

incubated for 1 h in 50 mM TEAB, acetonitrile (50:50) on a shaker

at room temperature. After incubation, the sample was centrifuged

shortly and the supernatant was discarded. Gel pieces were dried in

a vacuum centrifuge for 15 min and subsequently 30 ll of trypsin(10 ng/ll) in 20 mM TEAB pH 7.5 was added to cover the dried

gel pieces. The solution was incubated in 4°C for 30 min. After

incubation, the trypsin solution was replaced with 30 ll 20 mM

TEAB pH 7.5 and the tube was incubated at 37°C overnight.

Peptides from the digestion solution after incubation were recovered

in a low binding Eppendorf tube (Sorensen Bioscience), and the gel

pieces washed with 50% acetonitrile for 15 min to extract more

peptides. The washing solution was mixed with the recovered

peptides, and the peptide solution was lyophilized. Phosphopeptides

were purified by titanium dioxide (TiO2) chromatography (Larsen

et al, 2005). Lyophilized peptides were redissolved in loading buffer

for TiO2 chromatography (80% acetonitrile, 5% TFA, 1 M glycolic

acid), and 0.3 mg TiO2 beads (GL Science, Japan) were added to the

solution and incubated for 10 min. After incubation, the beads were

pelleted by centrifugation and the supernatant was removed. The

beads were washed once with 80% acetonitrile, 1% TFA and once

with 10% acetonitrile, 0.1% TFA. Phosphopeptides were eluted

using 1% ammonium hydroxide and desalted and concentrated prior

to LC-MS/MS using a Poros Oligo R3 micro-column (Engholm-Keller

et al, 2012).

For quantitative phosphopeptide analysis, the tryptic peptide

mixtures after in-gel digestion were labeled with iTRAQ 4 plex

(according to the manufacturer’s protocol) and the samples were

mixed prior to TiO2 enrichment as described above. The non-

phosphorylated peptides were concentrated and desalted using a

Poros Oligo R3 micro-column (Engholm-Keller et al, 2012). The

non-phosphorylated peptides were used to normalize the PAT1

protein level.

LC-MS/MS analysis was performed using an EASY-LC system

(Proxeon, Thermo Fischer Scientific) coupled to an LTQ-Orbitrap XL

mass spectrometer (Thermo Fisher Scientific) as described previ-

ously (Engholm-Keller et al, 2012). Peptides were separated using a

20 min gradient from 0–34% B-buffer (A-buffer: 0.1% formic acid;

B-buffer: 90% acetonitrile, 0.1% formic acid). The data-dependent

analysis was performed using one MS full scan in the area 300–

1,800 Da performed in the Orbitrap with 60,000 in resolution,

followed by the five most intense ions selected for MSMS (collision

induced dissociation) performed in the linear ion trap.

Raw data from the LTQ-Orbitrap-XL were processed using the

Proteome Discoverer (PD) program (Thermo Fisher Scientific,

Bremen, Germany). The data were searched against the Arabidopsis

database (33,596 sequences; 13,487,687 residues) using the Mascot

2.2 version database. The parameters for the database search were

as follows: Enzyme—trypsin; missed cleavages—2; MS mass accu-

racy—10 ppm; MSMS mass accuracy—0.8 Da; Variable modifica-

tions—Phosphorylation (S, T, Y), Oxidation (met), Propionamide

(C). The identified peptides were filtered for 1% false discovery

rate using the ‘Fixed Value PSM Validator’ in PD. All identified

phosphopeptides were manually validated. For iTRAQ-labeled phos-

phopeptides, the PD quantitation node was used and the data were

normalized based on the non-phosphorylated peptides.

Supplementary information for this article is available online:

http://emboj.embopress.org

AcknowledgementsWe thank Yuelin Zhang for providing summ2-8 and mkk1/2 summ2-8 seeds.

This work was supported by the Danish Research Council for independent

Research, Natural Sciences Grant #10-084139 (M.P.) & Postdoctoral Fellowship



54

#11-116368 (M.E.R.) & Technology and Production Sciences #11-106302. KP

was supported by an EMBO Long-Term Fellowship and a Marie Curie

International Incoming Fellowship. We thank Suksawad Vonvisuttikun for

technical help and Dr. Tsuyoshi Nakagawa (Shimane University) for providing

Gateway binary pGWB vectors. All confocal work was done at Center for

Advanced Bioimaging. MRL was supported by the Lundbeck Foundation (Junior

Group Leader Fellowship).

Author contributionsMER, MWR, KP, JM and MP conceived and designed the experiments. MER,

MWR KP, SL, MRL, JG, AMR, LS, WZ and GB performed experiments. MER,

MWR, JM and MP analyzed the data. MER, MWR, JM and MP wrote the

manuscript.

Conflict of interestThe authors declare that they have no conflicts of interest.

References

Andreasson E, Jenkins T, Brodersen P, Thorgrimsen S, Petersen NH, Zhu S,

Qiu J-L, Micheelsen P, Rocher A, Petersen M, Newman M-A, Nielsen HB,

Hirt H, Somssich IE, Mattsson O, Mundy J (2005) The MAP kinase

substrate MKS1 is a regulator of plant defense responses. EMBO J 24:

2579 – 2589

Andreasson E, Ellis B (2010) Convergence and specificity in the Arabidopsis

MAPK nexus. Trends Plant Sci 15: 106 – 113

Asai T, Tena G, Plotnikova J, Willmann MR, Chiu WL, Gómez-Gómez L, Boller

T, Ausubel FM, Sheen J (2002) MAP kinase signalling cascade in

Arabidopsis innate immunity. Nature 415: 977 – 983

Balagopal V, Parker R (2009) Polysomes, P bodies and stress granules: states

and fates of eukaryotic mRNAs. Curr Opin Cell Biol 21: 403 – 408

Bari�si�c-Jäger E, Krezcioch I, Hosiner S, Antic S, Dorner S (2013) HPat a

decapping activator interacting with the miRNA effector complex. PLoS

One 8: e71860

Bethke G, Unthan T, Uhrig JF, Poeschl Y, Gust AA, Scheel D, Lee J (2009)

Flg22 regulates the release of an ethylene response factor substrate from

MAP kinase 6 in Arabidopsis thaliana via ethylene signaling. PNAS 106:

674

Bethke G, Pecher P, Eschen-Lippold L, Tsuda K, Katagiri F, Glazebrook J, Scheel

D, Lee J (2012) Activation of the Arabidopsis thaliana mitogen-activated

protein kinase MPK11 by the flagellin-derived elicitor peptide, flg22. Mol

Plant Microbe Interact 25: 471 – 480

Blewett NH, Goldstrohm AC (2012) A eukaryotic translation initiation factor

4E-binding protein promotes mRNA decapping and is required for PUF

repression. Mol Cell Biol 32: 4181 – 4194

Boag PR, Atalay A, Robida S, Reinke V, Blackwell TK (2008) Protection of

specific maternal messenger RNAs by the P body protein CGH-1 (Dhh1/

RCK) during Caenorhabditis elegans oogenesis. J Cell Biol 182: 543 – 557

Boller T, Felix G (2009) A renaissance of elicitors: perception of

microbe-associated molecular patterns and danger signals by

pattern-recognition receptors. Annu Rev Plant Biol 60: 379 – 406

Bonnerot C, Boeck R, Lapeyre B (2000) The two proteins Pat1p (Mrt1p) and

Spb8p interact in vivo, are required for mRNA decay, and are functionally

linked to Pab1p. Mol Cell Biol 20: 5939 – 5946

Bouveret E, Rigaut G, Shevchenko A, Wilm M, Seraphin B (2000) A Sm-like

protein complex that participates in mRNA degradation. EMBO J 19:

1661 – 1671

Brader G, Djamei A, Teige M, Palva ET, Hirt H (2007) The MAP kinase kinase

MKK2 affects disease resistance in Arabidopsis. Mol Plant Microbe Interact

20: 589 – 596

Brengues M (2005) Movement of eukaryotic mRNAs between polysomes and

cytoplasmic processing bodies. Science 310: 486 – 489

Brodersen P, Petersen M, Nielsen H, Zhu S, Newman M-A, Shokat KM, Rietz S,

Parker J, Mundy J (2006) Arabidopsis MAP kinase 4 regulates salicylic acid-

and jasmonic acid/ethylene-dependent responses via EDS1 and PAD4.

Plant J 47: 532 – 546

Brodersen P, Sakcarelidze-Achard L, Bruun-Rasmussen M, Dunoyer P,

Yamamoto YY, Sieburth L, Voinnet O (2008) Widespread translational

inhibition by plant miRNAs and siRNAs. Science 320: 1185 – 1190

Buchan JR, Yoon J-H, Parker R (2011) Stress-specific composition, assembly

and kinetics of stress granules in Saccharomyces cerevisiae. J Cell Sci 124:

228 – 239

Chinchilla D, Bauer Z, Regenass M, Boller T, Felix G (2006) The Arabidopsis

receptor kinase FLS2 binds flg22 and determines the specificity of flagellin

perception. Plant Cell 18: 465 – 476

Chisholm ST, Coaker G, Day B, Staskawicz BJ (2006) Host-microbe interactions:

shaping the evolution of the plant immune response. Cell 124: 803 – 814

Coller J, Parker R (2005) General translational repression by activators of

mRNA decapping. Cell 122: 875 – 886

Deyholos MK, Cavaness GF, Hall B, King E, Punwani J, van Norman J, Sieburth

L (2003) VARICOSE, a WD-domain protein, is required for leaf blade

development. Development 130: 6577 – 6588

Droillard M-J, Boudsocq M, Barbier-Brygoo H, Laurière C (2004)

Involvement of MPK4 in osmotic stress response pathways in cell

suspensions and plantlets of Arabidopsis thaliana: activation by

hypoosmolarity and negative role in hyperosmolarity tolerance. FEBS

Lett 574: 42 – 48

Engholm-Keller K, Birck P, Størling J, Pociot F, Mandrup-Poulsen T, Larsen MR

(2012) TiSH–a robust and sensitive global phosphoproteomics strategy

employing a combination of TiO2, SIMAC, and HILIC. J Proteomics 75:

5749 – 5761

Gachon C, Saindrenan P (2004) Real-time PCR monitoring of fungal

development in Arabidopsis thaliana infected by Alternaria brassicicola and

Botrytis cinerea. Plant Physiol Biochem 42: 367 – 371

Gao M, Liu J, Bi D, Zhang Z, Cheng F, Chen S, Zhang Y (2008) MEKK1,

MKK1/MKK2 and MPK4 function together in a mitogen-activated protein

kinase cascade to regulate innate immunity in plants. Cell Res 18:

1190 – 1198

Garneau NL, Wilusz J, Wilusz CJ (2007) The highways and byways of mRNA

decay. Nat Rev Mol Cell Biol 8: 113 – 126

Glazebrook J (2005) Contrasting mechanisms of defense against biotrophic

and necrotrophic pathogens. Annu Rev Phytopathol 43: 205 – 227

Gloggnitzer J, Akimcheva S, Srinivasan A, Kusenda B, Riehs N, Stampfl H,

Bautor J, Dekrout B, Jonak C, Jimenez-Gomez JM, Parker JE, Riha K (2014)

Nonsense-mediated mRNA decay modulates immune receptor levels to

regulate plant antibacterial defense. Cell Host Microbe 16: 376 – 390

Goeres D, Van Norman JM, Zhang W, Fauver NA, Spencer ML, Sieburth L

(2007) Components of the Arabidopsis mRNA decapping complex are

required for early seedling development. Plant Cell 19: 1549 – 1564

Golisz A, Sikorski PJ, Kruszka K, Kufel J (2013) Arabidopsis thaliana LSM

proteins function in mRNA splicing and degradation. Nucleic Acids Res 41:

6232 – 6249

Gomez-Gomez L, Bauer Z, Boller T (2001) Both the extracellular leucine-rich

repeat domain and the kinase activity of FLS2 are required for flagellin

binding and signaling in Arabidopsis. Plant Cell 13: 1155 – 1163



55

Gregory BD, Lister R, OMalley R, Urich MA, Tonti-Phillipini J, Chen H, Millar

AH, Ecker JR (2008) A link between RNA metabolism and silencing

affecting Arabidopsis development. Dev Cell 14: 854 – 866

Grigg SP, Canales C, Hay A, Tsiantis M (2005) SERRATE coordinates shoot

meristem function and leaf axial patterning in Arabidopsis. Nature 437:

1022 – 1026

Haas G, Braun JE, Tritschler F, Nishihara T, Izaurralde E (2010) HPat provides

a link between deadenylation and decapping in metazoa. J Cell Biol 189:

289 – 302

Hatfield L, Beelman CA, Stevens A, Parker R (1996) Mutations in trans-acting

factors affecting mRNA decapping in Saccharomyces cerevisiae. Mol Cell

Biol 16: 5830 – 5838

Hino T, Tanaka Y, Kawamukai M, Nishimura K, Mano S, Nakagawa T (2011)

Two Sec13p homologs, AtSec13A and AtSec13B, redundantly contribute to

the formation of COPII transport vesicles in Arabidopsis thaliana. Biosci

Biotechnol Biochem 75: 1848 – 1852

Ichimura K, Casais C, Peck SC, Shinozaki K, Shirasu K (2006) MEKK1 is required for

MPK4 activation and regulates tissue-specific and temperature-dependent

cell death in Arabidopsis. J Biol Chem 281: 36969 – 36976

Jeong H-J, Kim YJ, Kim SH, Kim Y-H, Lee I-J, Kim YK (2011)

Nonsense-mediated mRNA decay factors, UPF1 and UPF3, contribute to

plant defense. Plant Cell Physiol 52: 2147 – 2156

Jiao X, Xiang S, Oh C, Martin CE, Tong L, Kiledjan M (2010) Identification of a

quality-control mechanism for mRNA 50-end capping. Nature 467:

608 – 611

Jones JDG, Dangl JL (2006) The plant immune system. Nature 444: 323 – 329

Kulkarni M, Ozgur S, Stoecklin G (2010) On track with P-bodies. Biochem Soc

Trans 38: 242 – 251

Larsen MR, Thingholm TE, Jensen ON, Roepstorff P, Jørgensen TJD (2005)

Highly selective enrichment of phosphorylated peptides from peptide

mixtures using titanium dioxide microcolumns. Mol Cell Proteomics 4:

873 – 886

Li S, Liu L, Zhuang X, Yu Y, Liu X, Cui X, Ji L, Pan Z, Cao X, Mo B, Zhang F,

Raikhel N, Jiang L, Chen X (2013) MicroRNAs inhibit the translation of

target mRNAs on the endoplasmic reticulum in Arabidopsis. Cell 153:

562 – 574

Marnef A, Maldonado M, Buguaut A, Balasubramanian S, Kress M, Weil D,

Standart N (2010) Distinct functions of maternal and somatic Pat1

protein paralogs. RNA 16: 2094 – 2107

Marnef A, Standart N (2010) Pat1 proteins: a life in translation, translation

repression and mRNA decay. Biochem Soc Trans 38: 1602 – 1607

Mészáros T, Helfer A, Hatzimasoura E, Magyar Z, Serazetdinova L, Rios G,

Bardóczy V, Teige M, Koncz C, Peck S, Bögre L (2006) The Arabidopsis MAP

kinase kinase MKK1 participates in defence responses to the bacterial

elicitor flagellin. Plant J 48: 485 – 498

Morel JB (2002) Fertile hypomorphic ARGONAUTE (ago1) mutants impaired in

post-transcriptional gene silencing and virus resistance. Plant Cell 14:

629 – 639

Motomura K, Le QT, Kumakura N, Fukaya T, Takeda A, Watanabe Y (2012)

The role of decapping proteins in the miRNA accumulation in Arabidopsis

thaliana. RNA Biol 9: 644 – 652

Munchel SE, Shultzaberger RK, Takizawa N, Weis K (2011) Dynamic profiling

of mRNA turnover reveals gene-specific and system-wide regulation of

mRNA decay. Mol Biol Cell 22: 2787 – 2795

Nakagawa T, Kurose T, Hino T, Kawamukai M, Niwa Y, Toyooka K, Matsuoka

K, Jinbo T, Kimura T (2007) Development of series of gateway binary

vectors, pGWBs, for realizing efficient construction of fusion genes for

plant transformation. J Biosci Bioeng 104: 34 – 41

Nikovics K, Blein T, Peaucelle A, Ishida T, Morin H, Aida M, Laufs P (2006) The

balance between the MIR164A and CUC2 genes controls leaf margin

serration in Arabidopsis. Plant Cell 18: 2929 – 2945

Nissan T, Rajyaguru P, She M, Song H, Parker R (2010) Decapping activators

in Saccharomyces cerevisiae act by multiple mechanisms. Mol Cell 39:

773 – 783

Olmedo G, Guo H, Gregory BD, Nourizadeh SD, Aguilar-Henonin L, Li H, An F,

Guzman P, Ecker JR (2006) ETHYLENE-INSENSITIVE5 encodes a 5 ‘-> 3 ‘

exoribonuclease required for regulation of the EIN3-targeting F-box

proteins EBF1/2. PNAS 103: 13286 – 13293

Ozgur S, Chekulaeva M, Stoecklin G (2010) Human Pat1b connects

deadenylation with mRNA decapping and controls the assembly of

processing bodies. Mol Cell Biol 30: 4308 – 4323

Park S-H, Chung PJ, Juntawong P, Bailey-Serres J, Kim YS, Jung H, Bang SW,

Kim Y-K, Do Choi Y, Kim J-K (2012) Posttranscriptional control of

photosynthetic mRNA decay under stress conditions requires 30 and 50

untranslated regions and correlates with differential polysome association

in rice. Plant Physiol 159: 1111 – 1124

Parker JE, Holub EB, Frost LN, Falk A, Gunn ND, Daniels MJ (1996)

Characterization of eds1, a mutation in Arabidopsis suppressing resistance

to Peronospora parasitica specified by several different RPP genes. Plant

Cell 8: 2033 – 2046

Parker R, Sheth U (2007) P bodies and the control of mRNA translation and

degradation. Mol Cell 25: 635 – 646

Pearson G, Robinson F, Gibson TB, Xu BE, Karandikar M, Berman K, Cobb MH

(2001) Mitogen-activated protein (MAP) kinase pathways: regulation and

physiological functions. Endocr Rev 22: 153 – 183

Perea-Resa C, Hernández-Verdeja T, López-Cobollo R, del Mar Castellano M,

Salinas J (2012) LSM proteins provide accurate splicing and decay of

selected transcripts to ensure normal Arabidopsis development. Plant Cell

24: 4930 – 4947

Petersen M, Brodersen P, Naested H, Andreasson E, Lindhart U, Johansen B,

Nielsen HB, Lacy M, Austin MJ, Parker JE, Sharma SB, Klessig DF, Martienssen

R, Mattsson O, Jensen AB, Mundy J (2000) ArabidopsisMAP kinase 4

negatively regulates systemic acquired resistance. Cell 103: 1111 – 1120

Pitzschke A, Schikora A, Hirt H (2009) MAPK cascade signalling networks in

plant defence. Curr Opin Plant Biol 12: 421 – 426

Potuschak T, Vansiri A, Binder BM, Vierstra RD, Genschik P (2006) The

exoribonuclease XRN4 is a component of the ethylene response pathway

in Arabidopsis. Plant Cell 18: 3047 – 3057

Qiu J-L, Fiil BK, Petersen K, Nielsen HB, Botanga CJ, Thorgrimsen S, Palma K,

Suarez-Rodriguez MC, Sandbech-Clausen S, Lichota J, Brodersen P, Grasser

KD, Mattsson O, Glazebrook J, Mundy J, Petersen M (2008a) Arabidopsis

MAP kinase 4 regulates gene expression through transcription factor

release in the nucleus. EMBO J 27: 2214 – 2221

Qiu J-L, Zhou L, Yun B-W, Nielsen HB, Fiil BK, Petersen K, MacKinlay J, Loake GJ,

Mundy J, Morris PC (2008b) Arabidopsis mitogen-activated protein kinase

kinases MKK1 and MKK2 have overlapping functions in defense signaling

mediated by MEKK1, MPK4, and MKS1. Plant Physiol 148: 212 – 222

Rasmussen MW, Roux M, Petersen M, Mundy J (2012) MAP kinase cascades

in Arabidopsis innate immunity. Front Plant Sci 3: 169

Ravet K, Reyt G, Arnaud N, Krouk G, Djouani E, Boucherez J, Briat J, Gaymard

F (2012) Iron and ROS control of the DownSTream mRNA decay pathway

is essential for plant fitness. EMBO J 31: 175 – 186

Rayson S, Arciga-Reyes L, Wooton L, de Torres Zabala M, Truman W, Grant

M, Davies B (2012a) A role for nonsense-mediated mRNA decay in plants:

pathogen responses are induced in Arabidopsis thaliana NMD mutants.

PLoS One 7: e31917



56

Rayson S, Ashworth M, de Torres Zabala M, Grant M, Davies B (2012b) The

salicylic acid dependent and independent effects of NMD in plants. Plant

Signal Behav 7: 1434 – 1437

Riehs-Kearnan N, Gloggnitzer J, Dekrout B, Jonak C, Riha K (2012) Aberrant

growth and lethality of Arabidopsis deficient in nonsense-mediated RNA

decay factors is caused by autoimmune-like response. Nucleic Acids Res

40: 5615 – 5624

Rodriguez-Cousino N, Lill R, Neupert W, Court D (1995) Identification and

initial characterization of the cytosolic protein Ycr77p. Yeast 11:

581 – 585

Rymarquis LA, Souret FF, Green PJ (2011) Evidence that XRN4, an Arabidopsis

homolog of exoribonuclease XRN1, preferentially impacts transcripts with

certain sequences or in particular functional categories. RNA 17: 501 – 511

Rzeczkowski K, Beuerlein K, Müller H, Dittrich-Breiholz O, Schneider H,

Kettner-Buhrow D, Holtmann H, Kracht M (2011) c-Jun N-terminal kinase

phosphorylates DCP1a to control formation of P bodies. J Cell Biol 194:

581 – 596

Salgado-Garrido J, Bragado-Nilsson E, Kandels-Lewis S, Seraphin B (1999) Sm

and Sm-like proteins assemble in two related complexes of deep

evolutionary origin. EMBO J 18: 3451 – 3462

Scheller N, Resa-Infante P, la Luna de S, Galao RP, Albrecht M, Kaestner L,

Lipp P, Lengauer T, Meyerhans A, Díez J (2007) Identification of PatL1, a

human homolog to yeast P body component Pat1. Biochim Biophys Acta

1773: 1786 – 1792.

Shi C, Baldwin I, Wu J (2012) Arabidopsis plants having defects in

nonsense-mediated mRNA decay factors UPF1, UPF2, and UPF3 show

photoperiod-dependent phenotypes in development and stress responses.

J Integr Plant Biol 54: 99 – 114

Suarez-Rodriguez MC, Adams-Phillips L, Liu Y, Wang H, Su S-H, Jester PJ,

Zhang S, Bent AF, Krysan PJ (2007) MEKK1 is required for flg22-induced

MPK4 activation in Arabidopsis plants. Plant Physiol 143: 661 – 669

Teige M, Scheikl E, Eulgem T, Doczi F, Ichimura K, Shinozaki K, Dangl JL, Hirt

H (2004) The MKK2 pathway mediates cold and salt stress signaling in

Arabidopsis. Mol Cell 15: 141 – 152

Tharun S, He WH, Mayes AE, Lennertz P, Beggs JD, Parker R (2000) Yeast

Sm-like proteins function in mRNA decapping and decay. Nature 404:

515 – 518

Tharun S, Parker R (2001) Targeting an mRNA for decapping: displacement of

translation factors and association of the Lsm1p-7p complex on

deadenylated yeast mRNAs. Mol Cell 8: 1075 – 1083

Tharun S, Muhlrad D, Chowdhury A, Parker R (2005) Mutations in the

Saccharomyces cerevisiae LSM1 gene that affect mRNA decapping and

30 end protection. Genetics 170: 33 – 46

Tharun S (2009) Lsm1-7-Pat1 complex. RNA Biol 6: 228 – 232

Thomas M, Loschi M, Desbats M, Boccaccio G (2011) RNA granules: the good,

the bad and the ugly. Cell Signal 23: 324 – 334

Totaro A, Renzi F, La Fata G, Mattioli C, Raabe M, Urlaub H, Achsel T (2011)

The human Pat1b protein: a novel mRNA deadenylation factor identified

by a new immunoprecipitation technique. Nucleic Acids Res 39: 635 – 647

Ubersax JA, Ferrell JE Jr (2007) Mechanisms of specificity in protein

phosphorylation. Nat Rev Mol Cell Biol 8: 530 – 541

Van Mullem V, Wery M, De Bolle X, Vandenhaute J (2003) Construction of a

set of Saccharomyces cerevisiae vectors designed for recombinational

cloning. Yeast 20: 739 – 746

Vogel F, Hofius D, Paulus KE, Jungkunz I, Sonnewald U (2011) The second

face of a known player: Arabidopsis silencing suppressor AtXRN4 acts

organ-specifically. New Phytol 189: 484 – 493

Wang X, Watt P, Louis E, Borts RH, Hickson I (1996) Pat1: a topoisomerase

II-associated protein required for faithful chromosome transmission in

Saccharomyces cerevisiae. Nucleic Acids Res 24: 4791 – 4797

Weber C, Nover L, Fauth M (2008) Plant stress granules and mRNA processing

bodies are distinct from heat stress granules. Plant J 56: 517 – 530

Xu J, Yang J-Y, Niu Q-W, Chua N-H (2006) Arabidopsis DCP2, DCP1, and

VARICOSE form a decapping complex required for postembryonic

development. Plant Cell 18: 3386 – 3398

Xu J, Chua NH (2009) Arabidopsis decapping 5 is required for mRNA

decapping, P-Body formation, and translational repression during

postembryonic development. Plant Cell 21: 3270 – 3279

Xu J, Chua N-H (2011) Processing bodies and plant development. Curr Opin

Plant Biol 14: 88 – 93

Xu J, Chua NH (2012) Dehydration stress activates Arabidopsis MPK6 to signal

DCP1 phosphorylation. EMBO J 31: 1975 – 1984

Yang L, Wu G, Poethig RS (2012) Mutations in the GW-repeat protein SUO

reveal a developmental function for microRNA-mediated translational

repression in Arabidopsis. PNAS 109: 315 – 320

Yoon J-H, Choi E-J, Parker R (2010) Dcp2 phosphorylation by Ste20 modulates

stress granule assembly and mRNA decay in Saccharomyces cerevisiae. J

Cell Biol 189: 813 – 827

Zhang J, Shao F, Li Y, Cui H, Chen L, Li H, Zou Y, Long C, Lan L, Chai J, Chai J,

Chen S, Tang X, Zhou J-M (2007) A Pseudomonas syringae effector

inactivates MAPKs to suppress PAMP-induced immunity in plants. Cell Host

Microbe 1: 175 – 185

Zhang Z, Wu Y, Gao M, Zhang J, Kong Q, Liu Y, Ba H, Zhou J, Zhang Y (2012)

Disruption of PAMP-induced MAP kinase cascade by a Pseudomonas

syringae effector activates plant immunity mediated by the NB-LRR protein

SUMM2. Cell Host Microbe 11: 253 – 263

Zipfel C (2009) Early molecular events in PAMP-triggered immunity. Curr

Opin Plant Biol 12: 414 – 420



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Figure S1: Pat1 alleles, protein and immunoblotting

Phylogenetic tree of PAT1 orthologues (neighbor-joining tree generated from MAFFT multiple alignment in Geneious

version 5.6.6 created by Biomatters (http://www.geneious.com/). AtPAT, Arabidopsis thaliana; CePAT, C. elegans; OsPAT1,

Oryza sativa; HPat, Drosophila melanogaster; HsPAT, Homo sapiens; ScPAT, Saccharomyces cerevisiae; XlPAT, Xenopus

laevis. RT-PCR analysis of PAT1, ACTIN8, PAT1H1 and PAT1H2 gene expression in Arabidopsis Col-0 seedlings.C. PAT1

gene structure. Promoter (grey box), exons (white boxes) and introns (black lines). Start codon is marked ATG. T-DNA

insertion sites of pat1-1 (Salk_040660) and pat1-2 (WiscDs_437D04) are marked with black triangles in exon. D. PAT1

contains N-terminal, Pro-rich, middle and Pat-C domains. Peptides for antibody generation are indicated by black

pentagons. Potential SP phosphosites are marked with black stars. Amino acid numbers are above each domain.

E. Anti-PAT1 immunoblotting of yeast protein extracts from pat1∆ transformed with AtPAT1 and untransformed pat1∆.

AtPAT1 corresponds to the 89 kDa band seen in the pat1∆ transformed with AtPAT1. A nonspecific band indicated by *.

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F. Co-IP between PAT1-GFP and DCP1-HA. Transgenic lines expressing DCP1-HA (1,2) or PAT1-GFP and DCP1-HA (3,4)

were treated with water (-) or 200nM flg22 (+) for 15 mins. Proteins were extracted and subjected to HA and GFP IPs.

Immunoblots of inputs (20 ug each, top panel), GFP IPs (middle panel) and HA IPs (bottom panel) were probed with anti-

GFP and anti-HA antibodies. GFP IPs were also probed with anti-MPK4 antibodies as a control for PAT1-GFP IPs.

Molecular weights indicated on the right in kDa.

G. The anti-PAT1 antibody does not cross-react with any protein in pat1-1 mutant total protein extracts. Protein extracts (35

µg) derived from wild-type Col-0 and pat1-1 probed with 1:250 dilution of anti-PAT1 and alkaline phosphatase-conjugated

anti-rabbit antibodies.

Figure S2: Mass spectrometry analysis of PAT1-GFP.

Colloidal Coomassie-stained SDS-PAGE of GFP IPs from Col-0

or Col-0/PAT1-GFP treated with water or flg22. Molecular weight

in kDa indicated on the left.

Figure S3: MPK3 does not phosphorylate PAT1 in vitro.

MPK3 was immunoprecipitated from Col-0 seedlings treated with water (-)

or 200 nM flg22 (+) for 10 minutes. MPK3 IPs were incubated with His6-

PAT1 or MBP for 30 mins at 37°C before SDS-PAGE. Autoradiogram (top

panel), Coomassie-stained gel for loading control (bottom). Molecular

weight in kDa indicated at right.

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Figure S4: Pat1 mutants have a leaf serration phenotype.

4-week-old (left) or 8-week-old (right) plants grown under short-day conditions.

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Figure S5: PAT1-GFP complements the pat1-1 phenotype.

A. Plants photographed at 5-weeks of growth in short-day conditions where PAT1-GFP complements the pat1-1 leaf

serration phenotype. B. The accumulation of PR1 and UGT87A2 transcripts in pat1-1 are reverted when introducing PAT1-

GFP under control of the native PAT1 promoter. Transcript levels were quantified by qRT-PCR using RNA extracted from

5-week-old plants grown in soil under short-day conditions. Fold change is represented relative to Col-0 and normalized to

ACT2. Standard error of the mean is indicated by error bars (n=3) and statistical significance determined by ANOVA

followed by Tukey's test is shown as letters above error bars.

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Figure S6: effect of flg22 treatment on PAT1 transcript and PAT1-GFP protein.

Confocal microscopy with root elongation zones of five-days-old Col-0/PAT1-GFP seedlings. Roots treated in DMSO show no

significant change in localization or amounts of GFP signal. The number of GFP foci in PAT1-GFP seedlings is clearly

induced upon treatment with 1μM flg22 on glass slides for 20 min (second panel). No reduction in GFP foci is seen after

treatment with DMSO for 60 min (bottom panel). The scale bar corresponds to 10 µm. qRT-PCR analysis of PAT1

transcripts in Col-0 seedlings treated with time course of 100 nM flg22 with time in minutes post-treatment as indicated.

UBQ10 was used for normalization. Expression is displayed as fold change (where Col-0 at t=0 mins is 1). Standard error of

the mean is indicated by error bars (n=3).

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Figure S7: pat1 summ2 mutant phenotype

A. 4-week-old plants grown under short-day

conditions.

B. qPCR analysis of SUMM2 transcripts in 4-

week-old Arabidopsis Col-0, pat1-1, summ2-8

and pat1-1 summ2-8 plants. UBQ10 was used for

normalization. Expression is displayed as fold

change (where Col-0 = 1). Standard error of the

mean is indicated by error bars (n=3).

Figure S8: Infection of pat1-1 summ2-8 mutants with necrotrophic fungus. B. cinerea drop inoculation infection of 4-week-old

Col-0, pat1-1, summ2-8 and pat1-1 summ2-8 plants sampled at 0, 2 and 3 days. Data are the average of 3 biological replicates.

Disease severity was measured as accumulation of the B. cinerea CUTINASE A transcript by qPCR relative to A. thaliana α-

SHAGGY KINASE (At5g26751).

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Figure S9: PAT1 is associated with LSM1, MPK4 and SUMM2 in N. benthamiana. Proteins were transiently co-expressed

(PAT1-HA with LSM1-GFP, MycGFP, MPK4-GFP or SUMM2-GFP) in N. benthamiana and tissue and harvested 2 days

post-infiltration. Anti-HA immunoblot of PAT1-HA IPs are shown in the top panel. Anti-GFP immunoblot of PAT1-HA IPs

are shown in the middle panels. The bottom panel show the presence of LSM1-GFP, Myc-GFP and MPK4-GFP in the input.

Although we were unable to detect SUMM2-GFP in the input SUMM2-GFP was easily detected in the enriched PAT1-HA IP.

Molecular weights are shown in kDa at left.

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Figure S10: Multiple alignments of key regions of PAT1 homologues from several kingdoms. Alignment used MAFFT

algorithm in Geneious version 5.6.6 created by Biomatters (http://www.geneious.com/). Pink boxes mark predicted SP sites;

yellow boxes mark phosphorylation sites identified by MS in this study.

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AOC3 is an MPK4 substrate and its overexpression

induce immunity against pst DC3000

Introduction

In response to pathogen perception plants rapidly instigate local defense mechanisms. Two systems

for detecting pathogens are: (i) Pathogen recognition receptors on the plant cell surface that

recognize conserved pathogenic structures termed pathogen associated molecular patterns (PAMPs)

and (ii) cytoplasmic immune receptors that recognize either injected pathogen effectors or

modifications of effector host targets (Rasmussen et al., 2012). Both mechanisms induce rapid

localized responses but can also induce systemic acquired resistance mediated by phytohormones

(Robert-Seilaniantz et al., 2011). The transduction of and/or responses to defense signaling in

Arabidopsis involves at least three phytohormones that mediate appropriate responses toward to

specific classes of pathogens. These hormones are salicylic acid (SA), jasmonic acid (JA) and

ethylene (ET) or their derivatives (Bari and Jones, 2008). In response to pathogen perception, plants

produce these hormones in ratios depending on the specific class of pathogen (Robert-Seilaniantz et

al., 2011). Plants usually respond to biotrophic pathogens feeding on living plant tissue by

producing SA, whereas necrotrophic pathogen feeding on dead tissue typically induces production

of JA and ET (Glazebrook, 2005). The lifestyle of many pathogens are not easily classified as either

biotrophic or necrotrophic. Therefore, the otherwise antagonistic SA and JA/ET pathways are likely

correlated to tailor response to specific pathogens (Adie et al., 2007). Some biotrophic plant

pathogens have developed ways of suppressing SA mediated defenses by inducing the antagonistic

JA signaling pathway. For example, this is accomplished by introducing the bacterial JA analog

coronatine into the plant cell leading to suppression of defense responses against biotrophic

pathogens (Cui et al., 2005; Laurie-Berry et al., 2006). Athough coronatine in some instances mimics

JA, this is not always true. For example, plants respond to coronatine by opening their stomata

allowing pathogens to enter the apoplastic space while JA induces stomatal closure (Melotto et al.,

2006; Suhita et al., 2004).

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The synthesis of defense related hormones including JA is upregulated in response to recognition of

specific pathogens. The first steps in the biosynthesis of JA occur in the chloroplast (Fig. 1). Here

α-linolenic acid present in the thylakoid membrane is oxygenated by a lipoxygenase (LOX) to yield

the fatty acid peroxide 13(S)‐HpOTrE. This peroxide is next dehydrated by ALLENE OXIDE

SYNTEHASE (AOS) to the unstable epoxide 12,13(S)-epoxyoctadecatrienoic acid which is further

modified by ALLENE OXIDE CYCLASE (AOC) to produce 9S,13S-12-oxo-phytodienoic acid

(OPDA) (Hamberg and Fahlstadius, 1990; Schaller et al., 2004; Vick and Zimmerman, 1979;

Zimmerman and Feng, 1978). In aqueous solutions the epoxide produced from AOS degrades

rapidly (half-life of less than 30 seconds at 0˚C) into mainly α- and γ-ketols while a minor fraction

undergoes cyclization producing a racemic mixture of OPDA (Hamberg, 1988; Ziegler et al., 1999).

This is in contrast to the AOC catalyzed conversion which produces only the optically pure

enantiomer 9S,13S-OPDA used in downstream JA biosynthesis (Hamberg and Fahlstadius, 1990).

Due to the short half-life of the AOS product, it is likely that the functions of AOS and AOC are

coupled in a complex, although no direct evidence of such an interaction has been shown.

Translocation of OPDA to the peroxisomes, where the remaining steps in the pathway occur, is not

fully understood. OPDA itself can function as a signaling compound and therefor its translocation

across the chloroplast envelope is likely to be tightly regulated (Dave and Graham, 2012).

Translocation from the cytosol to the peroxisome is actively facilitated by ABC transporters

(Gerhardt, 1983; Theodoulou et al., 2005). In the peroxisomes OPDA is reduced enzymatically by

an OPDA reductase (OPR) and subsequently undergoes three rounds of β-oxidation resulting in the

production of JA (Fig. 1) (Li et al., 2005; Schaller et al., 2000).

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Figure 1: The JA biosynthesis pathway.

In the chloroplasts α-linolenic acid is oxygenated by LOX to generate 13(S)-HpOTrE which is further modified by AOS to

yield the unstable epoxide 12,13(S)-epoxyoctadecatrienoic acid. In the absence of AOC the epoxide rapidly hydrolyzes into

mainly α- and γ-ketols while a minor fraction undergoes cyclization into a racemic mixture of OPDA. In contrast, AOC

catalyzed cyclization of the epoxide produces only the optically pure 9S,13S-OPDA which then is transported out of the

chloroplast to the cytosol by an unknown mechanism. From the cytosol 9S,13S-OPDA is actively transported into the

peroxisomes by an ABC transporter where it is reduced by OPR and undergoes three rounds of β-oxidation resulting in the

production of JA.

68

AOC displays a greater substrate specificity than AOS and it thus appears that AOC confers

additional specificity on the JA biosynthetic pathway (Schaller et al., 2008). The Arabidopsis

genome contain four genes encoding AOC enzymes: AOC1 (At3g25770), AOC2 (At3g25780),

AOC3 (At3g25760) and AOC4 (At1g13280). These four genes share great sequence identity (Fig.

2) and are most likely functionally redundant to each other as individual T-DNA loss-of-function

mutants do not exhibit JA-related phenotypes (Stenzel et al., 2012). Promoter analysis using a GUS

reporter system has also shown partially redundant promoter activities during development: (i) In

fully developed leaves AOC1, AOC2 and AOC3 promoter activity are apparent throughout all leaf

tissue, whereas AOC4 promoter activity are specific to vascular tissue; (ii) only AOC3 and AOC4

promoter activities are detected in roots; (iii) AOC1 and AOC4 promoter activities are detected

during flower development (Stenzel et al., 2012). In Arabidopsis AOC2 is the best characterized of

the four AOC enzymes and its crystal structure confirms previous findings that AOC forms a

functional trimer (Hofmann et al., 2006). The regulation of the JA biosynthesis pathway is not fully

understood, although positive feedback regulation seems able to induce accumulation of transcripts

encoding genes involved in the biosynthetic pathway of JA (Wasternack and Hause, 2013).

Interestingly all four AOC enzymes can perform both homo and heteromerization in vivo with each

other. This might serve as a level of regulation in JA biosynthesis since some spatial and temporal

promoter activities are observed for the different AOC promoters (Stenzel et al., 2012).

Figure 2: Alignment of AOC1-4 from Arabidopsis

Peptide sequences were aligned with “CLC Main Workbench” without their chloroplast transit peptides (first 81-84 aa).

Putative MAP kinase phosphorylation sites (SP/AP) are indicated with red background coloring. Secondary structures

according the crystal structure of AOC2 are indicated above the alignment.

69

One of the early responses to PAMP perception is activation of MAP Kinase (MPK) signaling

(Rasmussen et al., 2012). MPK4 activation is linked to recognition of the bacterial PAMP flagellin

and thus functions in transducing immune signaling through phosphorylation of downstream targets

(Qiu et al., 2008; Rodriguez et al., 2010; Roux et al., 2015). MPK4 is a target for the bacterial

effector protein AvrB indicating MPK4s pivotal role in immune signaling (Cui et al., 2010). This is

confirmed by the fact that SUPPRESSOR OF MKK1 MKK2 2 (SUMM2), a cytoplasmic immune

receptor, is activated in the absence of MPK4 or MPK4 activity (Zhang et al., 2012). In

investigating the molecular function of MPK4, a yeast two-hybrid screen was initiated searching for

MPK4 interacting proteins (Andreasson et al., 2005). AOC3 was identified in this screen as a full-

length cDNA clone. Here we characterize the interaction between AOC3 and MPK4. We show that

AOC3 is a substrate of MPK4 and that overexpressing AOC3 in Arabidopsis trigger enhanced

immunity. We also provide evidence that MPK4 is associated with the chloroplast, that may

indicate that MPK4 can regulate AOC3 activity/stability inside the chloroplast in response to

pathogens.

Results

AOC3 Interacts with MPK4 in planta.

In a previously conducted yeast two-hybrid screen we identified AOC3 as an interactor of MPK4.

To confirm the AOC3-MPK4 interaction in planta, we transiently expressed in Nicotiana

benthamiana AOC3-GFP along with MPK4-HA and then immunoprecipitated AOC3-GFP. MPK4-

HA was detected in AOC3-GFP immunoprecipitates from plants transiently expressing both

MPK4-HA and AOC3-GFP, but not in control plants only expressing MPK4-HA (Fig. 3). Thus,

MPK4 and AOC3 can be found in complex in planta.

We also generated double transgenic Arabidopsis lines in the mpk4-1 background expressing

MPK4 with a C-terminal HA tag under control of its native promoter and AOC3 tagged C-

terminally with either Myc or GFP under control of a 35S promoter. MPK4-HA were detected in

AOC3-Myc and AOC3-GFP immunoprecipitates only from double transgenic lines but not from

control lines expressing only MPK4-HA (Fig. 4).

70

Figure 3: MPK4 is in complex with AOC3 in planta.

MPK4-HA is detected in AOC3-GFP immunoprecipitates from N. benthamiana transiently expressing AOC3-GFP and

MPK-HA. Immunoblots of input and anti-GFP immunoprecipitates are probed with anti-HA and anti-GFP antibodies

Figure 4: MPK4 is in complex with AOC3 in Arabidopsis.

Immunoprecipitates from four-week-old plants expressing MPK4-HA and AOC3-GFP or AOC3-Myc. (A) MPK4-HA is

detected in AOC3-GFP immunoprecipitates. Immunoblots of input and anti-GFP immunoprecipitates are probed with anti-

HA and anti-GFP antibodies. (B) MPK4-HA is detected in AOC3-Myc immunoprecipitates. Immunoblots of input and anti-

Myc immunoprecipitates are probed with anti-HA and anti-Myc antibodies.

MPK4 is activated by treatment with the bacterial flagellin-derived flg22 peptide (Rodriguez et al.,

2010). We questioned whether activation of Arabidopsis immune responses including MPK

activation would affect the interaction between AOC3 and MPK4. We therefore pre-treated doubly

transgenic plants expressing AOC3-GFP and MPK4-HA for 10 minutes with 100nM flg22 before

immunoprecipitation of AOC3-GFP. MPK4-HA was detected in immunoprecipitations from both

flg22 and mock treated plants (Fig. 5). A less intense band was observed in the

immunoprecipitation from flg22 treated plants, but the experiment has not been repeated and

therefore it is not possible to conclude if the lower intensity arises from stochastic events or if the

affinity of the interactions decreases upon flg22 perception.

71

Figure 5: The AOC3-MPK4 interaction is independent of flg22.

Three-week-old plants grown in liquid media were incubated with or without 200 nM flg22 for 15 min. MPK4-HA were

detected in anti-GFP immunoprecipitates from both mock and flg22 treated double transgenic Arabidopsis plants.

Immunoblots of input and anti-GFP immunoprecipitates are probed with anti-HA and anti-GFP antibodies.

Over-expression of AOC3 leads to increased resistance

While mpk4-1 plants expressing MPK4-HA under its native promoter are complemented and thus

indistinguishable from wild type overexpression of AOC3-Myc or AOC3-GFP in these plants

caused dwarfism (Fig. 6A). Mpk4 mutants also exhibit dwarfism and we therefore asked if

overexpression of tagged AOC3 also caused accumulation of PR1 protein. Plants overexpressing

AOC3-Myc accumulated PR1 protein to a similar extent as the mpk4-1 mutant (Fig. 6B). AOC3-

GFP overexpressing plants also accumulated PR1 protein but to a lesser extent and mpk4

complemented lines had no detectable accumulation of PR1 protein (Fig. 6B). Since mpk4 plants

also exhibit increased resistance towards virulent pathogens, we next assayed the susceptibility of

AOC overexpressing plants. Both AOC3-GFP and AOC3-Myc overexpressing plant exhibited

increased resistance against pst DC3000 (Fig. 6C). Thus, overexpression of AOC3 interestingly

leads to accumulation of PR1 protein and induced resistance against pst DC3000.

AOC3 is an MPK4 substrate

Since MPK4 and AOC3 are found in complexes in planta, it is likely that AOC3 also represent an

MPK4 substrate. Therefore, we carried out in vitro kinase assays using immunoprecipitated flg22-

activated MPK4 or MPK3 or MPK6 to determine whether AOC3 is also an MPK4 specific

substrate. These MPKs were then incubated with recombinant His6-AOC3 protein lacking the

chloroplast transit peptide. Phosphorylated His6-AOC3 was detectable as a radioactive band around

20 kDa after incubation with flg22-activated MPK4 (Fig. 7), while MPK6 caused only low levels of

AOC3 phosphorylation and MPK3 did not significantly alter AOC3 phosphorylation. Each MPK

72

Figure 6: Overexpression of AOC3 induce resistance.

(A) Plants photographed at 4 weeks of growth in short day conditions, genotypes as indicated. (B) PR1 protein accumulation

is elevated in mpk4-1 complemented lines overexpressing AOC3-myc and AOC3-GFP. Four-week-old plants were used for

protein extraction and Bradford determined protein concentrations were adjusted to equal levels before SDS page (Samples

are run in duplicates). Immunoblots were probed with anti-PR1. (C) Tagged AOC3 lines are more resistant to colonization

by Pst DC3000 (OD600=0.001). Bacteria were syringe-infiltrated and samples taken 4 days post-infiltration. Data are shown

as log10-transformed colony-forming units/cm2 leaf tissue. Standard error of the mean is indicated by errors bars (n = 7).

Statistical significance between the mean values was determined by ANOVA followed by Tukey's test. Multiplicity adjusted P

values are shown for significantly different mean values.

73

Figure 6: AOC3 is phosphorylated in vitro by MPK4

MPK-3, -4 and -6 were immunoprecipitated from extracts of Col-0 seedlings treated with 200 nM flg22 for 10 min. For the

negative control (-), extracts were incubated with agarose beads without antibodies. Immunoprecipitates were incubated with

His6-AOC3 or MBP for 60 min at 30°C before SDS-PAGE. Autoradiogram (top panel), Coomassie-stained gel for loading

control (bottom).

was also incubated with the generic MPK substrate myelin basic protein (MBP) to verify their

activation (Fig. 7). Thus, AOC3 is an in vitro substrate of MPK4, if this is also true in vivo is the

subject of further research.

AOC3 contains 1 Ser-Pro (SP) and 1 Thr-Pro (TP) motif which are commonly phosphorylated by

MPKs (Pearson et al., 2001; Ubersax and Ferrell Jr, 2007). The SP motif is conserved in all 4

Arabidopsis AOC enzymes (Fig. 2) and is central for their enzyme activity (Hofmann et al., 2006;

Schaller et al., 2008). In AOC2 the SP motif (Ser-96,Pro-97) and two Asn residues (Asn-90,Asn-

118) are important for stabilizing the reactive oxygen in the epoxide group in catalyzing the

cyclization of 13-HPOT into OPDA (Hofmann et al., 2006; Schaller et al., 2008). Online database

searches (PhosPhAt and P3DB) showed that AOC2 from Arabidopsis, as well as AOC4 from

Glycine max (Soybean) and Medicago truncatula (barrel clover), are phosphorylated at this specific

SP motif (Durek et al., 2010; Yao et al., 2014). It is reasonable to believe that this phosphorylation

might regulate the activity of AOC enzymes as addition of the negatively charged phosphate group

to the SP motif could affect the stabilization of the epoxide group in the 13-HPOT substrate. To test

if MPK4 can phosphorylate the SP site we incubated a mutant version of His6-AOC3 with Ser-100

mutated to alanine (S100A) with MPK4 immunoprecipitates and measured the incorporation of

radioactive labeled AT32P (Fig. 8). There was no clear difference in the phosphorylation of AOC3

and AOC3-S100A meaning that (i) MPK4 does not phosphorylate AOC3-Ser100 in vitro or (ii) the

74

phosphorylation of an additional residue(s) mask the reduction in phosphorylation of the AOC3-

S100A mutant.

To investigate the MPK4 mediated phosphorylation of AOC3 we have cloned, expressed and

purified the additional phospho site mutants AOC3-T233A and AOC-S100A/T233A, but we have

not yet tested their specificity as MPK4 substrates.

Figure 6: AOC3A100A is phosphorylated by MPK4.

MPK4 was immunoprecipitated from extracts of Col-0 seedlings treated with 200 nM flg22 for 10 min. For the negative

control (-), extracts were incubated with agarose beads without antibodies. immunoprecipitates were incubated with His6-

AOC3 or His6-AOC3S100A for 60 min at 30°C before SDS-PAGE. Autoradiogram (top panel), Coomassie-stained gel for

loading control (bottom).

MPK4 associates with chloroplasts.

AOC3 is transcribed in the nucleus, translated in the cytosol and an N-terminal transit peptide

guides the protein to the chloroplast where it functions in JA biosynthesis (Schaller et al., 2008).

MPK4 has previously been shown to be present in the cytoplasm and nucleus, but no association to

plastids has so far been shown (Andreasson et al., 2005; Petersen et al., 2000, 4). A large scale

phosphoprotein analysis discovered that numerous chloroplast proteins are phosphorylated at

putative MAP kinase phosphorylation motifs (Reiland et al., 2009). However, no MAP kinase has

been shown to associate with chloroplasts. We therefore questioned where the interaction between

MPK4 and AOC3 occurs and investigated whether MPK4 associates with chloroplasts.

To answer these questions, we isolated intact chloroplasts from transgenic plants expressing MPK4-

HA and wild type Ler plants and probed for MPK4-HA by immunoblotting (Fig. 9). A clear,

distinct band for MPK4-HA was observed in the chloroplast fraction from MPK4-HA expressing

plants, but was absent in the control plants. We further probed for the presence of MPK6, another

immunity regulating MAPK (Rasmussen et al., 2012). MPK6 was detected only in the total protein

75

fraction and not in the chloroplast fraction (Fig. 9). To exclude the possibility that the chloroplast

fractions were contaminated with cytoplasmic protein we probed for the cytoplasmic protein

DECAPPING 1 (DCP1) (Fig. 9) (Xu et al., 2006, 2). We detected DCP1 only in the total protein

sample and not in the chloroplast fractions. This indicates that these fractions where not

contaminated with cytoplasmic proteins. We also probed for the presence of the chloroplast protein

COPPER/ZINC SUPEROXIDE DISMUTASE 2 (CDS2) to verify presence of chloroplastic

proteins (Fig. 9) (Pilon et al., 2011). CSD2 was observed in both total protein samples and the

chloroplast fractions. Whereas CSD2 is present in the chloroplast, CSD1 is a cytoplasmic protein

(Pilon et al., 2011). The CSD2 antibody cross-reacts with CSD1 and a lower band is observed in the

total protein samples but is absent in the chloroplast fractions. This band correlates to the size of

CSD1 (15 kDa). These results confirm that the chloroplast fractions are free of cytoplasmic

proteins.

Figure 9: MPK4 is detected in isolated chloroplasts.

Intact chloroplasts were isolated from leaf tissue of four-week-old plants expressing MPK4-HA under control of its native

promoter or from Ler wild type plants. Total protein samples and chloroplast fractions were then subjected to SDS-PAGE

and immunoblotting.

76

MPK4 does not have a chloroplast transit peptide. However, proteomic data suggest that up to 30%

of the protein species in the chloroplast lack encoded transit peptides and therefore are translocated

to the chloroplasts by unknown mechanisms (Armbruster et al., 2009). It is therefore possible that

MPK4 can translocate to the chloroplasts even in absence of a transit peptide. Alternatively MPK4

could also simply associate with the outer envelope of the chloroplast without actually being

translocated into the chloroplast.

Discussion

In this study, we identify AOC3 as an interactor and substrate of the immunity regulating MPK4 in

Arabidopsis. AOC3 is located in the chloroplasts where it catalyzes the production of the JA

precursor ODPA (Hamberg and Fahlstadius, 1990). Although the enzymatic steps in the

biosynthesis of JA are well understood, information regarding regulation is scarce. A possible point

of regulation might be controlling the availability of enzymes necessary in the pathway. LOX2

catalyzes the first step in the JA biosynthesis by converting α-linolenic acid into 13(S)‐HpOTrE

(Stenzel et al., 2003). LOX2 interacts with the eukaryotic translational initiating factor 4E (eIF4E)

in the cytosol (Freire et al., 2000). It is possible that this interaction sequesters LOX2 in the cytosol

thwarting the initial step in JA biosynthesis. Alternatively, it is also possible that LOX2 actually

down-regulates translation through its interaction with eIF4E. Confirming or rejecting one or both

of these hypotheses requires further investigation.

Regulation of the pathway is also likely to occur through phosphorylation. Numerous sites of

chloroplast proteins are mapped through phospho-proteome profiling including several motifs

thought to be phosphorylated by MAP kinases (Reiland et al., 2009). Online database searches

(PhosPhAt and P3DB) show that AOC2 from Arabidopsis as well as AOC4 from Glycine max

(Soybean) and Medicago truncatula (barrel clover) are phosphorylated at a conserved SP motif

(AOC3 Ser-100 Pro-101) (Durek et al., 2010; Yao et al., 2014). This SP motif is important for the

function of the AOC enzymes as it functions in stabilizing the reactive oxygen of the epoxide group

in its substrate when 13-HPOT cyclizes into OPDA (Hofmann et al., 2006; Schaller et al., 2008).

This however does not seem to occur though the activity of MPK4 since an SP to AP mutant was

still phosphorylated by MPK4 in vitro (Fig. 8). The database searches further showed that AOC4

from Medicago truncatula is phosphorylated on an SP site between α-helix 3 and α-helix 4. In

Arabidopsis AOC1-3 has a TP site between α-helix 3 and α-helix 4 while AOC4 has an EP site

putatively mimicking a phosphorylated motif (Fig. 2). It is likely that this motif can be

phosphorylated by MPK4, although this and its possible effects have to be tested experimentally.

77

To gain insight into where the interaction between AOC3 and MPK4 occurs, we investigated if

MPK4 associates with chloroplasts. MPK4 was clearly observed in immunoblots from isolated

chloroplast (Fig. 9). This, however, does not provide information of whether MPK4 is translocated

into the chloroplast or if it merely resides attached to the outer membrane. Therefore, to further

explore the location of the molecular interaction between AOC3 and MPK4 studies such as

bimolecular fluorescence could be applied for a more precise confirmation of where in the cell this

interaction occurs. Nonetheless, the presence of phosphorylated MAP kinase motifs in chloroplast

proteins sustains the idea of MPK4 functioning inside the chloroplast as well as in the cytosol and

nucleus (Andreasson et al., 2005; Reiland et al., 2009). If MPK4 resides inside the chloroplast, it

raises the question whether it is activated by perception of pathogens similar to its cytoplasmic

counterpart? And, if this is the case, can MPK4 regulate JA biosynthesis through phosphorylation

of AOC3 (and AOC1 and -2) upon pathogen perception?

MPK4 was initially considered to regulate JA induced gene transcription as the mpk4 mutant fails to

accumulate JA induced PDF1.2 transcripts (Petersen et al., 2000, 4). However, crossing the mpk4

mutant into an eds1 mutant background which blocks ETI restored the exogenous JA induced

accumulation of PFD1.2 transcripts (Brodersen et al., 2006). This implies that the blockage of JA

induced genes in the mpk4 mutant is a pleiotropic effect of activating ETI and not necessarily linked

to MPK4 lack of function. Further investigation of the phosphorylation of AOC3 by MPK4 may

shed light on a putative regulatory point in JA biosynthesis and how this converges with defense

signaling.

Materials and methods

See material and methods for “eIF4E-Thr6 is specifically phosphorylated in vitro by MPK4 and

form a complex in the nucleus in vivo.”

78

References

Adie, B. A. T., Pérez-Pérez, J., Pérez-Pérez, M. M., Godoy, M., Sánchez-Serrano, J.-J., Schmelz, E.

A., et al. (2007). ABA Is an Essential Signal for Plant Resistance to Pathogens Affecting JA

Biosynthesis and the Activation of Defenses in Arabidopsis. Plant Cell 19, 1665–1681.

doi:10.1105/tpc.106.048041.

Andreasson, E., Jenkins, T., Brodersen, P., Thorgrimsen, S., Petersen, N. H., Zhu, S., et al. (2005).

The MAP kinase substrate MKS1 is a regulator of plant defense responses. EMBO J 24,

2579–2589. doi:10.1038/sj.emboj.7600737.

Armbruster, U., Hertle, A., Makarenko, E., Zühlke, J., Pribil, M., Dietzmann, A., et al. (2009).

Chloroplast Proteins without Cleavable Transit Peptides: Rare Exceptions or a Major

Constituent of the Chloroplast Proteome? Mol. Plant 2, 1325–1335. doi:10.1093/mp/ssp082.

Bari, R., and Jones, J. D. G. (2008). Role of plant hormones in plant defence responses. Plant Mol.

Biol. 69, 473–488. doi:10.1007/s11103-008-9435-0.

Brodersen, P., Petersen, M., Nielsen, H. B., Zhu, S., Newman, M.-A., Shokat, K. M., et al. (2006).

Arabidopsis MAP kinase 4 regulates salicylic acid- and jasmonic acid/ethylene-dependent

responses via EDS1 and PAD4. Plant J. 47, 532–546. doi:10.1111/j.1365-

313X.2006.02806.x.

Cui, H., Wang, Y., Xue, L., Chu, J., Yan, C., Fu, J., et al. (2010). Pseudomonas syringae Effector

Protein AvrB Perturbs Arabidopsis Hormone Signaling by Activating MAP Kinase 4. Cell

Host Microbe 7, 164–175. doi:10.1016/j.chom.2010.01.009.

Cui, J., Bahrami, A. K., Pringle, E. G., Hernandez-Guzman, G., Bender, C. L., Pierce, N. E., et al.

(2005). Pseudomonas syringae manipulates systemic plant defenses against pathogens and

herbivores. Proc. Natl. Acad. Sci. U. S. A. 102, 1791–1796. doi:10.1073/pnas.0409450102.

Dave, A., and Graham, I. A. (2012). Oxylipin signaling: a distinct role for the jasmonic acid

precursor cis-(+)-12-oxo-phytodienoic acid (cis-OPDA). Plant Physiol. 3, 42.

doi:10.3389/fpls.2012.00042.

Durek, P., Schmidt, R., Heazlewood, J. L., Jones, A., MacLean, D., Nagel, A., et al. (2010).

PhosPhAt: the Arabidopsis thaliana phosphorylation site database. An update. Nucleic Acids

Res. 38, D828–D834. doi:10.1093/nar/gkp810.

Freire, M. A., Tourneur, C., Granier, F., Camonis, J., Amrani, A. E., Browning, K. S., et al. (2000).

Plant lipoxygenase 2 is a translation initiation factor-4E-binding protein. Plant Mol. Biol.

44, 129–140. doi:10.1023/A:1006494628892.

Gerhardt, B. (1983). Localization of β-oxidation enzymes in peroxisomes isolated from nonfatty

plant tissues. Planta 159, 238–246. doi:10.1007/BF00397531.

79

Glazebrook, J. (2005). Contrasting Mechanisms of Defense Against Biotrophic and Necrotrophic

Pathogens. Annu. Rev. Phytopathol. 43, 205–227.

doi:10.1146/annurev.phyto.43.040204.135923.

Hamberg, M. (1988). Biosynthesis of 12-oxo-10,15(Z)-phytodienoic acid: Identification of an

allene oxide cyclase. Biochem. Biophys. Res. Commun. 156, 543–550. doi:10.1016/S0006-

291X(88)80876-3.

Hamberg, M., and Fahlstadius, P. (1990). Allene oxide cyclase: A new enzyme in plant lipid

metabolism. Arch. Biochem. Biophys. 276, 518–526. doi:10.1016/0003-9861(90)90753-L.

Hofmann, E., Zerbe, P., and Schaller, F. (2006). The Crystal Structure of Arabidopsis thaliana

Allene Oxide Cyclase: Insights into the Oxylipin Cyclization Reaction. Plant Cell 18, 3201–

3217. doi:10.1105/tpc.106.043984.

Laurie-Berry, N., Joardar, V., Street, I. H., and Kunkel, B. N. (2006). The Arabidopsis thaliana

JASMONATE INSENSITIVE 1 Gene Is Required for Suppression of Salicylic Acid-

Dependent Defenses During Infection by Pseudomonas syringae. Mol. Plant. Microbe

Interact. 19, 789–800. doi:10.1094/MPMI-19-0789.

Li, C., Schilmiller, A. L., Liu, G., Lee, G. I., Jayanty, S., Sageman, C., et al. (2005). Role of β-

Oxidation in Jasmonate Biosynthesis and Systemic Wound Signaling in Tomato. Plant Cell

17, 971–986. doi:10.1105/tpc.104.029108.

Melotto, M., Underwood, W., Koczan, J., Nomura, K., and He, S. Y. (2006). Plant Stomata

Function in Innate Immunity against Bacterial Invasion. Cell 126, 969–980.

doi:10.1016/j.cell.2006.06.054.

Pearson, G., Robinson, F., Beers Gibson, T., Xu, B., Karandikar, M., Berman, K., et al. (2001).

Mitogen-Activated Protein (MAP) Kinase Pathways: Regulation and Physiological

Functions. Endocr. Rev. 22, 153–183. doi:10.1210/edrv.22.2.0428.


Arabidopsis MAP Kinase 4 Negatively Regulates Systemic Acquired Resistance. Cell 103,

1111–1120. doi:10.1016/S0092-8674(00)00213-0.

Pilon, M., Ravet, K., and Tapken, W. (2011). The biogenesis and physiological function of

chloroplast superoxide dismutases. Biochim. Biophys. Acta BBA - Bioenerg. 1807, 989–998.

doi:10.1016/j.bbabio.2010.11.002.

Qiu, J.-L., Zhou, L., Yun, B.-W., Nielsen, H. B., Fiil, B. K., Petersen, K., et al. (2008). Arabidopsis

Mitogen-Activated Protein Kinase Kinases MKK1 and MKK2 Have Overlapping Functions

in Defense Signaling Mediated by MEKK1, MPK4, and MKS1. Plant Physiol. 148, 212–

222. doi:10.1104/pp.108.120006.

Rasmussen, M. W., Roux, M., Petersen, M., and Mundy, J. (2012). MAP kinase cascades in

Arabidopsis innate immunity. Plant Proteomics 3, 169. doi:10.3389/fpls.2012.00169.

Reiland, S., Messerli, G., Baerenfaller, K., Gerrits, B., Endler, A., Grossmann, J., et al. (2009).

Large-Scale Arabidopsis Phosphoproteome Profiling Reveals Novel Chloroplast Kinase

Substrates and Phosphorylation Networks. Plant Physiol. 150, 889–903.

doi:10.1104/pp.109.138677.

80

Robert-Seilaniantz, A., Grant, M., and Jones, J. D. G. (2011). Hormone Crosstalk in Plant Disease

and Defense: More Than Just JASMONATE-SALICYLATE Antagonism. Annu. Rev.

Phytopathol. 49, 317–343. doi:10.1146/annurev-phyto-073009-114447.

Rodriguez, M. C. S., Petersen, M., and Mundy, J. (2010). Mitogen-Activated Protein Kinase

Signaling in Plants. Annu. Rev. Plant Biol. 61, 621–649. doi:10.1146/annurev-arplant-

042809-112252.

Roux, M. E., Rasmussen, M. W., Palma, K., Lolle, S., Regué, À. M., Bethke, G., et al. (2015). The

mRNA decay factor PAT1 functions in a pathway including MAP kinase 4 and immune

receptor SUMM2. EMBO J., e201488645. doi:10.15252/embj.201488645.

Schaller, F., Biesgen, C., Müssig, C., Altmann, T., and Weiler, E. W. (2000). 12-Oxophytodienoate

reductase 3 (OPR3) is the isoenzyme involved in jasmonate biosynthesis. Planta 210, 979–

984. doi:10.1007/s004250050706.

Schaller, F., Schaller, A., and Stintzi, A. (2004). Biosynthesis and metabolism of jasmonates. J.

Plant Growth Regul. 23, 179–199. doi:10.1007/BF02637260.

Schaller, F., Zerbe, P., Reinbothe, S., Reinbothe, C., Hofmann, E., and Pollmann, S. (2008). The

allene oxide cyclase family of Arabidopsis thaliana – localization and

cyclization. FEBS J. 275, 2428–2441. doi:10.1111/j.1742-4658.2008.06388.x.

Stenzel, I., Hause, B., Miersch, O., Kurz, T., Maucher, H., Weichert, H., et al. (2003). Jasmonate

biosynthesis and the allene oxide cyclase family of Arabidopsis thaliana. Plant Mol. Biol.

51, 895–911. doi:10.1023/A:1023049319723.

Stenzel, I., Otto, M., Delker, C., Kirmse, N., Schmidt, D., Miersch, O., et al. (2012). ALLENE

OXIDE CYCLASE (AOC) gene family members of Arabidopsis thaliana: tissue- and organ-

specific promoter activities and in vivo heteromerization*. J. Exp. Bot. 63, 6125–6138.

doi:10.1093/jxb/ers261.

Suhita, D., Raghavendra, A. S., Kwak, J. M., and Vavasseur, A. (2004). Cytoplasmic Alkalization

Precedes Reactive Oxygen Species Production during Methyl Jasmonate- and Abscisic

Acid-Induced Stomatal Closure. Plant Physiol. 134, 1536–1545.

doi:10.1104/pp.103.032250.

Theodoulou, F. L., Job, K., Slocombe, S. P., Footitt, S., Holdsworth, M., Baker, A., et al. (2005).

Jasmonic Acid Levels Are Reduced in COMATOSE ATP-Binding Cassette Transporter

Mutants. Implications for Transport of Jasmonate Precursors into Peroxisomes. Plant

Physiol. 137, 835–840. doi:10.1104/pp.105.059352.

Ubersax, J. A., and Ferrell Jr, J. E. (2007). Mechanisms of specificity in protein phosphorylation.

Nat. Rev. Mol. Cell Biol. 8, 530–541. doi:10.1038/nrm2203.

Vick, B. A., and Zimmerman, D. C. (1979). Substrate Specificity for the Synthesis of Cyclic Fatty

Acids by a Flaxseed Extract. Plant Physiol. 63, 490–494. doi:10.1104/pp.63.3.490.

Wasternack, C., and Hause, B. (2013). Jasmonates: biosynthesis, perception, signal transduction

and action in plant stress response, growth and development. An update to the 2007 review

in Annals of Botany. Ann. Bot. 111, 1021–1058. doi:10.1093/aob/mct067.

81

Xu, J., Yang, J.-Y., Niu, Q.-W., and Chua, N.-H. (2006). Arabidopsis DCP2, DCP1, and

VARICOSE Form a Decapping Complex Required for Postembryonic Development. Plant

Cell 18, 3386–3398. doi:10.1105/tpc.106.047605.

Yao, Q., Ge, H., Wu, S., Zhang, N., Chen, W., Xu, C., et al. (2014). P3DB 3.0: From plant

phosphorylation sites to protein networks. Nucleic Acids Res. 42, D1206–D1213.

doi:10.1093/nar/gkt1135.

Zhang, Z., Wu, Y., Gao, M., Zhang, J., Kong, Q., Liu, Y., et al. (2012). Disruption of PAMP-

Induced MAP Kinase Cascade by a Pseudomonas syringae Effector Activates Plant

Immunity Mediated by the NB-LRR Protein SUMM2. Cell Host Microbe 11, 253–263.

doi:10.1016/j.chom.2012.01.015.

Ziegler, J., Wasternack, C., and Hamberg, M. (1999). On the specificity of allene oxide cyclase.

Lipids 34, 1005–1015. doi:10.1007/s11745-999-0451-z.

Zimmerman, D. C., and Feng, P. (1978). Characterization of a prostaglandin-like metabolite of

linolenic acid produced by a flaxseed extract. Lipids 13, 313–316.

doi:10.1007/BF02533720.

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eIF4E-Thr6 is specifically phosphorylated in vitro by

MPK4 and form a complex in the nucleus in vivo.

Introduction

Translation of mRNAs into proteins is primarily regulated at the point of initiation (Jackson et al.,

2010). Canonical translational initiation in most eukaryotes involves scanning of the 5’ untranslated

region (UTR) for the start codon by a ribosomal preinitiation complex, which is comprised of the

40S ribosomal subunit end several eukaryotic initiation factors (eIFs) (Jackson et al., 2010;

Topisirovic et al., 2011). A preliminary step in the formation of the preinitiation complex is the

binding of eIF4F complex to the 5’cap of the mRNA (Merrick, 2015). The eIF4F complex comprise

the proteins eIF4E which is the actual 5’cap binding protein, the RNA helicase eIF4A and the

scaffold protein eIF4G (Merrick, 2015). At the center of this complex is eIF4G a multidomain

protein, which functions as a scaffold protein for both eIF4E and eIF4A (Merrick, 2015). When

eIF4G binds eIF4E it induce allosteric changes and enhance the affinity of eIF4E for the 5’cap of

the mRNA (Gross et al., 2003; Volpon et al., 2006). Intriguingly the binding of the 5’cap of the

mRNA also enhances the interaction between eI4E and eIF4G, thus stabilizing the complex (Volpon

et al., 2006). eIF4G also enhance the activity of the RNA helicase eIF4A and functions as a scaffold

protein that induce allosteric conformational changes to both eIF4A and eIF4E (Gross et al., 2003;

Schütz et al., 2008; Volpon et al., 2006).

eIF4G also interacts with eIF3 and poly-A binding protein (PABP). After the eIF4F complex has

bound the 5’cap of the mRNA eIF3 recruits the 40S ribosomal subunit and several other eIFs

resulting in the formation of the 48S ribosomal initiation complex that scan the 5’UTR for a

transitional start site (Jackson et al., 2010; Topisirovic et al., 2011). The binding of PABP by eIF4G

is thought to circularize the mRNA which is believed to enhance translation due to recycling of the

ribosome after ended translation (Topisirovic et al., 2011).

Initiation of translation is somehow conserved in all eukaryotes although differences occur. For

example, in contrast to the three-subunit eIF4F complex identified in mammals and plants, only a

two-subunit complex comprising eIF4G and eIF4E have been identified in yeast. However, eIF4A

helicase activity in yeast is believed to function in association with eIF4E and eIF4G (Mayberry et

al., 2011; Merrick, 2015). Plants also stand out as in both yeast and mammalian cell lines, but no

plants have eIF4E binding proteins (4E-BP) that regulate the availability of eIF4E by sequestration

thereby inhibiting the formation of the eIF4F complex (Lukhele et al., 2013; Peter et al., 2015). In

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mammals and yeast phosphorylation of 4E-BPs functions to regulate translational initiation and

phosphorylated 4E-BPs lose the affinity for eIF4E, promoting the formation of the eIF4F complex

and thereby translation (Merrick, 2015). How plants regulate translational initiation in absence of

4E-BPs is unclear. In mammals, a MAPK-interacting kinase (MNK) phosphorylates eIF4E at a

conserved residue (Ser-209) in the C-terminal region (Fig. 1). This phosphorylation upregulates

translation of a subset of genes involved in inflammation responses and tumor progression (Furic et

al., 2010). Using phosphoproteomic several other phosphorylated residues in human eIF4E has been

identified, but no function has been assigned to these phosphorylated residues (Fig.1)

(PhosphoSitePlus Database: Eto, 2010; Franz-Wachtel et al., 2012; Hornbeck et al., 2015; Li et al.,

2009; Rychlik et al., 1987; Sharma et al., 2014; Van Hoof et al., 2009; Zhou et al., 2013). The Ser-

209 of human eIF4E does not align to any Ser or Thr residues in the Arabidopsis eIF4E (Fig. 1), so

it is plausible that this specific phosphorylation is explicit for humans/mamels. Nonetheless one

phosphosite has been identified in Arabidopsis eIF4E (Fig. 1), but no function has been assigned to

it (PhosPhAt Database: Durek et al., 2010).

* At_eIF4E 1 MAVE-----DTPKSVVTEEAKPNSIENPIDRYHEEGDDAEEGEIAGGEGDGNVDESSKSG

Hs_eIF4E 1 MATVEPETTPTPNPPTTEEEKTESNQ----------------EVANPEH-----------

At_eIF4E 56 VPESHPLEHSWTFWFDNPAVKSKQTSWGSSLRPVFTFSTVEEFWSLYNNMKHPSKLAHGA

Hs_eIF4E 34 -YIKHPLQNRWALWFFKN---DKSKTWQANLRLISKFDTVEDFWALYNHIQLSSNLMPGC

At_eIF4E 116 DFYCFKHIIEPKWEDPICANGGKWTMTFPKEK----SDKSWLYTLLALIGEQFD-HGDEI

Hs_eIF4E 90 DYSLFKDGIEPMWEDEKNKRGGRWLITLNKQQRRSDLDRFWLETLLCLIGESFDDYSDDV

At_eIF4E 171 CGAVVNIRGKQERISIWTKNASNEAAQVSIGKQWKEFLDYNN--SIGFIIHED-AKKLDR

Hs_eIF4E 150 CGAVVNVRAKGDKIAIWTTECENREAVTHIGRVYKERLGLPPKIVIGYQSHADTATKSGS

At_eIF4E 228 NAKNAYTA

Hs_eIF4E 210 TTKNRFVV

Figure 1: Alignment of human and Arabidopsis peptide sequence

Sequences were aligned with T-Coffee and colored by BoxShade3.2. Known phospho-sites (PhosPhAt and PhosphoSitePlus)

are indicated by red background coloring and putative MPK phospo-sites are marked above the alignment with (*).

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The Arabidopsis genome contain four different eIF4E genes namely eIF4E (AT4G18040), eIF4E-B

(AT1G29550), eIF4E-C (AT1G29590) and eIF(ISO)4E (AT5G35620). eIF4EB and eIF4EC are

expressed at very low levels and are therefore not thought to function in general translation, but

expression of eIF4EB is upregulated in flowering tissue implying a possible role in reproduction

(Patrick et al., 2014; Rodriguez et al., 1998). eIF4E are expressed throughout all green tissue

whereas eIF(iso)4E are expressed mainly in flowers (Rodriguez et al., 1998). Albeit differential

expression patterns of eIF4E and eIF(iso)4E they are able to complement each other’s function in

Arabidopsis as individual loss-of-function mutants reassemble a wild type phenotype whereas the

double eif4e eif(iso)4e mutants are embryo lethal (Callot and Gallois, 2014). In Arabidopsis eIF4E

and eIF(iso)4E interacts with eIF4G and eIF(iso)4G respectively forming eIF4F and eIF(iso)4F

complexes (Patrick et al., 2012). It is therefore more likely that the complementation occurs through

the eIF4F and eIF(iso)4F complexes. In vitro assays shows that eIF4(iso)4E has an up to 10 fold

lower affinity for a 5’cap analog than eIF4E (Kropiwnicka et al., 2015). This raises the question of

how these proteins compete with each other in vivo for in translational initiation or perhaps it is

more appropriate to question whether they actually compete? Albeit they are able to complement

each other meaning that they are both able to initiate general translation, it is possible that they both

have favored affinity for a subset of differential mRNA species.

Some viruses depend on the host translational machinery for translation of their genome, and a few

of these have been shown to rely specifically on either eIF4E or eIF(iso)4E. Thus, mutations in

these proteins are often associated with loss of susceptibility (Sanfaçon, 2015). Genomic potyvirus

RNA contains a 5′ end covalently linked virus-encoded protein (VPg), which is required for virus

infectivity (Riechmann et al., 1992). The VPg linked to the viral genome interacts with the host

translational machinery on which it is dependent on translation of the viral genome (Wang and

Krishnaswamy, 2012). Therefore, mutations or loss-of-function of indispensable host factors

required by the virus can thwart viral infection (Diaz-Pendon et al., 2004). So far all mutations in

genes conferring recessive resistance in plants are found in eIFs and the vast majority are found in

genes coding for eIF4E or eIF(iso)4E (Wang and Krishnaswamy, 2012). Pytoviruses display

selective involvement of either eIF4E or eIF(iso)4E during infection for example Clover yellow

vein virus (ClYVV) dependents on the interaction between the viral VPg and the host eIF4E and not

eIF(iso)4E for proliferation and (Sato et al., 2005). Whereas both Turnip mosaic virus (TuMV) and

Lettuce mosaic virus (LMV) on the other hand rely on interactions between their viral VPg and host

eIF(iso)4E and not eIF4E (Duprat et al., 2002). In some cases the VPg compete with host 5’capped

mRNAs for their interaction with eIF4E or eI4(iso)4E, but because the specific motifs required for

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interaction differs for each pytovirus. Thus in some cases eIF4E or eIF(iso)4E interacts with both

host 5’capped mRNAs and viral VPg proteins. And therefore the is no consensus regarding whether

or not domains for binding 5’capped host mRNA and viral VPg proteins overlap (Charron et al.,

2008; Truniger and Aranda, 2009).

Immune signaling is often involve Mitogen Activated Protein kinases (MPKs). In Arabidopsis

MPK3, MPK4 and MPK6 are described as canonical defense regulating kinases (Rasmussen et al.,

2012). All three kinases are activated by perception of Pathogen Associated Molecular Patterns

(PAMPs) by plasma membrane spanning Pathogen Recognition Receptors (PPRs). Activated MPKs

can then transduce immune signaling through for example phosphorylation of defense related

transcription factors (Rasmussen et al., 2012). In plants, virulent pathogens can inject effector protein

directly into the host cell. These effectors can then modify the host to suppress immune responses.

Both MPK3, MPK4 and MPK6 are pathogenic effector targets, implying that they are important

immune regulators (Deslandes and Rivas, 2012). Successful plants have evolved resistance (R)

proteins that can directly sense pathogenic effector proteins or indirectly recognize their effect.

SUPRESSOR OF MKK1 MKK2 1 (SUMM2) surveillances the MPK4 pathway and is activated in

absence of MPK4 activity (Zhang et al., 2012). Activation of SUMM2 activates effector-

triggered immunity (ETI) which is somehow similar to PAMP-triggered immunity (PTI), but

defense response are both amplified and prolonged and can include localized programmed cell

death to effectively thwart pathogen growth (Cui et al., 2015; Zhang et al., 2012).

A screen for MPK substrates using functional protein microarrays published by Popescu et al.

(2009) identified 152 possible MPK4 substrates. However, by focusing on substrates

phosphorylated only by MPK4 we could reduce this list to 14 potential MPK4 specific substrates.

We scrutinized these 14 proteins and further eliminated three proteins that lacked the canonical

Ser/The-Pro MPK phosphorylation site (Pearson et al., 2001). We cloned the remaining list of

eleven proteins and co-expressed them in N. benthamiana with MPK4 to identify any possible

interactions by co-immunoprecipitation. We identified eIF4E as an MPK4 interacting protein and

further investigated its relation to MPK4 both in vitro and in Arabidopsis.

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Results

eIF4E is an MPK4 substrate

In the MPK substrates published by Popescu et al. (2009) we focused on eIF4E as a putative

substrate of MPK4 kinase activity. Not all substrates reported by Popescu et al. (2009) contained the

canonical Ser/Thr-Pro MPK phosphorylation motif (Pearson et al., 2001). We therefore first

performed an in vitro kinase experiment to confirm eIF4E as an MPK4 substrate. We

immunoprecipitated PAMP activated MPKs and incubated them with recombinant His6-eIF4E

protein purified from e.coli. Phosphorylated His6-eIF4E was observed as a radioactive band after

incubation with flg22 activated MPK4, while nor MPK3 or MPK6 resulted in His6-eIF4E

phosphorylation (Fig. 2). Each MPK was also incubated with the generic MPK substrate myelin

basic protein (MBP) to verify their activation (Fig. 2). The eIF4E peptide sequence contains one

putative MPK phosphorylation site (Thr6-Pro7). A version of eIF4E with Thr6 mutated to an

alanine (eIF4E-T6A) was also incubated with immunoprecipitated MPK-3, -4 and -6, this mutant

had significantly lower levels of phosphorylation after incubation with MPK4 (Fig. 2). Thus,

eIF4E-Thr6 is phosphorylated in vitro by MPK4 and not by MPK3 or MPK6. This phosphorylated

TP site aligns with a TP site in the human eIF4E, which is also phosphorylated (Fig. 1). The effect

of phosphorylating this TP site in human eIF4E is unknown, but given its homology to the TP site

in Arabidopsis eIF4E it might have a conserved regulatory function.

Figure 2: MPK4 specifically phosphorylates eIF4E in vitro.

MPK-3, -4 and -6 were immunoprecipitated from extracts of Col-0 seedlings treated with 200 nM flg22 for 10 min. For the

negative control (-), extracts were incubated with agarose beads without antibodies. Immunoprecipitates were incubated with

His6-eIF4E, His6-eIF4E-T6A or MBP for 60 min at 30°C before SDS-PAGE. Autoradiogram (top panel), Coomassie-stained

gel for loading control (bottom).

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eIF4E interacts with MPK4 in planta

To examine the interaction we generated transgenic Arabidopsis plants in an eif4e T-DNA loss-of-

function line (SALK_145583) expressing eIF4E-Myc under control of a 35S promoter. In anti-Myc

immunoprecipitations from transgenic eIF4E-Myc tissue, we detected a 40-kDa band corresponding

to MPK4 (Fig. 3). We questioned whether a non-phophorylatable form of eIF4E would affect the

interaction. We therefore generated transgenic plants expressing eIF4E-T6A-Myc under control of a

35S promoter in the eif4e T-DNA loss-of-function mutant. Anti-Myc immunoprecipitation from

transgenic eIF4E-T6A-Myc tissue detected a more intense band than from wild type eIF4E-Myc

(Fig. 3). Thus, the interaction between MPK4 and the non-phosphorylatable eIF4E-T6A is stronger

than the interaction with eIF4E. This may indicate the interaction with MPK4 is dependent on

whether or not eIF4E is phosphorylated.

Figure 3: MPK4 is in complex with eIF4E eIF4E-T6A in Arabidopsis.

Immunoprecipitates from five-week-old plants expressing eIF4E-Myc or the phospho-dead mutant eIF4E-T6A-Myc. MPK4

is detected in anti-Myc immunoprecipitates. Immunoblots of input and anti-Myc immunoprecipitates are probed with anti-

MPK4 and anti-Myc antibodies.

eif4e mutants does not exhibit autoimmunity similar to mpk4

eif4e plants exhibit normal growth and are indistinguishable from Col-0 plants (Fig. 4A).

Nevertheless, given that MPK4 is an important regulator of defense, we tested whether eIF4E may

also be involved in immunity. To this end, we first examined for altered bacterial resistance by

syringe infiltrating pst DC3000, into eif4e leaf tissue. However, there was no noticeable difference

in bacterial growth after four days when compared to Col-0 (Fig. 4B). Given that eIF4E and

eIF(iso)4E can complement each other, we hypothesized that if phosphorylation of eIF4E by MPK4

is important for defense then the non-phophorylatable eIF4E-T6A might dominantly inhibit defense

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regulatory functions normally mediated by eIF4E. We therefore also tested for altered resistance in

the eIF4E-T6A complementing lines by syringe infiltrating pst DC3000, but no noticeable

difference was observed in bacterial growth after four-days post infiltration between these plants,

Col-0 and eIF4E-Myc complementing lines (Fig. 4B)

This implies that phosphorylation of eIF4E-Thr6 is not critical for normal defense responses against

pst DC3000, nonetheless it is possible that eIF4E might be involved in defense responses against

other types of pathogens.

Figure 4: eif4e does not induce autoimmunity

(A) Photographs of five-week old plants, genotypes are as indicated.

(B)Four-week old plants we infiltrated with pst DC3000 (OD600=0.001). Bacteria were syringe-infiltrated and samples taken

4 days post-infiltration. Data are shown as log10-transformed colony-forming units/cm2 leaf tissue. Standard error of the

mean is indicated by errors bars (n = 4).

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ClYVV susceptibility is not dependent on eIF4E phosphorylation

Potyviruses including ClYVV and TuMV seize the host translational machinery to proliferate

themselves. ClYVV and TuMV depends on interactions with eIF4E and eIF(iso)4E respectively to

initiate translation of their genomic RNA (Duprat et al., 2002; Sato et al., 2005). Thus, mutations in

these genes can result in recessive resistance against viruses. We questioned whether

phosphorylation of eIF4E by MPK4 would affect eIF4E activity and thus have impact on virus

resistance against ClYVV. Given that mpk4 exhibits autoimmunity and suppression by summ2 is

only partial, we tested for ClYVV susceptibility in mkk1/2-summ2 plants. MKK1/2 are the upstream

MPKKs of MPK4. The mkk1/2 double mutant reassembles the phenotype of mpk4, however this

phenotype is fully suppressed by summ2, while MPK4 is not activated by PAMP perception. Col-0

was susceptible to both ClYVV and TuMV, whereas respectively eif4e and eif(iso)4e were resistant

(Fig. 5A+B).

Figure 5: ClYVV and TuMV susceptibility iss not dependent on MPK4 activation.

Five-week-old plants were inoculated with (A) ClYVV or (B) TuMV. Virus accumulation was assayed 24 dpi by ELISA.

Error bars indicate SEM (n=6). The red line indicates threshold for susceptibility equal to three times the mean value for

uninfected controls (n=3)

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Both summ2 and mkk1/2-summ2 were susceptible to ClYVV and TuMV, thus lack of MPK4

activity do not impair eIF4E and eIF(iso)4E mediated ClYVV or TuMV proliferation.

It is possible that other kinases can phosphorylate eIF4E-Thr6 in vivo, we therefore tested ClYVV

susceptibility in the complementing eIF4E-T6A-Myc line. Plants expressing either eIF4E-Myc or

eIF4E-T6A-Myc similar to Col-0 were susceptible to ClYVV (Fig. 6). We therefore conclude that

absence of MPK4 meditated eIF4E phosphorylation does not affect eIF4E mediated ClYVV

proliferation. Nevertheless, phosphorylation of eIF4E by MPK4 may regulate its activity in assays

not tested here and might still be important for proper regulation.

Figure 6: ClYVV susceptibility is not dependent phosphorylation eIF4E Thr6.

Five-week-old plants were inoculated with (A) ClYVV. Virus accumulation was assayed after 24 days by ELISA. Error bars

indicate SEM (n=6). The red line indicates threshold for susceptibility equal to three times the mean value for uninfected

controls (n=3)

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MPK4 and eIF4E interact in the nucleus.

Apart from its function in translational initiation eIF4E also function in export of mRNAs out of the

nucleus, in both yeast and humans up to 68% of eIF4E is present in the nucleus (Iborra et al., 2001;

Strudwick and Borden, 2002). eIF4E interacts with a secondary structure in the 3’UTR of specific

mRNA and promotes their export out of the nucleus (Culjkovic et al., 2006). We were interested in

where the MPK4-eIF4E interaction occurs in the cell and therefore employed a bimolecular

fluorescence complementation assay (BiFC). We cloned MPK4 with a C-terminal fusion of nYFP

and eIF4E with a C-terminal fusion of cYFP, both under control of a 35S promoter and co-

expressed them transiently in Nicotiana benthamiana. When expressed together we detected distinct

signal in the nucleus indicating reconstitution of YFP while no signal was observed when eIF4E-

cYFP were co-expressed MPK6-nYFP (Fig. 7). When eIF4E-T6A-nYFP was co-expressed with

MPK4-cYFP we observed an even more intense nuclear fluorescent signal compared to the eIF4E-

nYFP (Fig. 7), which is in agreement with the CoIP data (Fig. 3). Thus, MPK4 and eIF4E interacts

in the nucleus and the non-phophorylatable eIF4E-T6A gives rise to a stronger interaction.

Discussion

In this study, we characterize the translational initiation factor eIF4E as an interactor (Fig. 3+7) and

substrate (Fig. 2) of the immune regulating kinase MPK4. MPK4 specifically phosphorylates

eIF4E-Thr6, which align well with a phosphorylation site in human eIF4E (Fig. 1). Although no

function has been assigned to this phosphorylation, it is possible that its function can be regulatory

or serve as a (de)stabilizing factor. Given that MPK4 is an important immune regulator, we tested a

possible role of eIF4E functioning in immunity. However we did not find evidence of eIF4E

mediated resistance against pst DC3000 (Fig. 4B). Similar we did not find MPK4 activity (Fig. 5A)

or phosphorylation of eIF4E-Thr6 (Fig. 6) important in conferring resistance against the potyvirys

ClYVV that normally rely on eIF4E in Arabidopsis for its own proliferation. However, since we

only tested a very limited set of pathogens we cannot exclude the possibility that phosphorylation of

eIF4E can affect immunity in Arabidopsis.

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Figure 7: eIF4E interacts with MPK4 in the nucleus

Confocal images of yellow fluorescent protein complementation. eIF4E-cYFP and eIF4E-T6A-cYFP were was transiently co-

expressed with either MPK4-nYFP or MPK6-nYFP in N. benthamiana. Confocal microscopy on leaf disks was conducted 2

days post-infiltration. Merged pictures show overlay of brightfield, chlorophyll and eYFP. Blue arrows indicate nuclei and

the scale bar corresponds to 10 µm.

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The interaction between MPK4 and eIF4E occurs in the nucleus (Fig. 7). Up to 68% of eIF4E is

nuclei in both yeast and humans, in the nucleus, eIF4E serves different role than cytoplasmic

translational initiation, instead eIF4E function in specific mRNA transport out of the nucleus

(Culjkovic et al., 2006; Iborra et al., 2001; Strudwick and Borden, 2002). In humans overexpression

of eIF4E leads to enhanced translation of a subset of mRNAs with long highly structured 5’UTRs.

Interestingly these mRNAs are not upregulated at the transcriptional level, but rather actively

transported out of the nucleus to the cytoplasm for translation (Culjkovic et al., 2007). In humans

eIF4E mediated mRNA transport out of the nucleus depends on specific secondary structures in the

3’UTR distinct from bulk mRNA (Culjkovic et al., 2006). Thus, both structured 5’ and 3’UTR

influence eIF4E mediated translation (Osborne and Borden, 2015). We show that in Arabidopsis,

MPK4 interacts with eIF4E in the nucleus. It is therefore appealing to hypothesize that MKP4 can

somehow regulate eIF4E mediated mRNA transport through phosphorylation. However, to this end

we have no data showing eIF4E mediated transport in response to for example immunity. Further

examination of the relationship between MPK4 and eIF4E are needed to clarify this hypothesis.

Recently eIF4E has also been implicated in non-sense mediated decay (NMD) of mRNA in

mammalian cells in response to insulin (Park et al., 2015). eIF4E seemingly interacts with the NMD

factor Upstream Frameshift protein 1 (UPF1) as well as the general decapping factor Decapping 1

(DCP1) to promote NMD of eIF4E associated mRNAs (Park et al., 2015). We have recently shown

that MPK4 interacts with and phosphorylates the mRNA decay factor Protein Associated with

Topoisomerase II (PAT1) (Roux et al., 2015). PAT1 interacts with the cytoplasmic immune-

receptor SUMM2 which in absence of PAT instigates ETI (Roux et al., 2015). Similar, ETI is also

observed in NMD deficient mutants including upf1, suggesting a role of NMD in immunity (Jeong

et al., 2011; Rayson et al., 2012). Any possible link between MPK4 mediated phosphorylation of

eIF4E in Arabidopsis and mRNA decay are however still unclear and will be a subject for further

investigation.

Materials and methods

Plants growth conditions

Arabidopsis thaliana Col-0 accession was used as wild-type in all experiments, except if otherwise

stated. Salk lines were obtained from the Nottingham Arabidopsis Seed Centre (NASC). Lines

obtained from NASC include SALK_101850 (aoc3), SALK_145583 (eif4e). mkks1/2 summ2 was

a kind gift from Yuelin Zhang. Arabidopsis plants were grown in 9 × 9 cm pots at 22°C with a 8-h

94

photoperiod, or on plates containing Murashige–Skoog (MS) salts medium (Duchefa), 1% sucrose

and 1% agar with a 16-h photoperiod.

SDS-PAGE and immunoblotting

SDS-tris/glycine or bis/tris gels were prepared with either 8, 10 or 12% acrylamide. Proteins were

loaded and gels run at 100–150 V until the dye front reached the end of the gel. Gels were next

electroblotted onto PVDF membranes (GE Healthcare) by wet transfer for 1h at 70 V. Membranes

were rinsed in TBS and blocked for 30min in 5% (w/v) non-fat milk in TBS-Tween (0.1% (v/v)).

Membranes were incubated with primary antibodies for 1 h to overnight. Membranes were washed

3 × 10 min in TBS-Tween (0.1%) before 1-h incubation in secondary antibodies. Membrnaes were

incubated in chemiluminescent substrate (100 mM Tris/HCl pH 8.8, 1.25 mM luminol, 5.3 mM

hydrogenperoxide and 2 mM 4IPBA)(Haan and Behrmann, 2007) before exposure to a Sony a7S

DSLR camera(Khoury et al., 2010). For AP-conjugated secondary antibodies membranes were

incubated in NBT/BCIP (Roche) until bands were visible. For probing immunoblots with multiple

antibodies, stripping was carried out using Restore Western Blot Stripping Buffer (Pierce) for 15–

30 min, following three washes with TBS-Tween (0.1% (v/v)).

Antibodies

Primary antibodies were diluted in 1% (w/v) non-fat milk in TBS-Tween (0.1% (v/v)) to the

following dilutions:

Anti Host Dilution Suplier

Myc Mouse 1:1000 Santa Cruz

GFP Mouse 1:1000 Santa Cruz

HA Mouse 1:1000 Santa Cruz

MPK3 Rabbit 1:2000 Sigma



CSD2 Rabbit 1:4000 Agrisera

PR1 Rabbit 1:4000 Agrisera

DCP1 Rabbit 1:1000 Gift from

eIF4E Rabbit 1:5000 Gift from Jean-Luc Gallois

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Secondary antiboides were diluted in 1% (w/v) non-fat milk in TBS-Tween (0.1% (v/v)) to the

following dilutions:

anti-mouse-HRP 1:10.000 (Promega)

anti-Rabbit-HRP 1:10.000 (Promega)

ProteinA-HRP 1:1000 (GE Healthcare)

Bacterial infection assays

Pseudomonas syringae pv. tomato DC3000 (Pst DC3000) strains were grown in overnight culture in

NYG medium supplemented with appropriate antibiotics. Cells were harvested by centrifugation

and pellets resuspended in sterile water to indicated concentrations. Bacteria were infiltrated with a

needleless 1-mm syringe into adult leafs and maintained in growth chambers for 3-4 days. Samples

were taken using a cork-borer (6.5 mm) to cut leaf disks from infected leafs. Leaf disks were

ground in water, serially diluted and plated on NYG plates with appropriate selection. Plates were

incubated at 28°C and colonies counted 2–3 days later.

Transient expression in Nicotiana benthamiana

Agrobacterium tumefaciens strains were grown in LB medium supplemented with appropriate

antibiotics overnight. Cultures were spun down and resuspended in 10 mM MgCl2 to OD600 = 0.8.

For co-infiltrations bacterial cultures were mixed 1:1. Bacterial cultures were syringe-infiltrated into

3-week-old N. benthamiana leaves. Samples for protein extraction were harvested 2 days post-

infiltration.

Protein extraction and immunoprecipitation in Nicotiana benthamiana and Arabidopsis

Plant material were ground in liquid nitrogen and extraction buffer [10 mM phosphate pH 7.2, 150

mM NaCl, 5 mM DTT, 0.1 % Nonidet P-40, supplemented with EDTA-free protease inhibitor cocktail

form Roche or 110 mM potassium acetate, 100 mM NaCl, 2 mM MgCl, 0.5%(v/v) TritonX-100

supplemented with EDTA-free protease inhibitor cocktail form Roche]

Samples were clarified by 10 min centrifugation at 4°C and 13,000 rpm. Supernatants were adjusted

to 2 mg/ml protein and incubated 1-2 h at 4°C with GFPTrap-A beads (Chromotek), anti-Myc

magnetic beads (Miltenyi Biotech) or EZview™ Red Protein A Affinity Gel supplemented with

specific antibodies. Following incubation, beads were washed four times with extraction buffer,

before adding 6XSDS loading buffer [300 mM Tris HCL pH 6.8, 300 mM DTT, 6% SDS, 6 mM

EDTA, 0.03%(w/v) bromphenol blue, 60%(v/v) glycerol] and heating at 70°C for 10 min.

96

Purification of X7 polymerase

E.coli containing a plasmid encoding His tagged PfuX7 under control of a IPTG inducible promotor

was grown in liquid LB media (Nørholm, 2010). At OD600=0.4 IPTG was added to a final

concentration of 0.5mM. Cells where harvested by centrifugation and re-suspended in lysis buffer []

and lysed by 3-4 freeze/thaw cycles. Soluble protein were purified nickel spin columns () according

to manufactures instruction. The enzyme was desalted by dialysis o.n. against storage buffer [50%

glycerol, 100 mM Tris/HCl pH 8.0, 0.2 mM EDTA, 2 mM DTT, 0.2% NP40, 0.2% Tween 20.] and

stored at -20C.

Genotyping

Leaf tissue was used for Edwards DNA extraction (Edwards et al., 1991). PfuX7 polymerase were

used for genotyping PCR reactions in with a final concentration of 0.4 mM dATP, 0.4 mM dTTP,

0.4 mM dCTP, 0.4 mM dGTP, 20 mM Tris/HCl pH 8.8, 10 mM KCl, 6 mM (NH4)2SO4, 2 mM

MgSO4, 0.1 mg/ml BSA and 0.1% Triton X-100 (Nørholm, 2010). Cycling conditions included a

60 second 96°C template denaturation step followed by 30 cycles of 15 second 56-65°C annealing

step and a 15 second long primer extension step at 72°C. PCR products were electrophoresed on a

1% agarose gel in 0.5X TBE [45 mM Tris-borate/1 mM EDTA] with etBr and PCR products were

visualized by UV light. Primers designed using the iSect tool

(http://signal.salk.edu/isectprimers.html) were used to genotype lines for homozygosity.

Cloning and transgenic lines

Phusion Hot Start II High-Fidelity polymerase (Thermo) or PfuX7 (Nørholm, 2010) were used to

amplify PCR products for all subsequent cloning reactions.

MPK6

MPK6 was cloned into pETNR D-TOPO (Invitrogen) from genomic DNA without stop codon with

the following primers fwd “5’caccATGGACGGTGGTTCAGGTCA” reverse

“5’TTGCTGATATTCTGGATTGAAAGC”

AOC3

AOC3 without signal peptide was cloned into pETNR pOPINf for expression in E.coli from cDNA

DNA without start codon with the following primers fwd

“5’AAGTTCTGTTTCAGGGCCCGAGACCAAGTAAGATCCAAGAACTAA” reverse

“5’ATGGTCTAGAAAGCTTTAATTAGTAAAGTTACTTATAACTCCA”. For cloning AOC3-

S100A the following start primer where used

“5’AAGTTCTGTTTCAGGGCCCGCCAAGTAAGATCCAAGAACTAAACGTGTATGAGCTC

97

AACGAAGGAGATAGAAACgcTCCAGC”. For cloning AOC3-T235A the following revers

primer were used

“5’ATGGTCTAGAAAGCTTTAATTAGTAAAGTTACTTATAACTCCACTAGGCTCCATCGC

CTTAGCTTCCGGTGCCGGTTTCACATCCTTCGACGGCGCAACCGCCG”.

eIF4E

eIF4E was cloned into pETNR D-TOPO from genomic DNA without stop codon with the following

primers fwd “5’caccATGGCGGTAGAAGACACTCC”reverse

“5’AGCGGTGTAAGCGTTCTTTG “. For cloning eIF4E-T6A the following fwd primer was used

“5’caccATGGCGGTAGAAGACgCTCCCAAATC”. eIF4E was cloned into pOPINf for expression

in E.coli from cDNA without start codon with the following primers fwd

“5’AAGTTCTGTTTCAGGGCCCGGCGGTAGAAGACACTCCCAAA” reverse

“5’ATGGTCTAGAAAGCTTTAAGCGGTGTAAGCGTTCTTTGC”. For cloning eIF4E-T6A the

following start primer where used

“5’AAGTTCTGTTTCAGGGCCCGGCGGTAGAAGACgCTCCCAAATC”.

MPK6, AOC3 and eIF4E were recombined into binary gateway destination vectors for expression

in plants using LR Clonase II (Invitrogen). For expression of C-terminally tagged GFP, HA, Myc,

nYFP and cYFP construct pENTR clones were recombined into pGWB605, pGWB615, pGWB617,

pcYCW and pnYGW respectively (HINO et al., 2011; Nakagawa et al., 2007).

Clones were transformed into A. tumefaciens strain GV3101 and used for transient expression in N.

benthamiana or floral dipping in Arabidopsis.

Recombinant protein purification and in vitro kinase assays

For in vitro experiments, PAT1 protein was purified from E. coli. The AOC3 and eIF4E CDS was

cloned into pET15b (for an N-terminal His fusion) and transformed into E. coli BL21 (pLysS).

Protein expression was induced by overnight treatment with 0.5 mM IPTG at 37°C, added to cells

at OD600 = 0.6. Proteins were purified under native conditions Ni-NTA spin columns (Qiagen). For

kinase assay see Roux et al. (2015).

Virus inoculation and detection by ELISA

TuMV and ClYVV were propagated on turnip (Brassica rapa) and Nicotiana benthamiana before

virus inoculating of five-week-old Arabidopsis plants. Virus accumulation was assayed after 24

days by ELISA using respective viral antisera (DSMZ). Six plants per genotype were used in each

98

experiment. The susceptibility threshold is indicated for each experiment as a red line and refers to

an absorbance value at 405 nm in ELISA equal to three times the mean value for uninfected

controls.

References

Callot, C., and Gallois, J.-L. (2014). Pyramiding resistances based on translation initiation factors in

Arabidopsis is impaired by male gametophyte lethality. Plant Signal. Behav. 9.

doi:10.4161/psb.27940.

Charron, C., Nicolaï, M., Gallois, J.-L., Robaglia, C., Moury, B., Palloix, A., et al. (2008). Natural

variation and functional analyses provide evidence for co-evolution between plant eIF4E

and potyviral VPg. Plant J. 54, 56–68. doi:10.1111/j.1365-313X.2008.03407.x.

Cui, H., Tsuda, K., and Parker, J. E. (2015). Effector-Triggered Immunity: From Pathogen

Perception to Robust Defense. Annu. Rev. Plant Biol. 66, 487–511. doi:10.1146/annurev-

arplant-050213-040012.

Culjkovic, B., Topisirovic, I., and Borden, K. L. B. (2007). Controlling Gene Expression through

RNA Regulons: The Role of the Eukaryotic Translation Initiation Factor eIF4E. Cell Cycle

6, 65–69. doi:10.4161/cc.6.1.3688.

Culjkovic, B., Topisirovic, I., Skrabanek, L., Ruiz-Gutierrez, M., and Borden, K. L. B. (2006).

eIF4E is a central node of an RNA regulon that governs cellular proliferation. J. Cell Biol.

175, 415–426. doi:10.1083/jcb.200607020.

Deslandes, L., and Rivas, S. (2012). Catch me if you can: bacterial effectors and plant targets.

Trends Plant Sci. 17, 644–655. doi:10.1016/j.tplants.2012.06.011.

Diaz-Pendon, J. A., Truniger, V., Nieto, C., Garcia-Mas, J., Bendahmane, A., and Aranda, M. A.

(2004). Advances in understanding recessive resistance to plant viruses. Mol. Plant Pathol.

5, 223–233. doi:10.1111/j.1364-3703.2004.00223.x.

Duprat, A., Caranta, C., Revers, F., Menand, B., Browning, K. S., and Robaglia, C. (2002). The

Arabidopsis eukaryotic initiation factor (iso)4E is dispensable for plant growth but required

for susceptibility to potyviruses. Plant J. 32, 927–934. doi:10.1046/j.1365-

313X.2002.01481.x.

Durek, P., Schmidt, R., Heazlewood, J. L., Jones, A., MacLean, D., Nagel, A., et al. (2010).

PhosPhAt: the Arabidopsis thaliana phosphorylation site database. An update. Nucleic

Acids Res. 38, D828–D834. doi:10.1093/nar/gkp810.

Edwards, K., Johnstone, C., and Thompson, C. (1991). A simple and rapid method for the

preparation of plant genomic DNA for PCR analysis. Nucleic Acids Res. 19, 1349.

99

Eto, I. (2010). Upstream molecular signaling pathways of p27(Kip1) expression: effects of 4-

hydroxytamoxifen, dexamethasone, and retinoic acids. Cancer Cell Int. 10, 3.

doi:10.1186/1475-2867-10-3.

Franz-Wachtel, M., Eisler, S. A., Krug, K., Wahl, S., Carpy, A., Nordheim, A., et al. (2012). Global

detection of protein kinase D-dependent phosphorylation events in nocodazole-treated

human cells. Mol. Cell. Proteomics MCP 11, 160–170. doi:10.1074/mcp.M111.016014.

Furic, L., Rong, L., Larsson, O., Koumakpayi, I. H., Yoshida, K., Brueschke, A., et al. (2010).

eIF4E phosphorylation promotes tumorigenesis and is associated with prostate cancer

progression. Proc. Natl. Acad. Sci. 107, 14134–14139. doi:10.1073/pnas.1005320107.

Gross, J. D., Moerke, N. J., von der Haar, T., Lugovskoy, A. A., Sachs, A. B., McCarthy, J. E. G.,

et al. (2003). Ribosome Loading onto the mRNA Cap Is Driven by Conformational

Coupling between eIF4G and eIF4E. Cell 115, 739–750. doi:10.1016/S0092-

8674(03)00975-9.

Haan, C., and Behrmann, I. (2007). A cost effective non-commercial ECL-solution for Western blot

detections yielding strong signals and low background. J. Immunol. Methods 318, 11–19.

doi:10.1016/j.jim.2006.07.027.

HINO, T., TANAKA, Y., KAWAMUKAI, M., NISHIMURA, K., MANO, S., and NAKAGAWA,

T. (2011). Two Sec13p Homologs, AtSec13A and AtSec13B, Redundantly Contribute to the

Formation of COPII Transport Vesicles in Arabidopsis thaliana. Biosci. Biotechnol.

Biochem. 75, 1848–1852. doi:10.1271/bbb.110331.

Hornbeck, P. V., Zhang, B., Murray, B., Kornhauser, J. M., Latham, V., and Skrzypek, E. (2015).

PhosphoSitePlus, 2014: mutations, PTMs and recalibrations. Nucleic Acids Res. 43, D512–

D520. doi:10.1093/nar/gku1267.

Iborra, F. J., Jackson, D. A., and Cook, P. R. (2001). Coupled Transcription and Translation Within

Nuclei of Mammalian Cells. Science 293, 1139–1142. doi:10.1126/science.1061216.

Jackson, R. J., Hellen, C. U. T., and Pestova, T. V. (2010). The mechanism of eukaryotic translation

initiation and principles of its regulation. Nat. Rev. Mol. Cell Biol. 11, 113–127.

doi:10.1038/nrm2838.

Jeong, H.-J., Kim, Y. J., Kim, S. H., Kim, Y.-H., Lee, I.-J., Kim, Y. K., et al. (2011). Nonsense-

Mediated mRNA Decay Factors, UPF1 and UPF3, Contribute to Plant Defense. Plant Cell

Physiol. 52, 2147–2156. doi:10.1093/pcp/pcr144.

Khoury, M. K., Parker, I., and Aswad, D. W. (2010). Acquisition of chemiluminescent signals from

immunoblots with a digital SLR camera. Anal. Biochem. 397, 129–131.

doi:10.1016/j.ab.2009.09.041.

Kropiwnicka, A., Kuchta, K., Lukaszewicz, M., Kowalska, J., Jemielity, J., Ginalski, K., et al.

(2015). Five eIF4E isoforms from Arabidopsis thaliana are characterized by distinct features

of cap analogs binding. Biochem. Biophys. Res. Commun. 456, 47–52.

doi:10.1016/j.bbrc.2014.11.032.

100

Li, H., Ren, Z., Kang, X., Zhang, L., Li, X., Wang, Y., et al. (2009). Identification of tyrosine-

phosphorylated proteins associated with metastasis and functional analysis of FER in human

hepatocellular carcinoma cells. BMC Cancer 9, 366. doi:10.1186/1471-2407-9-366.

Lukhele, S., Bah, A., Lin, H., Sonenberg, N., and Forman-Kay, J. D. (2013). Interaction of the

Eukaryotic Initiation Factor 4E with 4E-BP2 at a Dynamic Bipartite Interface. Structure 21,

2186–2196. doi:10.1016/j.str.2013.08.030.

Mayberry, L. K., Allen, M. L., Nitka, K. R., Campbell, L., Murphy, P. A., and Browning, K. S.

(2011). Plant Cap-binding Complexes Eukaryotic Initiation Factors eIF4F and eIFISO4F

MOLECULAR SPECIFICITY OF SUBUNIT BINDING. J. Biol. Chem. 286, 42566–

42574. doi:10.1074/jbc.M111.280099.

Merrick, W. C. (2015). eIF4F: A Retrospective. J. Biol. Chem. 290, 24091–24099.

doi:10.1074/jbc.R115.675280.

Nakagawa, T., Suzuki, T., Murata, S., Nakamura, S., Hino, T., Maeo, K., et al. (2007). Improved

Gateway binary vectors: high-performance vectors for creation of fusion constructs in

transgenic analysis of plants. Biosci. Biotechnol. Biochem. 71, 2095–2100.

Nørholm, M. H. (2010). A mutant Pfu DNA polymerase designed for advanced uracil-excision

DNA engineering. BMC Biotechnol. 10, 21. doi:10.1186/1472-6750-10-21.

Osborne, M. J., and Borden, K. L. B. (2015). The eukaryotic translation initiation factor eIF4E in

the nucleus: taking the road less traveled. Immunol. Rev. 263, 210–223.

doi:10.1111/imr.12240.

Park, J., Ahn, S., Jayabalan, A. K., Ohn, T., Koh, H. C., and Hwang, J. Insulin signaling augments

eIF4E-dependent nonsense-mediated mRNA decay in mammalian cells. Biochim. Biophys.

Acta BBA - Gene Regul. Mech. doi:10.1016/j.bbagrm.2015.12.006.

Patrick, R. M., Browning, K. S., Patrick, R. M., and Browning, K. S. (2012). The eIF4F and

eIFiso4F Complexes of Plants: An Evolutionary Perspective, The eIF4F and eIFiso4F

Complexes of Plants: An Evolutionary Perspective. Int. J. Genomics Int. J. Genomics 2012,

2012, e287814. doi:10.1155/2012/287814, 10.1155/2012/287814.

Patrick, R. M., Mayberry, L. K., Choy, G., Woodard, L. E., Liu, J. S., White, A., et al. (2014). Two

Arabidopsis Loci Encode Novel Eukaryotic Initiation Factor 4E Isoforms That Are

Functionally Distinct from the Conserved Plant Eukaryotic Initiation Factor 4E. Plant

Physiol. 164, 1820–1830. doi:10.1104/pp.113.227785.

Pearson, G., Robinson, F., Beers Gibson, T., Xu, B., Karandikar, M., Berman, K., et al. (2001).

Mitogen-Activated Protein (MAP) Kinase Pathways: Regulation and Physiological

Functions. Endocr. Rev. 22, 153–183. doi:10.1210/edrv.22.2.0428.

Peter, D., Igreja, C., Weber, R., Wohlbold, L., Weiler, C., Ebertsch, L., et al. (2015). Molecular

Architecture of 4E-BP Translational Inhibitors Bound to eIF4E. Mol. Cell 57, 1074–1087.

doi:10.1016/j.molcel.2015.01.017.

Popescu, S. C., Popescu, G. V., Bachan, S., Zhang, Z., Gerstein, M., Snyder, M., et al. (2009).

MAPK Target Networks in Arabidopsis Thaliana Revealed Using Functional Protein

Microarrays. Genes Dev. 23, 80–92. doi:10.1101/gad.1740009.

101

Rasmussen, M. W., Roux, M., Petersen, M., and Mundy, J. (2012). MAP kinase cascades in

Arabidopsis innate immunity. Plant Proteomics 3, 169. doi:10.3389/fpls.2012.00169.

Rayson, S., Arciga-Reyes, L., Wootton, L., De Torres Zabala, M., Truman, W., Graham, N., et al.

(2012). A Role for Nonsense-Mediated mRNA Decay in Plants: Pathogen Responses Are

Induced in Arabidopsis thaliana NMD Mutants. PLoS ONE 7, e31917.

doi:10.1371/journal.pone.0031917.

Riechmann, J. L., Lain, S., and Garcia, J. A. (1992). Highlights and prospects of potyvirus

molecular biology. J. Gen. Virol. 73, 1–16. doi:10.1099/0022-1317-73-1-1.

Rodriguez, C. M., Freire, M. A., Camilleri, C., and Robaglia, C. (1998). The Arabidopsis thaliana

cDNAs coding for eIF4E and eIF(iso)4E are not functionally equivalent for yeast

complementation and are differentially expressed during plant development. Plant J. 13,

465–473. doi:10.1046/j.1365-313X.1998.00047.x.

Roux, M. E., Rasmussen, M. W., Palma, K., Lolle, S., Regué, À. M., Bethke, G., et al. (2015). The

mRNA decay factor PAT1 functions in a pathway including MAP kinase 4 and immune

receptor SUMM2. EMBO J., e201488645. doi:10.15252/embj.201488645.

Rychlik, W., Russ, M. A., and Rhoads, R. E. (1987). Phosphorylation site of eukaryotic initiation

factor 4E. J. Biol. Chem. 262, 10434–10437.

Sanfaçon, H. (2015). Plant Translation Factors and Virus Resistance. Viruses 7, 3392–3419.

doi:10.3390/v7072778.

Sato, M., Nakahara, K., Yoshii, M., Ishikawa, M., and Uyeda, I. (2005). Selective involvement of

members of the eukaryotic initiation factor 4E family in the infection of Arabidopsis

thaliana by potyviruses. FEBS Lett. 579, 1167–1171. doi:10.1016/j.febslet.2004.12.086.

Schütz, P., Bumann, M., Oberholzer, A. E., Bieniossek, C., Trachsel, H., Altmann, M., et al. (2008).

Crystal structure of the yeast eIF4A-eIF4G complex: An RNA-helicase controlled by

protein–protein interactions. Proc. Natl. Acad. Sci. 105, 9564–9569.

doi:10.1073/pnas.0800418105.

Sharma, K., D’Souza, R. C. J., Tyanova, S., Schaab, C., Wiśniewski, J. R., Cox, J., et al. (2014).

Ultradeep human phosphoproteome reveals a distinct regulatory nature of Tyr and Ser/Thr-

based signaling. Cell Rep. 8, 1583–1594. doi:10.1016/j.celrep.2014.07.036.

Strudwick, S., and Borden, K. L. B. (2002). The emerging roles of translation factor eIF4E in the

nucleus. Differentiation 70, 10–22. doi:10.1046/j.1432-0436.2002.700102.x.

Topisirovic, I., Svitkin, Y. V., Sonenberg, N., and Shatkin, A. J. (2011). Cap and cap-binding

proteins in the control of gene expression. Wiley Interdiscip. Rev. RNA 2, 277–298.

doi:10.1002/wrna.52.

Truniger, V., and Aranda, M. A. (2009). “Chapter 4 - Recessive Resistance to Plant Viruses,” in

Advances in Virus Research Natural and Engineered Resistance to Plant Viruses, Part I., ed.

G. L. and J. P. Carr (Academic Press), 119–231. Available at:

http://www.sciencedirect.com/science/article/pii/S0065352709075046 [Accessed December

17, 2015].

102

Van Hoof, D., Muñoz, J., Braam, S. R., Pinkse, M. W. H., Linding, R., Heck, A. J. R., et al. (2009).

Phosphorylation dynamics during early differentiation of human embryonic stem cells. Cell

Stem Cell 5, 214–226. doi:10.1016/j.stem.2009.05.021.

Volpon, L., Osborne, M. J., Topisirovic, I., Siddiqui, N., and Borden, K. L. (2006). Cap-free

structure of eIF4E suggests a basis for conformational regulation by its ligands. EMBO J.

25, 5138–5149. doi:10.1038/sj.emboj.7601380.

Wang, A., and Krishnaswamy, S. (2012). Eukaryotic translation initiation factor 4E-mediated

recessive resistance to plant viruses and its utility in crop improvement. Mol. Plant Pathol.

13, 795–803. doi:10.1111/j.1364-3703.2012.00791.x.




doi:10.1016/j.chom.2012.01.015.

Zhou, H., Di Palma, S., Preisinger, C., Peng, M., Polat, A. N., Heck, A. J. R., et al. (2013). Toward

a comprehensive characterization of a human cancer cell phosphoproteome. J. Proteome

Res. 12, 260–271. doi:10.1021/pr300630k.

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Concluding remarks

MPK4 was initially characterized as a negative regulator of defense, due to MPK4 the loss-of-

function mutant constitutively induction of defense responses (Petersen et al., 2000). MPK4 is

activated by perception of the flagellin-derived flg22 peptide by the pathogen recognition receptor

FLS2 (Rodriguez et al., 2010). FLS2 and its co-receptor BAK1 sequentially signals the activation of

the three tiered MAPK signaling cascade comprising MEKK1, MKK1/2 and MPK4 (Chinchilla et

al., 2007; Gao et al., 2008; Qiu et al., 2008a). Inactive MPK4 is bound to MKS1 and the WRKY33

transcription factor, upon activation, MPK4 phosphorylates MKS1, which release WRKY33 from

the complex to induce transcription of the defense gene PAD3 (Andreasson et al., 2005; Qiu et al.,

2008a, 2008b). The function of MPK4 as a negative regulator of defense is in contrast to MPK4

positively inducing transcription of PAD3. The MPK4 loss-of-function defense phenotype is

suppressed my loss-of-function of EDS1 (Brodersen et al., 2006), which is essential for fully

functional effector-triggered immunity (Falk et al., 1999). This indicated that the mpk4 defense

phenotype was due to inappropriate activation of ETI and not due to loss of negative suppression of

defense. Zhang et al. (2012) identified the R-protein SUMM2, which surveillance the MPK4

pathway and induce ETI upon disruption of this pathway. This endorse the hypothesis of the mpk4

defense phenotype arising from inappropriate activation of ETI and enables the analysis of the true

in vivo function of MPK4 without pleiotropic effects in the mpk4 loss-of-function mutant.

In the previous sections, we have described and characterized three novel MPK4 substrates (i)

PAT1 a protein functioning in decapping of mRNAs which was previously undescribed in plants.

(ii) AOC3 functioning in JA biosynthesis and (iii) eIF4E with a dual function in both translational

initiation and export of specific mRNAs out of the nucleus. Both PAT1 and AOC3 seems to be

regulated through immune signaling whereas we were not able to position eIF4E in immunity.

PAT1 is particular interesting given that its loss-of-function defense phenotype is dependent on

SUMM2. PAT1 and SUMM2 interacts in planta and it is possible that SUMM2 guards a specific

branch in MPK4 mediated immunity comprising PAT1. Equally interesting is the link between

MPK4 and the JA biosynthesis component AOC3. The possible regulatory role that MPK4 have on

AOC3 is still not clear, but the in vivo interaction between these two proteins prompted us to test if

MPK4 is present in chloroplast. To this end, we have shown that MPK4, as the first MAPK so far

shown, associates with the chloroplasts. For eIF4E, we have not found a putative role in MPK4

mediated defense, however we localized the interaction to take place in the nucleus. Given the dual

function of eIF4E in both translation and mRNA export, it is still not clear what its connection to

MPK4 is, but it is possible that MPK4 regulate either translation or mRNA export or both in

104

response to pathogens. With the findings presented here in this thesis, MPK4 is evidently a

multifunctional MAPK functioning spatial and temporal in numerous cellular processes.

References

Andreasson, E., Jenkins, T., Brodersen, P., Thorgrimsen, S., Petersen, N. H., Zhu, S., et al. (2005).

The MAP kinase substrate MKS1 is a regulator of plant defense responses. EMBO J 24,

2579–2589. doi:10.1038/sj.emboj.7600737.

Brodersen, P., Petersen, M., Nielsen, H. B., Zhu, S., Newman, M.-A., Shokat, K. M., et al. (2006).

Arabidopsis MAP kinase 4 regulates salicylic acid- and jasmonic acid/ethylene-dependent

responses via EDS1 and PAD4. Plant J. 47, 532–546. doi:10.1111/j.1365-

313X.2006.02806.x.

Chinchilla, D., Zipfel, C., Robatzek, S., Kemmerling, B., Nurnberger, T., Jones, J. D. G., et al.

(2007). A flagellin-induced complex of the receptor FLS2 and BAK1 initiates plant defence.

Nature 448, 497–500. doi:10.1038/nature05999.

Falk, A., Feys, B. J., Frost, L. N., Jones, J. D. G., Daniels, M. J., and Parker, J. E. (1999). EDS1, an

essential component of R gene-mediated disease resistance in Arabidopsis has homology to

eukaryotic lipases. Proc. Natl. Acad. Sci. U. S. A. 96, 3292–3297.

Gao, M., Liu, J., Bi, D., Zhang, Z., Cheng, F., Chen, S., et al. (2008). MEKK1, MKK1/MKK2 and

MPK4 function together in a mitogen-activated protein kinase cascade to regulate innate

immunity in plants. Cell Res 18, 1190–1198.


Arabidopsis MAP Kinase 4 Negatively Regulates Systemic Acquired Resistance. Cell 103,

1111–1120. doi:10.1016/S0092-8674(00)00213-0.

Qiu, J. L., Fiil, B. K., Petersen, K., Nielsen, H. B., Botanga, C. J., Thorgrimsen, S., et al. (2008a).

Arabidopsis MAP kinase 4 regulates gene expression through transcription factor release in

the nucleus. EMBO J. 27, 2214–2221.

Qiu, J.-L., Zhou, L., Yun, B.-W., Nielsen, H. B., Fiil, B. K., Petersen, K., et al. (2008b).

Arabidopsis Mitogen-Activated Protein Kinase Kinases MKK1 and MKK2 Have

Overlapping Functions in Defense Signaling Mediated by MEKK1, MPK4, and MKS1.

Plant Physiol. 148, 212–222. doi:10.1104/pp.108.120006.

Rodriguez, M. C. S., Petersen, M., and Mundy, J. (2010). Mitogen-Activated Protein Kinase

Signaling in Plants. Annu. Rev. Plant Biol. 61, 621–649. doi:10.1146/annurev-arplant-

042809-112252.




doi:10.1016/j.chom.2012.01.015.

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