The Decoy Substrate of a Pathogen Effector and a Pseudokinase Specify Pathogen-Induced Modified-Self Recognition and Immunity in Plants

Article

The Decoy Substrate of a Pathogen Effector and a Pseudokinase Specify Pathogen-Induced ModifiedSelf Recognition and Immunity in Plants Graphical Abstract

Authors Guoxun Wang, Brice Roux, Feng Feng, ..., Chaozu He, Laurent D. Noe¨l, Jian-Min Zhou

Correspondence [email protected] (L.D.N.), [email protected] (J.-M.Z.)

In Brief A plant pathogen effector, AvrAC, uridylylates a host kinase to promote virulence. Wang et al. find that plants have evolved a decoy substrate which upon uridylylation by AvrAC is recognized by a pseudokinase-immune receptor complex to trigger immunity. Thus, decoy substrates and pseudokinases specify and expand immune capacity in plants.

Highlights d

BIK1 paralog PBL2 is uridylylated by X. campestris effector AvrAC/XopAC

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PBL2 uridylylation is not required for AvrAC-mediated virulence but triggers immunity

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AvrAC recognition requires the NLR ZAR1 and pseudokinase RKS1 that are in a complex

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The uridylylated PBL2 is specifically recruited to RKS1 to trigger immunity

Wang et al., 2015, Cell Host & Microbe 18, 285–295 September 9, 2015 ª2015 Elsevier Inc. http://dx.doi.org/10.1016/j.chom.2015.08.004

Cell Host & Microbe

Article The Decoy Substrate of a Pathogen Effector and a Pseudokinase Specify Pathogen-Induced Modified-Self Recognition and Immunity in Plants Guoxun Wang,1,9 Brice Roux,2,3,9 Feng Feng,1,9 Endrick Guy,2,3 Lin Li,4 Nannan Li,4 Xiaojuan Zhang,1 Martine Lautier,2,3,5 Marie-Franc¸oise Jardinaud,2,3,6 Matthieu Chabannes,2,3,8 Matthieu Arlat,2,3,5 She Chen,4 Chaozu He,7 Laurent D. Noe¨l,2,3,* and Jian-Min Zhou1,* 1State Key Laboratory of Plant Genomics, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, No. 1 West Beichen Road, Beijing 100101, China 2INRA, Laboratoire des Interactions Plantes-Micro-organismes (LIPM), UMR 441, F-31326 Castanet-Tolosan, France 3CNRS, Laboratoire des Interactions Plantes-Micro-organismes (LIPM), UMR 2594, F-31326 Castanet-Tolosan, France 4National Institute of Biological Sciences, Zhongguancun Life Science Park, Changping, Beijing 102206, China 5Universite ´ de Toulouse, UPS, F-31062 Toulouse, France 6INPT-Universite ´ de Toulouse, ENSAT, Avenue de l’Agrobiopole, Auzeville-Tolosane, F-31326 Castanet-Tolosan, France 7Hainan Key Laboratory for Sustainable Utilization of Tropical Bioresources, College of Agriculture, Hainan University, Haikou 570228, China 8Present address: CIRAD, UMR BGPI, F-34398 Montpellier, France 9Co-first author *Correspondence: [email protected] (L.D.N.), [email protected] (J.-M.Z.) http://dx.doi.org/10.1016/j.chom.2015.08.004

SUMMARY

In plants, host response to pathogenic microbes is driven both by microbial perception and detection of modified-self. The Xanthomonas campestris effector protein AvrAC/XopAC uridylylates the Arabidopsis BIK1 kinase to dampen basal resistance and thereby promotes bacterial virulence. Here we show that PBL2, a paralog of BIK1, is similarly uridylylated by AvrAC. However, in contrast to BIK1, PBL2 uridylylation is specifically required for host recognition of AvrAC to trigger immunity, but not AvrAC virulence. PBL2 thus acts as a decoy and enables AvrAC detection. AvrAC recognition also requires the RKS1 pseudokinase of the ZRK family and the NOD-like receptor ZAR1, which is known to recognize the Pseudomonas syringae effector HopZ1a. ZAR1 forms a stable complex with RKS1, which specifically recruits PBL2 when the latter is uridylylated by AvrAC, triggering ZAR1-mediated immunity. The results illustrate how decoy substrates and pseudokinases can specify and expand the capacity of the plant immune system.

INTRODUCTION Plants rely on cell-surface-localized pattern recognition receptors (PRRs) and intracellular NOD-like receptors (NLRs) for pathogen detection (Dodds and Rathjen, 2010; Maekawa et al., 2011; Monaghan and Zipfel, 2012). The former are comprised of receptor kinases (RKs) and receptor-like proteins that are functionally analogous to Toll-like receptors in animals and directly perceive molecular patterns derived from invading microbes or released

from the plant upon infection and set off pattern-triggered immunity (PTI). Pathogenic microbes often deliver into the host cell effector proteins for virulence (Feng and Zhou, 2012). As a result of host-pathogen coevolution, the effectors are monitored by plants in a highly specific manner and confer effector-triggered immunity (ETI) when cognate NLRs are present (Cui et al., 2015; Jones and Dangl, 2006). Recent reports show that bacterial pathogen effector proteins are also detected by animal intracellular immune receptors including NLRs and pyrin (Keestra et al., 2013; Mu¨ller et al., 2010; Yarbrough et al., 2009), highlighting similarities between plant and animal innate immunity. Plant NLRs recognize pathogen effectors either directly or indirectly (Cui et al., 2015; Jones and Dangl, 2006). Pathogen effectors often possess enzymatic activities that modify host proteins to their benefit. Several plant NLRs indirectly recognize pathogen effectors by interacting with other host proteins when the latter are modified by effectors (Axtell and Staskawicz, 2003; Chung et al., 2011; Kim et al., 2005; Liu et al., 2011; Shao et al., 2003). These modified proteins can either be virulence targets of the effectors or presumed decoys that mimic virulence targets (van der Hoorn and Kamoun, 2008; Zhou and Chai, 2008). Animal intracellular immune receptors may also detect pathogen effectors by indirectly interacting with a modified host protein, although this has not been convincingly shown (Vanaja et al., 2015). Thus the nature of host protein modification by pathogen effectors and mechanisms by which NLRs recognize pathogen effectors hold a key to our understanding of host-pathogen coevolution. One class of such effector proteins contains the FIC (filamentation induced by cAMP) domain that is shared by several bacterial pathogens infecting plants and animals. The animal bacterial pathogen FIC domain effectors IbpA, VopS, and AnkX adenylylate or phosphocholinate human Rho GTPase for virulence (Mukherjee et al., 2011; Worby et al., 2009; Yarbrough et al., 2009). Interestingly, the modification of a subfamily of Rho by VopS and several other bacterial effectors indirectly triggers

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pyrin inflamasome activation (Xu et al., 2014). The Xanthomonas campestris pv. campestris (Xcc) effector AvrAC/XopAC (Xu et al., 2008) uridylylates and inhibits several plant receptor-like cytoplasmic kinases belonging to family VII (RLCK VII), including BIK1 and RIPK (Feng et al., 2012). BIK1 is a critical component of multiple plant PRR complexes, whereas RIPK is necessary for ETI against Pseudomonas syringae effector AvrB (Liu et al., 2011; Lu et al., 2010; Zhang et al., 2010). While the inhibition of BIK1 dampens the BIK1-mediated PTI and RIPK-mediated ETI in mesophyll tissues (Feng et al., 2012), AvrAC is recognized in some Arabidopsis ecotypes, including Col-0, and triggers ETI in vascular tissues (Xu et al., 2008). Interestingly, a homolog of BIK1, PBL2, is required for ETI against Xcc carrying avrAC (Guy et al., 2013). However, the genetic and molecular mechanisms underlying AvrAC recognition remain unknown. Here we show evidence that PBL2 acts as a decoy for AvrAC enzymatic activity, as it is solely required for AvrAC-dependent ETI in vascular tissues but not virulence function of AvrAC in mesophyll tissues. The recognition of AvrAC also requires the Arabidopsis NLR protein ZAR1 and a pseudokinase belonging to RLCK XII, RKS1. ZAR1 and RKS1 exist in a preformed complex that specifically recruits PBL2 only when the latter is uridylylated by AvrAC. In this complex the recruitment of uridylylated PBL2 by RKS1 is required for the activation of ZAR1-dependent ETI, indicating a specific and indirect recognition of modified decoy by the plant pseudokinase RKS1. In addition, ZAR1 is known to form a complex with another RLCK XII pseudokinase, ZED1, to recognize the Pseudomonas syringae effector HopZ1a. We show that RKS1 and ZED1 display high specificities in the recognition of AvrAC and HopZ1a, respectively, suggesting that these pseudokinases act as sensors for distinct host protein modifications and confer expanded recognition specificities to ZAR1. RESULTS PBL2 Acts as a BIK1 Decoy in AvrAC Recognition We previously showed that PBL2 interacts with AvrAC (Guy et al., 2013b). In vitro uridylylation assay was used to test whether AvrAC uridylylates PBL2 similarly to BIK1, RIPK, or PBL1 (Feng et al., 2012). The PBL2 recombinant protein was uridylylated by AvrAC, but not the catalytic-deficient mutant AvrACH469A (Figure 1A). PBL2 Ser253 and Thr254 are conserved residues in the activation loop of members of the RLCK VII family. We previously showed that these conserved residues were uridylylated by AvrAC in BIK1 and RIPK. The PBL2S253A/T254A mutant protein was not uridylylated by AvrAC in vitro (Figure 1A), suggesting that Ser253 and Thr254 are major uridylylation sites in PBL2. Mass spectrum analysis on the AvrAC-modified PBL2 confirmed that Ser253 and Thr254 were indeed uridylylated by AvrAC (Figure 1B). We further conducted complementation experiments to determine if the uridylylation is required for the AvrAC-triggered ETI in vascular tissues by introducing PBL2 wild-type and uridylylation site mutants under its native promoter as stable transgenes into pbl2 plants. As indicated by disease symptom and bacterial growth assays (Figures 1C and 1D), only the wild-type (WT) PBL2, but not PBL2S253A, PBL2T254A, and PBL2S253A/T254A, restored full vascular resistance to Xcc. These experiments indicated that both uridylylation sites were

essential for the AvrAC-induced ETI in plants. The results raised the possibility for PBL2 being a BIK1 decoy, since BIK1 is required for virulence in mesophyll tissues but not vascular ETI triggered by AvrAC (Feng et al., 2012; Guy et al., 2013b). To test this hypothesis, we performed competitive index assays on WT, bik1, and pbl2 plants by directly infiltrating Xcc bacteria into mesophyll tissues. The avrAC-deletion strain (XccB186DavrAC) multiplied at a lower level compared to the WT strain (XccB186) on WT and pbl2 plants, indicating that PBL2 is not needed to convey avrAC virulence functions. In contrast, both strains multiplied to similar levels on bik1 plants (Figure 1E), indicating that BIK1 alone is sufficient to mediate avrAC virulence functions, a result consistent with previous findings (Feng et al., 2012). Thus the results support that PBL2 is a BIK1 decoy for AvrAC recognition, since it contributes solely to AvrAC ETI in vascular tissues. ZAR1 NLR Protein and RKS1 Pseudokinase Are Required for AvrAC Recognition To study avrAC recognition in the plant independently from bacterial infection and other Xcc type III effectors, we investigated the effects of inducible expression of avrAC in stable transgenic Col-0 Arabidopsis seedlings. AvrAC expression resulted in seedling growth arrest (Figure 2A and see Figures S1A–S1D available online) in a manner dependent on PBL2 but not RIPK (Figure 2B), thus linking AvrAC-induced developmental phenotypes with AvrAC-triggered immunity independent from bacterial infection and other Xcc type III effectors. We therefore took advantage of this seedling growth arrest and screened for suppressors of XopAC phenotypes (sxc) in a nonsaturated EMS-mutagenized population of an avrAC transgenic line. Three mutants, sxc3, sxc5, and sxc7, were insensitive to avrAC expression (Figure 2A). All three mutants inoculated with WT Xcc displayed disease symptoms (Figure 2C) and increased levels of bacterial growth in leaves compared to parental lines (Figure 2D). Map-based cloning of these mutations was achieved by deep sequencing of DNA bulks of F2 sxc backcrossed plants growing normally on inducing medium (Figures S2A and S3A). Additional genetic characterizations demonstrated that the sxc phenotype was caused by mutations in RKS1/ZRK1 and ZAR1 (Figures 2B, S2B, and S3B). ZAR1 (HopZ-activated resistance 1) is an NLR containing an N-terminal coiled-coil (CC) domain, a central NBARC domain, and a C-terminal LRR domain and is required for the recognition of the Pseudomonas syringae effector HopZ1a (Lewis et al., 2010). In vitro, HopZ1a acetylates the RLCK XII ZED1, a pseudokinase required for HopZ1a-induced ETI (Lewis et al., 2013). RKS1 is a close relative of ZED1 and has recently been shown to confer broad-spectrum quantitative resistance to Xcc (Huard-Chauveau et al., 2013). sxc5 caused a Pro359Leu substitution in a highly conserved motif of the NB-ARC domain of ZAR1, whereas sxc7 led to a Pro816Leu substitution in the last LRR of ZAR1 (Figure S2E). sxc3 carried the Leu179Phe substitution in RKS1, a position highly conserved in RLCKs and RKs (Figures S3E and S3F). Importantly, wound inoculation of zar1-1, zar1-2, zar1-6, and rks1-1 with both WT and the DavrAC mutant strains of Xcc strain 8004 showed that these mutants were susceptible, in contrast to the WT Col-0 plants, which were resistant to the WT Xcc strain but susceptible to the DavrAC strain (Figures 2E, 2F, S2C, S2D, S3C, and S3D). In addition, an

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Figure 1. PBL2 Is a Decoy for AvrAC (A) AvrAC uridylylates PBL2 in vitro primarily at Ser253 and Thr254. Recombinant His-PBL2 and His-PBL2S253A/T254A proteins were incubated with GSTAvrAC or GST-AvrACH469A in the presence of a32P-UTP and subjected to SDS-PAGE and autoradiography. The differential migration of His-PBL2 and HisPBL2S253A/T254A in SDS-PAGE might be caused by differential autophosphorylation of these proteins, since His-PBL2S253A/T254A lacks a functional activation loop. (B) PBL2 Ser253 and Thr254 are uridylylated by AvrAC. Tandem mass spectrum of a tryptic peptide of His-PBL2 coexpressed with GST-AvrAC. The b and y ions are marked and displayed along the peptide sequence on top of the graph. (C and D) PBL2 Ser253 and Thr254 are required for avrAC-specified resistance to Xcc. Plants of the indicated genotypes were wound-inoculated in the main vein of leaves with Xcc8004 (WT) and Xcc8004DavrAC (DAC), which led to vascular infection. Disease symptoms of representative leaves were photographed (C), and bacterial growth assay was conducted (D). (E) PBL2 is not required for avrAC virulence. Competitive index assay was performed on plants of the indicated genotype by infiltrating leaf mesophyll with an equal mixture of XccB186 and XccB186DavrAC. Statistical groups were determined using a parametric Tukey test (adjusted p < 0.01) and are indicated by different letters. See also Figure S4.

independent screen of a collection of Col-0 T-DNA insertion mutants for candidate NLRs identified a single mutant, zar1-6, which was susceptible to both WT and the DavrAC mutant strains of Xcc strain 8004 (Table S1). Altogether, these results demonstrate the direct involvement of ZAR1, RKS1, and PBL2 in the recognition of avrAC in vascular tissues. We further tested if ZAR1 and RKS1 contribute to AvrAC virulence functions in mesophyll tissues by conducting competitive index assay (Figure S4). Mutations in zar1-1 and rks1-1 did not impact avrAC-induced virulence, confirming that, similar to PBL2 (Figure 1E), ZAR1 and RKS1 are only important for AvrAC recognition in vascular tissues.

The Genetics of AvrAC Recognition Can Be Recapitulated in Arabidopsis Protoplasts We sought to employ a protoplast-based transient expression system to facilitate the analysis of AvrAC recognition by PBL2, ZAR1, and RKS1 proteins. Coexpression of AvrAC with PBL2, RKS1, and ZAR1 in protoplasts isolated from pbl2, rks1, or zar1 plants, respectively, led to cell death indicative of ETI activation, whereas coexpression of only two host proteins failed to induce cell death (Figure S5A). Similarly to what was observed in sxc5 and sxc7 mutants, ZAR1P359L and ZAR1P816L mutant proteins were completely unable to trigger cell death when coexpressed with AvrAC, PBL2, and RKS1 (Figure S5B). ZAR1

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Figure 2. ZAR1 and RKS1 Are Required for avrAC Recognition (A) sxc mutants are resistant to AvrAC-induced growth inhibition. (B) pbl2, zar1, and rks1 are insensitive to the AvrAC-induced growth inhibition. (C and D) sxc mutants are susceptible to Xcc8004 as indicated by the disease index assay (C) and bacterial growth assay (D). (E and F) pbl2, zar1, and rks1 plants display full susceptibility to Xcc8004 as indicated by bacterial growth assay (E) and disease symptoms (F). Shown are Arabidopsis plants of the indicated genotype following wound inoculation of main leaf vein with Xcc8004 (WT) or Xcc8004DavrAC (DAC). Statistical groups were determined using a parametric Tukey test (adjusted p < 0.01) and are indicated by different letters. See also Table S1 and Figures S1–S3.

Lys195 is a residue conserved in the NB-ARC domain predicted to be required for nucleotide binding and NLR function (Ade et al., 2007). Coexpression of the ZAR1K195N mutant with AvrAC, PBL2, and RKS1 failed to trigger AvrAC ETI in protoplasts (Figure S5C). Finally, PBL2S253A, PBL2T254A, and PBL2S253A/T254A were all unable to trigger cell death when coexpressed with RKS1, ZAR1, and AvrAC (Figure S5D), indicating that both uridylylation sites were required for the AvrAC-triggered cell death in protoplasts. Thus the protoplast assay fully recapitulates the genetics of avrAC-triggered immunity in plants and justified its use as a tool to dissect further the molecular mechanisms of AvrAC recognition. RKS1 and ZAR1 Exist in a Preformed Complex We first performed pairwise coimmunoprecipitation (coIP) assays to test if and how AvrAC, PBL2, RKS1, and ZAR1 proteins interacted with each other. Consistent with PBL2 being a substrate of AvrAC, AvrAC interacted with PBL2, but not RKS1 nor ZAR1 (Figure 3A). Consistent with these results, in vitro uridylylation assay indicated that, unlike PBL2, RKS1 is not uridylylated by AvrAC (Figure S6A). ZAR1 interacted with RKS1, but not AvrAC, PBL2, BIK1, or RIPK (Figures 3A, 3B, S6B, and S6C). The ZAR1-RKS1 interaction in plants was also detected in a split-luciferase assay (Figure S6D). However, RKS1 failed to interact with RPM1, a different CC-NLR (Figure S6B), indicating that RKS1 does not generally interact with CC-NLRs. The ZAR1-RKS1 interaction was not affected by the presence of AvrAC (Figure S6E), indicating that ZAR1 and RKS1 exist in a preformed complex. We next examined the impact of the ZAR1P359L and ZAR1P816L sxc mutations on ZAR1-RKS1 inter-

action. While ZAR1P359L was still capable of interacting with RKS1, ZAR1P816L failed to interact with RKS1 (Figure 3C), thus suggesting the involvement of the ZAR1 LRR domain in RKS1-interaction and potentially explaining the mutant phenotype of sxc7. Deletion analyses indicated that the LRR domain of ZAR1 is necessary and sufficient for the interaction with RKS1 (Figures 3D and 3E). Furthermore, the NB-ARC mutant protein ZAR1K195N still interacted with RKS1 (Figure 3C). These results indicated that the ZAR1 LRR domain is required for RKS1-interaction, whereas the NB-ARC domain likely functions in signal transduction. We further generated transgenic plants expressing epitope-tagged RKS1 and ZAR1 under the control of their native promoters. CoIP experiment showed that the tagged RKS1 and ZAR1 indeed interacted in Arabidopsis plants (Figure 3F). AvrAC Induces the Recruitment of PBL2 to RKS1-ZAR1 Complex While PBL2 and RKS1 did not interact by themselves, coexpression of the WT AvrAC, but not the catalytic mutant AvrACH469A, induced a strong PBL2-RKS1 interaction (Figure 4A), indicating that AvrAC enzymatic activity is necessary for the PBL2-RKS1 interaction. In contrast to the WT RKS1, RKS1L179F failed to interact with PBL2 in the presence of AvrAC (Figure 4B). However, RKS1L179F did not affect RKS1-ZAR1 interaction (Figure 4C), indicating that RKS1 Leu179 is specifically required for the AvrAC-induced recruitment of PBL2 to RKS1. To determine if the bacterially delivered AvrAC similarly induces the recruitment of PBL2 to RKS1, we generated transgenic plants expressing epitope-tagged RKS1 and PBL2 under the control of their native promoters. Inoculation of these plants with the WT Xcc, but not the strain lacking avrAC (DAC), specifically induced PBL2-RKS1 interaction in plants (Figure 4D). Protoplast-based coIP assays were performed to address whether AvrAC induces PBL2-ZAR1 interaction. The NB-ARC mutant protein ZAR1K195N was used instead of WT ZAR1 in

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Figure 3. RKS1 and ZAR1 Exist in a Preformed Complex (A) AvrAC interacts with PBL2, but not RKS1 and ZAR1. (B) RKS1 interacts with ZAR1, but not AvrAC or PBL2. (C) ZAR1 Pro816 is required for the interaction with RKS1. (D) Schematic presentation of ZAR1 deletion constructs. (E) The ZAR1 LRR domain is required and sufficient for the interaction with RKS1. (F) RKS1 and ZAR1 interact in stable transgenic plants. CoIP assays were performed in Col-0 protoplasts transfected with the indicated constructs (A–E) or in plants carrying the indicated transgenes under the control of their native promoters (F). See also Figures S5 and S6.

this experiment to avoid cell death when all components are coexpressed (Figure S5C). While AvrAC failed to induce PBL2ZAR1K195N interaction in the absence of RKS1, it did when RKS1 was coexpressed, suggesting the formation of a PBL2RKS1-ZAR1 complex (Figure 4E). Again, the PBL2-ZAR1 interaction required an enzymatically active AvrAC. While AvrAC stimulated the PBL2-ZAR1K195N and PBL2-ZAR1P359L interactions, it failed to induce the PBL2-ZAR1P816L interaction (Figure 4F), indicating that a ZAR1-RKS1 interaction is prerequisite for the AvrAC-induced PBL2-ZAR1 interaction. We further tested if the AvrAC-induced recruitment of PBL2 to RKS1 is mediated by specific uridylylation on PBL2 Ser253 and Thr254. CoIP assay showed that, in contrast to the WT PBL2 protein, PBL2S253A, PBL2T254A, and PBL2S253A/T254A were all unable to interact with RKS1 upon AvrAC coexpression (Figure 4G), indicating that the double uridylylation of Ser253 and Thr254 was required for the AvrAC-induced recruitment of PBL2 to RKS1. Together these results indicate that RKS1 in a stable complex with ZAR1 recruits PBL2 when the latter is uridylylated by AvrAC at Ser253 and Thr254.

PBL2 Kinase Activity Is Not Required for AvrAC Recognition We tested if PBL2 kinase activity is relevant to uridylylation and ETI triggered by AvrAC. PBL2 uridylylation was correlated with a protein mobility-shift assay which was used to monitor posttranslational modification of various PBL2 mutants. PBL2K124E, in which the ATP-binding site is mutated, did not appear to be affected in the uridylylation (Figure 5A), suggesting that the PBL2 kinase activity is not required for its modification by AvrAC. Protoplast viability assay indicated that WT PBL2 and PBL2K124E were equally capable of inducing cell death when coexpressed with RKS1 and ZAR1, indicating that the PBL2 kinase activity is not required for AvrAC-induced cell death (Figure 5B). In support of this observation, PBL2D219A, PBL2G247R, PBL2Y262A, and PBL2K124A, which were predicted to be defective in kinase activity or lacking major phosphorylation sites (Lin et al., 2014), were similarly capable of inducing death in protoplasts when coexpressed with ZAR1, RKS1, and AvrAC (Figure 5B). Coexpression of these PBL2 variants with AvrAC resulted in similar mobility shift of PBL2 proteins, suggesting that they were not affected

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Figure 4. AvrAC Induces the Recruitment of PBL2 to RKS1 (A) The AvrAC uridylylate-transferase activity induces a PBL2-RKS1 interaction in protoplasts. (B) RKS1 Leu179 is critical for the AvrAC-induced PBL2-RKS1 interaction in protoplasts. (C) RKS1 Leu179 is not required for RKS1-ZAR1 interaction. (D) The bacterially delivered AvrAC induces PBL2-RKS1 interaction in plants. (E) AvrAC induces PBL2-ZAR1 interaction in a RKS1-dependent manner. (F) ZAR1 Pro816 is required for the AvrAC-induced PBL2-ZAR1 interaction. (G) Uridylylation at Ser253 and Thr254 is required for the recruitment of PBL2 to RKS1. A ZAR1K195N mutant, which is abolished in ATP binding and signaling, was used to avoid cell death induction (E and F). For protoplast-based coIP assays, Col-0 (C) or zar1 (A, B, and E–G) protoplasts were transfected with the indicated constructs. For AvrAC-induced PBL2-RKS1 interaction in plants (D), plants carrying the indicated transgenes under the control of native promoters were inoculated with the WT Xcc or the mutant Xcc strain lacking avrAC (DAC) prior to coIP assay. Note that AvrAC-HA was not detected in some of the coIP experiments (A, B, and E–G) because of short exposures used for immune detection, indicating that the PBL2 interactions with RKS1 and ZAR1 were much stronger than the PBL2-AvrAC interaction.

in uridylylation (Figure 5C). Together these results support that PBL2 kinase activity is not required for uridylylation nor for recognition of AvrAC, consistent with PBL2 being a mere decoy. Single RLCK XII and VII Genes RKS1 and PBL2 Specifically Mediate AvrAC Recognition The aforementioned results and previous reports suggested that RLCK VII and RLCK XII members play multiple roles in plant-bacteria interactions, including PTI, ETI, and effector-triggered sus-

ceptibility (Feng et al., 2012; Guy et al., 2013b; Huard-Chauveau et al., 2013; Lewis et al., 2013; Liu et al., 2011, 2013; Lu et al., 2010; Shao et al., 2003; Zhang et al., 2010). We tested if, similarly to RKS1 and ZED1, additional RLCK XII members would also interact with ZAR1. As shown in Figure 6A, the three tested ZRKs (ZRK3, ZRK6, and ZRK15) all interacted with ZAR1, indicating that ZAR1 can interact with multiple RLCK XII. Pro816 appeared to be essential for ZAR1 interaction with RLCK XII members, as ZAR1P816L compromised the interaction with ZED1, ZRK3, ZRK6,

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Figure 5. PBL2 Kinase Activity Is Not Required for the AvrAC-Triggered Cell Death (A) AvrAC induces band-shift of both WT PBL2 and the kinase-dead mutant PBL2K124E in protoplasts. (B) Kinase-dead and phosphosite mutants of PBL2 showed wild-type AvrAC-induced cell death in protoplast viability assay. Data are represented as mean ± SEM. (C) AvrAC induces band-shift of PBL kinase-deficient and phosphosite mutants. Protoplasts were isolated from Col-0 (A and C) or pbl2 (B) plants and transfected with the indicated constructs, and analyzed by immunoblot (A and C) or cell death assays (B).

and ZRK15 as observed for RKS1 (Figure S7A). Unlike RKS1, however, ZED1 was unable to recruit PBL2 upon AvrAC induction (Figure 6B) and did not participate in avrAC ETI (Figure S5A), indicating that RKS1 is specifically involved in the recruitment of uridylylated PBL2 to the ZAR1 complex. Protoplast viability assay showed that among the 13 RLCK XII members tested, RKS1 is the only one mediating avrAC ETI (Figures 7C and S7B). Thus, RKS1 seems to be the only RLCK XII required for AvrAC recognition. We also tested whether RLCK VII members other than PBL2 might be recruited by RKS1 in the presence of AvrAC. In contrast to PBL2, BIK1 and RIPK were not recruited to RKS1 (Figure 6B), although BIK1 and RIPK are uridylylated in vitro by AvrAC in the conserved serine and threonine of the activation loop (Feng et al., 2012). These results indicate that PBL2 is a specific RLCK VII member recruited to RKS1 upon uridylylation by AvrAC. Consistent with this possibility, inoculation of Arabidopsis mutants for 39 RLCK VII members with Xcc showed that PBL2 was the only RLCK VII tested genetically required for AvrAC ETI (Figure 6D; Table S2). Lack of Genetic Overlap between AvrAC and HopZ1a Recognition We tested in parallel to AvrAC recognition the involvement of RLCK XII and RLCK VII members in HopZ1a ETI. While zar1-1and zed1 mutants were defective in bacterial growth restriction and HR when challenged with P. syringae expressing hopZ1a, the rks1-1 and pbl2 mutants were indistinguishable from WT Col-0 plants (Figures 6E and S7C), indicating that ZAR1 and ZED1, but not RKS1 and PBL2, are required for HopZ1a ETI. In addition, none of the 39 RLCK VII members tested were required for HopZ1a ETI in HR assays (Table S2). The results thus demonstrated a high specificity of RLCK VII and RLCK XII members involved in the recognition of distinct effectors, namely ZED1 for HopZ1a and PBL2 and RKS1 for AvrAC. DISCUSSION In this study we show that a CC-NLR (ZAR1), a specific RLCK XII member (RKS1), and a specific RLCK VII member (PBL2) act

together to mediate AvrAC-induced ETI against Xcc. ZAR1 and RKS1 form a preactivation complex independently of pathogen challenge. The uridylylation of PBL2 at both Ser253 and Thr254 by AvrAC triggers the PBL2RKS1 interaction and thus the assembly of an ETI-competent PBL2-RKS1-ZAR1 complex (Figure 7). Unlike previously reported effector targets that are directly guarded by NLRs (Axtell and Staskawicz, 2003; Dodds and Rathjen, 2010; Kim et al., 2005; Maekawa et al., 2011), PBL2 is indirectly guarded by ZAR1 through the adaptor protein RKS1. Importantly, PBL2 is an unlikely virulence target of AvrAC, because PBL2 plays a minimum role, if any, in PTI signaling (Zhang et al., 2010). Furthermore, PBL2 has no measurable contribution to AvrAC virulence functions in mesophyll tissues, and BIK1 alone can explain all the AvrAC virulence functions in Arabidopsis (Figure 1E). Thus our findings and previous work performed on AvrAC ETI present an example of decoy in which the RKS1-ZAR1 complex guards PBL2 modification by AvrAC to indirectly protect the infected plant tissues from inhibition of BIK1 and PTI suppression (Figure 7; van der Hoorn and Kamoun, 2008; Zhou and Chai, 2008). The utilization of a decoy to recognize pathogen effectors may be a common theme in plant NLRs. Indeed, recent reports show that the Arabidopsis RRS1 NLR uses its WRKY DNA-binding domain as an effector target decoy to sense pathogen interference with host defensive WRKY transcription factors (Le Roux et al., 2015; Sarris et al., 2015). Thus, the modification of a decoy by an effector can be sensed indirectly by a preformed NLR complex, as shown in this study, or directly by an NLR containing an integrated decoy (Le Roux et al., 2015; Sarris et al., 2015). This study indicates that quantitative disease resistance can be achieved by a typical R gene-mediated effector-triggered immunity as speculated (Lewis et al., 2014). Here, the RKS1mediated quantitative resistance to Xcc might be due to the suboptimal induction of plant defenses leading to partial resistance without macroscopic HR (Jones and Dangl, 2006; Poland et al., 2009). In support of this hypothesis, AvrAC recognition triggers strong cell death in protoplasts upon overexpression of RKS1, ZAR1, and PBL2. Several plant NLRs confer resistance to multiple pathogens (Birker et al., 2009; Milligan et al., 1998; Narusaka et al., 2009; Periyannan et al., 2013; Rossi et al., 1998; Takken et al.,

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Figure 6. Specificity of PBL and ZRK Family Members in avrAC and hopZ1a Recognition (A) ZAR1 interacts with multiple ZRK-RLCK XII family members in coIP assay. (B) Specificity of the AvrAC-induced recruitment of PBL2 to RKS1 in coIP assay. (C) Specificity of ZRK family members in the AvrAC-induced cell death in protoplast viability assay. Data are represented as mean ± SE. (D) Other PBLs-RLCK VII are not required for vascular disease resistance to avrAC in bacterial growth assay. (E) Specificity of ZED1 in hopZ1a recognition as indicated by bacterial growth assay. Statistical groups were determined using a parametric Tukey test (adjusted p < 0.01) and are indicated by different letters. See also Figure S7 and Table S2.

2006; Vos et al., 1998), likely by recognizing unrelated effectors (Bisgrove et al., 1994; Kim et al., 2002; Narusaka et al., 2009). Recent advances show that the ability of an NLR to recognize multiple effectors/pathogens played an important role in the evolution of plant NLRs (Karasov et al., 2014). The molecular basis for the expanded recognition specificities, however, is poorly understood. ZAR1 is one of the few ancient and evolutionary conserved NLRs present in all Arabidopsis ecotypes and crucifers plants examined to date (Peele et al., 2014). Our results show that, in addition to P. syringae, ZAR1 also confers resistance to Xcc. In this case, the association with two RLCK XII members, RKS1 and ZED1, respectively, allowed ZAR1 to confer distinct recognition specificities against unrelated effectors. Though the RKS1 and ZED1 pseudokinases are unlikely bona fide kinases (Huard-Chauveau et al., 2013; Lewis et al., 2013), they could function as scaffold/adaptor proteins as proposed for RKS1 in this study (Lewis et al., 2013). All the RLCK XII members tested were able to interact with ZAR1, suggesting that other RLCK XII members could confer additional recognition specificities to ZAR1 and thus maximize NLR surveillance capa-

bilities. This may be analogous to the tomato kinases Pto and Fen, which interact with the Prf NLR and confer overlapping but differential recognition specificities (Mathieu et al., 2014). Our findings add to the growing list of RLCK VII members guarded by NLRs (Ade et al., 2007; Liu et al., 2011; Shao et al., 2003), supporting that RLCK VII members are a hub targeted by multiple pathogen effectors and have played an important role in the arms race during plant-pathogen coevolution. FIC domain effectors are utilized by both animal and plant bacterial pathogens. Whereas AvrAC targets RLCK VII, the animal pathogen FIC domain effectors are all known to target Rho GTPases. Interestingly, Rho GTPases are also targeted by multiple animal pathogen effectors that do not possess the FIC domain (Aktories, 2011), indicating that Rho family proteins are a hub targeted by various animal bacterial pathogens. Not surprisingly, multiple Rho-targeting effectors activate NLR or pyrin inflamasomes (Keestra et al., 2013; Xu et al., 2014). These findings thus highlight remarkable analogy of host-pathogen coevolution in both plant and animal systems. It is worth noting that NLRs and pyrin do not appear to directly interact with the Rho-modifying effectors.

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Figure 7. Model for AvrAC and HopZ1aMediated ETI in Arabidopsis Xanthomonas AvrAC was already reported to inhibit BIK1 RLCK VII by uridylylation to dampen PTI. AvrAC also modifies other RLCK VII such as the PBL2 decoy, thus triggering its recruitment to a pre-existing complex made of the RLCK XII RKS1 and the NLR ZAR1 causing ETI. During recognition of HopZ1a, the RLCK XII ZED1 guardee is acetylated directly by HopZ1a, which activates ZAR1dependent ETI.

Future research is needed to determine if these intracellular immune receptors recognize effectors through an interaction with effector-modified Rho proteins. Furthermore, a thorough analysis of specific roles of individual Rho family members in pathogen virulence and effector recognition will answer whether the decoy model also applies to the animal innate immunity.

In Vitro Uridylylation Assay The assay was performed as previously described (Feng et al., 2012). Briefly, GST-tagged AvrAC and AvrACH469A, and His-tagged RKS1, PBL2, and PBL2 S253A/T254A proteins were affinity purified and used for the uridylylation assay. Approximately 400 ng of GST–AvrAC or GST–AvrACH469A was incubated with 2 mg of His–RKS1, His– PBL2, or His– PBL2S253A/T254A in a 20 ml reaction buffer containing 25 mM Tris-HCl (pH 7.5), 25 mM MgCl2, 500 mM UTP, 1 mM DTT, a-32P UTP (5 mCi) for 30 min at 30
C. The reaction was stopped by the addition of SDS loading buffer. The products were then separated on 10% NuPAGE gel (Invitrogen) and detected by autoradiography.

EXPERIMENTAL PROCEDURES Ethyl Methanesulfonate Mutagenesis and the sxc Mutant Screen Seeds of a Col-0 pER8:avrAC transgenic line were mutagenized as described (Weigel and Glazebrook, 2002). Individual M2 families were screened for normal growth on MS/10 medium supplemented with 5 mM b-estradiol (Murashige and Skoog, 1962). Mutants that did not carry any mutation in the avrAC coding sequence and did not exhibit any detectable differences in AvrAC accumulation were renamed sxc1, sxc3, sxc5, and sxc7 for suppressor of XopAC/AvrAC and kept for further molecular and genetic studies. Map-Based Cloning of sxc Mutations by Deep Sequencing sxc3, sxc5, and sxc7 lines were backcrossed with the Col-0 pER8:avrAC parental line. Individual plants of the corresponding F2 populations showing normal development in the presence of 5 mM b-estradiol were harvested and subjected to DNA extraction and deep DNA sequencing. DNA sequencing of paired ends reads (2 3 100 bp) and subsequent bioinformatic analyses allowed the identification of SNP positions in each sxc mutants (Figures S2 and S3). Bacterial Inoculation Xcc pathogenicity was assayed by wound inoculation of the main leaf vein of 4-week-old Arabidopsis plants with a bacterial suspension at 108 cfu/mL essentially as described (Meyer et al., 2005). Disease indices were scored 8 days postinoculation: 0–1, no symptoms; 1–2, weak chlorosis; 2–3, strong chlorosis; 3–4, necrosis. Alternatively, 5-week-old plants were piercing-inoculated with Xcc bacteria at 5 3 108 cfu/mL, and photographs were taken 7 days later. To detect hopZ1a-mediated HR, 5-week-old Arabidopsis plant leaves were hand-infiltrated using a needleless syringe containing a P. syringae suspension at 5 3 107cfu/mL, and hypersensitive response was scored 20 hr later. Each assay was performed at least three times. For bacterial growth assay, 5-week-old Arabidopsis leaves were hand infiltrated using a needleless syringe with P. syringae bacteria at 106 cfu/mL or wound inoculation of the main vein of leaves with Xcc at 2 3 108 cfu/mL. The bacterial population was measured in the leaves at 0, 4, or 5 days postinoculation as described (Katagiri et al., 2002). For virulence assay, 5-week-old Arabidopsis plants were infiltrated in mesophyll tissues using a needleless syringe with a mixture of wild-type XccB186 and XccB186DavrAC mutant bacteria at 1:1 ratio and a final concentration of 106 cfu/mL. Bacterial titers for each strain were measured in the leaf 4 days after inoculation, and the ratio of the mutant versus wild-type bacteria (competitive index) was determined as described (Feng et al., 2012).

Uridylylation Site Identification The PBL2-His and GST-AvrAC constructions were coexpressed in E. coli BL21. The protein was purified by Ni-NTA affinity chromatography, separated on 10% NuPAGE gel (Invitrogen), and stained with Coomassie brilliant blue. The excised bands were destained for nano-LC-MS/MS analysis as described (Feng et al., 2012). Coimmunoprecipitation and Split-Luciferase Complementation Assay For coIP experiments in protoplasts, protoplasts from selected genetic backgrounds were transfected with the desired plasmids and incubated overnight as described (He et al., 2007). Total proteins were extracted and subjected to anti-FLAG immunoprecipitation as previously described (Zhang et al., 2010). The immunoprecipitates were separated on a 10% SDS-PAGE gel and detected by anti-HA and anti-Flag immunoblot. For coIP assay in plants, 4-week-old transgenic plants were used for protein extraction. For AvrAC-induced PBL2-RKS1 interaction, leaves of 4-week-old transgenic plants were infiltrated with Xcc8004 or Xcc8004DavrAC mutant bacteria at 2 3 108 cfu/mL, and leaf samples were taken at 4 hr after inoculation. Total protein extracts were subject to anti-FLAG IP and analyzed by immunoblot. Split-luciferase complementation assays were performed as previously described (Chen et al., 2008). Agrobacterium tumefaciens strain GV3101 containing the indicated constructs was infiltrated into expanded leaves of N. benthamiana by a needleless syringe. The plants were covered with plastic bags and incubated in the growth room for 36 hr before LUC activity measurements. SUPPLEMENTAL INFORMATION Supplemental Information includes seven figures, two tables, and Supplemental Experimental Procedures and can be found with this article at http:// dx.doi.org/10.1016/j.chom.2015.08.004. AUTHOR CONTRIBUTIONS G.W. performed protein-protein interaction analyses, site-directed mutagenesis, construction of transgenic plants, and cell death assays. M.C., L.D.N., and E.G. constructed avrAC transgenic lines and performed seedling growth inhibition assays. B.R. and E.G. performed the genetic screen and subsequent analyses of sxc mutants. G.W. and B.R. performed disease resistance

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analyses. G.W., F.F., and X.Z. performed the screen of NLR, RLCK VII, and RLCK XII mutant collections for AvrAC recognition. L.L., N.L., and S.C. performed mass spectrum analysis of PBL2 uridylylation. M.-F.J. performed statistical analyses. M.L., M.A., and C.H. assisted manuscript preparation. L.D.N. and J.-M.Z. supervised the research and wrote the manuscript.

Arabidopsis receptor-like cytoplasmic kinase genes PBL2 and RIPK. PLoS ONE 8, e73469.

ACKNOWLEDGMENTS

Huard-Chauveau, C., Perchepied, L., Debieu, M., Rivas, S., Kroj, T., Kars, I., Bergelson, J., Roux, F., and Roby, D. (2013). An atypical kinase under balancing selection confers broad-spectrum disease resistance in Arabidopsis. PLoS Genet. 9, e1003766.

We wish to thank Ce´line Remblie`re (LIPM, Toulouse) for plant transformation and Claudette Icher (LIPM, Toulouse) for production of the mutagenized population. We are grateful to the INRA MIGALE bioinformatics platform (http://migale.jouy.inra.fr) for providing computational resources through its Galaxy server. We also wish to acknowledge Darrell Desveaux (University of Toronto, Canada), Wenbo Ma (University of California, Riverside), Jun Liu (Institute of Microbiology, CAS), and Dominique Roby (LIPM, Toulouse, France) for providing biological materials. J.-M.Z. was funded by the Strategic Priority Research Program of the Chinese Academy of Sciences (grant number XDB11020200), Chinese Ministry of Science and Technology (2015CB910200), and Chinese Natural Science Foundation (31320103909 and 31230007), and State Key Laboratory of Plant Genomics. F.F. was supported by a grant from Chinese Natural Science Foundation (31370293). E.G. was supported by a PhD grant from the French Ministry of National Education and Research and French Guyana and L.D.N. by a grant from the Agence Nationale de la Recherche (Xopaque, ANR-10-JCJC-1703-01). The LIPM is part of the LABEX TULIP (ANR-10-LABX-41; ANR-11-IDEX-0002-02). Received: July 1, 2015 Revised: July 31, 2015 Accepted: August 12, 2015 Published: September 9, 2015 REFERENCES Ade, J., DeYoung, B.J., Golstein, C., and Innes, R.W. (2007). Indirect activation of a plant nucleotide binding site-leucine-rich repeat protein by a bacterial protease. Proc. Natl. Acad. Sci. USA 104, 2531–2536. Aktories, K. (2011). Bacterial protein toxins that modify host regulatory GTPases. Nat. Rev. Microbiol. 9, 487–498. Axtell, M.J., and Staskawicz, B.J. (2003). Initiation of RPS2-specified disease resistance in Arabidopsis is coupled to the AvrRpt2-directed elimination of RIN4. Cell 112, 369–377. Birker, D., Heidrich, K., Takahara, H., Narusaka, M., Deslandes, L., Narusaka, Y., Reymond, M., Parker, J.E., and O’Connell, R. (2009). A locus conferring resistance to Colletotrichum higginsianum is shared by four geographically distinct Arabidopsis accessions. Plant J. 60, 602–613. Bisgrove, S.R., Simonich, M.T., Smith, N.M., Sattler, A., and Innes, R.W. (1994). A disease resistance gene in Arabidopsis with specificity for two different pathogen avirulence genes. Plant Cell 6, 927–933. Chen, H., Zou, Y., Shang, Y., Lin, H., Wang, Y., Cai, R., Tang, X., and Zhou, J.-M. (2008). Firefly luciferase complementation imaging assay for proteinprotein interactions in plants. Plant Physiol. 146, 368–376. Chung, E.-H., da Cunha, L., Wu, A.-J., Gao, Z., Cherkis, K., Afzal, A.J., Mackey, D., and Dangl, J.L. (2011). Specific threonine phosphorylation of a host target by two unrelated type III effectors activates a host innate immune receptor in plants. Cell Host Microbe 9, 125–136. Cui, H., Tsuda, K., and Parker, J.E. (2015). Effector-triggered immunity: from pathogen perception to robust defense. Annu. Rev. Plant Biol. 66, 487–511. Dodds, P.N., and Rathjen, J.P. (2010). Plant immunity: towards an integrated view of plant-pathogen interactions. Nat. Rev. Genet. 11, 539–548. Feng, F., and Zhou, J.-M. (2012). Plant-bacterial pathogen interactions mediated by type III effectors. Curr. Opin. Plant Biol. 15, 469–476. Feng, F., Yang, F., Rong, W., Wu, X., Zhang, J., Chen, S., He, C., and Zhou, J.-M. (2012). A Xanthomonas uridine 50 -monophosphate transferase inhibits plant immune kinases. Nature 485, 114–118. Guy, E., Lautier, M., Chabannes, M., Roux, B., Lauber, E., Arlat, M., and Noe¨l, L.D. (2013). xopAC-triggered immunity against Xanthomonas depends on

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