Cell Host & Microbe

Article The FLS2-Associated Kinase BIK1 Directly Phosphorylates the NADPH Oxidase RbohD to Control Plant Immunity Lei Li,1,5 Meng Li,1,2,3,5 Liping Yu,1 Zhaoyang Zhou,1 Xiangxiu Liang,1 Zixu Liu,1 Gaihong Cai,3 Liyan Gao,4 Xiaojuan Zhang,1 Yingchun Wang,4 She Chen,3 and Jian-Min Zhou1,* 1Center for Genome Biology and State Key Laboratory of Plant Genomics, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China 2College of Life Sciences, Peking University, Beijing 100871, China 3National Institute of Biological Sciences, Beijing 102206, China 4Center for Molecular Systems Biology and State Key Laboratory of Molecular Developmental Biology, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China 5These authors contributed equally to this work *Correspondence: [email protected] http://dx.doi.org/10.1016/j.chom.2014.02.009

SUMMARY

The Arabidopsis immune receptor FLS2 senses the bacterial flagellin epitope flg22 to activate transient elevation of cytosolic calcium ions, production of reactive oxygen species (ROS), and other signaling events to coordinate antimicrobial defenses, such as stomatal closure that limits bacterial invasion. However, how FLS2 regulates these signaling events remains largely unknown. Here we show that the receptor-like cytoplasmic kinase BIK1, a component of the FLS2 immune receptor complex, not only positively regulates flg22-triggered calcium influx but also directly phosphorylates the NADPH oxidase RbohD at specific sites in a calcium-independent manner to enhance ROS generation. Furthermore, BIK1 and RbohD form a pathway that controls stomatal movement in response to flg22, thereby restricting bacterial entry into leaf tissues. These findings highlight a direct role of the FLS2 complex in the regulation of RbohD-mediated ROS production and stomatal defense.

INTRODUCTION Plants are equipped with a variety of immune receptors that sense the invasion of numerous pathogenic microbes (Boller and Felix, 2009; Dodds and Rathjen, 2010; Spoel and Dong, 2012; Monaghan and Zipfel, 2012). A major class of the plant immune receptors are the cell surface-localized pattern-recognition receptors (PRRs) that detect conserved microbial molecular patterns, such as fungal chitin or bacterial lipopolysaccharides, peptidoglycans, elongation factor-Tu, and flagellin, and trigger an array of defenses, enabling plants to ward off the majority of potential pathogens (Kaku et al., 2006; Chinchilla et al., 2006; Zipfel et al., 2006; Miya et al., 2007; Wan et al., 2008; Willmann et al., 2011). PRRs also recognize endogenous molecular

patterns such as oligogalacturonides and small peptides that are generated during pathogen infection (Brutus et al., 2010; Krol et al., 2010; Yamaguchi et al., 2010). Thus PRRs form the first layer of the plant surveillance system that ensures immediate activation of plant immunity upon first contact with pathogens. A well-known PRR is the Arabidopsis receptor kinase FLS2, which recognizes a conserved 22 amino acid N-terminal sequence of the bacterial flagellin protein (flg22). FLS2 contains an extracellular leucine-rich repeat (LRR) domain, a transmembrane domain, and a cytoplasmic kinase domain (Chinchilla et al., 2006). The LRR domain perceives flg22 and rapidly recruits another LRR receptor-like kinase called BAK1 (Chinchilla et al., 2007; Heese et al., 2007; Schulze et al., 2010). Recent advances demonstrate that BAK1 is a coreceptor of flg22, and intermolecular interactions between the FLS2 LRR domain, flg22, and the BAK1 LRR domain initiate the activation of the PRR complex (Sun et al., 2013). After exposure to flg22 and other microbial molecular patterns, the plant cell undergoes a rapid activation of MAP kinase (MPK) cascades (Nu¨hse et al., 2000), a burst of reactive oxygen species (ROS) controlled by the NADPH oxidase RbohD (Nu¨hse et al., 2007; Zhang et al., 2007), and a transient influx of calcium ion from the apoplast (Blume et al., 2000; Lecourieux et al., 2002), with the latter essential for the ROS production (Ogasawara et al., 2008; Ranf et al., 2011; Segonzac et al., 2011). The identity of the calcium channel operating downstream of PRRs remains unclear, but cyclic nucleotide-gated ion channels, ionotropic glutamate receptor-like channels, and calcium pumps have all been implicated to play a role in regulating cytosolic calcium concentration in defenses (Ma et al., 2009, 2013; Kwaaitaal et al., 2011; Frei dit Frey et al., 2012). Phosphoproteomic studies have shown that RbohD is phosphorylated at multiple sites required for the FLS2-mediated ROS production (Benschop et al., 2007; Nu¨hse et al., 2007). A number of calcium-dependent protein kinases (CPKs) including CPK4, CPK5, CPK6, and CPK11 have been shown to regulate ROS production during immune signaling (Kobayashi et al., 2007; Boudsocq et al., 2010; Dubiella et al., 2013; Gao et al., 2013), but these CPKs do not appear to account for all the phophosites identified in RbohD.

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example, FLS2 plays a major role in the bacterium-induced stomatal closure that restricts bacterial entry (Melotto et al., 2006). This stomatal defense also involves ROS generated by RbohD and other downstream components (Mersmann et al., 2010; Macho et al., 2012; Zeng et al., 2011; Montillet et al., 2013), but a mechanistic link between FLS2 and downstream signaling remains unclear. Here we show that RbohD is a component of the FLS2 immune receptor complex and that BIK1 phosphorylates RbohD at Ser39 and Ser343. In addition, BIK1 also positively regulates the flg22induced increase of cytosolic calcium. Together, the BIK1-specific phosphorylation of RbohD and calcium increase control the flg22-induced ROS production and the stomatal defense. RESULTS

Figure 1. BIK1 Positively Regulates Calcium Signaling (A) BIK1 positively contributes to flg22-induced calcium influx. Aequorin reporter plants of the indicated genetic background were treated with 1 mM flg22, and luminescence was measured immediately. Arrow indicates the time when flg22 was administered (data are represented as mean ± SEM; n = 8; three biological repeats). (B) BIK1 and PBL1 are not required for flg22-induced CPK5 activation. An HAtagged CPK5 was expressed in protoplasts isolated from WT (Col-0) or bik1 pbl1 double mutant. Upon stimulation with 1 mM flg22, protein was analyzed by anti-HA immunoblot. The positions of unphosphorylated (CPK5) and phosphorylated CPK5 (CPK5-P) are indicated. (C) The dominant-negative mutant BIK1K105E inhibits the flg22-induced CPK5 phosphorylation in protoplasts.

BIK1 and PBL1 are two highly homologous receptor-like cytoplasmic kinases that play an additive role in defenses by associating with the unstimulated FLS2 and other PRRs (Lu et al., 2010; Zhang et al., 2010). Upon flg22 recognition, BIK1 and PBL1 become phosphorylated. The importance of BIK1 and PBL1 in plant immunity is further indicated by the findings that they are required for resistance against necrotroph fungal pathogens and ethylene-induced defenses (Veronese et al., 2006; Laluk et al., 2011; Liu et al., 2013) and are attacked by at least two pathogen effector proteins (Zhang et al., 2010; Feng et al., 2012). We have shown previously that BIK1 and PBL1 play a positive role in the RbohD-dependent ROS production, but are not required for MPK activation (Zhang et al., 2010; Feng et al., 2012). How BIK1 and PBL1 mediate immune signaling remains unknown. In addition, we know little about how immune signaling alters plant physiology to limit pathogen progression. For

BIK1 Contributes to flg22-Induced Calcium Influx Because calcium signaling is known to be essential for RbohD activation (Ogasawara et al., 2008; Ranf et al., 2011; Segonzac et al., 2011), we determined if BIK1 is involved in the flg22triggered calcium influx. An aequorin transgene (Knight et al., 1996) was introduced into the bik1 mutant background by crossing. Plants homozygous for both the aequorin transgene and bik1 were analyzed for the flg22-induced aequorin luminescence. The WT aequorin line exhibited a rapid and strong luminescence indicative of calcium influx
2–5 min after flg22 treatment (Figure 1A), and this is consistent with a previous study (Ranf et al., 2011). In contrast, the bik1 aequorin line showed only half of calcium influx, indicating that BIK1 plays a positive role in flg22-triggered calcium ion influx. This finding prompted us to examine if the bik1 pbl1 double mutant impacts the activation of CPK5, which is known to be activated upon flg22 stimulation and reported to activate RbohD (Dubiella et al., 2013). When expressed in protoplasts, the flg22 treatment led to the accumulation of a slow-migrating form of CPK5 in SDS PAGE indicative of phosphorylation accompanied by a reduction of the unphosphorylated form (Figure 1B). This flg22-induced CPK5 phosphorylation also occurred in the bik1 pbl1 double mutant protoplasts. We reasoned that CPK5 may be activated by a modest level of cytosolic calcium existing in the bik1 pbl1 double mutant. Because BIK1 and PBL1 belong to a large family of proteins, it is possible that additional members of this family may contribute to calcium influx required for CPK5 activation. We therefore overexpressed in protoplasts the BIK1K105E mutant which lacked the kinase activity and was known to act in a dominant-negative manner to inhibit defenses (Zhang et al., 2010). Indeed, the overexpression of BIK1K105E protein prevented CPK5 activation (Figure 1C), supporting a role of additional BIK1 family proteins in CPK5 activation, likely by controlling calcium influx. The BIK1K105E mutant does not accumulate to a high level in stable transgenic plants, however, preventing us from testing if the inhibition of CPK5 activation impact immune responses in plants. RbohD Directly Interacts with BIK1 and FLS2 The results described above indicated a role of BIK1, and likely other BIK1 family proteins, in calcium signaling. The findings appeared to be consistent with the possibility that BIK1 and PBL1 regulate RbohD indirectly through CPK5. However, the largely normal CPK5 activation but severely compromised

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Figure 2. BIK1 Interacts with RbohD (A) BIK1 interacts with RbohD in vitro. A His-tagged N-terminal fragment of RbohD (His-RbohDNT) and a GST-tagged BIK1 or BIK1K105E recombinant proteins were affinity purified, and the proteinprotein interaction was tested by a GST pull-down assay. CBB, Coomassie brilliant blue staining. (B) BIK1 and FLS2 interact with RbohD in N. benthamiana. The indicated constructs were transiently expressed in N. benthamiana, and luciferase complementation imaging assay was performed. Nluc, N-terminal fragment of firefly luciferase; Cluc, C-terminal fragment of firefly luciferase; EV, empty vector. (C) BIK1 and FLS2 interact with RbohD in Arabidopsis protoplasts. The indicated constructs were expressed in WT protoplasts, and coIP assay was performed using an anti-HA antibody. (D) BIK1 interacts with RbohD in Arabidopsis plants. Plants carrying the BIK1-HA transgene under the native BIK1 promoter (NP::BIK1-HA) and 35S::FLAG-RbohD were treated with (+) or without () flg22, and total protein extract was subject to coIP. See also Figure S1 and Table S1.

ROS production in the bik1 pbl1 double mutant suggests a CPK5-independent mechanism for ROS regulation by BIK1 and PBL1. To elucidate this mechanism, we sought to identify BIK1-interacting proteins. The BIK1-FLAG fusion protein was expressed in Arabidopsis protoplasts, and the immune complex was isolated by immunoprecipitation. The isolated proteins were subject to LC-MS/MS analysis to identify candidate BIK1-interacting proteins. In total, we reproducibly identified 62 proteins, including BIK1, in two independent experiments where BIK1-FLAG was expressed, but not in control protoplasts lacking BIK1-FLAG (see Table S1 available online). One of the candidate proteins is RbohD and is the focus of this study. We first sought to verify its interaction with BIK1 using multiple assays. As shown in Figure 2A, the N-terminal cytoplasmic fragment of RbohD was able to bind both WT and ATP-bindingdeficient BIK1 protein (BIK1K105E) in GST pull-down assays, indicating that BIK1 can directly interact with RbohD N-terminal fragment in vitro. This RbohD fragment also interacted with the kinase domain of FLS2 in vitro (Figure S1A). Split-luciferase complementation assays in Nicotiana benthaminana indicated that both BIK1 and FLS2, but not BAK1, can interact with RbohD in vivo (Figures 2B and S1B), indicating that RbohD specifically interacts with the unstimulated FLS2 receptor complex. The BIK1-RbohD and FLS2-RbohD interactions were also detected in coimmunoprecipitation (coIP) assays when a FLAG-tagged RbohD was coexpressed with BIK1-HA or FLS2-HA in WT protoplasts regardless of the orientation of the FLAG tag fused to RbohD (Figures 2C, S1C, and S1D). CoIP experiments using Arabidopsis plants carrying a 35S::FLAG-RbohD transgene and a BIK1-HA transgene under the control of the BIK1 native promoter (Zhang et al., 2010) confirmed that the BIK1-RbohD interaction can be detected in Arabidopsis plants (Figure 2D). In some experiments, a slow-migrating form of FLAG-RbohD suggestive of phosphorylation was observed when the protoplasts were treated with flg22 (Figures S1C and S1D). This coincides with the flg22-induced phosphorylation of BIK1-HA, which

also appears as a slow-migrating band. Interestingly, the flg22 treatment reduced BIK1-RbohD and FLS2-RbohD interactions, indicating that RbohD dissociates from the FLS2 complex upon activation (Figures 2C, 2D, S1C, and S1D). However, the flg22-induced dissociation was not detected when a BIK1K105E mutant was used, indicating that an active BIK1 is required for the dissociation. The BIK1-RbohD interaction was also detected in fls2 and bak1 mutant protoplasts, indicating that BIK1 interacts with RbohD independent of FLS2 and BAK1 (Figure S1D). However, neither the flg22-induced dissociation nor BIK1 phosphorylation was detected in fls2 and bak1 mutant protoplasts, further supporting that the flg22-induced activation of the receptor complex and BIK1 phosphorylation is required for the dissociation. Deletion analysis was carried out to determine the RbohD sequence required for BIK1-interaction. CoIP assays indicated that the minimal sequence is located between amino acids 126 and 294, as indicated by coIP assays (Figure S1E). BIK1 Phosphorylates RbohD at Ser39 and Ser343 We next sought to determine if RbohD is a substrate of BIK1. As indicated by immunoblot with anti-phospho-Ser/Thr antibodies, the recombinant protein of the RbohD N-terminal fragment became strongly phosphorylated after incubation with the WT but not BIK1K105E recombinant protein (Figure 3A), indicating that BIK1 can directly phosphorylate RbohD at Ser or Thr in vitro. The phosphorylated RbohD was subject to mass spectrum analysis to determine phosphorylation sites in RbohD. Three amino acid residues, Ser39, Ser343, and Ser347, were readily identified as residues phosphorylated by BIK1 (Table S2; Figure S2A and S2B). These sites corresponded to several flg22induced RbohD phosphorylation sites reported by previous phosphoproteomic studies (Benschop et al., 2007; Nu¨hse et al., 2007). To determine if BIK1 is responsible for the phosphorylation of these sites in vivo, we further developed phosphopeptidespecific antibodies recognizing these phosphorylated residues

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stantiate a role of BIK1 in RbohD phosphorylation in the plant cell, we overexpressed BIK1-HA along with FLAG-RbohD in the bik1 pbl1 double-mutant protoplasts. This led to a constitutive phosphorylation of Ser39, which was further enhanced upon the flg22 treatment (Figure 3C). The flg22 treatment allowed the detection of Ser343 but not Ser347 phosphorylation in protoplasts overexpressing BIK1-HA, indicating that BIK1 also positively regulates the phosphorylation of Ser343 in vivo. To determine if the BIK1-specific RbohD phosphorylation occurs in seedlings, we generated stable transgenic plants expressing FLAG-RbohD under the control of a native promoter. Figure 3D shows that Ser39 was strongly phosphorylated upon flg22 treatment, indicating that the phosphorylation observed in the protoplast assay faithfully reflects the molecular event in plants. Together these results support that BIK1 directly phosphorylates RbohD at Ser39, and likely Ser343, in vivo.

Figure 3. BIK1 Phosphorylates RbohD at Ser39, Ser343, and Ser347 (A) BIK1 phosphorylates the N-terminal fragment of RbohD. The RbohDNT recombinant protein was incubated with BIK1 in an in vitro kinase assay, and the phosphorylation of RbohD was detected with immunoblot with antiphosphoSer/Thr antibodies (pSpT), or antibodies that specifically recognize phospho-Ser39 (pS39), phospho-Ser343 (pS343), phospho-Ser347 (pS347), or a dually phosphorylated Ser343 and Ser347 (pS343pS347). (B) Flg22 induces Ser39 phosphorylation in a BIK1 PBL1-dependent manner. FLAG-RbohD was expressed in protoplasts of the indicated genotype, the FLAG-RbohD protein was affinity purified with anti-FLAG antibodies, and Ser39 phosphorylation was detected by anti-pSer39 immunoblot. Total FLAGRbohD protein was detected by anti-FLAG immunoblot. (C) BIK1 overexpression enhances the phosphorylation of Ser39 and Ser343. The phosphorylation of specific sites in RbohD was determined by immunoblot with the indicated antibodies. Total FLAG-RbohD and BIK1-HA proteins were determined by immunoblot with anti-FLAG and anti-HA immunoblot. (D) Flg22 induces Ser39 phosphorylation independent of calcium influx. Transgenic plants expressing FLAG-RbohD were treated with 10 mM LaCl3 for 10 min prior to elicitation with 1 mM flg22, and Ser39 phosphorylation was determined by immunoblot. See also Figure S2 and Table S2.

in RbohD. The antibodies specifically recognized Ser39 and Ser343 phosphorylation in RbohD recombinant protein that had been incubated with the WT BIK1, but not BIK1K105E protein (Figure 3A). The anti-phospho-Ser347 antibodies detected a much weaker signal than did the anti-phospho-Ser39 and antiphospho-Ser343 antibodies, suggesting that Ser347 was phosphorylated at a lower level or that the antibodies were simply not sensitive. These antibodies were used to determine the role of BIK1 and PBL1 in RbohD phosphorylation in the plant cell. A FLAG-RbohD fusion protein was expressed in protoplasts isolated from WT and bik1 pbl1 double mutant plants. RbohD Ser39 was mostly unphosphorylated in nonstimulated WT protoplasts but became strongly phosphorylated upon stimulation with flg22 (Figure 3B), indicating that this residue is specifically phosphorylated in an flg22-dependent manner. In contrast, the Ser39 phosphorylation was abolished in the bik1 pbl1 doublemutant protoplasts, indicating that BIK1 and PBL1 are indeed required for the flg22-induced phosphorylation of Ser39 in the plant cell. The phosphorylation of Ser343 and Ser347 in protoplasts could not be detected (data not shown). To further sub-

The Ser39 Phosphorylation Is Calcium Independent Overexpression of CPK5 in Arabidopsis plants was reported to enhance the phosphorylation of Ser39 of RbohD (Dubiella et al., 2013). However, it is not clear if CPK5 is capable of phosphorylating Ser39 directly. In seedlings, the flg22-induced Ser39 phosphorylation was completely insensitive to calcium channel blocker LaCl3 (Figure 3D), while the same treatment completely abolished the flg22-induced ROS production (Figure S2C). The LaCl3 treatment was also unable to block Ser39 phosphorylation in protoplasts (Figure S2D). These results indicated that the flg22-induced phosphorylation on Ser39 is independent of calcium signaling and unlikely caused by CPK5. The normal BIK1specific phosphorylation of RbohD but a lack of ROS production in the presence of calcium channel blocker indicated that the phosphorylation by BIK1 is not sufficient to activate RbohD. The Phosphorylation of Ser39, Ser343, and Ser347 Contributes to RbohD Regulation by BIK1 Ser343 and Ser347 have been shown to play a crucial role in flg22-induced activation of RbohD (Nu¨hse et al., 2007), but a role of Ser39 has not been tested. To determine if the BIK1-specific phosphorylation of Ser39 plays a role in RbohD activation, we substituted Ser39 along with Ser343 and Ser347 with Ala, which blocks phosphorylation, or Asp, which often mimicks phosphorylation, either individually or in combinations, and introduced the mutant forms of RbohD into rbohD plants. As indicated in Figures 4A and S3, the flg22-induced H2O2 production was reduced to
50% in the RbohDS39A mutant compared to plants transformed with the WT RbohD, indicating that Ser39 phosphorylation is required for the full activation of RbohD. Consistent with a previous report (Nu¨hse et al., 2007), the RbohDS343A,S347A and RbohDS39A,S343A,S347A mutants were completely abolished in the RbohD-mediated H2O2 production, indicating that these residues are essential for RbohD activation. In contrast, the phosphomimetic RbohDS39D and RbohDS39D,S343D,S347D mutants restored the flg22-induced H2O2 production to a similar level as did by the WT RbohD tansgene (Figures 4B and S3). However, the RbohDS39D,S343D,S347D mutant exhibited no H2O2 production in the absence of flg22 treatment, suggesting that the phosphorylation of these residues is required, but not sufficient, for flg22-induced H2O2 production. We tested the significance of Ser39, Ser343, and Ser347

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merely caused by an elevated expression of RbohD. Thus, we conclude that the phosphorylation of Ser39, Ser343, and potentially Ser347 contributes to RbohD activation by BIK1 and PBL1. However, it should be noted that although RbohDS39D,S343D,S347D fully restored the flg22-induced H2O2 production to the rbohD mutant, it did not fully restore H2O2 production to the bik1 pbl1 double mutant, indicating that the phosphorylation of these residues only partially accounts for the defect of H2O2 production to bik1 pbl1.

Figure 4. BIK1 Regulates RbohD Activation through Phosphosites (A and B) Phosphosites are required for the full activation of RbohD oxidative burst. The indicated RbohD mutant constructs were transformed into the rbohD mutant plants, and two independent T2 transgenic lines for each construct were examined for flg22-induced H2O2 production (RLU, relative luminescence units; data are represented as mean ± SEM; different letters indicate significant difference; Student’s t test, p < 0.05; n R 4; three biological repeats). (C) Phosphomimetic RbohD mutants are significantly enhanced in the flg22induced H2O2 production in bik1 pbl1 plants. WT RbohD, RbohDS39D, or RbohDS39D,S343D,S347D transgene was introduced into bik1 pbl1 plants, and T2 transgenic lines were examined for flg22-induced H2O2 production (data are represented as mean ± SEM; different letters indicate significant difference; Student’s t test, p < 0.05; n R 4; three biological repeats). See also Figure S3.

phosphorylation in RbohD regulation by BIK1 and PBL1. The WT RbohD, RbohDS39D, and RbohDS39D,S343D,S347D mutants were transformed into the bik1 pbl1 double mutant, and two independent lines were tested for the flg22-induced H2O2 production. The flg22-induced H2O2 production in the bik1 pbl1 double mutant was reduced to 35% compared to that in WT plants. The H2O2 production was restored to 57%–62% in the two lines expressing RbohDS39D and to 67%–75% in the two lines expressing RbohDS39D,S343D,S347D (Figures 4C). In contrast, the lines expressing the WT RbohD had only 38%–46% H2O2 production. The accumulation of the RbohD protein in the RbohDS39D and RbohDS39D,S343D,S347D lines was similar to or less than that in the WT RbohD lines (Figure S3), indicating that the effect of RbohDS39D and RbohDS39D,S343D,S347D was not

BIK1 and RbohD Phosphorylation Control Stomatal Defense We examined if the mutations of the RbohD phosphosites affected stomatal closure in response to flg22. Consistent with previous reports (Mersmann et al., 2010; Macho et al., 2012), the rbohD mutant failed to close stomata in response to flg22, whereas the line carrying the WT RbohD transgene was fully responsive (Figure 5A). The line carrying RbohDS39A had slightly greater stomatal aperture but was statistically insignificant compared to WT plants. The lines carrying RbohDS343A,S347A and RbohDS39A,S343A,S347A were completely insensitive to flg22. In contrast, lines carrying RbohDS39D and RbohDS39D,S343D,S347D were fully restored in the flg22-induced stomatal closure (Figure 5B). These results indicate that the combined phosphorylation of these sites is required for the flg22-induced stomatal closure. FLS2 is a major immune receptor in the guard cell controlling the bacterium-induced stomatal defense (Zeng and He, 2010). We tested if BIK1 is a missing link between FLS2 and the RbohD-mediated stomatal defense. Indeed, stomata of the bik1 mutant are completely unable to respond to flg22 (Figures 5C and S4A). When treated with ABA, however, the bik1 mutant, bik1 pbl1 double mutant and WT plants displayed indistinguishable stomatal closure (Figure S4B), indicating that BIK1 plays a specific role in stomata defense, but not the ABA-regulated stomatal movement. We further tested if the RbohD phosphomimetic mutants were able to restore stomatal closure to bik1 pbl1. The bik1 pbl1 RbohDS39D stomatal aperture showed a small but statistically significant reduction in response to flg22 (Figure 5D), whereas the bik1 pbl1 RbohD stomata were completely insensitive to flg22. The stomatal closure was more pronounced in bik1 pbl1 RbohDS39D,S343D,S347D plants, but less than that in WT plants. Similar results were obtained when these plants were treated with P. syringae hrcC bacteria (Figure 5E), which are considered to contain a collection of microbial molecular patterns. To determine if the reduced H2O2 generation accounted for the defects of stomatal closure in bik1 pbl1 plants, we treated plants with H2O2 at different concentrations. Application of H2O2 strongly induced stomatal closure in all plants (Figure 5F), supporting an importance of H2O2 generation in BIK1-mediated stomatal closure. Interestingly, while the treatment induced stomatal closure similarly in WT and rbohD plants, it only did so partially in bik1 and bik1 pbl1 plants, suggesting that, in addition to RbohD, BIK1 and PBL1 must regulate other signaling components to control stomatal movement in response to bacterial infection. These results presented above support that BIK1 and RbohD together constitute a pathway through which FLS2 regulates

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Figure 5. RbohD Phosphorylation Contributes to BIK1-Regulated Stomatal Closure (A and B) RbohD Ser39, S343, and S347 are required for flg22-induced stomatal closure. The flg22-induced stomatal closure was determined in the rbohD T2 transgenic lines complemented with RbohD (line 2), RbohDS39A (line 1), RbohDS343A,347A (line 1), RbohDS39A,343A,347A (line 2), RbohDS39D (line 1), and RbohDS39D,343D,347D (line 4). (C) The bik1 mutant is abolished in the flg22-induced stomatal closure. (D and E) Phosphomimetic RbohD promotes flg22- and bacterium-induced stomatal closure in the bik1 pbl1 background. bik1 pbl1 T2 transgenic plants containing the WT RbohD transgene (line 1), RbohDS39D (line 2), and RbohDS39D,343D,347D (line 2) were treated with flg22 (D) or P. syringae hrcC- bacteria (E), and stomatal aperture was determined 1 hr later. (F) H2O2 induces stomatal closure in bik1 and bik1 pbl1 plants. The leaf epidermis was treated with H2O2 at the indicated concentrations, and stomatal aperture was measured 1 hr later. At least six peels from four different plants were examined for each treatment. * and ** (D) indicate significant difference at p < 0.05 and 0.01, respectively (two-way ANOVA and Student’s t test; data are represented as mean ± SEM; n R 30; three biological repeats). Different letters (A and E) indicate significant difference at p < 0.01 (one-way ANOVA, Tukey’s test; data are represented as mean ± SEM; n R 30; three biological repeats).

stomatal closure in response to bacterial infection. Indeed, we have shown previously that the bik1 mutant supported greater growth of the P. syringae hrcC strain only when spray-inoculated but not infiltrated into leaf tissues (Zhang et al., 2010). Figure 6A shows that, 4 days after being spray-inoculated with this strain, bacterial populations in bik1, pbl1, and bik1 pbl1 leaves were significantly greater than that in WT plants, indicating that both BIK1 and PBL1 are required for restricting bacterial infection. Bacterial populations in fls2 and rbohD plants were significantly greater than that in WT plants but less than that in bik1 pbl1 plants. The RbohDS39D,S343D,S347D transgene significantly reduced the bacterial population in bik1 pbl1 plants, whereas the WT RbohD and RbohDS39D transgenes failed to restore stomatal defense to bik1 pbl1 plants (Figure 6B). These results support that the phosphorylation of multiple sites in RbohD plays an important role in BIK1- and PBL1-mediated stomatal defense. DISCUSSION In summary, we show that RbohD is part of the dynamic immune receptor complex and directly interacts with FLS2 and BIK1 prior

to activation. BIK1 directly phosphorylates RbohD at Ser39 and Ser343. BIK1 and PBL1 are required for flg22-induced RbohD Ser39 phosphorylation in plants and protoplasts, and BIK1 overexpression can enhance both Ser39 and Ser343 phosphorylation in protoplasts. The functional significance of the Ser39 phosphorylation in RbohD activation was demonstrated by the finding that RbohDS39A is unable to fully activate H2O2 production in response to flg22, whereas RbohDS39D is able to restore the flg22-induced H2O2 production. We also show that BIK1 is required for flg22-induced stomatal closure and that phosphorylation of multiple sites in RbohD contributes to the BIK1-mediated stomatal defense to bacterial infection. Calcium signaling plays a crucial role in RbohD activation and MPK activation (Ranf et al., 2011; Segonzac et al., 2011). Two lines of evidence indicate that the BIK1 family proteins play an important role in calcium signaling. The flg22-induced calcium influx is reduced to half in the bik1 mutant compared to WT plants, indicating that BIK1 is required for optimum calcium signaling. Furthermore, overexpression of the dominant-negative mutant BIK1K105E blocked the flg22-induced phosphorylation of CPK5, suggesting that additional members of BIK1 family may act additively in regulating calcium signaling. A future

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Figure 6. BIK1 and RbohD Phosphorylation Regulate Stomatal Defense (A) BIK1, PBL1, and RbohD are required for stomatal defense. Plants of the indicated genotypes were spray-inoculated with P. syrigae hrcC- bacteria. The bacterial population in the leaf was determined 4 days postinoculation (data are represented as mean ± SEM; different letters indicate significant difference; Student’s t test, p < 0.05; n R 8; three biological repeats). (B) The RbohDS39D,S343D,S347D mutant transgene restores stomatal defense to bik1 pbl1 plants. The bacterial growth was determined in two independent T2 transgenic lines, and the bacterial population in the leaf was determined 4 days postinoculation (data are represented as mean ± SEM; different letters indicate significant difference; Student’s t test, p < 0.05; n R 8; three biological repeats). (C) Model depicting RbohD activation by flg22.

challenge will be to identify additional BIK1 substrates responsible for the calcium signaling. Although BIK1 positively regulates calcium influx, BIK1 directly phosphorylates RbohD in vitro in a buffer lacking calcium. Furthermore, the flg22-indcued RbohD Ser39 phosphorylation in plants occurs in the presence of LaCl3, indicating that BIK1 phosphorylates RbohD independent of the calcium signaling. This result also indicates that the Ser39 phosphorylation is unlikely to be caused by CPK5. Thus BIK1 and CPKs appear to phosphorylate RbohD at distinct sites. As shown in our study, the Ser39 and likely Ser343 phosphorylation is BIK1 specific and calcium independent, whereas the Ser148 phosphorylation shown in previous reports is likely calcium dependent and CPK specific (Kobayashi et al., 2007; Dubiella et al., 2013; Gao et al., 2013). It should be noted, however, that CPK5 is known to play a role in amplifying ROS, and could phosphorylate other sites at a later stage.

Results from a previous report (Nu¨hse et al., 2007) and this study indicate that RbohD phosphorylation at Ser39, Ser343, and Ser347 are required but not sufficient for RbohD activation. The phosphomimetic RbohDS39D,S343D,S347D mutant confers flg22-induced but not constitutive H2O2 production. Likewise, the LaCl3 treatment does not affect Ser39 phosphorylation but abolishes the flg22-inducd H2O2 production. It is likely that additional regulation mediated by the calcium signal is required for the activation of RbohD. Indeed, it has been shown that the EF-hand located in the N-terminal region of RbohD is required for RbohD activation (Ogasawara et al., 2008). Thus, the BIK1and CPK5-specified phosphorylations and direct calcium binding to the EF-hand may act in a coordinated manner to regulate RbohD activation, ensuring a tight control of ROS signals during immune responses. Several lines of evidence indicate that FLS2, BIK1, and RbohD form a pathway controlling stomatal defense. As shown in previous reports, FLS2 and RbohD are required for the flg22-induced stomatal closure (Melotto et al., 2006; Mersmann et al., 2010; Macho et al., 2012). We show here that BIK1 is also required for the flg22-induced stomatal closure. All three genes are required to restrict P. syringae hrcC bacterial infection when spray-inoculated. Phosphomimetic RbohD mutants were capable of at least partially restoring the flg22-induced H2O2 production, stomatal closure, and resistance to bacteria in the bik1 pbl1 background. These results indicate that the phosphorylation of RbohD at Ser39, Ser343, and perhaps Ser347 acts downstream of BIK1 and PBL1 to regulate H2O2 production and stomatal defense. It should be noted, however, the bik1 and bik1 pbl1 stomata are only partially responsive to exogenous H2O2 application, suggesting that additional components are necessary for optimum stomatal defense. We propose a model in which RbohD is regulated by at least two parallel mechanisms during flg22 signaling (Figure 6C). Upon the activation of the FLS2 receptor complex, BIK1 and PBL1 directly phosphorylate RbohD at S39 and Ser343 independent of calcium signaling. In the second pathway, the elevated cytosolic calcium which also involves BIK1, PBL1, and additional proteins further activates RbohD through binding to EF-hand and a CPK-mediated phosphorylation of additional sites, such as Ser148, activating RbohD and defenses. EXPERIMENTAL PROCEDURES Plant Materials and Constructs Arabidopsis plants used in this study include Col-0, bik1, bik1/pbl1, rbohD, fls2, and bak1-4 (Zhang et al., 2007, 2010). The plants were grown in the growth room at 23 C at 70% relative humidity with 10/14 hr day/night photoperiod for 4 weeks before protoplasts isolation. To generate full-length and truncated FLAG-RbohD constructs, the corresponding fragments were PCR amplified from cDNA and inserted between KpnI and BstBI of pUC-35S-FLAG-RBS vector (Zhang et al., 2010). To generate the His-RbohDNT construct, the N-terminal region (bp 1–1,128) was amplified from cDNA and inserted into pET28a. BIK1-HA, BIK1(K105E)HA, GST-BIK1, GST-BIK1(K105E) were described previously (Zhang et al., 2010). To generate constructs of Cluc-RbohD, BIK1-Nluc, FLS2-Nluc, BAK1-Nluc, the cDNA were amplified and cloned into Cluc-pCAMBIA1300 or Nluc-pCAMBIA1300 as previously described (Zhang et al., 2010). To generate the RbohD mutant transgenic plants, a native RbohD promoter of 2,041 bp in length and the cDNA were PCR amplified and cloned into pCAMBIA1300. Desired RbohD mutant plasmids were generated by

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site-directed mutagenesis, and the resulting constructs were introduced into rbohD plants by Agrobacterium-mediated transformation. Oxidative Burst Assay Leaves of 4-week-old plant were sliced into 1 mm strips and incubated in water overnight then treated with 100 nM flg22 in 200 ml buffer containing 20 mM luminol and 10 mg/ml horseradish peroxidase. The luminescence was recorded by the GLOMAX96 Luminometer (Promega). Coimmunoprecipitation Assay The protoplasts were transfected with the indicated plasmids, incubated overnight, and then treated with H2O or 1 mM flg22 for 10 min. Total protein was extracted for coIP with the extraction buffer (50 mM HEPES [pH 7.5], 150 mM KCl, 1 mM EDTA, 0.5% Trition-X 100, 1 mM DTT, proteinase inhibitor cocktail). For anti-FLAG IP, total protein was incubated with 50 ml agaroseconjugated anti-FLAG antibody (Sigma) for 4 hr and washed seven times with washing buffer (50 mM HEPES [pH 7.5], 150 mM KCl, 1 mM EDTA, 0.5% Trition-X 100, 1 mM DTT). The bound protein was eluted with 60 ml of 0.5 mg/ml 3 3 FLAG peptide for 1 hr. For anti-HA IP, the protein was precleared with protein A agarose for 1 hr, followed by an incubation with 2 mg anti-HA antibody (CWBIO) and protein A agarose for 4 hr. After washing, the protein was separated by SDS-PAGE and detected by anti-HA and antiFLAG immunoblot. GST Pull-Down and In Vitro Phosphorylation Assays GST, GST-BIK1, GST-BIK1K105E, and His-RbohDNT were expressed in E. coli and purified using the glutathione agarose beads (GE Healthcare). For GST pull-down assay, 5 mg His-RbohDNT and 10 mg each GST, GST-BIK1, GSTBIK1K105E were incubated with 30 ml glutathione agarose beads in a buffer containing 25 mM Tris-HCl (pH 7.5), 100 mM NaCl, and 1 mM DTT for 1 hr. The beads were washed seven times with the washing buffer containing 25 mM Tris-HCl (pH 7.5), 100 mM NaCl, 1 mM DTT, and 0.1% Trition-X 100. The bound protein was eluted with an elution buffer containing 25 mM Tris-HCl (pH 7.5), 100 mM NaCl, 1 mM DTT, and 15 mM GSH. His-RbohDNT was detected by anti-His (TianGen) immunoblot. For in vitro phosphorylation assay, 200 ng GST, GST-BIK1, or GSTBIK1K105E protein was incubated with 2 mg His-RbohDNT as substrate in a 20 ml reaction buffer containing 25 mM Tris-HCl (pH 7.5), 10 mM MgCl2, and 1 mM DTT and 100 mM ATP for 30 min at 30 C. The reaction was stopped by adding the SDS loading buffer. The protein phosphorylation was detected by anti-phospho-Ser/Thr antibodies or anti-phosphopepetide antibodies (Abmart). Split-Luciferase Complementation Assay The assay was performed as previously described (Chen et al., 2008). Agrobacterium tumefaciens strain GV3101 containing the indicated plasmids was infiltrated into expanded leaves of N. benthamiana and incubated in the growth room for 48 hr before the LUC activity measurement. For the CCD imaging and LUC activity measurement, 1 mM luciferin was sprayed onto the leaves. The cooled CCD imaging apparatus was used to capture the LUC image. Relative LUC activity per cm2 infiltrated leaf area was calculated. Each data point contains at least four replicates, and three independent experiments were carried out. Mass Spectrometric Analysis for BIK1-Interacting Proteins To identify BIK1-interacting proteins, BIK1-FLAG was expressed in Arabidopsis protoplasts, resuspended in 15 ml IP buffer (50 mM HEPES [pH 7.5], 50 mM NaCl, 0.2% Triton X-100, 1 mM DTT, 1 3 protease inhibitor cocktail). Debris was removed from the lysate by centrifugation at 14,000 rpm for 10 min. The supernatant was filtered through a 0.22 mm low-protein binding filter (Millipore) and incubated with 50 ml anti-FLAG agarose beads (Sigma). The mixture was incubated for 4 hr, and the immunocomplex was washed essentially as described (Liu et al., 2009). Immunocomplexes were eluted in 100 ml 1 mg/ml 3 3 FLAG peptide (Sigma). The eluted proteins were loaded onto a single lane on a 4%–10% SDS-PAGE gel (Invitrogen). Proteins were run 5 mm into the separating gel and stained with colloidal. Protein bands on the SDS-PAGE gel were destained and digested in-gel with sequencing grade trypsin (10 ng/mL trypsin, 50 mM ammonium bicarbonate [pH 8.0]) at

37 C overnight. Peptides were sequentially extracted with 5% formic acid/ 50% acetonitrile and 0.1% formic acid/75% acetonitrile and then concentrated to
20 ml. The extracted peptides were separated by an analytical capillary column (50 mm 3 10 cm) packed with 5 mm spherical C18 reversed-phase material (YMC, Kyoyo, Japan). An Agilent 1100 series binary pumps system (Agilent Technologies) was used to generate the following HPLC gradient: 0%–5% B in 5 min, 5%–40% B in 70 min, and 40%–100% B in 10 min (A = 0.2 M acetic acid in water, B = 0.2 M acetic acid/70% acetonitrile). The eluted peptides were sprayed into a LTQ mass spectrometer (Thermo Fisher Scientific) equipped with a nano-ESI ion source. The mass spectrometer was operated in data-dependent mode with one MS scan followed by five MS/MS scans for each cycle. Database searches were performed on an in-house Mascot server (Matrix Science Ltd., London, UK) against IPI (International Protein Index) Arabidopsis protein database. Methionine oxidation was set as variable modification. Phosphosite Identification The His-RbohDNT recombinant protein was coexpressed with GST or GSTBIK1 in E. coli, purified by Ni-NTA affinity chromatography, separated by 10% NuPAGE gel, and stained with Coomassie brilliant blue. The band for His-RbohDNT was excised from SDS-PAGE gel, and in-gel digestion was performed using a well-established protocol with slight modifications (Shevchenko et al., 2006). Briefly, the protein embedded in gel slices was reduced with 10 mM DTT and alkylated with 55 mM iodoacetamide, and then digested overnight with sequencing grade trypsin (Sigma) at 37 C. The tryptic peptides were analyzed by LC-MS/MS using LTQ-Orbitrap elite mass spectrometer with enabled multistage activation. Peptide identification and phosphosites assignment were performed with the Proteome Discoverer software (version 1.3) (Thermo Fisher). The Arabidopsis thaliana proteome sequences (Uniprot) were used as the database and the mass tolerances were set to 10 PPM for precursor and 0.5 Da for fragment ions for the database search. Phosphosite Antibodies Phosphosite-specific antibodies were custom-made through Abmart. Briefly, a phosphopeptide and a control peptide were synthesized for each phosphosite, and rabbits were immunized with the phosphopeptide conjugated to keyhole limpet hemocyanin (KLH) carrier. The polyclonal antiserum was purified by affinity chromatography using phosphopeptide, and the eluate was passed through the column coupled with control peptides to remove nonspecific antibodies. For anti-pSer39, the phosphopeptide is [DRGAF(pS)GPLGR], and the control peptide is [DRGAFSGPLGR]. For anti-pSer343, the phosphopeptide is [DSRIL(pS)QMLSQ], and the control peptides are [LSQML(pS) QKLRP] and [DSRILSQMLSQKLRP]. For anti-pSer347, the phosphopeptide is [LSQML(pS)QKLRP], and the control peptides are [DSRILSQMLSQKLRP] and [DSRIL(pS)QMLSQ]. For anti-pSer343pSer347, the phosphopeptide is [DSRIL(pS)QML(pS)QKLRP], and the control peptides are [DSRILSQMLS QKLRP], [DSRIL(pS)QMLSQ], and [LSQML(pS)QKLRP]. Calcium Influx Assay For aequorin luminescence measurements, the aequorin transgene (Knight et al., 1996) was introduced into the bik1 mutant by crossing, and homozygous progenies in the F3 generation were used. Leaf discs of 2-week-old plants were placed in 96-well plates in 1 mM coelenterazine in the dark for 6 hr. The luminescence was recorded by microplate reader (PerkinElmer) after treatment with 1 mM flg22. Stomatal Aperture Measurement Plants were kept under light for 2 hr to ensure that most stomata were opened before treatment. Leaf peels were collected from the abaxial side of 5-weekold plant leaves and floated in buffer (10 mM MES [pH 6.15], 10 mM KCl, 10 mM CaCl2). The stomata were observed after treated with 10 mM flg22, ABA, or mock solution for 1 hr using a microscope (ZEISS). The stomatal aperture was measured using ZEN lite software. SUPPLEMENTAL INFORMATION Supplemental Information includes two tables and four figures and can be found with this article at http://dx.doi.org/10.1016/j.chom.2014.02.009.

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ACKNOWLEDGMENTS We thank Dr. Yan Guo for sharing Arabidopsis materials. J.-M.Z. and Z.Z. were funded by the Chinese Natural Science Foundation (31230007), the Chinese Ministry of Science and Technology (2011CB100700), and the State Key Laboratory of Plant Genomics (2013B0125-02, 2013C0125-03). Received: October 15, 2013 Revised: January 12, 2014 Accepted: February 20, 2014 Published: March 12, 2014

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