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. Author manuscript; available in PMC: 2014 Nov 7.
Published in final edited form as: Plant J. 2013 Dec 9;77(2):235–245. doi: 10.1111/tpj.12381

Pseudomonas syringae Effector HopF2 Suppresses Arabidopsis Immunity by Targeting BAK1

Jinggeng Zhou 1,#, Shujing Wu 2,3,#, Xin Chen 2, Chenglong Liu 2, Jen Sheen 4, Libo Shan 2, Ping He 1,†
PMCID: PMC4224013  NIHMSID: NIHMS635222  PMID: 24237140

Summary

Pseudomonas syringae delivers a plethora of effector proteins into host cells to sabotage immune responses and modulate physiology to favor infection. We have previously shown that P. syringae pv. tomato DC3000 effector HopF2 suppresses Arabidopsis innate immunity triggered by multiple microbe-associated molecular patterns (MAMP) at the plasma membrane. We show here that HopF2 possesses distinct mechanisms in the suppression of two branches of MAMP-activated MAP kinase (MPK) cascades. Besides blocking MKK5 (MPK kinase 5) activation in the MEKK1/MEKKs-MKK4/5-MPK3/6 cascade, HopF2 targets additional component(s) upstream of MEKK1 in the MEKK1-MKK1/2-MPK4 cascade and plasma membrane-localized receptor-like cytoplasmic kinase BIK1 and its homologs. We further show that HopF2 directly targets BAK1, a plasma membrane-localized receptor-like kinase involved in multiple MAMP signaling. The interaction between BAK1 and HopF2 or two other P. syringae effectors AvrPto and AvrPtoB, was confirmed in vivo and in vitro. Consistent with BAK1 as a physiological target of AvrPto, AvrPtoB and HopF2, the strong growth defects or lethality associated with ectopic expression of these effectors in wild-type Arabidopsis transgenic plants were largely alleviated in bak1 mutant plants. Thus, our results provide genetic evidence to further support that BAK1 is a physiological target of AvrPto, AvrPtoB and HopF2. Identification of BAK1 as an additional target of HopF2 virulence not only explains HopF2 suppression of multiple MAMP signaling at the plasma membrane, but also supports the notion that pathogen virulence effectors act through multiple targets in host cells.

Keywords: Bacterial effector, pattern-triggered immunity, BAK1, BIK1, MAPK cascade, Pseudomonas syringae, Arabidopsis thaliana

Introduction

Plants have evolved robust immune systems to protect them from pathogen invasions. Plant innate immunity is initiated with recognition of conserved pathogen- or microbe-associated molecular patterns (PAMPs or MAMPs) through membrane-localized receptor-like kinases (RLKs) or receptor-like proteins (RLPs) (Jones and Dangl, 2006; Boller and Felix, 2009). Pattern-triggered immunity (PTI) plays a pivotal role in defense against a broad spectrum of potential pathogens (Jones and Dangl, 2006; Boller and Felix, 2009). A 22-amino-acid peptide from the N-terminus of bacterial flagellin, flg22, can be perceived by Arabidopsis RLK flagellin-sensing 2 (FLS2), and induces FLS2 association with another plasma membrane-localized RLK BAK1 (Chinchilla et al., 2007; Heese et al., 2007). BAK1 was originally isolated as a RLK interacting with plant growth hormone brassinosteroid (BR) receptor BRI1 (Li et al., 2002; Nam and Li, 2002). BAK1 with a relatively short extracellular leucine-rich repeat (LRR) domain is not involved in flagellin nor BR perception (Kinoshita et al., 2005; Chinchilla et al., 2007). Notably, BAK1 is required for signaling triggered by multiple MAMPs, including bacterial elongation factor Tu (EF-Tu), flagellin, harpin Z (HrpZ), lipopolysaccharide (LPS), peptidoglycan (PGN), necrosis-inducing Phytophthora protein 1(NPP1), oomycete elicitor INF1 and bacterial cold-shock protein in Arabidopsis and Nicotiana benthamiana (Chinchilla et al., 2007; Heese et al., 2007; Shan et al., 2008). In addition to FLS2, BAK1 has been shown to hetero-dimerize with EFR, a RLK for EF-Tu and PEPR1/2, a RLK for plant endogenous signal Pep1/2 (Postel et al., 2010; Roux et al., 2011). BAK1 is able to directly phosphorylate a plasma membrane-localized receptor-like cytoplasmic kinase (RLCK) BIK1 (Lu et al., 2010b). In non-elicited cells, BIK1 interacts with BAK1, FLS2, EFR, and PEPR1/2 (Lu et al., 2010b; Zhang et al., 2010; Liu et al., 2013). Flg22 induces rapid phosphorylation of BIK1 which further transphosphorylates FLS2-BAK1, and leads to its dissociation from FLS2-BAK1 complex (Lu et al., 2010b; Zhang et al., 2010; Cao et al., 2013). As a step toward attenuation of immune responses, flg22 induces FLS2 endocytosis in vesicles within ~30 minutes, and leads to FLS2 degradation (Robatzek et al., 2006; Beck et al., 2012). Protein ubiquitination often directs target proteins for degradation through the proteasome or vacuole, or mediates receptor intracellular endosomal sorting. FLS2 is targeted by plant U-box containing E3 ubiquitin ligases PUB12 and PUB13 (Lu et al., 2011). BAK1 phosphorylates PUB12/13 upon flg22 elicitation and promotes FLS2-PUB12/13 association for ligand-induced FLS2 degradation. Despite specific recognition of MAMPs by their corresponding receptors, diverse MAMPs often elicit largely overlapping responses, including ion fluxes across the plasma membrane leading to membrane depolarization and medium alkalinization, production of reactive oxygen species (ROS), cytoplasmic calcium transients, callose deposition, stomatal closure, expression of defense-related genes and activation of mitogen-activated protein kinase (MAPK) cascades and Ca2+-dependent protein kinases (CDPKs) (Boller and Felix, 2009; Tena et al., 2011; Schwessinger and Ronald, 2012).

Successful pathogens evolved the ability to interfere with plant physiology and immunity to favor infection. Pseudomonas syringae is a Gram-negative phytobacterial pathogen that causes a wide range of diseases, including blights, leaf spots, and galls, in different plant species and is also a model system in molecular plant pathology (Preston, 2000). Extensive genetic and genomic studies of P. syringae have identified many key virulence determinants, including global virulence regulators, the type III secretion system (TTSS), phytotoxins and exopolysaccharides (Block et al., 2008). In particular, P. syringae delivers around 30 effectors into plant cells through TTSS, and many of these effectors target important host components to sabotage plant immunity (Speth et al., 2007; Block et al., 2008; Gohre and Robatzek, 2008; Lewis et al., 2009; Hann et al., 2010). The P. syringae effector HopU1 is a mono-ADP-ribosyltransferase (ADP-RT) and targets several Arabidopsis RNA-binding proteins including GRP7 (Fu et al., 2007). Interestingly, GRP7 interacts with both translational components and MAMP receptors FLS2 and EFR, suggesting its role in plant immunity (Nicaise et al., 2013). In addition, GRP7 directly binds to FLS2 and EFR transcripts, and this binding was blocked by HopU1 to modulate FLS2 protein level (Nicaise et al., 2013). Two sequence-distinct effectors, AvrPto and AvrPtoB, are potent suppressors of multiple MAMP signaling by targeting RLKs, including BAK1 and FLS2 (de Torres et al., 2006; He et al., 2006; Gohre and Robatzek, 2008; Shan et al., 2008; Xiang et al., 2008; Gimenez-Ibanez et al., 2009). AvrPtoB possesses an E3 ubiquitin ligase activity and target certain RLKs including FLS2 and CERK1 for degradation (Gohre et al., 2008; Gimenez-Ibanez et al., 2009). Other Pseudomonas effectors target components downstream of MAMP receptor complexes. For example, HopAI1 targets MPK3, MPK4 and MPK6 to disrupt MAPK activation upon MAMP perception (Zhang et al., 2007; Zhang et al., 2012). Interestingly, inactivation of MPK4 by HopAI1 activates nucleotide binding leucine-rich repeat (NB-LRR) protein SUMM2-mediated defense responses (Zhang et al., 2012). AvrRpt2 promotes auxin responses to facilitate pathogen virulence by stimulating the turnover of Aux/IAA proteins, the key negative regulators in auxin signaling (Cui et al., 2013). Effector-mediated suppression of PTI signaling could also be overcome by NB-LRR mediated effector-triggered immunity (ETI). HopM1 targets and degrades a member of the ARF family of guanine nucleotide exchange factors, including AtMIN7, involved in vesicle trafficking (Nomura et al., 2006). Activation of ETI signaling by AvrRpt2, AvrPphB, and HopA1, prevents HopM1-mediated degradation of AtMIN7 to suppress HopM1 virulence activity (Nomura et al., 2011).

We previously reported a P. syringae pv tomato DC3000 effector, HopF2, suppresses Arabidopsis innate immunity at the plasma membrane (Wu et al., 2011). Similar with AvrPto, HopF2 possesses a putative myristoylation modification motif which is required for its plasma membrane-localization and virulence activity in Arabidopsis, tobacco and tomato (Shan et al., 2000; Robert-Seilaniantz et al., 2006; Wu et al., 2011). HopF2 could suppress immune responses triggered by multiple MAMPs, including flg22, elf18, LPS, PGN, HrpZ and chitin (Wu et al., 2011). Structural analysis of HopF2 homolog AvrPphF from P. syringae pv. phaseolicola has identified several conserved surface-exposed residues, and mutational analysis indicates that the corresponding residues in HopF2 are required for its virulence and MAMP suppression activity (Shan et al., 2004; Singer et al., 2004; Wang et al., 2010; Wu et al., 2011). It has been shown that RIN4, a component involved in both PTI and ETI, is targeted and suppressed by HopF2 (Wilton et al., 2010). HopF2 also targets MAPK kinase 5 (MKK5) and suppresses MKK5 phosphorylation of downstream MPK3/6 through its ADP-ribosyltransferase activity (Wang et al., 2010). Interestingly, HopF2 suppresses flg22-induced BIK1 phosphorylation, an event likely acts upstream or independently of MAPK cascades in flg22 signaling. HopF2 did not directly interact with BIK1 nor affected BIK1 kinase activity (Wu et al., 2011), suggesting that HopF2 targets additional host proteins upstream of BIK1 in flg22 signaling. We extended this study and report here that HopF2 blocks flg22-induced phosphorylation of two BIK1 homologs, PBS1 and PBL1, and HopF2 virulence is associated with its suppression of BIK1 phosphorylation. Consistent with its suppression upstream of BIK1, HopF2 did not affect MPK4 activation by MKK1/2 or MEKK1. Importantly, HopF2 directly interacts with BAK1 in vivo and in vitro in an FLS2-independent manner. We have previously shown that BAK1 is also a virulence target of AvrPto and AvrPtoB (Shan et al., 2008). Recently the crystal structure of AvrPtoB/BAK1 complex has been solved (Cheng et al., 2011). The interaction between BAK1 and AvrPto or AvrPtoB was confirmed with in vivo co-immunoprecipitation (co-IP) and bimolecular fluorescence complementation (BiFC) assays, and in vitro pull-down assay in this study. Expression of AvrPto, AvrPtoB or HopF2 under the control of constitutive 35S promoter leads to lethality or causes severe growth defects in Arabidopsis wild-type (WT) plants likely due to their strong virulence. Significantly, the growth defects/lethality caused by ectopic expression of AvrPto, AvrPtoB or HopF2 were dramatically reduced in bak1 mutant plants, further supporting that BAK1 is their physiological target.

Results

HopF2 virulence is associated with its suppression of BIK1 phosphorylation

Flg22-induced BIK1 phosphorylation is evidenced by a mobility shift on the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel (Lu et al., 2010b). The mobility shift of HA epitope-tagged BIK1 is blocked by co-expression of green fluorescence protein (GFP)-tagged HopF2 in Arabidopsis protoplasts, suggesting that HopF2 suppresses flg22-induced BIK1 phosphorylation (Fig. 1A) (Wu et al., 2011). It has been reported that several BIK1 homologs, including PBL1 and PBS1, are also quickly phosphorylated upon flg22 treatment (Lu et al., 2010a; Zhang et al., 2010). Interestingly, HopF2 also blocks flg22-induced phosphorylation of PBL1 and PBS1 (Fig. 1A). BIK1 and PBS1 are plasma membrane-associated RLCKs. Thus, our data are consistent with that HopF2 functions at the plant plasma membrane through a putative myristoylation modification and the myristoylation motif is required for its virulence functions (Robert-Seilaniantz et al., 2006; Wu et al., 2011). These data suggest that HopF2 suppresses flg22-mediated signaling at an immediate early step upstream of BIK1 phosphorylation in the FLS2/BAK1 receptor complex.

Figure 1. HopF2 suppresses flg22-induced phosphorylation of BIK1 and homologs.

Figure 1

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(A) HopF2 blocks flg22-induced mobility shift of BIK1 and homologs. Arabidopsis protoplasts were co-transfected with HA-tagged BIK1, PBL1 or PBS1 and GFP-tagged HopF2 for 10 hr and treated with 1 μM flg22 for 10 min. (B) Conserved surface residues of HopF2 are required for its suppression of flg22-induced BIK1 phosphorylation. Protoplasts were co-transfected with FLAG-tagged BIK1 and HA-tagged HopF2 or its mutants, and treated with flg22 as in (A).

Structure analysis of HopF1 (AvrPphF) from P. syringae pv phaseolicola, a homolog of HopF2, identified several conserved surface-exposed residues which are required for its virulence and avirulence functions in beans (Singer et al., 2004). The corresponding residues in HopF2 are essential for its suppression of flg22-induced expression of pFRK1::LUC (the FRK1 promoter fused with luciferase reporter) (Wu et al., 2011). In particular, HopF2 R71A and D175A mutants lost the ability to suppress flg22-induced pFRK1::LUC activation (Fig. S1) (Wu et al., 2011). To determine whether these residues are also essential for HopF2 suppression of BIK1 phosphorylation, we examined the flg22-induced mobility shift of BIK1 in the presence of various HopF2 mutants. Significantly, HopF2 R71A and D175A mutants failed to suppress flg22-induced BIK1 phosphorylation (Fig. 1B). Consistent with their suppression actions on flg22-induced pFRK1::LUC and MAPK activation (Fig. S1) (Wang et al., 2010; Wu et al., 2011), the S89A mutant had minor effect whereas H96A and E97A mutants had no effect on the HopF2 suppression of flg22-induced BIK1 phosphorylation. Taken together, our data suggest that HopF2 virulence is associated with its suppression of BIK1 phosphorylation.

Distinct mechanisms of HopF2 suppression of two branches of flg22-induced MAPK cascades

MAPK activation is one of the early signaling events following MAMP recognition in both plants and animals (Barton and Medzhitov, 2003; Nurnberger et al., 2004; Tena et al., 2011). Accumulating evidence suggests that the perception of flg22 activates two branches of MAPK cascades in Arabidopsis, MEKK1/MEKKs-MKK4/5-MPK3/6 and MEKK1-MKK1/2-MPK4 (Fig. 2A) (Tena et al., 2011). It has been reported that HopF2 directly targets and blocks MKK5 function, thereby suppressing downstream MPK3 and MPK6 activation (Wang et al., 2010). In addition, HopF2 is able to suppress flg22-induced MPK4 activation, which is mediated through the MEKK1 and MKK1/2 cascade (Wu et al., 2011). Surprisingly, HopF2 did not directly affect MKK1 and MKK2 activity (Fig. 2B). As shown in Fig. 2B, the constitutively active form of Myc epitope tagged MKK1 and MKK2 (MKK1ac-Myc and MKK2ac-Myc) activated MPK4-HA in Arabidopsis protoplasts with an immuno-complex kinase assay. Expression of HopF2 did not affect activation of MKK1ac or MKK2ac on MPK4 (Fig. 2B). Furthermore, HopF2 did not interfere with the MEKK1-mediated activation of MPK4 (Fig. 2C). The data suggest that HopF2 suppresses flg22-induced MPK4 activation upstream of MEKK1-MKK1/2, which is consistent with HopF2 suppression of flg22-induced BIK1 phosphorylation. In agreement with the previous report (Wang et al., 2010), HopF2 functions on MKK5 to suppress MPK3 activation. As shown in Fig. 2D, expression of HopF2 diminished active MKK5ac-mediated MPK3 activity (Fig. 2D). Thus, in addition to MKK5, HopF2 also targets additional component(s) upstream of MEKK1 and BIK1, probably immediately after flagellin perception by FLS2/BAK1 receptor complex.

Figure 2. HopF2 suppresses two branches of flg22-induced MAPK cascades.

Figure 2

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(A) Scheme of two branches of MAPK cascades in Arabidopsis flagellin signaling. (B) HopF2 does not suppress MKK1/2-mediated MPK4 activation. Arabidopsis protoplasts were co-transfected with myc-tagged constitutively active form of MKK1/2 (MKK1ac-myc/MKK2ac-myc), HA-tagged MPK4 (MPK4-HA) and GFP-tagged HopF2 (HopF2-GFP). MPK4-HA was immunoprecipitated by an α-HA antibody and subjected to an immunocomplex kinase assay using myelin basic protein as a substrate. (C) HopF2 does not suppress MEKK1-mediated MPK4 activation. HA-tagged MEKK1 (MEKK1-HA) was coexpressed with MPK4-HA and HopF2-GFP, and MPK4-HA kinase activity was detected in an immunocomplex kinase assay as in (B). (D) HopF2 suppresses MKK5-mediated MPK3 activation. MKK5ac-myc was coexpressed with MPK3-HA and HopF2-GFP, and MPK3-HA kinase activity was detected in an immunocomplex kinase assay as in (B).

HopF2 interacts with BAK1

HopF2 suppresses pFRK1::LUC activation by multiple MAMPs, including elf18, PGN, LPS and HrpZ. Since BAK1 is involved in signaling activated by multiple MAMPs, we tested whether HopF2 might directly interact with BAK1. Interestingly, similar with AvrPto and AvrPtoB, HopF2 co-immunoprecipitated with BAK1 in Arabidopsis wild-type protoplasts (Fig. 3A). The association between BAK1 and HopF2, AvrPto or AvrPtoB was also detected in fls2 mutant protoplasts, indicating that this association is independent of FLS2 (Fig. 3A). To further confirm the in vivo association of HopF2 and BAK1 in intact plants, we transformed the HA-tagged HopF2 under the control of Dexamethasone (DEX) inducible promoter (DEX::HopF2-HA) into the pBAK1::BAK1-GFP transgenic plants. HopF2-HA co-immunoprecipitated BAK1-GFP as detected with α-HA antibody upon α-GFP antibody immunoprecipitation (Fig. 3B). In addition, AvrPto-HA also co-immunoprecipitated with BAK1-GFP in transgenic plants carrying DEX::AvrPto-HA and pBAK1::BAK1-GFP as detected with α-HA antibody upon α-GFP antibody immunoprecipitation (Fig. 3C). Consistently, bimolecular fluorescence complementation (BiFC) assay also indicated the in vivo association of HopF2 and BAK1 (Fig. 3D). The fluorescence signal was detected when HopF2-nYFP (the amino-terminal part of YFP fused with HopF2) was co-expressed with BAK1-cYFP (the carboxy-terminal part of YFP fused with BAK1) (Fig. 3D). Similarly, the in vivo AvrPto and BAK1 association was observed with co-transfection of BAK1-cYFP and AvrPto-nYFP in protoplasts (Fig. 3D). None of the individual constructs emitted fluorescence signals in protoplasts. Furthermore, HopF2 or AvrPto protein fused to glutathione-S-transferase (GST) immobilized on agarose beads purified from E. coli specifically pulled down BAK1-FLAG expressed from protoplasts (Fig. 3E), suggesting a direct interaction between BAK1 and HopF2 or AvrPto. Thus, our results not only provide evidence that BAK1 is a target of HopF2, but also verify our previous finding that AvrPto and AvrPtoB interact with BAK1 (Shan et al., 2008).

Figure 3. HopF2, AvrPto and AvrPtoB interact with BAK1.

Figure 3

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(A) HopF2 and AvrPto/B interact with BAK1 in Arabidopsis protoplasts. The α-HA co-IP was performed with protoplasts co-expressing FLAG-tagged BAK1 and HA-tagged AvrPto, AvrPtoB or HopF2, and the immunoprecipitated proteins were analyzed in Western blot with an α-FLAG antibody. HopF2 (B) and AvrPto (C) interact with BAK1 in Arabidopsis plants. pBAK1::BAK1-GFP transgenic seedlings with DEX-inducible effector transgene were treated with 5 μM DEX for 12 hr and subjected to an α-GFP co-IP assay, and the immunoprecipitated proteins were analyzed in Western blot with an α-HA antibody. (D) The BiFC assays for HopF2-BAK1 or AvrPto-BAK1 interactions in Arabidopsis protoplasts. The various BiFC constructs were transfected into protoplasts and the fluorescence were visualized under a confocal microscope. Bar=50 μm. (E) The pull-down assays for HopF2-BAK1 or AvrPto-BAK1 interactions. GST, GST-AvrPto and GST-HopF2 were expressed individually in E. coli, purified with glutathione agarose, and used to pull-down the proteins from protoplasts expressing FLAG-tagged BAK1. The pull-downed proteins were analyzed in Western blot with an α-FLAG antibody.

HopF2 interacts with BAK1 via transmembrane and kinase domain

BAK1 consists of an extracellular domain, a single transmembrane (TM) domain, a juxtamembrane (J) domain and a kinase (K) domain (Li et al., 2002; Nam and Li, 2002). Using a yeast split-ubiquitin assay and co-immunoprecipitation assay, we previously reported that BAK1’s transmembrane and kinase domains (BAK1TJK) are required for its interaction with AvrPto (Shan et al., 2008). Similar with AvrPto, HopF2 immuno-precipitated with BAK1TJK in protoplasts co-transfected with HopF2-HA and BAK1TJK-FLAG (Fig. 4A). In addition, GST-AvrPto or GST-HopF2 fusion proteins pulled down BAK1TJK-FLAG expressed from protoplasts (Fig. 4B). These data suggested that HopF2 associates with BAK1 via the transmembrane domain and kinase domain. The data are consistent with the observation that HopF2 functions inside plant cells, and the plasma membrane localization is critical for its function to suppress flg22 signaling.

Figure 4. Transmembrane, juxtamembrane and kinase domains of BAK1(BAK1TJK) are sufficient for BAK1-HopF2 or BAK1-AvrPto interaction.

Figure 4

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(A) The α-HA co-IP was performed with protoplasts co-expressing FLAG-tagged BAK1TJK with AvrPto-HA or HopF2-HA, and the immunoprecipitated proteins were analyzed in Western blot with an α-FLAG antibody. (B) The pull-down assay was performed by using GST, GST-AvrPto and GST-HopF2 proteins expressed in E. coli, purified with glutathione agarose to bind the total proteins from protoplasts expressing BAK1TJK-FLAG.

BAK1 is a physiological target of AvrPto, AvrPtoB and HopF2

In addition to the above biochemical analyses, we also examined the BAK1 dependence of AvrPto, AvrPtoB and HopF2 growth perturbation in transgenic plants. The strong growth perturbation of AvrPto prevented the generation of viable transgenic plants carrying AvrPto under the control of constitutive 35S promoter in Col-0 WT plants as we previously reported (Shan et al., 2008). The occasionally survived transgenic plants with detectable AvrPto protein expression showed dwarfed stature with small, round, and thick leaves; short petioles; and short inflorescences (Fig. 5A and 5B) (Shan et al., 2008). We never obtained viable seeds from 35S::AvrPto-HA transgenic plants in Col-0 background. The similar phenotype was observed when we transformed 35S::AvrPto-HA construct into fls2 mutant plants. Interestingly, when the same 35S::AvrPto-HA construct was transformed into bak1-4 mutant (a BAK1 null mutant), several transgenic lines with detectable AvrPto-HA protein expression have been obtained and displayed much alleviated growth defects compared with transgenic plants in WT Col-0 background. The 35S::AvrPto-HA transgenic plants in bak1-4 mutant had longer petioles, bigger leaves, and longer inflorescence with viable seeds than 35S::AvrPto-HA transgenic plants in Col-0 background (Fig. 5A and 5B). These physiological and genetic data suggest that BAK1 is a virulence target of AvrPto and strongly support our early observation that AvrPto targets to BAK1 to suppress PTI signaling (Shan et al., 2008).

Figure 5. Ectopic expression of AvrPto in bak1-4 mutant plants.

Figure 5

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(A) Phenotype of 4-week-old 35S::AvrPto-HA/Col-0 and 35S::AvrPto-HA/bak1-4 transgenic plants. The expression of AvrPto protein was shown with an α-HA Western blot. (B) Phenotype of 10-week-old 35S::AvrPto-HA/Col-0 and 35S::AvrPto-HA/bak1-4 transgenic plants.

Previously, we and others found that AvrPto can be associated with FLS2 (Shan et al., 2008; Xiang et al., 2008). We also transformed 35S::AvrPto-HA into Arabidopsis WS ecotype which carries a natural mutation on FLS2 gene (Gomez-Gomez et al., 1999). However, unlike bak1-4 plants, we never obtained any viable transgenic plants with detectable AvrPto expression. In contrast, many transgenic plants with strong and constitutive AvrPto expression were obtained repeatedly in the bak1-1 mutant in the WS background. Significantly, multiple lines of 35S::AvrPto-HA transgenic plants in the bak1-1 mutants were almost indistinguishable with bak1-1 mutant plants at the early developmental stage (Fig. 6A). The 35S::AvrPto-HA transgenic plants in bak1-1 mutants displayed moderately reduced stem length and apical dominance at the late developmental stage compared with bak1-1 mutant (Fig. S2). The ameliorated growth defects of AvrPto in bak1-1 and bak1-4 mutants support that BAK1 is a physiological target of AvrPto. Similarly, we never obtained any viable 35S::AvrPtoB-HA or 35S::HopF2-HA transgenic plants in Col-0 or WS background with detectable protein expression, whereas many 35S::AvrPtoB-HA or 35S::HopF2-HA transgenic plants in bak1-1 background could survive and set seeds (Fig. 6B and 6C), supporting that BAK1 is also a physiological target of AvrPtoB and HopF2, consistent with our biochemical data of direct BAK1-AvrPtoB and BAK1-HopF2 interactions.

Figure 6. Ectopic expression of AvrPto, AvrPtoB or HopF2 in bak1-1 mutant plants.

Figure 6

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Phenotype of 4-week-old WS, bak1-1, 35S::AvrPto-HA/bak1-1 (A), 35S::AvrPtoB-HA/bak1-1 (B) and 35S::HopF2-HA/bak1-1 (C) plants. The expression of corresponding effector proteins is shown with an α-HA Western blot.

DISSCUSSION

To achieve infections, successful pathogens have evolved deliberate virulence mechanisms to suppress host immunity and interfere with host physiological responses. The P. syringae type III effector HopF2 is injected into plant cells and blocks immune responses triggered by multiple MAMPs (Wu et al., 2011). Here we show that HopF2 directly interacts with the plasma membrane-resident RLK BAK1-a signaling partner of multiple MAMP receptors (Fig. 7). This conclusion was supported by our comprehensive Co-IP, BiFC and pull-down assays. The rapid heterodimerization of BAK1 with different MAMP receptors, including FLS2, EFR, and PEPR1/2, constitutes an initial step in PTI signaling (Chinchilla et al., 2007; Heese et al., 2007; Postel et al., 2010; Schulze et al., 2010; Roux et al., 2011). By targeting BAK1, our data explain the observations that HopF2 suppresses diverse early signaling events triggered by multiple MAMPs, including BIK1 phosphorylation, MAPK activation and immune gene expression. Our data are also consistent with the requirement of membrane localization for HopF2 virulence activity (Shan et al., 2004; Robert-Seilaniantz et al., 2006; Wu et al., 2011). Interestingly, HopF2 possesses distinct mechanisms in the suppression of two branches of MAMP-activated MAPK cascades. Besides direct blocking MKK5 in the MEKK1/MEKKs-MKK4/5-MPK3/6 cascade, HopF2 also functions at the plasma membrane and targets BAK1 upstream of the MEKK1-MKK1/2-MPK4 cascade as well as BIK1 and its homologs. In this study, we also confirmed our previous finding that BAK1 interacts with AvrPto and AvrPtoB with Co-IP, BiFC and pull-down assays (Shan et al., 2008). Importantly, the strong growth defects associated with AvrPto transgene in WT Arabidopsis plants were largely alleviated in bak1 mutant plants, providing genetic and physiological evidence of BAK1 as a virulence target of AvrPto. Thus, BAK1 is a virulence target of three sequence distinct bacterial effectors AvrPto, AvrPtoB and HopF2.

Figure 7. A model of multiple host targets of HopF2.

Figure 7

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Plant innate immunity includes pattern-triggered immunity (PTI) and effector-triggered immunity (ETI). Perception of bacterial flagellin by FLS2 activates FLS2/BAK1/BIK1 complex phosphorylation and two branches of MAPK cascades, MEKK1-MKK1/2-MPK4 and MEKK1/MEKKs-MKK4/5-MPK3/6 in PTI signaling. Bacterial type III secretion system (TTSS) effectors AvrB, AvrRpm1 and AvrRpt2 modify host RIN4 protein, which is sensed by corresponding RPM1 and RPS2 proteins to activate ETI signaling. Bacterial effector proteins have the ability to suppress both PTI and ETI signaling. AvrPto and AvrPtoB target BAK1 to suppress PTI signaling. HopF2 suppresses PTI signaling by targeting BAK1 and MKK5, and suppresses ETI signaling by targeting RIN4.

BIK1 is rapidly phosphorylated upon flg22 perception and is directly phosphorylated by BAK1 (Lu et al., 2010b; Zhang et al., 2010). Consistently, flg22-induced BIK1 phosphorylation depends on BAK1 (Lu et al., 2010b). Although the detailed mechanisms remain elusive, the current model suggests that BIK1 together with its homologs functions upstream or independent of MAPK cascades in flagellin signaling (Lu et al., 2010b; Zhang et al., 2010). Recently, it has been shown that a rice BIK1 homolog OsRLCK185 acts upstream of MAPK cascades in chitin- and peptidoglycan-induced plant immunity (Yamaguchi et al., 2013). Genetic analyses also indicate that RLCK SSP (Short suspensor) acts upstream of YDA (a MAPK kinase kinase, MEKK)-MPK3/6 cascade in the embryonic patterning process (Bayer et al., 2009). Nevertheless, HopF2 suppression of flg22-induced phosphorylation of BIK1 and its homologs suggests that HopF2 targets an immediate early step in flagellin signaling. Importantly, HopF2 virulence function is associated with its suppression of BIK1 phosphorylation. Notably, HopF2 did not interact with BIK1 or affect BIK1 in vitro kinase activity (Wu et al., 2011). All these observations are consistent with HopF2 targeting BAK1, which functions upstream of BIK1.

It has been reported that HopF2 targets MKK5 and likely other MKKs to block flg22-triggered signaling (Wang et al., 2010). The HopF2 homolog, HopF1 (AvrPphF) from P. syringae pv. phaseolicola was shown to possess a marginally structural similarity to the catalytic domain of bacterial diphtheria toxin, an ADP-ribosyltransferase, though no ADP-ribosyltransferase activity was detected (Singer et al., 2004). Wang et al., reported that HopF2 directly ADP-ribosylates MKK5 and blocks MKK5 kinase activity in vitro (Wang et al., 2010). The complex MAPK signaling plays pivotal roles in transmitting MAMP signaling (Tena et al., 2011; Meng and Zhang, 2013). Two parallel MAPK cascades consisting of MEKK1/MEKKs-MKK4/5-MPK3/6 and MEKK1-MKK1/2-MPK4 have been proposed to function downstream of MAMP receptor complex. Intriguingly, HopF2 did not directly interfere with MKK1 and MKK2 activity. Furthermore, HopF2 did not interfere with MEKK1-mediated MPK4 activation. Suppressing MKK5 but not MKK1/2 activity could not explain the suppression function of HopF2 in flg22-induced MPK4 activation. Thus, HopF2 likely has additional target upstream of the MEKK1-MKK1/2-MPK4 cascade. The identification of plasma membrane-resident BAK1 as a HopF2 target is consistent with these observations.

In addition, HopF2 has also been found to directly interact with Arabidopsis RIN4, an important component in both PTI and ETI responses (Wilton et al., 2010). Three additional P. syringae type III effectors AvrRpt2, AvrRpm1 and AvrB directly target and modify RIN4, which is sensed by the corresponding NB-LRR receptors RPS2 and RPM1 to initiate ETI signaling (Mackey et al., 2002; Axtell and Staskawicz, 2003; Mackey et al., 2003). AvrRpt2 cleaves RIN4 which is suppressed by HopF2, thereby inhibiting AvrRpt2-mediated ETI responses (Wilton et al., 2010). Thus, HopF2 suppresses both PTI and ETI responses through targeting BAK1/MKK5 and RIN4 respectively (Fig. 7). This is consistent with the emerging theme that a single effector protein is able to target multiple host proteins to suppress innate immune signaling at multiple steps (Mukhtar et al., 2011). It also appears that multiple seemingly distinct effectors could target the same host protein. In addition to AvrRpt2, AvrRpm1 and AvrB that target RIN4, we found here that AvrPto, AvrPtoB and HopF2 target BAK1 (Fig. 7). We have previously showed that AvrPto and AvrPtoB suppress early defense signaling triggered by multiple MAMPs upstream of MAPK cascades, and proposed that AvrPto and AvrPtoB may target cell surface-resident RLKs that initiate MAMP signaling (He et al., 2006). We further reported that AvrPto and AvrPtoB interacted with BAK1 and other RLKs, including FLS2, to block the initiation of MAMP signaling (Shan et al., 2008). Biochemical and crystal structural analysis of AvrPtoB-BAK1 complex indicates that AvrPtoB250-359 is sufficient for BAK1 interaction (Cheng et al., 2011). In this study, we demonstrated in parallel experiments HopF2-BAK1, AvrPto-BAK1 and AvrPtoB-BAK1 interactions in transient assays and transgenic plants in vivo with Co-IP and BiFC assays and in vitro with a GST pull-down assay. More importantly, the various growth defects associated with constitutive expression of HopF2, AvrPto and AvrPtoB in Arabidopsis WT plants are significantly alleviated in the bak1 mutant plants. These biochemical, genetic and phenotypic results unambiguously support our data that HopF2, AvrPto and AvrPtoB target BAK1 for their virulence functions. We also predicted that additional targets exist since AvrPto and AvrPtoB also suppress BAK1-independent immune signaling. Indeed, AvrPtoB is able to target Arabidopsis and tomato chitin receptor CERK1 to promote bacterial virulence (Gimenez-Ibanez et al., 2009; Zeng et al., 2012). Thus AvrPto and AvrPtoB likely target multiple RLKs to impede plant immune signaling. It is possible that pathogenic bacteria have evolved the strategy to secure the infection by targeting the key components in plant immunity with multiple virulence factors. This is also consistent with that only minute amount of individual effectors is delivered into host cells and multiple effectors may function synergistically or in a specific hierarchy to exhibit virulence activity.

MATERIALS AND METHODS

Plant materials and growth conditions

The bak1-1 (WS background) and bak1-4 (Col-0 background) mutants were reported previously (Li et al., 2002; Lu et al., 2010b). Arabidopsis plants were grown in soil (Metro Mix 360) in a growth chamber at 23°C, 65% relative humidity, 75 μE m−2 s−1 light and with a 12-hr photoperiod for 4 weeks before protoplast isolation. To grow Arabidopsis seedlings, the seeds were surface sterilized with 50% bleach for 15 min, and then placed on the plates with half-strength Murashige and Skoog medium (½ MS) containing 0.5% sucrose, 0.8% agar and 2.5 mm MES at pH 5.7. The plates were first stored at 4°C for 3 days in the dark for seed stratification, and then moved to the growth chamber.

Plasmid construction and generation of transgenic plants

The constructs of HopF2, AvrPto, AvrPtoB, MAPKs, MKK, MEKK1, BIK1 and BAK1 in plant expression vector or protein expression vector were previously reported (He et al., 2006; Shan et al., 2008; Lu et al., 2010b; Wu et al., 2011). BAK1, AvrPto and HopF2 were sub-cloned into the modified BiFC vectors with BamHI and StuI digestion. The AvrPto-, AvrPtoB or HopF2- transgenic plants were generated by Agrobacterium tumefaciens-mediated transformation in Col-0, WS, bak1-1 or bak1-4 plants with the corresponding construct under the control of a constitutive cauliflower mosaic virus 35S promoter with an HA epitope tag. Dexamethasone (DEX)-inducible AvrPto-HA and HopF2-HA transgenic plants were previously reported (He et al., 2006; Wu et al., 2011). Transgenic plants carrying both DEX-inducible HopF2-HA and pBAK1::BAK1-GFP were generated by transforming HopF2 construct into the pBAK1::BAK1-GFP transgenic plants. Transgenic plants carrying both DEX-inducible AvrPto-HA and pBAK1::BAK1-GFP were previously reported (Shan et al., 2008). The transgenic plants were confirmed by Western blot with an α-HA or α-GFP antibody.

Co-immunoprecipitation (co-IP) assay

Protoplast isolation and transfection were performed as described (He et al., 2007). For protoplast co-IP assay, the total proteins from 2 × 105 transfected protoplasts were isolated with 0.5 ml of extraction buffer (10 mM HEPES [pH 7.5], 100 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% Triton X-100, and 1 X protease inhibitor cocktail from Roche). The samples were vortexed vigorously for 30 s, and then centrifuged at 13,000 rpm for 10 min at 4 °C. The supernatant was inoculated with an α-HA antibody for 2 hr and was further inoculated with agarose beads for another 2 hr at 4 °C with gentle shaking. The beads were collected and washed three times with washing buffer (10 mM HEPES [pH 7.5], 100 mM NaCl, 1 mM EDTA, 10% glycerol, 0.1% Triton X-100, and 1 X protease inhibitor cocktail) and once more with 50 mM Tris-HCl [pH 7.5]. Bound protein was released from beads by boiling in SDS-PAGE sample loading buffer and analyzed by Western blot with an α-FLAG antibody.

For co-IP assay in plants, 7-day-old seedlings grown on ½ MS medium plates were treated with 5 μM DEX for overnight to induce HopF2 or AvrPto expression and were then ground with liquid nitrogen. The total proteins from 50 seedlings were isolated with 1 mL of extraction buffer. The samples were centrifuged twice at 13,000 rpm for 10 min at 4 °C to remove cell debris. The supernatant was subjected into an α-GFP co-IP assay and the immunoprecipitated proteins were analyzed by Western blot with an α-HA antibody.

GST pull-down assay

GST, GST-AvrPto and GST-HopF2 were individually expressed in E. coli BL21 strain and purified with standard glutathione agarose. 2 × 105 protoplasts were transfected with full length or truncated version of BAK1 construct tagged with an FLAG epitope at its C-terminus. The total proteins were isolated with 0.5 ml of extraction buffer. The samples were vortexed vigorously for 30 s, and then centrifuged at 13,000 rpm for 10 min at 4°C. The supernatant was inoculated with prewashed GST or GST-tagged protein for 2 hr at 4°C with gentle shaking. The beads were harvested and washed 3 times with washing buffer and once with 50 mM Tris·HCl [pH7.5]. Bound protein was released from beads by boiling in SDS-PAGE sample loading buffer and analyzed by Western blot with an α-FLAG antibody.

BiFC assay

Arabidopsis protoplasts were co-transfected with various BiFC constructs as indicated in the figures. Complementation of fluorescence signal was visualized under a confocal microscope (Leica Microsystems CMS GmbH) 18 hr after transfection. Following are the filter sets used for excitation (Ex) and emission (Em): GFP, 488 nm (Ex)/BP505 nm to 530 nm (Em); chlorophyll, 543 nm (Ex)/LP650 nm (Em); bright field, 633 nm. Images were captured in a multichannel mode, and were analyzed and processed with Leica LAS AF Life and Adobe Photoshop (Adobe Systems).

Immunocomplex kinase assays

2 × 105 protoplasts transfected with various DNA constructs were lysed with 0.5 ml of IP buffer (50 mM Tris-HCl [pH 7.5], 150 mM NaCl, 5 mM EDTA, 1 mM DTT, 2 mM NaF, 2 mM Na3VO3, 1% Triton, and protease inhibitor cocktail). The samples were vortexed vigorously for 30 s, and then centrifuged at 13,000 rpm for 10 min at 4°C. The supernatant was incubated with an α-HA antibody for 2 hr and then with protein-G–agarose beads for another 2 hr at 4°C with gentle shaking. The beads were harvested and washed once with IP buffer and once with kinase buffer (20 mM Tris-HCl [pH 7.5], 10 mM MgCl2, 5 mM EGTA, 100 mM NaCl, and 1 mM DTT). The kinase reactions were performed in 20 μl of kinase buffer with 2 μg of myelin basic protein as a substrate, 0.1 mM cold ATP, and 5 μCi of [32P]-γ-ATP at room temperature for 1 hr with gentle shaking. The phosphorylation of proteins was analyzed by 12% SDS-PAGE.

Supplementary Material

Acknowledgments

We thank Dr. Jianmin Li for pBAK1-BAK1-GFP transgenic plant seeds. This work was supported by NIH (R01 GM70567) to J.S., NSF (IOS-1030250) to L.S., NIH (R01GM092893) to P.H.

Footnotes

AUTHOR CONTRIBUTION

Conceived and designed the experiments: JZ, SW, JS, LS, PH; Performed the experiments: JZ, SW, XC, CL; Analyzed the data: JZ, SW, JS, LS, PH; Wrote the paper: JZ, LS, PH.

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