Plant Immunity Triggered by Microbial Molecular Signatures

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1 Molecular Plant Advance Access published August 16, 2010 Molecular Plant Pages 1 11, 2010 REVIEW ARTICLE Plant Immunity Triggered by Microbial Molecular Signatures Jie Zhang 1 and Jian-Min Zhou National Institute of Biological Sciences, 7 Science Park Road, Zhongguancun Life Science Park, Changping District, Beijing , China ABSTRACT Pathogen/microbe-associated molecular patterns (PAMPs/MAMPs) are recognized by host cell surfacelocalized pattern-recognition receptors (PRRs) to activate plant immunity. PAMP-triggered immunity (PTI) constitutes the first layer of plant immunity that restricts pathogen proliferation. PTI signaling components often are targeted by various Pseudomonas syringae virulence effector proteins, resulting in diminished plant defenses and increased bacterial virulence. Some of the proteins targeted by pathogen effectors have evolved to sense the effector activity by associating with cytoplasmic immune receptors classically known as resistance proteins. This allows plants to activate a second layer of immunity termed effector-triggered immunity (ETI). Recent studies on PTI regulation and P. syringae effector targets have uncovered new components in PTI signaling. Although MAP kinase (MAPK) cascades have been considered crucial for PTI, emerging evidence indicates that a MAPK-independent pathway also plays an important role in PTI signaling. Key words: INTRODUCTION Disease resistance; plant microbe interactions; signal transduction; PAMP; innate immunity; receptor. To effectively ward off pathogenic microbes, plants must recognize the intruders and activate a battery of defenses that collectively arrest the pathogen. Unlike vertebrate animals that possess both acquired immunity and innate immunity, plants rely solely on innate immunity. The long history of plant pathogen associations led to the evolution of multiple surveillance mechanisms in the plant. For instance, plants are equipped to sense evolutionarily conserved microbial molecular signatures, collectively called pathogen-associated molecular patterns (PAMPs) or microbe-associated molecular patterns (MAMPs), and activate immune responses (Ausubel, 2005; Bittel and Robatzek, 2007; Boller and Felix, 2009). PAMPtriggered immunity (PTI) is thought to be an ancient form of innate immunity (Chisholm et al., 2006; Jones and Dangl, 2006). Research in the last 5 years showed that pathogens adapted to their host plants are capable of inhibiting PTI by delivering virulence effector proteins into host cells (Abramovitch et al., 2006; Boller and He, 2009; Cui et al., 2009; Grant et al., 2006; Zhou and Chai, 2008). To counteract, plants have evolved cytoplasmic immune receptors, classically called resistance (R) proteins, to detect some of the effector protein activities inside the plant cell and trigger disease resistance. Effector-triggered immunity (ETI) is highly specific and often accompanies hypersensitive response (HR). Interestingly, R proteins often sense effector activities indirectly through other host proteins of diverse biochemical properties. Although PTI and ETI employ distinct immune receptors, they seem to use a similar signaling network (Tsuda et al., 2009) and activate a largely overlapping set of genes (Navarro et al., 2004; Zipfel et al., 2006). In addition to PAMPs and effectors, the activity of pathogen lytic enzymes often releases plant cell wall and cutin fragments that can act as damage-associated molecular patterns (DAMPs) to trigger immune responses (Boller and Felix, 2009; Denoux et al., 2008; Lotze et al., 2007). In this review, we will focus on the latest understanding of PTI signaling mechanisms, primarily using Arabidopsis Pseudomonas syringae as a model plant pathogen system. PAMPs AND PRRs PAMPs include a growing list of microbial molecules: lipooligosaccharides of gram-negative bacteria, bacterial flagellin, bacterial Elongation Factor-Tu (EF-Tu), glucans and glycoproteins from oomycetes, chitin from fungus cell wall, etc. A recent addition is a sulfated peptide called Ax21 from Xanthomonas 1 To whom correspondence should be addressed. zhangjie@nibs.ac.cn, fax , tel ª The Author Published by the Molecular Plant Shanghai Editorial Office in association with Oxford University Press on behalf of CSPP and IPPE, SIBS, CAS. doi: /mp/ssq035 Received 31 March 2010; accepted 26 May 2010

2 2 Zhang & Zhou d Plant Innate Immunity Signaling oryzae oryzae (Xoo), which causes bacterial blight on rice (Lee et al., 2009). Often, these molecules play essential roles in the fitness of microbes, making the pathogens less likely to evade the detection by simple mutations in these molecules. PAMPs are detected by pattern recognition receptors (PRRs), typically cell surface-localized receptor kinases or LRR-RLP proteins (Fritz-Laylin et al., 2005). Well known PRRs in plants include: the flagellin receptor FLS2 and EF-Tu receptor EFR from Arabidopsis, rice chitin binding protein CEBiP, Arabidopsis chitin receptor CERK1, and the rice receptor-like kinase XA21 that recognizes Xoo Ax21. Readers are referred to the excellent review by Boller and Felix (2009) for additional candidates for PRRs and PAMPs. This review focuses on PRRs involved in plant interactions with bacterial pathogens. The bacterial flagellar protein is one of the best characterized PAMPs. Flagellin purified from P. syringae pv. tabaci is able to induce medium alkalinization in plant cell cultures from tomato, tobacco, potato and Arabidopsis (Felix et al., 1999), and growth inhibition of Arabidopsis seedlings (Gómez-Gómez et al., 1999). Flagellin perception also induces the production of reactive oxygen species (ROS), activation of mitogenactivated protein kinases (MAPKs), callose deposition at the cell wall, and expression of defense-related genes (Gómez- Gómez et al., 1999). A conserved N-terminal 22-amino-acid peptide of flagellin, flg22, is responsible for the elicitor activity (Felix et al., 1999). Genetic studies led to the isolation of FLS2, which encodes a receptor kinase required for flagellin perception (Gómez-Gómez et al., 1999). Subsequent biochemical studies unequivocally demonstrated that FLS2 is the receptor for flg22 (Chinchilla et al., 2006). Similar to flg22, elf18, an acetylated N-terminal 18-aminoacid peptide of EF-Tu, induces growth inhibition of Arabidopsis seedling, a rapid oxidative burst and defense-related gene expression (Zipfel et al., 2006). Reverse genetic and biochemical studies elegantly demonstrated that EFR, a receptor kinase highly homologous to FLS2, is the receptor for EF-Tu (Zipfel et al., 2006). The rice chitin elicitor-binding protein (CEBiP) was isolated by biochemical purification (Kaku et al., 2006). CEBiP contains extracellular LysM motifs for chitin-binding but lacks an intracellular kinase domain. RNAi experiment showed that CEBiP is required for chitin-induced defenses in rice. The Arabidopsis CERK1, which contains three LysM motifs in the extracellular domain and an intracellular Ser/Thr kinase domain, was shown to be required for chitin perception (Miya et al., 2007; Wan et al., 2008) and bind directly to chitin in vitro (Iizasa et al., 2010). CERK1 homologs are present in rice. It is possible that CERK1 forms a heterodimer with CEBiP to bind chitin. Interestingly, CERK1 was also found to play an important role in disease resistance to P. syringae bacteria (Gimenez-Ibanez et al., 2009), raising the possibility that it also mediates the perception of an unknown bacterial PAMP. Xa21, which encodes a receptor-like kinase, was first cloned as a rice resistance gene conferring resistance to Xoo and was thought to activate ETI. A series of recent work convincingly showed that Ax21, a sulfated protein secreted by Xoo type I secretion system, is the elicitor inducing Xa21-mediated resistance. A tyrosine-sulfated 17-amino acid synthetic peptide corresponding to the N-terminus of Ax21 is fully active in eliciting Xa21-mediated resistance. Cross-linking experiments suggested that Ax21 directly binds XA21. Ax21 is conserved in most species of Xanthomonas, and the tyrosine sulfation is required for its recognition by XA21 (Shen et al., 2002; Lee et al., 2009). Thus, XA21 is a pattern recognition receptor for Ax21. These findings are of conceptual importance, because they reveal that a protein previously classified as an R protein is, in fact, a PRR mediating strong PTI resistance. ROLE OF PTI IN PLANT DISEASE RESISTANCE PAMP-induced defenses in plants have been widely documented in early literature (Ebel and Mithofer, 1998; Hammerschmidt, 1999), but the importance of PTI in plant disease resistance was largely overlooked because of a lack of genetic evidence. The significance of PTI in plant disease resistance was clearly demonstrated only in the last decade. Mutations in PRRs often compromise PTI defense responses and overall resistance to pathogens. For example, Arabidopsis plants lacking FLS2 are completely defective in flg22-induced ROS accumulation, MAPK activation, and defense gene expression (Asai et al., 2002; Gómez-Gómez et al., 1999). fls2 plants display enhanced susceptibility to a virulent strain of P. syringae, at least when spray-inoculated (Zipfel et al., 2004). The FLS2-mediated resistance to this strain is largely attributed to PAMP-induced guard cell closure that limits bacterial entry into the leaf tissue (Melotto et al., 2006). Likewise, efr mutants are completely abolished in all responses to elf18 and show enhanced susceptibility to Agrobacterium tumefaciens (Zipfel et al., 2006). cerk1 mutants not only are insensitive to chitin treatment and display enhanced susceptibility to fungal pathogens (Miya et al., 2007; Wan et al., 2008), but also are more susceptible to P. syringae bacteria (Gimenez-Ibanez et al., 2009). In addition, the flagellin gene flic-induced defenses partially account for Arabidopsis non-host resistance to a P. syringae pv. tabaci strain, a non-adapted pathogen on Arabidopsis (Li et al., 2005). Finally, the discovery that XA21 Ax21 defines a new pair of PRR PAMP interaction indicates that the activation of PTI defenses can effectively restrict adapted pathogen. In several investigated plant pathogen systems, PTI appears to give rise to mild disease resistance, which is in contrast to strong disease resistance conferred by ETI. However, it is incorrect to conclude that PTI plays a lesser role in disease resistance. We now know that PTI is largely inhibited by effector proteins of adapted pathogens. Abrogation of PTI by transgenic expression of several P. syringae effectors renders Arabidopsis plants highly susceptible to normally nonpathogenic P. syringae strains (Hauck et al., 2003; Li et al., 2005; Kim et al., 2005). Moreover,

3 Zhang & Zhou d Plant Innate Immunity Signaling 3 many of these effector proteins often target important signaling components of PTI pathways (Fu et al., 2007; Gimenez- Ibanez et al., 2009; Göhre et al., 2008; Nomura et al., 2006; Xiang et al., 2008; Zhang et al., 2007). These findings re-enforce the importance of PTI in plant immunity (Boller and He, 2009; Cui et al., 2009) and indicate that PTI can be a highly effective defense barrier against non-adapted pathogens. The potential utility of PTI in the improvement of crop plant disease resistance is nicely demonstrated by the heterologous expression of EFR in solanaceous plants (Lacombe et al., 2010). EFR appears to be specific to the family of Brassicaceae. Overexpression of Arabidopsis EFR in Nicotiana benthamiana and tomato confers broad-spectrum resistance to multiple bacterial pathogens include P. syringae and Ralstonia solanecearum (Lacombe et al., 2010). Thus, cross-family transfer of PRRs can be an attractive strategy for improving disease resistance in crop plants. PTI SIGNAL TRANSDUCTION MECHANISM The Arabidopsis genome contains more than 600 receptorlike kinases (RLKs). RLKs not only play crucial roles in plant immunity, but also perceive developmental cues to control cell differentiation and plant growth (Morris and Walker, 2003; Johnson and Ingram, 2005; Hord et al., 2008; Smet et al., 2009). Among these, the brassinosteroid (BR) signaling pathway and the pathway governing stomatal patterning are two best understood pathways. Progress made in BR signaling and guard cell development has immensely advanced our understanding of how receptor kinases perceive external signal and regulate diverse cellular processes. The Brassinosteroid Signaling Pathway Brassinosteroids (BRs) are phytohormones regulating diverse processes in plant growth and development. BRs are perceived by the receptor kinase BRI1 to initiate a phosphorylationmediated signaling. A complete BR signaling pathway from the cell surface to the nucleus has emerged in the last decade (Gendron and Wang, 2007; Kim et al., 2009; Li, 2005). BRI1 directly associates with a BRI1 kinase inhibitor called BKI1 in unstimulated cells (Wang and Chory, 2006). This interaction is inhibitory to BR signaling, perhaps by preventing the association of BRI1 with its co-receptor BAK1 (see below). BRs directly bind the extracellular leucine-rich repeat domain of BRI1 to activate the BRI1 kinase activity. This triggers a dissociation of BKI1 from BRI1 (Wang and Chory, 2006) and promotes the association of BRI1 with another receptor-like kinase, BAK1 (Li et al., 2002; Nam and Li, 2002). The BRI1 BAK1 association results in cross-phosphorylation between BRI1 and BAK1 a molecular event believed to be critical for the activation of the receptor complex (Kinoshita et al., 2005; Wang and Chory, 2006; Wang et al., 2008). Phospho-proteomics identified a class of closely related receptor-like cytoplasmic kinases (RLCKs) designated BSKs that are substrates of BRI1 (Tang et al., 2008). BSKs constitutively interact with BRI1 and are phosphorylated by BRI1 upon the stimulation by BRs. The phosphorylated BSKs then dissociate from BRI1 to stimulate downstream components (Tang et al., 2008). Major downstream components include the GSK3-like kinase BIN2, the Kelch-repeats-containing protein phosphatase BSU1, and transcription factors BZR1 and BZR2/BES1 (He et al., 2002, 2005; Yin et al., 2002, 2005). BZR1 and BZR2/BES1 directly bind to BR-responsive gene promoters to regulate their expression. BIN2 negatively regulates BR signaling by phosphorylating BZR1 and BZR2/BES1, resulting in reduced binding to their target gene promoters (He et al., 2002; Vert and Chory, 2006; Yin et al., 2002). The phosphorylation was also reported to cause cytoplasmic retention and turnover of BZR1 (Gampala et al., 2007). Phosphorylated BSK1 was reported to bind BSU1 to promote the dephosphorylation of a conserved phosphortyrosine residue in BIN2 (Kim et al., 2009), which may lead to the inactivation of BIN2 and the accumulation of unphosphorylated BZR1 and BZR2/BES1 in the nucleus (Figure 1). Regulatory Pathway for Stomatal Development The development of stomata structures that control gas exchange and water loss is also tightly regulated by RLKs. The pattern of stomatal development strictly follows a one-cell rule in dicots. That is, stomata are separated by at least one non-stomatal cell in the epidermis. This precise positioning is achieved by sensing extracellular peptide signals (Figure 1). The epidermal patterning factors EPF1 and EPF2 negatively control stomatal patterning (Hara et al., 2007; Hunt and Gray, 2009). In contrast, the recently identified stomagen, a cysteinerich peptide, is a positive intercellular signal for stomatal development (Sugano et al., 2010). The functions of EPF1, EPF2, and stomagen all depend on the leucine-rich repeatcontaining receptor-like protein TMM (Hara et al., 2007; Hunt and Gray, 2009; Sugano et al., 2010). These findings indicate that EPF1, EPF2, and stomagen may be ligands that competitively bind TMM, although a direct binding remains to be demonstrated. The function of TMM also requires the ERECTA family RLKs ER, ERL1, and ERL2 in the negative regulation of stomatal development, likely by heterodimerization between TMM and an ERECTA family RLK (Nadeau, 2009; Shpak et al., 2005; Sugano et al., 2010). Downstream of the receptor complex, a MAP kinase cascade acts as a cell fate switch to regulate stomatal development. This MAPK cascade is consisted of the MAP kinase kinase kinase YODA, MAP kinase kinases MKK4 and MKK5, and MAP kinases MPK3 and MPK6 (Bergmann et al., 2004; Wang et al., 2007). Further downstream, three related transcription factors SPCH, MUTE, and FAMA sequentially determine stomatal cell lineage from meristemoid mother cell, meristemoid cell, guard mother cell, and guard cell (Lampard et al., 2008; MacAlister et al., 2007; Nadeau, 2009; Ohashi-Ito and Bergmann, 2006; Pillitteri et al., 2007). It is not known how the TMM receptor complex activates the MAP kinase pathway and how MPK3 and MPK6 regulate the three transcription factors.

4 4 Zhang & Zhou d Plant Innate Immunity Signaling Figure 1. A Model of BR Signaling Pathway and Stomatal Development Pathway. Left: BR signaling pathway. BR induces the formation of active receptor complex composed of the receptor BRI1 and co-receptor BAK1. BRI1 then directly phosphorylates BSKs. The activated BSKs directly interact with BSU1, a protein phosphatase, to inactivate the BIN2 kinase. The inactivation of BIN2 allows the unphosphorylated BZR1 and BES1 to regulate the expression of BR-responsive genes and plant development. It still is debated, however, whether BSU1 can directly regulate BES1 independently of BIN2 and whether BIN2 can be inactivated independently of BSU1. Figure adapted from Kim et al., Right: Stomatal development pathway. Both positive (stomagen) and negative (EPF1 and EPF2) signals regulating stomatal development are believed to be perceived by TMM together with other ERECTA family RLKs. The MAPK cascade consisted of YODA-MKK4/5-MPK3/6 that acts downstream to negatively regulate stomatal development. Transcription factors SPCH, FAMA, and MUTE positively regulate stomatal development, likely by acting downstream of the MAPK cascade. Activation of PRR Complexes Our understanding of PTI signaling mechanisms is rudimental. The best studied PTI signaling pathway is the FLS2 pathway. FLS2 contains both an extracellular leucine-rich-repeat (LRR) domain and a cytoplasmic serine/threonine protein kinase domain (Gómez-Gómez et al., 1999). Flg22 perception by FLS2 triggers an interaction between FLS2 and BAK1 (Chinchilla et al., 2007; Heese et al., 2007). The flg22 induced FLS2-BAK1 association occurs within seconds and is accompanied by increased phosphorylation on both FLS2 and BAK1 (Schulze et al., 2010). BAK1 probably acts as a co-activator of the receptor complex to enhance various signaling pathways (Wang et al., 2008). bak1 mutants are largely impaired in flg22-induced defense responses and show enhanced susceptibility to P. syringae (Chinchilla et al., 2007; Heese et al., 2007). The BAK1 kinase activity is required for FLS2-mediated signaling but not flg22- induced association of FLS2 and BAK1 (Schulze et al., 2010). BAK1 is also required for EFR-mediated signaling (Chinchilla et al., 2007), and a ligand-induced EFR BAK1 interaction was reported recently (Schulze et al., 2010). Furthermore, BAK1 forms a complex with a newly identified RLK BIR1 to negatively regulate cell death and defense responses (Gao et al., 2009). It is likely that BAK1 and/or its homologs are required for the activation of additional receptor kinases, such as CERK1. Extensive genetic screens have so far failed to identify new components in PTI signaling pathways. However, several recent genetic screens uncovered an important role of endoplasmic reticulum quality control (ER-QC) in EFR receptor maturation (Nekrasov et al., 2009; Saijo et al., 2009). The Arabidopsis stromal-derived factor-2 (SDF2) is required for elf18-induced seedling growth inhibition and oxidative burst, and is also required for EFR biogenesis. SDF2 localizes to endoplasmic reticulum, and forms a complex with ER-QC components Hsp40 ERdj3B and the Hsp70 BiP (Nekrasov et al., 2009). Independent studies also identified additional ER-QC components required for EFR accumulation and signaling. These include calreticulin3 (CRT3), UDPglucose glycoprotein glucosyl transferase (UGGT), HDEL receptor family member (ERD2b), and STT3A, a subunit of the ER-resident oligosaccharyltransferase (OST) complex (Häweker et al., 2009; Li et al., 2009; Lu et al., 2009; Saijo et al., 2009). Taken together, these results strongly demonstrated a role of ER-QC in EFR biogenesis and elf18-triggered signaling. This is reminiscent of the regulation of BRI1 maturation by ER-QC proteins (Hong et al., 2009; Jin et al., 2007, 2009). ER-QC appears to be a common regulatory mechanism for receptor kinases, as it has been suggested by Caplan et al. (2009) that ER-QC is also required for the maturation of IRK in N gene-mediated resistance. Interestingly, the ER-QC mutants described above do not affect FLS2-mediated signaling, suggesting that not all PRRs are subjected to regulation by ER-QC. BIK1, A Cytoplasmic Receptor-Like Kinase in PTI Signaling By using the P. syringae effector protein AvrPphB as a molecular probe, Zhang et al. (2010) identified a number of PBS1-like (PBL) RLCKs, including BIK1 and several other PBLs, as new components in PTI signaling pathways. AvrPphB is a cysteine protease known to cleave PBS1, an RLCK associated with the R protein RPS5. This cleavage allows Arabidopsis plants to detect AvrPphB as an avirulence protein and trigger ETI (Ade et al., 2007; Shao et al., 2003). In rps5 mutants, overexpression of AvrPphB resulted in an inhibition of PAMP-induced gene expression, ROS production, and callose deposition, likely by targeting host proteins in addition to PBS1 (Zhang et al., 2010). Indeed, AvrPphB is capable of cleaving BIK1 and a number of PBL proteins. Further genetic studies demonstrated that the bik1 mutant is severely compromised in defense responses induced by flg22, elf18, and chitin, indicating that BIK1 plays a critical role in the integration of signals from multiple PRRs. The bik1 mutant is significantly compromised in PAMP-induced resistance to P. syringae bacteria.

5 Zhang & Zhou d Plant Innate Immunity Signaling 5 PBL1, PBL2, and PBS1 also appear to additively contribute to PAMP-signaling, as their corresponding mutants showed slightly reduced PAMP-induced responses (Zhang et al., 2010). In vitro and in vivo studies showed that BIK1 forms a complex with unstimulated FLS2 in plants, and flg22 induces a rapid phosphorylation of BIK1 in both an FLS2- and BAK1- dependent manner (Zhang et al., 2010). BIK1 is also capable of interacting with EFR and CERK1 in protoplasts a result consistent with the requirement of BIK1 for elf18- and chitin-induced responses. In addition to phosphorylation, flg22 also induces a dissociation of BIK1 from FLS2 (Zhang et al., 2010), which is probably required for the activation of other unknown components in PTI signaling. These results were further confirmed by an independent study by Lu et al. (2010). The flg22-induced BIK1 phosphorylation and BIK1-FLS2 dissociation is reminiscent of the BR-induced phosphorylation of BSKs and BSK-BRI1 dissociation. Interestingly, PBLs and BSKs belong to two distinct subfamilies of RLCKs, suggesting that BRI1 and PRRs use analogous mechanisms to regulate downstream components. PAMP-Perception Activates Two MAPK Cascades Arabidopsis genome contains 20 MAPKs, among which MPK3, MPK4, and MPK6 are rapidly activated in response to PAMPs (Pitzschke et al., 2009a). Genetic and molecular studies indicated that the three MPKs represent two distinct MAPK cascades regulating plant immunity. MPK4, its upstream MAP kinase kinases MKK1 and MKK2, and the MAP kinase kinase kinase MEKK1 form a cascade that negatively regulates defenses in Arabidopsis, because loss-offunction mutations in this cascade result in constitutive activation of defenses and dwarfed plants (Gao et al., 2008; Ichimura et al., 2006; Suarez-Rodriguez et al., 2007; Mészáros et al., 2006; Qiu et al., 2008a; Pitzschike et al., 2009b). How MPK4 regulates plant immunity remains largely unknown. Nonetheless, MPK4 was reported to interact with its substrate MKS1; the latter interacts with WRKY transcription factors WRKY25 and WRKY33 (Andreasson et al., 2005). MPK4, MKS1, and WRKY33 form a complex in the nucleus, and the flg22-induced MPK4 activation was shown to release WRKY33 from the complex. This enables WRKY33 to directly activate the transcription of PAD3, which encodes a cytochrome P450 involved in camalexin biosynthesis (Qiu et al., 2008b). However, the functions of MKS1 and WRKY33 do not appear to account for the dramatic phenotype of the loss-of-function mpk4 mutants. Other substrates or interacting proteins mediating MPK4 function remain to be identified. A recent study showed that the P. syringae effector AvrB interacts with and stimulates the activity of MPK4, thereby perturbing hormone signaling and enhancing plant susceptibility in the absenceofcognaterproteinsrpm1andtao1(cui et al., 2010). This process is assisted by the molecular chaperone HSP90 and its co-chaperones SGT1 and RAR1. AvrB is also known to interact with and induce the phosphorylation of the RPM1- interacting protein RIN4 (Mackey et al., 2002). Interestingly, RIN4 can be phosphorylated by MPK4 in vitro and is required for AvrB to induce plant susceptibility to P. syringae. It remains to be determined whether the phosphorylation of RIN4 by MPK4 plays a role in regulating plant immunity. MPK3, MPK6, MKK4, and MKK5 are thought to form a cascade that positively regulates plant defenses (Pitzschke et al., 2009). The two MPKs and two MKKs are functionally redundant, and the mpk3/mpk6 and mkk4/mkk5 double mutants are lethal, making it difficult to examine their role in plant disease resistance. Nonetheless, MPK3 is required for camalexin accumulation upon Botrytis cinerea infection, and the mpk3 mutant exhibits enhanced disease susceptibility (Ren et al., 2008). Likewise, inactivation of MPK3 and MPK6 by the P. syringae effector HopAI1 and inactivation of MKKs by the P. syringae effector HopF2 severely impair PAMP-induced defenses and render plants highly susceptible to nonpathogenic P. syringae bacteria (Wang et al., 2010; Zhang et al., 2007). Yeast two-hybrid screen identified VIP1 as asubstrateofmpk3(djamei et al., 2007). VIP1 encodes a bzip transcriptionfactorthatisinitiallyidentifiedasahostprotein interacting with the Agrobacterium virulence protein VirE2. This interaction is believed to assist the nuclear import of the Agrobacterium transfer DNA, a key step in genetic transformation (Djamei et al., 2007). A PAMP-induced phosphorylation of VIP1 by MPK3 was shown to be necessary for the nuclear entry of VIP1. VIP1 appears to play a role in defense gene regulation, as suggested by its ability to transactivate PR1 promoter in protoplasts (Djamei et al., 2007). More than 1000 plant genes are transcriptionally activated by PAMPs. It remains to be determined to what extent VIP1 contributes to the transcription program activated by PAMPs. It is likely that additional transcription factors are involved in the transcription reprogramming in PTI. It was reported that MKK1/MKK2 can interact with MEKK1 at the plasma membrane, and MPK4 interacts with MKK1/ MKK2 both at the plasma membrane and in the nucleus (Gao et al., 2008). In addition, the plasma membrane-localized P. syringae effector AvrB interacts with MPK4 both in vivo and in vitro (Cui et al., 2010; Nimchuk et al., 2000). These findings raise the possibility that MPKs could be activated at the plasma membrane, although a direct interaction of PRRs or their coreceptors with MAPK cascade components has not been reported. It is conceivable that other membrane-associated proteins downstream of PRRs may mediate the activation of MAPK cascades. In addition to MKS1, WRKY 33, and VIP1, a significant number of WRKY transcription factors and some TGA transcription factors were identified as in vitro substrates for MPKs through protein microarrays (Popescu et al., 2009). This provides a useful resource for future studies, although the in vivo phosphorylation of these individual candidates by MPKs and their functions in defense signaling remain to be determined. Calcium Signaling in PTI It has long been known that intracellular Ca 2+ homeostasis is involved in defense signaling (Lecourieux et al., 2006). PAMP perception leads to membrane potential depolarization and an increase in cytoplasmic Ca 2+ concentration (Aslam et al.,

6 6 Zhang & Zhou d Plant Innate Immunity Signaling 2008; Lecourieux et al., 2006; Jeworutzki et al., 2010). Consistent with an important role of PAMP-induced calcium signaling, Xanthomonas capestris exoplolysaccharides (EPS) were shown to chelate extracellular calcium, thereby inhibiting PAMP-induced defense gene expression and enhancing virulence (Aslam et al., 2008). Most recently, calcium-dependent protein kinases CDPK4, CDPK5, CDPK6, and CDPK11 were reported to mediate PAMP-triggered defense responses, including gene expression and ROS production (Boudsocq et al., 2010). cpk5/cpk6 double mutant and cpk5/cpk6/cpk11 triple mutant are compromised in flg22-induced disease resistance to P. syringae. It remains to be determined how these CDPKs are regulated and how they regulate defense responses. PERSPECTIVES Approaches to the Identification of New PTI Signaling Components Forward genetics and reverse genetics were used extensively to identify mutants insensitive to PAMP-induced responses. These led to the successful isolation of several important PRRs such as FLS2, EFR, CERK1, and the co-receptor BAK1 (Chinchilla et al., 2006, 2007; Heese et al., 2007; Miya et al., 2007; Wan et al., 2008). In addition, extensive genetic screens for elf18- insensitive mutants have identified a number of genes involved in ER-QC of EFR (Häweker et al., 2009; Li et al., 2009; Lu et al., 2009; Nekrasov et al., 2009; Saijo et al., 2009). However, the genetic approach has failed to identify additional PTI signaling components downstream of PRRs. As suggested by the partial phenotypes of the bik1 and pbl mutants (Zhang et al., 2010), components downstream of PRRs may act additively, making it difficult to identify these components by traditional genetic screens. Because virulence effectors from P. syringae and other pathogens often target PTI signaling components, it should be feasible to use these effectors as a molecular probe to identify host proteins involved in PTI signaling. This is particularly true for pathogens such as P. syringae, which contains more than 30 effectors that are functionally distinct. The successful identification of MIN7, GRP7, and BIK1 as important components in plant immunity have demonstrated the power of this approach (Fu et al., 2007; Nomura et al., 2006; Zhang et al., 2010). Figure 2. PAMP-Triggered Signal Transduction Pathways and their Modulation by P. syringae Effectors. PRRs form a complex with BIK1 and PBLs in the absence of PAMPs. The perception of PAMPs, such as flagellin, EF-Tu, and chitin, induces an interaction between BAK1 and PRRs such as FLS2 and EFR. An interaction of CERK1 with BAK1 or BAK1 homologs remains to be demonstrated. This interaction leads to cross-phosphorylation of PRRs and BAK1, thereby activating the PRR complex. PTI signaling pathways likely bifurcate immediately downstream of PRRs. BIK1 and other PBLs associate with unstimulated PRRs (gray) and are phosphorylated immediately after the activation of PRRs (color) by PAMPs. The phosphorylated BIK1 and PBLs then dissociate from PRRs to regulate downstream components independently of MAPK cascades. PRR substrates responsible for MAPK activation remain to be identified. P. syrinage effector proteins that inhibit or activate various PTI signaling components are also indicated.

7 Zhang & Zhou d Plant Innate Immunity Signaling 7 As illustrated by the work on BSK proteins (Tang et al., 2008), quantitative proteomics can be applied to isolate signaling components downstream of receptor kinases. For example, sample pre-fractionation followed by two-dimensional difference gel electrophoresis (2-D DIGE) and mass spectrometry was carried out to successfully identify BRI1 substrate proteins BSK1, BSK2, and BSK3 (Tang et al., 2008). This holds promise in the isolation of novel signaling components in PTI. In an earlier proteomic study, several Arabidopsis proteins, including AtPHOS43, AtPHOS32, and AtPHOS34, were shown to be rapidly phosphorylated upon flg22 treatment (Peck et al., 2001). AtPHOS32 was recently shown to be a substrate of MPK3 and MPK6 (Merkouropoulos et al., 2008). In another study aimed at the isolation of plasma membrane-localized, phosphorylated proteins following flg22 treatment, 19 proteins were identified, five of which were predicted to localize to plasma membrane (Nühse et al., 2003). It is encouraging to note that one of the phospho-proteins identified in these studies is the NADPH oxidase RbohD that is required for PAMP-induced ROS production (Nühse et al., 2007; Zhang et al., 2007). Two flg22-induced phosphorylation sites in RbohD are found to be required for RbohD activation (Nühse et al., 2007). It will be important to determine whether other phosphor-proteins identified in these studies play a role in PTI signaling. A Model for PTI Signaling As described above, MAPK cascades play important roles in PTI regulation. Previous models have placed MAPK cascades as central components in the PTI signaling pathways (Boller and He, 2009; Chisholm et al., 2006; Pitzschke et al., 2009). However, multiple lines of evidence indicate the presence of a MAPK-independent PTI signaling pathway. Several ER-QC mutants displaying intact MAPK activation following PAMP perception are compromised in PTI defenses. Mutations in RSW3, which encodes the GIIa subunit of ER lumen enzyme, diminish elf18-induced disease resistance and gene expression but have little effect on MAPK activation and ROS production (Burn et al., 2002). The bik1 mutant is significantly compromised in PAMP-induced resistance, but not the flg22-induced MAPK activation (J. Zhang and J.M. Zhou, unpublished results). Likewise, transgenic plants expressing AvrPphB, which is capable of cleaving BIK1 and several PBL proteins, also show intact flg22-induced MAPK activation (J. Zhang and J.M. Zhou, unpublished results). The Arabidopsis dde2/ein2/pad4/sid2- quadruple mutant is largely impaired in flg22-induced resistance, but flg22-induced MAPK activation is comparable to wild-type plants (Tsuda et al., 2009). Conversely, transient expression of the P. syringae effector HopAI1, a potent inhibitor of MPKs (Zhang et al., 2007), does not affect the flg22-induced BIK1 phosphorylation (T. Xiang and J.M. Zhou, unpublished results). It is possible that PRR complexes may phosphorylate functionally distinct substrates to activate diverse downstream responses. As illustrated in Figure 2, we propose that one or more unknown substrates may be responsible for the activation of MAPK cascades in a way analogous to the stomatal development pathway, whereas BIK1 and some of the PBL proteins are activated independently of MAPK, probably by regulating the newly identified CDPK-mediated pathway. Although it remains unknown whether BIK1 is directly phosphorylated by FLS2, the ligand-induced phosphorylation of BIK1 and subsequent FLS2 BIK1 dissociation are highly analogous to the activation of BRI1 substrates BSKs upon BR stimulation (Tang et al., 2008; Zhang et al., 2010). 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