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Tomato MAPKKKe is a positive regulator of cell-deathsignaling networks associated with plant immunity

Shiri Melech-Bonfil and Guido Sessa*

Department of Molecular Biology and Ecology of Plants, Tel-Aviv University, 69978 Tel-Aviv, Israel

Received 6 July 2010; accepted 26 July 2010; published online 8 October 2010.*For correspondence (fax 972 3 640 9380; e-mail [email protected]).

SUMMARY

Mitogen-activated protein (MAP) kinase cascades are fundamental components of the signaling pathways

associated with plant immunity. Despite the large number of MAP kinase kinase kinases (MAPKKK) encoded in

the plant genome, only very few of them have an assigned function. Here, we identified MAPKKK gene of

tomato (Solanum lycopersicum), SlMAPKKKe, which is required for hypersensitive response cell death and

disease resistance against Gram-negative bacterial pathogens. Silencing of SlMAPKKKe compromised tomato

resistance to Xanthomonas campestris and Pseudomonas syringae strains, resulting in the appearance of

disease symptoms and enhanced bacterial growth. In addition, silencing of NbMAPKKKe in Nicotiana

benthamiana plants significantly inhibited the cell death triggered by expression of different R gene/effector

gene pairs. Conversely, overexpression of either the full-length SlMAPKKKe gene or its kinase domain in

N. benthamiana leaves caused pathogen-independent activation of cell death that required an intact kinase

catalytic domain. Moreover, by suppressing the expression of various MAPKK and MAPK genes and

overexpressing the SlMAPKKKe kinase domain, we identified a signaling cascade acting downstream of

SlMAPKKKe that includes MEK2, WIPK and SIPK. Additional epistasis experiments revealed that SIPKK

functions as a negative regulator of SlMAPKKKe-mediated cell death. Our results provide evidence that

SlMAPKKKe is a signaling molecule that positively regulates cell death networks associated with plant

immunity.

Keywords: tomato, Xanthomonas campestris pv. vesicatoria, Pseudomonas syringae pv. tomato, MAP kinase,

plant immunity, virus-induced gene silencing.

INTRODUCTION

A localized cell death, referred to as the hypersensitive

response (HR), often occurs in resistant plants challenged by

avirulent pathogens (Greenberg and Yao, 2004). The HR is

typically triggered upon recognition of microbial virulence

proteins, designated as pathogen effectors, by resistance (R)

proteins (Martin et al., 2003; Caplan et al., 2008). In addition

to the HR, immune responses activated by R proteins

include changes in ion fluxes, production of reactive oxygen

and nitrogen species, cell wall fortification and accumulation

of pathogenesis-related proteins (Hammond-Kosack and

Jones, 1996). A similar set of immune responses, but gen-

erally excluding the HR, are also activated in plants upon

recognition of conserved molecular structures of pathogenic

microbes by transmembrane pattern-recognition receptors

(PRRs) (Boller and Felix, 2009).

In recent years it has become clear that mitogen-

activated protein (MAP) kinase cascades participate in the

activation of plant immune responses mediated by R and

PRR proteins (Pedley and Martin, 2005; Pitzschke et al.,

2009). The MAP kinase core module includes three protein

kinases sequentially activated through phosphorylation by

their upstream component: MAP kinase kinase kinase

(MAPKKK), MAP kinase kinase (MAPKK) and MAP kinase

(MAPK). These three kinase modules function as molecular

switches that turn on the expression of specific sets of

genes, resulting in the activation of cellular responses.

Components of MAPK cascades are particularly abundant

in higher plants: there are 60 putative MAPKKKs, 10

MAPKKs and at least 20 MAPKs in Arabidopsis (Ichimura

et al., 2002). Functional information is only available for a

ª 2010 The Authors 379Journal compilation ª 2010 Blackwell Publishing Ltd

The Plant Journal (2010) 64, 379–391 doi: 10.1111/j.1365-313X.2010.04333.x

limited number of these proteins (Colcombet and Hirt,

2008).

MAPKKKs represent the most numerous and structurally

heterogeneous component of MAPK modules (Ichimura

et al., 2002). However, only a few MAPKKKs associated with

plant immunity have been identified to date (Pedley and

Martin, 2005; Pitzschke et al., 2009). For example, the

Nicotiana benthamiana MAPKKK NPK1 and its downstream

MAPKK MEK1 and MAPK Ntf6 were shown to be required for

resistance to the tobacco mosaic virus (TMV) mediated by

the R protein N (Jin et al., 2002; Liu et al., 2004). Similarly,

Solanum lycopersicum (tomato) MAPKKKa is required for

the HR mediated by the R protein Pto, and was proposed to

activate two MAP kinase modules (del Pozo et al., 2004). Our

knowledge of MAPKKKs acting downstream of PRRs is

limited to the MAPKKK MEKK1 from Arabidopsis, which is

activated upon the sensing of flagellin by the FLS2 receptor

(Asai et al., 2002). Finally, the Arabidopsis Raf-like MAPKKK

EDR1 was found to negatively regulate plant immunity (Frye

et al., 2001).

As model systems for the investigation of signaling

pathways involved in plant immunity, we study interactions

of tomato plants with the Gram-negative bacteria Pseudo-

monas syringae pv. tomato (Pst) and Xanthomonas cam-

pestris pv. vesicatoria (Xcv), which are the causal agents of

bacterial speck and spot diseases, respectively (Jones et al.,

1998; Pedley and Martin, 2003). Resistance to Pst in tomato is

mediated by the R protein Pto, a Ser/Thr protein kinase,

which interacts directly with the Pst effectors AvrPto and

AvrPtoB (Scofield et al., 1996; Tang et al., 1996; Kim et al.,

2002). The nucleotide binding site/leucine-rich repeats pro-

tein Prf is also required for resistance to Pst, and associates

with Pto in a high molecular weight complex (Salmeron

et al., 1996; Mucyn et al., 2006). Several proteins that

participate in signaling pathways downstream of the Pto/

Prf complex have been identified, including a Ser/Thr

protein kinase, an F-box E3 ligase, transcription factors

and, as described above, components of MAP kinase

cascades (Zhou et al., 1995; 1997; Ekengren et al., 2003; del

Pozo et al., 2004; Mayrose et al., 2006).

Signaling components involved in tomato resistance to

Xcv are largely unknown. Our knowledge is limited to

candidate proteins encoded by genes that are differentially

expressed in resistant plants challenged by avirulent Xcv

strains (Bonshtien et al., 2005; Balaji et al., 2007). Here, by a

virus-induced gene silencing (VIGS) screen of candidate

genes, we identified a tomato MAPKKK, SlMAPKKKe, which

is required for disease resistance against Xcv and Pst

bacteria, and is a positive regulator of cell death. In addition,

we provide evidence that NbMAPKKKe is required for

eliciting HR downstream of various R gene/effector gene

pairs in N. benthamiana plants. Finally, we dissected an

SlMAPKKKe-activated MAP kinase cascade, and identified a

negative regulator of SlMAPKKKe-mediated cell death.

These results demonstrate that SlMAPKKKe functions as a

key regulator of signaling networks activated by R proteins

to promote cell death and plant immunity.

RESULTS

SlMAPKKKe is required for tomato resistance to Xcv

expressing avrXv3

Plants of the tomato line Hawaii 7981 are resistant to Xcv

bacterial strains expressing the avrXv3 effector gene (Scott

et al., 1995). To identify components of signaling pathways

involved in tomato disease resistance to Xcv, we used the

VIGS technique to silence candidate genes in Hawaii 7981

plants. VIGS experiments were performed using a tobacco

rattle virus (TRV)-based vector (Liu et al., 2002), and included

genes that are differentially expressed during the tomato

resistance response to Xcv, or encoding components

of MAP kinase cascades (Balaji et al., 2007) (Table S1).

Silenced plants were inoculated with 5 · 104 colony-forming

units (cfu) ml)1 of an avirulent Xcv T2 strain expressing

avrXv3 [T2(avrXv3)], or were mock-inoculated, and were

then screened for breakdown of resistance. As a control,

plants were infected with an empty TRV vector and inocu-

lated either with the avirulent Xcv T2(avrXv3) strain or with a

virulent Xcv T2 strain. As shown in Figure 1(a), silencing the

tomato SlMAPKKKe gene using TRV that contains a unique

fragment of the gene (nucleotides 3009–3684, hereafter

TRV:3¢MAPKKKe; Figure 3a) resulted in the consistent

appearance of disease symptoms upon inoculation with Xcv

T2(avrXv3). Symptoms in these plants were qualitatively

similar to those observed in TRV-only control plants inocu-

lated with virulent Xcv T2, whereas no symptoms were

detected in TRV-only control plants inoculated with Xcv

T2(avrXv3), or in mock-inoculated SlMAPKKKe-silenced

plants (Figure 1a). In addition, 4 days after inoculation the

Xcv T2(avrXv3), the bacterial population was significantly

higher in plants silenced for SlMAPKKKe than in control

plants, yet lower than growth of Xcv T2 in control plants

(P < 0.05; Figure 1b). These results indicate that SlMAPKKKeis required for tomato disease resistance to Xcv strains

expressing avrXv3. Interestingly, the silencing of

SlMAPKKKe also caused a moderate inhibition of plant

growth, suggesting that this gene may also play a role in

developmental processes (Figure 2a).

To confirm that the above phenotypes directly result from

the silencing of SlMAPKKKe, we repeated the experiments

using another unique fragment of the gene (nucleotides

898–1308, hereafter TRV:5¢MAPKKKe; Figure 3a). In agree-

ment with our previous findings, silenced plants developed

disease symptoms upon inoculation with the aviru-

lent Xcv T2(avrXv3) strain, and supported significantly

higher bacterial populations than control plants (P < 0.05;

Figure 1). Tomato plants silenced with TRV:5¢MAPKKKe did

not produce any visible disease symptoms in the absence of

380 Shiri Melech-Bonfil and Guido Sessa

ª 2010 The AuthorsJournal compilation ª 2010 Blackwell Publishing Ltd, The Plant Journal, (2010), 64, 379–391

a pathogen (Figure 1a), but were slightly inhibited in growth

(Figure 2a). Importantly, quantitative RT-PCR (qRT-PCR)

analysis indicated that endogenous transcript levels of

SlMAPKKKe were reduced by approximately 70% in plants

silenced with either fragment of the gene (Figure S1a). As a

negative control, in the same plants we measured transcript

levels of the SlNPK1 gene (homolog of NPK1; Jin et al.,

2002), which does not show any sequence similarity to the

SlMAPKKKe fragments used for VIGS, but displays the

highest nucleotide identity to SlMAPKKKe among tomato

homologs of MAPKKK genes involved in plant immunity. As

shown in Figure S1(a), SlNPK1 transcript levels were only

slightly altered in SlMAPKKKe-silenced plants. Together with

the observation that SlMAPKKKe is a single-copy gene in the

tomato genome, as predicted by genome sequencing data

available in the Sol Genomics Network (SGN) database

(http://solgenomics.net) and confirmed by Southern blot

analysis (Figure S2), these results indicate that the break-

down of disease resistance to Xcv bacteria expressing

avrXv3 was a result of the specific silencing of SlMAPKKKe.

SlMAPKKKe belongs to the A4 subgroup of plant MEKKs

We isolated a full-length cDNA clone of SlMAPKKKe by

5¢ RACE PCR using a partial sequence of the gene retrieved in

the SGN database. The SlMAPKKKe open reading frame

encodes a polypeptide of 1401 amino acids with an N-ter-

minal kinase domain and two armadillo (Arm) repeat

domains (Coates, 2003) (Figure 3a). Phylogenetic analysis,

which included proteins from different plant species that

show high identity to SlMAPKKKe (Table S2), revealed that

(a)

(b)

Figure 1. Silencing of SlMAPKKKe compromises tomato (Solanum lycoper-

sicum) resistance to Xcv expressing avrXv3.

(a) Bacterial spot symptoms in leaves of Hawaii 7981 plants infected with TRV

only or silenced for SlMAPKKKe with fragments from its 3¢ (TRV:3¢MAPKKKe)or 5¢ (TRV:5¢MAPKKKe) regions. Photographs were taken 5 days after infection

with the virulent strain Xcv T2, with the avirulent strain Xcv T2 (avrXv3) or

after mock inoculation, as indicated.

(b) Bacterial populations in leaves treated as described in (a) were determined

at 1 h (0 days post-inoculation, dpi) and 4 dpi. Bars represent the average and

SE of at least eight independent plants. Letters represent groupings of

statistical significance based on analysis of variance (ANOVA) and compari-

sons for all pairs using the Tukey–Kramer honestly significance difference test

(HSD; P < 0.05). The experiment was repeated four times with similar results.

(a)

(b)

Figure 2. Phenotype of plants silenced for SlMAPKKKe or NbMAPKKKe.(a) Phenotype of 5-week-old Hawaii 7981 tomato (Solanum lycopersicum)

plants silenced for SlMAPKKKe with fragments from its 5¢ (TRV:5¢MAPKKKe) or

3¢ (TRV:3¢MAPKKKe) regions, or infected with empty TRV.

(b) Phenotype of Nicotiana benthamiana plants silenced for NbMAPKKKe with

TRV:3¢MAPKKKe or infected with empty TRV.

SlMAPKKKe regulates plant cell death 381


SlMAPKKKe has closely related homologs in dicot plants,

including a cell division control protein from Ricinus com-

munis (Figure 3b). Homologous proteins with the highest

percentage of identity to SlMAPKKKe were found in the

tomato wild species Solanum pimpinellifolium (99%), in

N. benthamiana (90%) and in Vitis vinifera (grape; 74%). As

expected, the evolutionary relationship of SlMAPKKKe to

proteins from the moss Physcomitrella patens and monocot

plants is more distant than to dicot plants (Figure 3b).

SlMAPKKKe homologs belong to the MEKK class or

‘group A’ of plant MAPKKKs, and they are the only members

of subgroup A4 (Ichimura et al., 2002). In fact, they do not

show any significant similarity to other MAPKKKs in any part

of their sequence besides the kinase domain. Within its

kinase domain SlMAPKKKe shares 46, 45 and 42% amino

acid identity with the immunity-related MAPKKK NPK1 from

tobacco, MEKK1 from Arabidopsis and MAPKKKa from

tomato, respectively.

SlMAPKKKe is required for tomato resistance to different

bacterial pathogens

We next assessed whether SlMAPKKKe plays a role in sig-

naling pathways activated by different R proteins or is spe-

cific to the resistance of tomato against Xcv expressing

avrXv3. To this aim, we used the tomato lines Hawaii 7998

and Rio Grande PtoR (RG-PtoR). Hawaii 7998 plants carry

three non-dominant R genes and are resistant to Xcv strains

expressing avrRxv (Whalen et al., 1993), whereas RG-PtoR

plants carry the Pto R gene and are resistant to Pst strains

expressing avrPto and avrPtoB (Pedley and Martin, 2003).

Plants of the two lines were silenced for SlMAPKKKe with

TRV:3¢MAPKKKe, and, as a positive control, RG-PtoR plants

were also silenced for the Prf gene that is required for Pto-

mediated resistance (Salmeron et al., 1996). SlMAPKKKe-silenced plants were then inoculated with corresponding

avirulent bacterial strains: Hawaii 7998 plants were infil-

trated with 5 · 104 cfu ml)1 of an Xcv T3 strain expressing

avrRxv, whereas RG-PtoR plants were dipped in

5 · 107 cfu ml)1 of the Pst strain DC3000. As an additional

control, Hawaii 7998 plants infected with empty TRV were

inoculated with a virulent Xcv T3 strain (5 · 104 cfu ml)1).

Plants were monitored for the appearance of disease

symptoms and for bacterial growth over 5 days. As shown in

Figure 4(a), the silencing of SlMAPKKKe compromised

resistance of both lines, as manifested by the appearance of

typical disease symptoms that were qualitatively similar

to those developed in the respective positive controls (i.e.

Hawaii 7998 infected with TRV-only and inoculated with

Xcv T3; RG-PtoR silenced for Prf and inoculated with Pst

DC3000). In addition, a significant increase of bacterial

populations was observed in leaves of SlMAPKKKe-silenced

plants compared with TRV-only infected leaves (P < 0.05;

Figure 4b). The degree of SlMAPKKKe silencing was

assessed by qRT-PCR and estimated as 80 and 70% in

Hawaii 7998 and RG-PtoR plants, respectively (Figure S1b).

These results suggest that SlMAPKKKe is required for tomato

disease resistance against different bacterial pathogens.

(a)

(b)

Figure 3. Structure of SlMAPKKKe and its phylogenetic relationship to

homologous proteins from different plant species.

(a) Schematic representation of the SlMAPKKKe sequence. Numbers above

and below the sequence represent amino acid (aa) and nucleotide (nt)

positions, respectively. The kinase domain (kinase), two armadillo repeats

(arm) and positions of DNA fragments used for silencing (5¢MAPKKKe and

3¢MAPKKKe) are indicated.

(b) A maximum-likelihood phylogenetic tree was created based on the

alignment of SlMAPKKKe with homologous proteins from different plant

species. Proteins included in the alignment and their GenBank accession

numbers are listed in Table S2. Kinase domain sequences of the immunity-

related NPK1 from tobacco (Nicotiana tabacum), MEKK1 from Arabidopsis

and MAPKKKa from tomato (Solanum lycopersicum) were used as

out-groups in this analysis. The scale bar above the tree represents branch

length measured by the number of amino acid replacements per position.

Bootstrap values are shown for each branch as a percentage of 100 replicates.



NbMAPKKKe is required for eliciting cell death triggered

by different R gene/effector gene pairs in N. benthamiana

plants

To determine whether MAPKKKe is involved in eliciting cell

death associated with disease resistance in N. benthami-

ana plants, we tested the effect of NbMAPKKKe silencing

on cell death triggered by the co-expression of the tomato

R protein Pto and the Pst effector AvrPto (Tang et al.,

1996). N. benthamiana plants are particularly suited for

such experiments because of the high uniformity and effi-

ciency of VIGS, and the conservation of signaling pathways

acting downstream to Pto-AvrPto recognition in tomato

and N. benthamiana (Liu et al., 2002; del Pozo et al., 2004).

Plants were silenced with TRV:3¢MAPKKKe or TRV:Prf, and

4 weeks later their leaves were infiltrated with Agrobacte-

rium strains for the expression of Pto or AvrPto alone, and

for co-expression of Pto and AvrPto. At this time, qRT-PCR

analysis revealed that in plants silenced with TRV:3¢MAP-

KKKe, transcript abundance of NbMAPKKKe was reduced

by 65%, and there was no effect on expression of the

NbNPK1 gene used as a negative control (Figure S1c). As

shown in Figure 5(a) and Table 1, the silencing of

NbMAPKKKe significantly reduced the percentage of leaves

developing cell death upon co-expression of Pto and Avr-

Pto, compared with TRV-only infected leaves (30 versus

76%). As expected, no cell death was observed when Pto or

AvrPto were expressed alone or in plants silenced for the

Prf gene. To confirm that silencing of NbMAPKKKe caused

a reduction of cell death, we measured electrolyte leakage

in leaves silenced for NbMAPKKKe and Prf, or infected with

empty TRV. Development of cell death caused by the

co-expression of Pto or AvrPto was visible 2 days after

Agrobacterium infiltration in control plants, and was

associated with increased ion leakage (Figure 5b). In con-

trast, leaves silenced for NbMAPKKKe or Prf, which showed

no or weak cell death as a result of Pto and Pto or AvrPto

co-expression, had reduced ion leakage compared with

control plants. Similarly, no ion leakage was observed in

control plants expressing Pto alone (Figure 5b). These

results are consistent with the involvement of NbMAPKKKein signaling pathways mediating cell death triggered by

Pto-AvrPto recognition. It is noteworthy that N. benthami-

ana plants silenced for NbMAPKKKe did not show any

spontaneous cell death phenotype, but instead displayed

a growth inhibition phenotype, as observed in tomato

(Figure 2b).

We next examined whether NbMAPKKKe is a compo-

nent of cell death signaling pathways activated by addi-

tional R protein/effector protein pairs, such as those

involved in the interaction between tomato plants and

the fungal pathogen Cladosporium fulvum (Rivas and

Thomas, 2005). To this aim, N. benthamiana leaves

silenced for NbMAPKKKe were infiltrated with Agrobacte-

rium strains co-expressing Cf4 and Avr4, or Cf9 and Avr9.

As shown in Table 1, the silencing of NbMAPKKKe reduced

the percentage of leaves developing cell death upon

co-expression of Cf4/Avr4 or Cf9/Avr9 compared with

control leaves (54 versus 86% and 46 versus 92%, respec-

tively). Together, our results suggest that NbMAPKKKeparticipates in signaling pathways mediating the elicitation

of cell death triggered by different R/effector protein

recognition events.

(a)

(b)

Figure 4. Silencing of SlMAPKKKe compromises tomato (Solanum lycoper-

sicum) resistance to Xcv expressing avrRxv and to Pst DC3000.

(a) Bacterial spot and speck symptoms in SlMAPKKKe-silenced Hawaii 7998

and Rio Grande PtoR plants, respectively. Hawaii 7998 plants were infected

with TRV only or TRV:3¢MAPKKKe, whereas Rio Grande PtoR plants were

infected with TRV-only, TRV:3¢MAPKKKe or TRV:Prf. Hawaii 7998 plants were

then inoculated with Xcv T3 or Xcv T3 (avrRxv) strains, whereas Rio Grande

PtoR plants were inoculated with Pst DC3000. Photographs were taken 5 days

after inoculation.

(b) Bacterial populations in leaves of plants treated as described above were

determined at 1 h (0 days post-inoculation, dpi) and 4 dpi. Bars represent the

average and SE of at least eight independent plants. Letters in bars represent

groupings of statistical significance based on ANOVA and comparisons for all

pairs using the Tukey–Kramer honestly significant difference test (P < 0.05).

Experiments were repeated four times with similar results.



SlMAPKKKe is not involved in cell death associated with

tomato disease susceptibility

Once determined that in N. benthamiana plants

NbMAPKKKe plays a role in cell death associated with plant

immunity, we tested whether this gene is also involved in

cell death associated with tomato disease susceptibility, as

reported previously for MAPKKKa (del Pozo et al., 2004). To

this aim, we monitored bacterial growth and development

of spot and speck disease symptoms in tomato plants

silenced for SlMAPKKKe, and inoculated with virulent Xcv

and Pst strains, respectively. For silencing, Hawaii 7981,

Hawaii 7998 and RG-PtoS lines were infected with

TRV:3¢MAPKKKe or TRV only. Four weeks later, Hawaii 7981

and Hawaii 7998 plants were inoculated with virulent Xcv T2

and Xcv T3 strains, respectively. At this time, RG-PtoS plants

were inoculated with the Pst strain DC3000. As shown in

Figure 6, SlMAPKKKe-silenced plants displayed disease

symptoms and supported bacterial populations similar to

control plants. These results indicate that SlMAPKKKe is not

involved in cell death observed during the progression of

disease symptoms caused by the compatible interactions

between tomato plants and Xcv or Pst bacteria.

Overexpression of SlMAPKKKe causes pathogen-indepen-

dent cell death

To test whether overexpression of SlMAPKKKe causes cell

death, we used Agrobacterium to transiently express the

full-length SlMAPKKKe protein or only its kinase domain in

active (KDe) or inactive (KDe)) forms under the control of an

estradiol-inducible system in N. benthamiana leaves. The

inactive form of the SlMAPKKKe kinase domain was

obtained by substituting the essential lysine in the ATP-

binding site (Lys49) with an arginine. As positive controls,

we expressed tomato MAPKKKa or its kinase domain alone

(KDa), which were previously shown to cause cell death in

N. benthamiana leaves (del Pozo et al., 2004). As negative

controls, we expressed a kinase-deficient form of KDa(KDa)) or infiltrated leaves with Agrobacterium carrying an

empty vector. As shown in Figure 7(a), overexpression of

SlMAPKKKe or KDe resulted in cell death in N. benthamiana

leaves within 72 and 48 h after estradiol application,

respectively. This was slightly slower than the cell death

caused by MAPKKKa and KDa that, as previously reported

(del Pozo et al., 2004), appeared 48 and 36 h after estradiol

application, respectively. However, cell death was not

observed following the expression of the inactive

SlMAPKKKe kinase domain (KDe)), indicating that the kinase

activity of SlMAPKKKe is required for the activation of cell

death. Similar results were also obtained in tomato plants

Table 1 Silencing of NbMAPKKKe reduces the hypersensitiveresponse (HR) cell death activated by co-expression of differentR protein/effector protein pairs in Nicotiana benthamiana plants

Silencingconstruct

Totalleavesinfiltrated

LeavesdevelopingHR

% of leavesdevelopingHR

Pto/AvrPtoTRV empty 48 36 76TRV:3¢MAPKKKe 45 12 30TRV:Prf 42 0 0

Cf4/Avr4TRV empty 35 30 86TRV:3¢MAPKKKe 35 19 54

Cf9/Avr9TRV empty 37 34 92TRV:3¢MAPKKKe 41 19 46

(a)

(b)

Figure 5. Silencing of NbMAPKKKe impairs Pto/AvrPto-mediated cell death in

Nicotiana benthamiana plants.

(a) Elicitation of Pto/AvrPto-mediated cell death in NbMAPKKKe-silenced

N. benthamiana leaves. Plants were infected with TRV-only, TRV:3¢MAPKKKeor TRV:Prf. Leaves of silenced plants were then inoculated with Agrobacte-

rium for the expression of Pto, AvrPto or Pto, and AvrPto. Photographs were

taken 3 days after agroinfiltration.

(b) Quantification of cell death in N. benthamiana leaves treated as described

above by measuring electrolyte leakage before (day 0) and 2 or 3 days after

agroinfiltration. Bars represent the average conductivity and SE of leaf

samples from at least five independent plants.



for KDe and KDe) (Figure S3). Expression of SlMAPKKKe,

KDe and KDe) was confirmed by immunoblot analysis

(Figure 7b,c). In this analysis, the full-length SlMAPKKKe did

not run at the predicted molecular weight size (160 kDa),

possibly because of post-translational modifications occur-

ring in the protein. These results support the conclusion that

SlMAPKKKe is a positive regulator of cell death that activates

signal transduction pathway(s) by phosphorylation.

MEK2, WIPK and SIPK act downstream of SlMAPKKKe,

whereas SIPKK is a negative regulator of SlMAPKKKe-

mediated cell death

To identify components of MAP kinase cascade(s) activated

by SlMAPKKKe, we used epistasis experiments that combine

a gain-of-function cell death assay (overexpression of the

SlMAPKKKe kinase domain) with loss of function based on

VIGS of MAPKKs and MAPKs that are known components of

immunity-related cascades (Ekengren et al., 2003; Pedley

and Martin, 2005). Our assumption was that SlMAPKKKe-

mediated cell death would be suppressed by silencing key

MAPKKs or MAPKs acting downstream of SlMAPKKKe. In

these experiments, N. benthamiana plants were silenced for

MEK1, MEK2 or SIPKK (MAPKKs) and for WIPK, SIPK or

(a)

(b)

(c)

Figure 6. Silencing of SlMAPKKKe does not affect cell death associated with

disease susceptibility in tomato (Solanum lycopersicum) plants.

Hawaii 7981 (a), Hawaii 7998 (b) and Rio Grande PtoS (c) plants were silenced

with TRV:3¢MAPKKKe or TRV only. Rio Grande PtoS plants were then

inoculated with Pst strain DC3000, whereas Hawaii 7981 and Hawaii 7998

plants were inoculated with the Xcv T2 and Xcv T3 strains, respectively.

Photographs (left) were taken 5 days after infection. Bacterial populations

(right) in infected leaves were determined at 1 h (0 days post-inoculation, dpi)

and 4 dpi. Bars represent the averages and SEs of four independent plants.

Letters represent groupings of statistical significance based on ANOVA and

comparisons for all pairs using the Tukey–Kramer honestly significant

difference test (P < 0.05). Experiments were repeated three times with similar

results.

(a)

(b)

(c)

Figure 7. Overexpression of SlMAPKKKe in Nicotiana benthamiana elicits cell

death.

(a) Cell death in N. benthamiana leaves infiltrated with Agrobacterium

(OD600 = 0.06) strains carrying an empty vector (EV) or expressing

SlMAPKKKe, SlMAPKKKe kinase domain in active (KDe) and inactive form

(KDe)), MAPKKKa, and MAPKKKa kinase domain in active (KDa) and inactive

form (KDa)). Proteins were tagged with a double hemagglutinin (HA) epitope,

and their expression was estradiol-inducible. Photographs were taken 48 h

after estradiol application.

(b,c) Expression of SlMAPKKKe, KDe and KDe) in N. benthamiana leaves

monitored by immunoblot analysis (left) with anti-HA antibody in leaf extracts

sampled 6 h after estradiol application. Ponceau staining of membranes

(right) confirmed similar protein loading.



NTF6 (MAPKs). As a negative control, we included plants

infected with the TRV empty vector. The efficiency and

specificity of silencing was assessed by qRT-PCR analysis

that measured the expression of each target gene and its

closest homolog in silenced plants relative to control plants.

This analysis indicated a decrease of at least 60% in tran-

script abundance of each gene targeted by VIGS, whereas

the abundance of the homologous genes remained at wild-

type levels in all instances (Figure S4). In addition, no

spontaneous cell death was observed in any of the silenced

plants. The kinase domain of SlMAPKKKe (KDe) and its

kinase deficient form (KDe)) were then transiently expressed

in the silenced leaves via Agrobacterium infiltration. As

shown in Figure 8(a,b), cell death was typically observed in

control plants expressing KDe 72 h after estradiol applica-

tion, whereas cell death was not observed in leaf areas

expressing KDe), or infiltrated with Agrobacterium carrying

an empty vector (not shown). However, silencing of MEK2,

WIPK and SIPK significantly inhibited cell death caused by

the expression of KDe. Conversely, in leaves silenced for

SIPKK, cell death mediated by KDe developed earlier, and

was significantly enhanced compared with control leaves

(Figure 8b,c). Finally, the silencing of MEK1 and NTF6 did

not affect KDe) mediated cell death. These results suggest

that MEK2, WIPK and SIPK act downstream of SlMAPKKKein signaling pathways activating immunity-associated cell

death. On the other hand, SIPKK appears to function as a

negative regulator of SlMAPKKKe-mediated cell death. A

role for MEK1 and NTF6 downstream of SlMAPKKKe is

unlikely, but it cannot be excluded because of the residual

transcript levels of these genes in the silenced plants

(Figure S4).

DISCUSSION

In this study, we established an important role for

SlMAPKKKe in signaling pathways mediating immune

responses to Xcv and Pst phytopathogenic bacteria. In

addition, by loss- and gain-of-function experiments, we

provided evidence that SlMAPKKKe is a key regulator of cell

death associated with plant immunity. Epistasis analysis

positioned SlMAPKKKe in a cell death signaling pathway,

upstream of MEK2 and WIPK/SIPK, and defined SIPKK as a

negative regulator of SlMAPKKKe-mediated cell death.

In tomato plants, SlMAPKKKe is required for disease

resistance pathways that are activated by different types of

genetic interactions (Figure 9). These include both quantita-

tive resistance to Xcv strains expressing avrRxv, which is

recognized by three plant R genes (Whalen et al., 1993; Yu

et al., 1995), and classical gene-for-gene interactions medi-

ating resistance to Xcv strains expressing avrXv3 (Astua-

Monge et al., 2000), as well as resistance to Pst strains

expressing avrPto and avrPtoB (Pedley and Martin, 2003).

(a) (b)

(c)

Figure 8. Identification of an MAPKK and MAPKs acting downstream of SlMAPKKKe.

(a) Nicotiana benthamiana plants were infected with TRV only or were silenced for the indicated MAPKK and MAPK genes. Leaves were then infiltrated with

Agrobacterium (OD600 = 0.02) for overexpression of the SlMAPKKKe kinase domain in active (KDe) or inactive (KDe)) form from an estradiol-inducible system. Black

circles mark infiltrated areas. Photographs were taken 72 h after induction with estradiol.

(b) Quantification of cell death caused by treatments described in (a) 72 h after estradiol application by using the following scoring system: 2, full cell death; 1, partial

cell death; and 0, no cell death. Cell death in plants silenced for the indicated genes is reported as a percentage of that observed in TRV-only infected plants. Data

represent the averages of at least 50 leaves from at least three independent experiments � SEs. Letters represent groupings of statistical significance based on

ANOVA and comparisons for all pairs using the Tukey–Kramer honestly significant difference test (P < 0.05).

(c) Quantification of KDe-mediated cell death in SIPKK-silenced plants 48 and 72 h after estradiol application. Cell death is reported as the percentage of that

observed in TRV-only infected plants 72 h after estradiol application. Data represent the average of 72 leaves from four independent experiments � SEs.



Components of resistance signaling are generally catego-

rized into two groups based on whether they act down-

stream of a single or multiple structural classes of R proteins

(Martin et al., 2003). SlMAPKKKe can be ascribed to the latter

group because silencing of the corresponding gene

impaired cell death mediated either by the Ser/Thr kinase

Pto (Martin et al., 1993), which acts in concert with the

cytoplasmic NBS-LRR R-like protein Prf (Salmeron et al.,

1996), or by the transmembrane LRR-containing R protein

Cf9 (Jones et al., 1994). Although silencing of SlMAPKKKecompromised disease resistance, in SlMAPKKKe-silenced

plants the appearance of disease symptoms and bacterial

growth remained at lower levels than in susceptible control

plants. A similar partial breakdown of resistance was

observed when the expression of several genes involved in

Pto-mediated resistance to Pst was silenced (Ekengren et al.,

2003; Mayrose et al., 2006). This phenomenon can be

ascribed either to residual expression of SlMAPKKKe in the

silenced plants, or more likely it may indicate that several

pathways contribute additively to the overall disease resis-

tance phenotype.

Besides its effect on disease resistance, the silencing of

SlMAPKKKe and NbMAPKKKe resulted in a certain degree of

growth inhibition in tomato and N. benthamiana plants,

respectively. Developmental phenotypes were previously

observed upon silencing or introducing mutations in several

genes that encode positive and negative regulators of plant

immunity (e.g. MEKK1, NPK1, MPK3, MPK4, MPK6, SGT1b,

RIN4 and TIP49a; Petersen et al., 2000; Jin et al., 2002;

Mackey et al., 2002; Whalen, 2005; Suarez-Rodriguez et al.,

2007; Wang et al., 2007). The growth inhibition phenotypes

caused by the silencing of SlMAPKKKe and NbMAPKKKemay be a side effect of imbalance in the plant immune

system, or may indicate that these genes also play roles in

the cellular processes associated with plant development. In

support of the latter possibility, a double mutant combina-

tion of the AtMAP3Ke1 and AtMAP3Ke2 genes caused pollen

lethality in Arabidopsis (Chaiwongsar et al., 2006). In addi-

tion, the BnMAP3Ke1 gene from Brassica napus was shown

to partially complement a loss-of-function mutation in the

yeast cell cycle-related gene cdc7 (Jouannic et al., 2001). It is

not unprecedented that signaling components that are

involved in plant immunity also play roles in other cellular

processes (Martin et al., 2003; Whalen, 2005). For example,

the tobacco MAPKKK NPK1 was shown to be required for

disease resistance and cytokinesis (Nishihama et al., 2001;

Jin et al., 2002), and to negatively regulate auxin signaling

(Kovtun et al., 1998). Similarly, the TIP49a protein was

shown to be a negative regulator of disease resistance and

to be required for meristem establishment, as well as for

sporophyte and female gametophyte viability (Holt et al.,

2002). It is tempting to hypothesize that SlMAPKKKe con-

nects stress response metabolism with normal growth

physiology.

Overexpression of SlMAPKKKe results in the activation of

pathogen-independent cell death, whereas silencing of

NbMAPKKKe inhibits cell death activated by the co-expres-

sion of Pto and AvrPto in N. benthamiana plants. This

evidence defines SlMAPKKKe as a positive regulator of

R protein-mediated cell death. Epistasis analysis in

N. benthamiana leaves indicates that MEK2, SIPK and WIPK

are required for SlMAPKKKe-mediated cell death. Based on

these data, we propose that after sensing either Xcv or Pst

bacteria, SlMAPKKKe receives the input signal for the

induction of cell death and activates MEK2, SIPK and WIPK

as components of a pro-cell death MAP kinase module

(Figure 9). A MEK2, SIPK/WIPK MAP cascade activated by

an unknown MAPKKK was previously implicated in disease

resistance to TMV (Jin et al., 2003). This MAPK module was

associated with cell death, as constitutively active forms of

MEK2 or SIPK were shown to induce pathogen-independent

cell death (Yang et al., 2001; Zhang and Liu, 2001). In

addition, by overexpression of a constitutively active MEK2

and biochemical analysis, MEK2 was confirmed as the

upstream MAPKK of SIPK and WIPK (Yang et al., 2001).

Our results strongly support that SlMAPKKKe can function

as an upstream MAPKKK to MEK2 in this cascade.

Figure 9. Model for mitogen-activated protein (MAP) kinase cascades acti-

vating hypersensitive response (HR) cell death upon recognition of Pst or Xcv

effectors by corresponding tomato (Solanum lycopersicum) R proteins. The

model is based on an analysis of cell death triggered by overexpression of

either SlMAPKKKe (this work) or MAPKKKa (del Pozo et al., 2004) in Nicotiana

benthamiana plants silenced for various MAPKK and MAPK genes.

SlMAPKKKe activates a MAP kinase module, which is negatively regulated

by SIPKK through its interaction with SIPK or activation of MPK4. MAPKKKa,

which is regulated by a 14-3-3 protein (Oh et al., 2010), activates two distinct

and possibly sequential cascades. Dotted lines indicate possible connections

between proteins or cascades.



MAPKKKa was previously shown to participate in signal-

ing pathways activated by Pto (del Pozo et al., 2004).

Because silencing of either MAPKKKa or NbMAPKKKe com-

promises Pto-mediated elicitation of cell death in N. benth-

amiana plants, these two MAPKKKs appear to be both

required for disease resistance, rather than act redundantly.

In support of this conclusion, MAPKKKa or SlMAPKKKesilencing is sufficient to compromise resistance to Pst

strains in N. benthamiana and tomato plants, respectively.

However, it should be noted that, in contrast to MAPKKKa,

SlMAPKKKe is not involved in cell death associated with

disease susceptibility. Based on VIGS and epistasis analysis

in N. benthamiana, del Pozo et al. (2004) proposed that two

distinct and interconnected MAP kinase cascades act down-

stream of MAPKKKa (Figure 9). The first cascade consists of

MAPKKKa, MEK2 and SIPK, and was proposed to sequen-

tially activate a second MAP kinase module that includes

MEK1 and Ntf6. Notably, SlMAPKKKe and MAPKKKa share

MEK2 and SIPK as downstream components. Conversely,

WIPK is required for cell death elicited only by SlMAPKKKe,

and not by MAPKKKa, whereas MEK1 and Ntf6 are specific

downstream components of MAPKKKa. Based on these

observations, we propose that upon their activation, MAP-

KKKa and SlMAPKKKe transfer input signals to MEK2 that in

turn activates SIPK (Figure 9). Consistent with the inter-

relationship between SIPK and WIPK proposed by Liu et al.

(2003), activation of SIPK by SlMAPKKKe-MEK2 might

induce transcription of the WIPK gene. Once newly synthe-

sized WIPK accumulates, it is then activated by MEK2 and

contributes along with SIPK to cell death by phosphorylation

of downstream proteins. On the other hand, SIPK activated

by MAPKKKa-MEK2 could lead to the activation of an

additional MAPKKKa-dependent MAP kinase cascade,

including an unknown MAPKKK, MEK1 and Ntf6 that con-

tribute to cell death. A scenario including de novo protein

synthesis in pathways activated by SlMAPKKKe, but not by

MAPKKKa, is consistent with the later development of cell

death observed upon overexpression of SlMAPKKKe, com-

pared with overexpression of MAPKKKa. However, although

not required for MAPKKKa-mediated cell death, WIPK could

still play a role downstream of MAPKKKa as the WIPK-

homolog MPK3 was found to be activated by overexpression

of MAPKKKa in tomato plants (Pedley and Martin, 2004).

Finally, it remains to be established what the nature of the

signals transferred by MAPKKKa and SlMAPKKKe to MEK2

is, and how SIPK output specificity is controlled.

An additional important result of our epistasis analysis is

that silencing of the SIPKK gene accelerates cell death

activated by overexpression of SlMAPKKKe. This obser-

vation strongly suggests that SIPKK acts as a negative

regulator of cell death activated by the SlMAPKKKe/MEK2/

SIPK-WIPK module. SIPKK could exert its negative effect

acting directly on SIPK, as these two proteins were shown to

physically interact in a yeast two-hybrid system (Liu et al.,

2000). In support of this possibility, SIPK activity was

suppressed in tobacco plants by overexpression of SIPKK,

either in the wild-type or in a constitutively active form

(Gomi et al., 2005). Alternatively, SIPKK may negatively

regulate cell death by acting in a MAPK signaling pathway

parallel with the SlMAPKKKe-activated module. Consistent

with such a mechanism, in tobacco plants SIPKK was shown

to activate MPK4, the Arabidopsis homolog of which is a

negative regulator of salicylic acid-dependent plant defense

responses (Petersen et al., 2000).

Additional research is required to fill the gap between the

recognition of AvrPto by the Pto/Prf complex and activation

of SlMAPKKKe- and MAPKKKa-initiated MAP kinase mod-

ules. A first important step in this direction was the

identification and functional characterization of TFT7, a

14-3-3 protein that interacts with MAPKKKa and regulates

its signaling ability (Oh et al., 2010). In the future it will be

important to determine whether SlMAPKKKe is also regu-

lated by TFT7, what other molecules control its activity, and

how the output signals of SlMAPKKKe- and MAPKKKa-

dependent cascades eventually lead to the activation of cell

death.

EXPERIMENTAL PROCEDURES

Bacterial strains and plant material

The bacterial strains used are: Xcv T3, Xcv T3 expressing avrRxv(Bonshtien et al., 2005), Xcv T2 strain 5746 (Xcv T2), and Xcv T2carrying the avrXv3 gene (Gibly et al., 2004); Pst strain DC3000(Buell et al., 2003); Agrobacterium tumefaciens strains GV2260 andEHA105.

The tomato cultivars used are: Hawaii 7981 (Scott et al., 1995),Hawaii 7998 (Whalen et al., 1993), RG-PtoR (Pto/Pto and Prf/Prf) andRG-PtoS (pto/pto and Prf/Prf) (Pedley and Martin, 2003). Plants weregrown using standard glasshouse practices (25 � 2�C and 16-hlight/8-h dark).

Plant inoculation

For inoculation with Xcv strains, 5-week-old plants were vacuuminfiltrated with bacterial cultures prepared as follows: Xcv strainswere grown overnight at 28�C in nutrient yeast glycerol (NYG) liquidmedium (Daniels et al., 1984), with the addition of 50 lg ml)1

kanamycin for Xcv T2 (avrXv3) and Xcv T3 (avrRxv). Bacteria werepelleted, washed twice with 10 mM MgCl2, and diluted to a con-centration of 5 · 104 cfu ml)1 in 10 mM MgCl2 and 0.005% (v/v)Silwett-L77.

For inoculation with Pst DC3000, 5-week-old plants were dippedfor 1 min in a bacterial suspension prepared as follows: cultureswere grown overnight at 28�C in King’s B (KBM) plates (Martinet al., 1993) in the presence of 100 lg ml)1 rifampicin. On the day ofinfection, bacteria were scooped from the plates, suspended in10 mM MgCl2 and diluted to a concentration of 5 · 107 cfu ml)1 in10 mM MgCl2 and 0.025% (v/v) Silwett-L77. Bacterial populationsin infected plants were determined as described in the supplemen-tary experimental procedures (Appendix S1).

Virus-induced gene silencing plasmids and procedures

Plasmids pTRV1, pTRV2, pTRV2:SlPrf, pTRV2:NbWIPK, pTRV2:NbSIPK, pTRV2:NtMEK1, pTRV2:NtMEK2 and pTRV2:NbSIPKK have



been previously described (Liu et al., 2002; Ekengren et al., 2003).To generate pTRV2 derivatives for VIGS of candidate genes intomato, cDNA fragments of the genes to be silenced werePCR-amplified from tomato cDNA by using the primers listed inTable S1. For VIGS of NbNTF6 (GenBank accession no. AY547494)in N. benthamiana, a corresponding cDNA fragment wasPCR-amplified from N. benthamiana cDNA using the followingprimers: 5¢-GCTCTAGAAGTTCCAAGACAACCCTTTTC-3¢ and5¢-GCGGGATCCTAGAGCCTCGTTCCATATGAG-3¢. PCR-amplifiedproducts were inserted into the multiple cloning site of pTRV2 asXbaI–BamHI fragments, and the identity of each construct wasconfirmed by sequencing. Plasmids were then transformed intoAgrobacterium strain GV2260, and plants were inoculated asdescribed in Appendix S1.

RNA isolation and quantitative RT-PCR analysis

Total RNA was isolated from leaf samples (50 mg) using the SVTotal RNA Isolation kit (Promega, http://www.promega.com). RNAsamples (2 lg) were reverse-transcribed and used for quantitativePCR analysis as described in Appendix S1 and Table S3.

Cloning and mutagenesis of the SlMAPKKKe gene

Based on partial sequences of an SlMAPKKKe expressed sequencetag (EST) that was retrieved from the SGN tomato EST database(SGN-U153362), an SlMAPKKKe full-length cDNA clone was ampli-fied by 5¢-RACE with the BD SMART� RACE cDNA Amplificationkit (BD Biosciences, Clontech, http://www.bdbiosciences.com),inserted at the SalI restriction site of the cloning vector pBlueScriptKS+, and sequenced (GenBank accession no. GU192457).

Site-directed mutagenesis of SlMAPKKKe was performed inpBlueScript KS+ containing the SlMAPKKKe coding region fusedto a double hemagglutinin (HA) epitope. Mutation of Lys49 to Arg inthe SlMAPKKKe kinase catalytic domain (KDe; from amino acid19–273) was introduced using the Quickchange kit (Stratagene,http://www.stratagene.com) and the following oligonucleotides:5¢-GGAGACTTTGTTGCAATTAGACAAGTTTCTCTGGAG-3¢ and 5¢-CTC-CAGAGAAACTTGTCTAATTGCAACAAAGTCTCC-3¢.

Alignment and phylogenetic tree analysis

To identify SlMAPKKKe homologs from different plant species, aBlastX search was performed in the National Center for Biotech-nology Information (NCBI) database by using the SlMAPKKKeamino acid sequence as a query. Proteins retrieved with thehighest identity to SlMAPKKKe (Table S2) were selected foranalysis. Homologs to SlMAPKKKe were also identified in themaize EST database (http://compbio.dfci.harvard.edu/tgi/plant.html), and in the S. pimpinellifolium genome draft (http://solge-nomics.net). A multiple sequence alignment of the retrievedproteins was generated by using MUSCLE v3.0 (Edgar, 2004). Amaximum-likelihood phylogenetic tree was created with PHYML(Guindon and Gascuel, 2003) using the LG replacement modelwith four discrete rate categories to model among-site rate vari-ation and the best of SPR and NNI as the tree search algorithm(Le and Gascuel, 2008). The tree was rooted using kinasedomains of tomato MAPKKKa, tobacco NPK1 and ArabidopsisMEKK1 as out-groups. Branch supports were computed using 100bootstrap replicates.

Agrobacterium-mediated transient expression

Constructs for Agrobacterium-mediated expression of the genepairs Pto/avrPto, Cf4/avr4 and Cf9/avr9 driven by the CaMV 35Spromoter were as previously described (Sessa et al., 2000; Van derHoorn et al., 2000), and transformed into Agrobacterium EHA105

by electroporation. For estradiol-inducible expression, full-lengthSlMAPKKKe or its kinase catalytic domain in the wild-type (KDe) ormutant (KDe)) form, were cloned with a C-terminal double HAepitope tag into the pER8 binary vector as XhoI-SpeI fragments, andtransformed into Agrobacterium GV2260. Constructs for theexpression of full-length MAPKKKa or its kinase catalytic domainin the wild-type (KDa) or mutant (KDa)) form were as previouslydescribed (del Pozo et al., 2004). Agrobacterium strains were grownovernight at 28�C in LB medium and used for transient expression,as described in Appendix S1.

Ion leakage measurements

Three discs (1 cm in diameter) were sampled from infiltrated areasfor each plant and floated in 50-ml tubes containing 15 ml double-distilled water for 4 h at room temperature (24�C) with gentleshaking. Conductivity was measured with an autoranging EC/TempMeter TH-2400 (El-Hamma Instruments, http://www.elhamma.com).

Protein extraction and immunoblotting

Proteins were extracted by grinding three leaf discs (1 cm indiameter) in extraction buffer (137 mM NaCl, 2.7 mM KCl, 10 mM

Na2HPO4, 1.8 mM KH2PO4, 1 mM PMSF, 5 lg ml)1 leupeptin and5 lg ml)1 aprotinin). Extracts were cleared by centrifugation andthe supernatant was collected. Protein samples (40 lg) were ana-lyzed by immunoblotting and chemiluminescence visualization(Amersham Biosciences, http://www.gelifesciences.com) usingmonoclonal HA antibodies (1:1000) (Roche Diagnostics, http://www.roche.com/diagnostics) and horseradish peroxidase-conju-gated anti-mouse secondary antibodies (1:5000) (Sigma-Aldrich,http://www.sigmaaldrich.com).

ACKNOWLEDGEMENTS

We thank S.P. Dinesh-Kumar for providing TRV VIGS vectors,Gregory B. Martin for providing TRV2 constructs, Chank-Sik Oh forfruitful suggestions and discussions, Itay Mayrose for assistancewith phylogenetic analysis, Naomi Ori, Maya Mayrose and mem-bers of the Sessa lab for critical comments on the manuscript. Thiswork was supported by the United States–Israel Binational Agri-cultural Research and Development Fund (BARD; grant no. IS-4159-08C). SM is the recipient of an Eshkol fellowship from the IsraeliMinistry of Science & Technology.

SUPPORTING INFORMATION

Additional Supporting Information may be found in the onlineversion of this article:Figure S1. Silencing efficiency and specificity in plants silenced forSlMAPKKKe and NbMAPKKKe.Figure S2. Southern blot analysis of SlMAPKKKe in the tomato(Solanum lycopersicum) genome.Figure S3. Overexpression of the SlMAPKKKe kinase domain intomato (Solanum lycopersicum) plants.Figure S4. Silencing efficiency and specificity in Nicotiana benth-amiana plants silenced for selected MAPKK and MAPK genes.Table S1. Genes targeted by virus-induced gene silencing (VIGS) inHawaii 7981 tomato (Solanum lycopersicum) plants and primersused for their PCR amplification.Table S2. Proteins included in the phylogenetic analysis.Table S3. Primers used for quantitative RT-PCR analysis.Appendix S1. Supplementary experimental procedures.Please note: As a service to our authors and readers, this journalprovides supporting information supplied by the authors. Suchmaterials are peer-reviewed and may be re-organized for onlinedelivery, but are not copy-edited or typeset. Technical support



issues arising from supporting information (other than missingfiles) should be addressed to the authors.

REFERENCES

Asai, T., Tena, G., Plotnikova, J., Willmann, 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. Nature, 415, 977–983.

Astua-Monge, G., Minsavage, G.V., Stall, R.E., Davis, M.J., Bonas, U. and

Jones, J.B. (2000) Resistance of tomato and pepper to T3 strains of Xan-

thomonas campestris pv. vesicatoria is specified by a plant-inducible avi-

rulence gene. Mol. Plant Microbe Interact. 13, 911–921.

Balaji, V., Gibly, A., Debbie, P. and Sessa, G. (2007) Transcriptional analysis

of the tomato resistance response triggered by recognition of the

Xanthomonas type III effector AvrXv3. Funct. Integr. Genomics, 7, 305–

316.

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.

Bonshtien, A., Lev, A., Gibly, A., Debbie, P., Avni, A. and Sessa, G. (2005)

Molecular properties of the Xanthomonas AvrRxv effector and global

transcriptional changes determined by its expression in resistant tomato

plants. Mol. Plant Microbe Interact. 18, 300–310.

Buell, C.R., Joardar, V., Lindeberg, M. et al. (2003) The complete genome

sequence of the Arabidopsis and tomato pathogen Pseudomonas syringae

pv. tomato DC3000. Proc. Natl. Acad. Sci. USA, 100, 10181–10186.

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.

Chaiwongsar, S., Otegui, M.S., Jester, P.J., Monson, S.S. and Krysan, P.J.

(2006) The protein kinase genes MAP3Ke1 and MAP3Ke2 are required for

pollen viability in Arabidopsis thaliana. Plant J. 48, 193–205.

Coates, J.C. (2003) Armadillo repeat proteins: beyond the animal kingdom.

Trends Cell Biol. 13, 463–471.

Colcombet, J. and Hirt, H. (2008) Arabidopsis MAPKs: a complex signalling

network involved in multiple biological processes. Biochem. J. 413, 217–

226.

Daniels, M.J., Barber, C.E., Turner, P.C., Cleary, W.G. and Sawczyc, M.K.

(1984) Isolation of mutants of Xanthomonas campestris pv. campestris

showing altered pathogenicity. J. Gen. Microbiol. 130, 2447–2455.

Edgar, R.C. (2004) MUSCLE: multiple sequence alignment with high accuracy

and high throughput. Nucleic Acids Res. 32, 1792–1797.

Ekengren, S.K., Liu, Y., Schiff, M., Dinesh-Kumar, S.P. and Martin, G.B. (2003)

Two MAPK cascades, NPR1, and TGA transcription factors play a role in

Pto-mediated disease resistance in tomato. Plant J. 36, 905–917.

Frye, C.A., Tang, D. and Innes, R.W. (2001) Negative regulation of defense

responses in plants by a conserved MAPKK kinase. Proc. Natl. Acad. Sci.

USA, 98, 373–378.

Gibly, A., Bonshtien, A., Balaji, V., Debbie, P., Martin, G.B. and Sessa, G.

(2004) Identification and expression profiling of tomato genes differentially

regulated during a resistance response to Xanthomonas campestris pv.

vesicatoria. Mol. Plant Microbe Interact. 17, 1212–1222.

Gomi, K., Ogawa, D., Katou, S. et al. (2005) A mitogen-activated protein

kinase NtMPK4 activated by SIPKK is required for jasmonic acid signaling

and involved in ozone tolerance via stomatal movement in tobacco. Plant

Cell Physiol. 46, 1902–1914.

Greenberg, J.T. and Yao, N. (2004) The role and regulation of programmed

cell death in plant-pathogen interactions. Cell Microbiol. 6, 201–211.

Guindon, S. and Gascuel, O. (2003) A simple, fast, and accurate algorithm to

estimate large phylogenies by maximum likelihood. Syst. Biol. 52, 696–704.

Hammond-Kosack, K.E. and Jones, J.D. (1996) Resistance gene-dependent

plant defense responses. Plant Cell, 8, 1773–1791.

Holt, B.F. 3rd, Boyes, D.C., Ellerstrom, M., Siefers, N., Wiig, A., Kauffman, S.,

Grant, M.R. and Dangl, J.L. (2002) An evolutionarily conserved mediator of

plant disease resistance gene function is required for normal Arabidopsis

development. Dev. Cell 2, 807–817.

Ichimura, K., Shinozaki, K., Tena, G. et al. (2002) Mitogen-activated protein

kinase cascades in plants: a new nomenclature. Trends Plant Sci. 7, 301–

308.

Jin, H.L., Axtell, M.J., Dahlbeck, D., Ekwenna, O., Zhang, S.Q., Staskawicz, B.

and Baker, B. (2002) NPK1, an MEKK1-like mitogen-activated protein kinase

kinase kinase, regulates innate immunity and development in plants. Dev.

Cell, 3, 291–297.

Jin, H., Liu, Y., Yang, K.Y., Kim, C.Y., Baker, B. and Zhang, S. (2003) Function of

a mitogen-activated protein kinase pathway in N gene-mediated resistance

in tobacco. Plant J. 33, 719–731.

Jones, D.A., Thomas, C.M., Hammond-Kosack, K.E., Balint-Kurti, P.J. and

Jones, J.D. (1994) Isolation of the tomato Cf-9 gene for resistance to

Cladosporium fulvum by transposon tagging. Science, 266, 789–793.

Jones, J.B., Stall, R.E. and Bouzar, H. (1998) Diversity among Xanthomonads

pathogenic on pepper and tomato. Annu. Rev. Phytopathol. 36, 41–58.

Jouannic, S., Champion, A., Segui-Simarro, J.M. et al. (2001) The protein

kinases AtMAP3Ke1 and BnMAP3Ke1 are functional homologues of

S. pombe cdc7p and may be involved in cell division. Plant J. 26, 637–649.

Kim, Y.J., Lin, N.C. and Martin, G.B. (2002) Two distinct Pseudomonas effector

proteins interact with the Pto kinase and activate plant immunity. Cell, 109,

589–598.

Kovtun, Y., Chiu, W.L., Zeng, W. and Sheen, J. (1998) Suppression of auxin

signal transduction by a MAPK cascade in higher plants. Nature, 395, 716–

720.

Le, S.Q. and Gascuel, O. (2008) An improved general amino acid replacement

matrix. Mol. Biol. Evol. 25, 1307–1320.

Liu, Y., Zhang, S. and Klessig, D.F. (2000) Molecular cloning and character-

ization of a tobacco MAP kinase kinase that interacts with SIPK. Mol. Plant

Microbe Interact. 13, 118–124.

Liu, Y., Schiff, M., Marathe, R. and Dinesh-Kumar, S.P. (2002) Tobacco Rar1,

EDS1 and NPR1/NIM1 like genes are required for N-mediated resistance to

tobacco mosaic virus. Plant J. 30, 415–429.

Liu, Y., Jin, H., Yang, K.Y., Kim, C.Y., Baker, B. and Zhang, S. (2003) Interaction

between two mitogen-activated protein kinases during tobacco defense

signaling. Plant J. 34, 149–160.

Liu, Y., Schiff, M. and Dinesh-Kumar, S.P. (2004) Involvement of MEK1

MAPKK, NTF6 MAPK, WRKY/MYB transcription factors, COI1 and CTR1 in

N-mediated resistance to tobacco mosaic virus. Plant J. 38, 800–809.

Mackey, D., Holt, B.F. 3rd, 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.

Martin, G.B., Brommonschenkel, S.H., Chunwongse, J., Frary, A., Ganal,

M.W., Spivey, R., Wu, T.Y., Earle, E.D. and Tanksley, S.D. (1993) Map-based

cloning of a protein-kinase gene conferring disease resistance in tomato.

Science, 262, 1432–1436.

Martin, G.B., Bogdanove, A.J. and Sessa, G. (2003) Understanding the func-

tions of plant disease resistance proteins. Annu. Rev. Plant Biol. 54, 23–61.

Mayrose, M., Ekengren, S.K., Melech-Bonfil, S., Martin, G.B. and Sessa, G.

(2006) A novel link between tomato GRAS genes, plant disease resistance

and mechanical stress response. Mol. Plant Pathol. 7, 593–604.

Mucyn, T.S., Clemente, A., Andriotis, V.M., Balmuth, A.L., Oldroyd, G.E.,

Staskawicz, B.J. and Rathjen, J.P. (2006) The tomato NBARC-LRR protein

Prf interacts with Pto kinase in vivo to regulate specific plant immunity.

Plant Cell, 18, 2792–2806.

Nishihama, R., Ishikawa, M., Araki, S., Soyano, T., Asada, T. and Machida, Y.

(2001) The NPK1 mitogen-activated protein kinase kinase kinase is a reg-

ulator of cell-plate formation in plant cytokinesis. Genes Dev. 15, 352–363.

Oh, C.S., Pedley, K.F. and Martin, G.B. (2010) Tomato 14-3-3 protein 7 posi-

tively regulates immunity-associated programmed cell death by enhancing

protein abundance and signaling ability of MAPKKKa. Plant Cell, 22, 260–

272.

Pedley, K.F. and Martin, G.B. (2003) Molecular basis of Pto-mediated resis-

tance to bacterial speck disease in tomato. Annu. Rev. Phytopathol. 41,

215–243.

Pedley, K.F. and Martin, G.B. (2004) Identification of MAPKs and their possible

MAPK kinase activators involved in the Pto-mediated defense response of

tomato. J. Biol. Chem. 279, 49229–49235.

Pedley, K.F. and Martin, G.B. (2005) Role of mitogen-activated protein kinases

in plant immunity. Curr. Opin. Plant Biol. 8, 541–547.

Petersen, M., Brodersen, P., Naested, H. et al. (2000) Arabidopsis MAP kinase

4 negatively regulates systemic acquired resistance. Cell, 103, 1111–1120.

Pitzschke, A., Schikora, A. and Hirt, H. (2009) MAPK cascade signalling net-

works in plant defence. Curr. Opin. Plant Biol. 12, 421–426.

del Pozo, O., Pedley, K.F. and Martin, G.B. (2004) MAPKKKa is a positive

regulator of cell death associated with both plant immunity and disease.

EMBO J. 23, 3072–3082.



Rivas, S. and Thomas, C.M. (2005) Molecular interactions between tomato

and the leaf mold pathogen Cladosporium fulvum. Annu. Rev. Phytopa-

thol. 43, 395–436.

Salmeron, J.M., Oldroyd, G.E., Rommens, C.M., Scofield, S.R., Kim, H.S.,

Lavelle, D.T., Dahlbeck, D. and Staskawicz, B.J. (1996) Tomato Prf is

a member of the leucine-rich repeat class of plant disease resistance

genes and lies embedded within the Pto kinase gene cluster. Cell, 86, 123–

133.

Scofield, S.R., Tobias, C.M., Rathjen, J.P., Chang, J.H., Lavelle, D.T.,

Michelmore, R.W. and Staskawicz, B.J. (1996) Molecular basis of gene-for-

gene specificity in bacterial speck disease of tomato. Science, 274, 2063–

2065.

Scott, J.W., Jones, J.B., Somodi, G.C. and Stall, R.E. (1995) Screening tomato

accessions for resistance to Xanthomonas campestris pv. vesicatoria, race

T3 HortScience, 30, 579–581.

Sessa, G., D’Ascenzo, M. and Martin, G.B. (2000) Thr38 and Ser198 are Pto

autophosphorylation sites required for the AvrPto-Pto-mediated hyper-

sensitive response. EMBO J. 19, 2257–2269.

Suarez-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 required for

flg22-induced MPK4 activation in Arabidopsis plants. Plant Physiol. 143,

661–669.

Tang, X., Frederick, R.D., Zhou, J., Halterman, D.A., Jia, Y. and Martin, G.B.

(1996) Initiation of plant disease resistance by physical interaction of Avr-

Pto and Pto kinase. Science, 274, 2060–2063.

Van der Hoorn, R.A., Laurent, F., Roth, R. and De Wit, P.J. (2000) Agroinfil-

tration is a versatile tool that facilitates comparative analyses of Avr9/Cf-9-

induced and Avr4/Cf-4-induced necrosis. Mol. Plant Microbe Interact. 13,

439–446.

Wang, H., Ngwenyama, N., Liu, Y., Walker, J.C. and Zhang, S. (2007) Stomatal

development and patterning are regulated by environmentally responsive

mitogen-activated protein kinases in Arabidopsis. Plant Cell, 19, 63–73.

Whalen, M.C. (2005) Host defence in a developmental context. Mol. Plant

Pathol. 6, 347–360.

Whalen, M.C., Wang, J.F., Carland, F.M., Heiskell, M.E., Dahlbeck, D., Min-

savage, G.V., Jones, J.B., Scott, J.W., Stall, R.E. and Staskawicz, B.J. (1993)

Avirulence gene avrRxv from Xanthomonas campestris pv. vesicatoria

specifies resistance on tomato line Hawaii 7998. Mol. Plant Microbe Inter-

act. 6, 616–627.

Yang, K.Y., Liu, Y. and Zhang, S. (2001) Activation of a mitogen-activated

protein kinase pathway is involved in disease resistance in tobacco. Proc.

Natl. Acad. Sci. USA, 98, 741–746.

Yu, Z.H., Wang, J.F., Stall, R.E. and Vallejos, C.E. (1995) Genomic localization

of tomato genes that control a hypersensitive reaction to Xanthomonas

campestris pv. vesicatoria (Doidge) Dye. Genetics, 141, 675–682.

Zhang, S. and Liu, Y. (2001) Activation of salicylic acid-induced protein kinase,

a mitogen-activated protein kinase, induces multiple defense responses in

tobacco. Plant Cell, 13, 1877–1889.

Zhou, J., Loh, Y.T., Bressan, R.A. and Martin, G.B. (1995) The tomato gene Pti1

encodes a serine/threonine kinase that is phosphorylated by Pto and is

involved in the hypersensitive response. Cell, 83, 925–935.

Zhou, J., Tang, X. and Martin, G.B. (1997) The Pto kinase conferring resistance

to tomato bacterial speck disease interacts with proteins that bind a cis-

element of pathogenesis-related genes. EMBO J. 16, 3207–3218.

Accession numbers: Newly deposited sequence data from this article can be found in the EMBL/GenBank data libraries under

accession numbers GU192457 for SlMAPKKKe, and GU205153 for NbMAPKKKe.



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