Plant immunity

Plant Signaling & Behavior 6:6, 794-799; June 2011; ©2011 Landes Bioscience

Plant immunity

Evolutionary insights from PBS1, Pto and RIN4 Shuguo Hou,* Yifei Yang, Daoji Wu and Chao Zhang School of Municipal and Environmental Engineering; Shandong Jianzhu University; Jinan, China

Key words: plant immunity, evolution, Pto, PBS1, RIN4

Two layers of plant immune systems are used by plants to defend against phytopathogens. The first layer is pathogenassociate molecular patterns (PAMPs)-triggered immunity (PTI), which is activated by plant cell-surface pattern recognition receptors (PRRs) upon perception of microbe general elicitors. The second layer is effector-triggered immunity (ETI), which is initiated by specific recognition of pathogen type III secreted effectors (T3SEs) with plant intracellular resistance (R) proteins. Current opinions agree that ETI was evolved from PTI, and the impetus for the evolution of plant immunity is pathogen T3SEs, which exhibit virulence functions through blocking PTI, but show avirulence functions for triggering ETI. A decoy model was put forward and explained that the avirulence targets of pathogen T3SEs were evolved as decoys to compete with the virulence targets for binding with pathogen T3SEs. However, little direct evidence for the evolutionary mode has been offered. Here we reviewed the recent progresses about Pto, PBS1 and RIN4 to present our viewpoints about the evolution of plant immunity.

Introduction Interaction between plants and pathogenic microbes is just like a combination. As outcomes, susceptibility and resistance are determined by the power of the opposing sides. On one side, pathogens proliferate in intracellular spaces of plants (called the apoplast) to obtain nutrition from plants. Once entry into the apoplast, pathogens usually inject so-called Type III secreted effectors (T3SEs) into plant cells by using a Type III secretion system (T3SS). The secreted T3SEs attribute to the pathogenesis of plant pathogens through disturbing normal physiological and biochemical processes of plants.1,2 On the other side, plants rely on an elaborate defense system, plant immune system, counteracting pathogens.3 Two major layers of plant immunity, pathogen-associate molecular patterns (PAMPs)-triggered immunity (PTI) and effector-triggered immunity (ETI), have been defined and extensively studied.4,5 PTI is initiated by the perception of microbe conserved PAMPs, such as bacterial flagellin and elongation factor Tu (EF-Tu), with specific plant cell *Correspondence to: Shuguo Hou; Email: [email protected] Submitted: 01/20/11; Revised: 02/13/11; Accepted: 02/14/11 DOI: 10.4161/psb.6.6.15143

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surface pattern-recognition receptors (PRRs), and it usually activates some early resistance responses, including stomatal closure, activation of mitogen-activated protein kinase (MAPK) cascades, transcription of resistance-related genes, reactive oxygen species (ROS) production and callose deposition.4-7 ETI was thought as an accelerated and amplified PTI response. It is activated by plant intracellular resistance (R) proteins after specific perception of pathogenic T3SEs, and is associated with programmed cell death, a response which is referred to as the hypersensitive response (HR).4,5,8 Pathogen T3SEs are like double-edged swords. They trigger ETI to display aviruelnce (Avr) functions in plant with their corresponding R protein on one hand, but exhibit virulence activities in plants in absence of the corresponding R proteins on the other. Great progresses have confirmed that the virulence functions of most pathogen T3SEs are performed by suppressing the plant PTI and ETI.1,9 For examples, HopAI1 was shown to be a phosphothreonine lyase which inactivates MAPK3 and MAPK6, disabling the cell wall defense and transcriptional activation of PAMPs response genes.10 HopU1 was found to ADP-ribosylate glycine-rich RNA-binding proteins (GRPs), GRP7 especially, to quell host immunity by affecting RNA metabolism and plant defense transcriptome.11 In rps2 Arabidopsis, AvrRpt2 inhibits AvrRpm1-triggered ETI through RIN4 cleavage.12 HopF2 was recently reported to target and ADP-ribosylate both MPK kinase 5 (MKK5) and RIN4 in Arabidopsis to block PTI and AvrRpt2trigerred ETI.13,14 In view of the cases that pathogen T3SEs can suppress plant PTI, and that plant ETI is just activated upon recognition of pathogen T3SEs with plant R proteins, a famous “Zigzag” model was brought forward to elucidate the evolution of plant immunity.4,5 The model explained that PTI was primarily evolved to recognize general feature of plant pathogens but subsequently suppressed by pathogen acquired T3SEs, then R proteins were evolved by plants to recognize pathogen T3SEs to initiate the advanced ETI response. Despite the evolutionary model was firmly confirmed by identifications and functional interpretations of more and more effectors which can block PTI, little is known about the detailed relationships or residual evolutionary footprints between these two immunity phages. As we know, Avr effectors also show virulence functions in plants in absence of their corresponding R protein by suppressing PTI. Therefore, the virulence targets of these Avr effectors are important regulators of PTI signaling pathway. For a same effector, whether its virulence

Plant Signaling & Behavior

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target just is its avirulence targets? If not, what is the relationship between the two kind of host proteins? Recently, several studies answered these questions and revealed the evolutionary manner of plant immunity by using comparative biological and biochemical methods. Here, we will give evolutionary insights from the recent researches about Pto, PBS1 and RIN4. Pto Pto was thought as the first R gene indentified from tomato species, and it confers a gene-for-gene resistance to Pseudomonas syringae containing AvrPto.15,16 It was revealed that AvrPtotriggered resistance is actually initiated by the nucleotide binding leucine-rich repeat (NB-LRR) type R protein Prf, which can monitor Pto, the direct target of AvrPto. Therefore, Pto was redefined as the avirulence target of AvrPto.17 AvrPto is able to induce Pto/Prf dependent ETI in tomato, but strongly inhibits PTI in Arabidopsis and susceptible tomato lacking Pto.18 Therefore, Pto is not the virulence target of AvrPto. AvrPto can block flg22induced MAPK activation upstream of MAPK kinase kinase (MAPKKK), suggesting a very early signal component(s) in PTI pathway was targeted by the effector.18 Subsequently, the targets just were proved to be multiple PRRs, including Arabidopsis Flagellin-sensitive 2 (FLS2) and EF-Tu receptor (EFR) and tomato FLS2 homolog. AvrPto acts as a kinase inhibitor, and the kinase inhibition activity of AvrPto is necessary for its virulence function.19 FLS2, an ancient LRR-type receptor-like kinase for specific recognition of bacterial flagellin, is highly conserved in plants. In contrast, Pto, a receptor-like cytoplasmic kinase (RLCK), presents only in a few wild tomato species, suggesting recent evolution of this protein. Through different emergence with FLS2, Pto has high structurally similar to the kinase domain of FLS2.19 And, the interaction between AvrPto and FLS2 shares similar sequence requirements with the interaction between AvrPto and Pto. Crucially, Pto competes with FLS2 for AvrPto binding in vitro and in vivo.19,20 Therefore, an evolutionary relationship between the two protein was naturally established that Pto was evolved from FLS2. Another P. syringae effector AvrPtoB also induces Pto/Prf dependent ETI in tomato and block flg22-induced MAPK activation upstream of MAPKKK in Arabidopsis,18 but AvrPtoB is structurally distinct from AvrPto. AvrPtoB encodes a protein with three functionary domains which include the first 307 amino acids in its N-terminus (AvrPtoB1–307), the middle region containing 80 amino acids (AvrPtoB308–387), and the C-terminus (AvrPtoB388–553) encoding a E3 ubiquitin ligase.21 These three regions confer different roles to the virulence and avirulence of AvrPtoB. For avirulence functions, AvrPtoB1–307 is responsible for the induction of Pto/Prf dependent ETI, AvrPtoB308–387 can activate Fen/Prf dependent ETI, and the C-terminal E3 ubiqutin ligase has not been indicated with a function on ETI induction.22,23 For virulence functions, AvrPtoB1–307 promotes ethylene-associated virulence in susceptible tomato, AvrPtoB308–387 is responsible for the PTI inhibition,22 and the C-terminal E3 ubiqutin ligase can inhibit PTI and Fen/Prf dependent ETI

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through inducing ubiquitination and elimination of FLS2 and Fen respectively.23,24 But the C-terminal E3 ubiqutin ligase can not inhibit the Pto/Prf dependent ETI for the reason that Pto can escape from the degradation induced by the E3 ubiqutin ligase.25 From all the cases above, a coevolution theory between AvrPtoB and its host receptors was revealed that the functionary regions of AvrPtoB were generally originated through a shuffling process called terminal re-assortment, and plant receptors were evolved correspondingly to recognize the different forms of AvrPtoB in different evolutionary stage. In the first stage, AvrPtoB1–387, existing as an original form of AvrPtoB, exhibited its virulence function mainly by inhibiting PTI, and additionally owned an ability to promote ethylene-associated virulence by interaction with another host protein. Susceptible tomato plant subsequently evolved some FLS2 kinase domain-like proteins, and Fen, one of the evolved proteins, can recognize the AvrPtoB30–387 region to trigger Prf-mediated ETI. In the second stage, a E3 ubiquitin ligase integrated with the AvrPtoB1–387 and generated the full-length AvrPtoB. Acquired E3 ubiquitin ligase domain of the effector abolished Fen/Prf dependent ETI through ubiquitinating and inducing elimination of Fen. The FLS2 also can be degraded because its kinase domain is structurally similar to Fen. At the last, a E3 ubiquitin ligase insensitive kinase, Pto, was evolved to restore Prf-activated ETI (Fig. 1). Though Fen kinase shares structural similarity with Pto and the kinase domain of FLS2, it does not interact with AvrPto.20 A reasonable explanation for this condition is that AvrPto was evolved separately from AvrPtoB, and it was generated after the emergence of Fen kinase to keep the ability of pathogen for PTI inhibition. In a resistant tomato variety, Pto and Fen are located within a cluster of five kinase homologs.26 Another Pto homolog in the cluster, PtoC, does not induce HR but has similar interaction mode with AvrPtoB truncation (AvrPtoB1–307) with Pto. Furthermore, PtoC is insensitive to ubiquitination by AvrPtoB, suggesting it probably is evolved as a transitional form between Fen and Pto to competes with Fen for AvrPtoB binding and relieving the degradation of Fen.27 Taken together, the total evolutionary processes of plant receptors and the pathogen effectors involved above were shown in Figure 1. PBS1 PBS1 also is a RLCK. It can be cleaved by P. syringae effector AvrPphB, a cysteine protease and the cleavage triggers the RPS5mediated ETI.28,29 In Arabidopsis lacking RPS5, AvrPphB inhibits PTI and its protease activity is required for the PTI inhibition function. PBS1 is cleaved by AvrPphB, but the cleavage of PBS1 does not appear to account for the PTI-inhibition activity of AvrPphB, because pbs1 mutants showed only minimal defects in PTI defenses.30 Therefore, some other substrates of AvrPphB cysteine protease were suggested to be the genuine virulence targets of this effector. AvrPphB cleaves PBS1 after the sequence “GDK,” a motif that defines AvrPphB substrate specificity.28 More than twenty proteins (named PBS1-like proteins, PBLs) fit this feature in Arabidopsis, and at least eight of them were identified to be cleaved by AvrPphB. Of these, botrytis-induced


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Figure 1. Coevolution of P. syringae effectors and Pto-associated immunity. FLS2-mediated PTI is a high conserved immune signaling pathway in plants. N-terminal region of P. syringae effector AvrPtoB, an initial pattern of AvrPtoB, suppress FLS2-mediated PTI in Arabidopsis and tomato. Fen kinase and R protein Prf were next evolved by tomato to relieve PTI suppression, and initiate ETI. AvrPto and E3 ligase domain of AvrPtoB were subsequently evolved by pathogen to restore PTI suppression activity, and counter Fen-mediated ETI. Finally, Pto kinase was generated in resistant tomato to recognize AvrPto and AvrPtoB to restore ETI.

Figure 2. Evolution of PBS1-RPS5 mediated ETI. Recognition of PAMPs by transmembrane PRRs triggers PTI responses, which usually mediated by receptor-like cytoplasmic kinases (RLCKs) and MAPK cascades. Pathogens deliver T3SE avrPphB to suppress PTI responses through targeting RLCK BIK1. Plant evolved BIK1-like proteins, which act redundant signaling mediators, to relieve the PTI inhibition by avrPphB. PBS1 at last was evolved by plant to trigger the ETI resistance together with R protein RPS5.

kinase 1 (BIK1) has been confirmed to play a major role in mediating PTI, PBS1 and other PBLs, display minor but functionally additive effects in PTI defenses.30,31 The data suggested that an original protein, maybe BIK1 itself, was targeted by AvrPphB to inhibit PTI, then some PBS1-like kinases were generally evolved from the original virulence target to relieve the opposing pressure of PTI inhibition, until the final generation of PBS1 with an ability on interaction with the NB-LRR R protein RPS5 and keeping an inactive state of its partner R protein. Once PBS1 was evolved, its cleavage would activate RPS5 to initiate the advanced ETI response (Fig. 2). AvrPphB was originally indentified from the P. syringae pv. phaseolicola, the causal agent of halo blight of bean. AvrPphB activates ETI in bean cultivar containing R3 resistance gene,

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but displays virulence function in susceptible bean cultivars without the marching R gene.32 Therefore, identifications the avirulence and virulence targets of AvrPphB in bean cultivars and analyzing correlations of the targets will thoroughly reveal the evolutionary process of plant immunity against this effector. RIN4 RIN4 was indentified to be an avirulence target of P. syringae effectors AvrRpt2, AvrRpm1 and AvrB in Arabidopsis. AvrRpt2, a cysteine protease, can cleave RIN4 and lead to RIN4 elimination and AvrRpm1 and AvrB can induce phosphorylation of RIN4. The cleavage and phosphorylation of RIN4 have been shown to be required for activations of R proteins RPS2 and


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Figure 3. Evolution of RIN4-RPM1/RPS2 mediated ETI. (A) RIN4 plays as a negative regulator of PTI pathway through mimicking a signal mediator of PTI, and plants keep a normal PTI by controlling an appropriate expression of RIN4; (B) PTI response is enhanced in plants without RIN4; (C) PTI response is reduced in plants with RIN4 overexpression. (D) Evolutionary process of RIN4-RPM1/RPS2 mediated ETI. Initially, plant PTI signaling pathway was initiated by PRRs upon specific perception of microbe PAMPs, and pathogen effectors AvrRpm1 and AvrRpt2 can block the PTI through targeting the mediators of this pathway. RIN4 was subsequently evolved to structurally mimic the targets of pathogen effectors, and compete with the targeted components for binding pathogen effectors. Finally, R protein RPM1 and RPS2 were evolved to initiate ETI through monitoring AvrRpm1/AvrRpt2induced modification of RIN4.

RPM1 respectively.33-35 Furthermore, genetic results showed that Arabidopsis RIN4 also plays as a negative regulator of PTI, because RIN4 overexpression lines exhibit increased PAMPtriggered cell wall thickening and rin4 rps2 double mutant lines shows slightly enhanced PTI response.36 AvrRpt2, AvrRpm1 and AvrB exhibit virulence activities in Arabidopsis lacking their marching R proteins, and the activity of cysteine protease for AvrRpt2 is also responsible for the virulence function of the effector.37,38 Although RIN4 is targeted by AvrRpm1, AvrB and AvrRpt2, RIN4 is not required for the virulence functions of the three effectors in Arabidopsis. On the contrary, the host protein negatively regulates AvrRpt2 virulence function.39,40 This scenario also is applied for RIN4 homologs of soybean (Glycine max). In soybean, P. syringae pv. glycinea effector AvrB targets all four RIN4 homologs (named GmRIN4a-d), and triggers RPG-b-mediated resistance. While, both GmRIN4a- and


GmRIN4b-silenced rpg1-b plants support more growth of AvrB expressing P. syringae, than the virulent strain and GmRIN4asilenced rpg1-b plants consistently accumulate higher levels of AvrB bacteria than the control plants.41 P. syringae pv. tomato DC3000 effector HopF2 was recently confirmed to abolish AvrRpt2-trigerred ETI through inhibiting RIN4 cleavage by AvrRpt2.13 HopF2 has ADP-ribosyltransferase activity, and Arabidopsis RIN4 also was indicated to be ADPribosylated by HopF2 in vitro. Furthermore, HopF2 strongly inhibits PTI responses, and the inhibition was just confirmed to be accomplished through interaction with an ADP-ribosylating MKK5.14 Although RIN4 is also responsible for the virulence function of HopF2, it is not required for the PTI inhibition by HopF2 in Arabidopsis.13 Also, our recent studies found that HopF1, HopF2 homolog in P. syringae pv. phaseolicola, inhibits PTI responses in RIN4 independent manner in common bean


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(Phaseolus vulgaris). On the contrary, silencing of RIN4 homologs of bean slightly enhanced the virulence of HopF1 (Hou S et al, unpublished data). Current opinion tends to believe that RIN4 serves as a decoy to mimic the primary virulence target of RIN4-interacted effectors to compete for binding with these effectors. The mimicry therefore relieves the virulence effect on the genuine target.42,43 RIN4 may just be a mimic in structure but not in function, and it could competitively bind not only with the virulence effectors but also with the signal components in PTI. In other words, RIN4 can interact with PTI signal components, but it has no function on mediation of the PTI signaling. That may be the reason why plants overexpressing RIN4 shows subdued PTI responses and increased bacterial propagation, and RIN4 silencing plants display enhanced PTI responses and reduced bacterial growth (Fig. 3A). Now that both RIN4 and MKK5 were targeted and ADP-ribosylated by HopF2, RIN4 was naturally presumed to be the mimic of MKK5. Though it is difficult for us to establish a direct relationship between the two classes of proteins based on current studies, a little evidence also was offered in some studies. First, RIN4 can be phosphorylated in AvrB and AvrRpm1 dependent manners, but AvrB and AvrRpm1 have no kinase activates, suggesting that a host kinase, maybe MAPKKK, was employed to accomplish the modification. Second, MAPK4 interacts with strongly phosphorylates RIN4 protein in an AvrB-dependent manner, confirming the possibility for RIN4 as a substrate of kinase.44 RIN4 is a highly conserved protein in plants. It is present in most detected monocot and dicot, such as tomato, tobacco, soybean, common bean and lettuce. And, all RIN4 homologs from multiple species usually contain domains required for cleavage by AvrRpt2 and interaction with AvrB. However, different RIN4 homologs from different or the same plant spices play different roles for resistance mediation. As introduced above, soybean contains at least four RIN4-like proteins (GmRIN4a-d). The four RIN4-like proteins play different roles in mediating PTI and ETI responses. GmRIN4a, but not GmRIN4b, negatively regulates AvrB virulence activity in the absence of soybean R protein RPG1-B. GmRIN4b, but not GmRIN4a, complements the Arabidopsis rin4 mutation. Both GmRIN4a and GmRIN4b bind AvrB, but only GmRIN4b binds RPG1-B, and silencing GmRIN4b abrogates RPG1-B-derived resistance to P. syringae expressing AvrB.41 Although, only one homolog of RIN4 exists in Arabidopsis, Arabidopsis has multiple proteins with the sequence of AvrRpt2cleavage site in RIN4, and these proteins probably play important function on PTI mediation. Overall, it was supposed that RIN4 homolog originally did as a positive regulator of PTI. During the process of evolution, plant(s) gradually generated diversified RIN4 homologs through both mutational changes and gene amplification. Among the newly evolved RIN4 homologs, some acquired the ability for R protein interaction and ETI initiation, but lost the function for PTI mediation (Fig. 3B). Conclusions and Perspectives Pto, PBS1 and RIN4 as avirulence targets of P. syringae effectors were put together here to elucidate the evolutionary principle of

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plant immunity. The answers for two questions are discussed here to summarize our general viewpoints. The first question is how and when did the three avirulence targets of effectors evolve? Two theoretical evolutionary patterns were suggested as introduced previously. The first possibility is that avirulence targets were evolved from original targets of pathogen T3SEs for virulence.42 This point was strongly supported by our discussion above about Pto and PBS1, but it can not be verified from RIN4 before targets for the virulence functions of AvrRpt2 and AvrRpm1 were indentified. The second possibility is that avirulence targets may be recruited to function in effector perception from another signaling pathway.42 It is difficult for us to understand this deduction because the recruitment of an essential signal component to be targeted by pathogen T3SE also is not favorable to the fitness of plant. The avirulence targets also were not evolved later than the emergence of their R protein partners because most R proteins usually have no effects on plant ETI activation alone, and some R proteins, such as RPS2, were constitutively active in absence of its avirulence target partner.38 This point is well illustrated by PBS1-RPS5, RIN4-RPS2/RPM1 and Fen/Prf. Here, we suggested that Pto was possibly generated later than R protein Prf and its generation just improved the Fen/Prf against AvrPtoB and AvrPto. Of course, the avirulence targets and its R protein partners possibly came into being at the same time. In the course of evolution, they generally developed a delicate immune switch, which normally keeps closed position, but opens after avirulence targeted was modified by pathogen effector. However, based on the last conjecture, it is difficult to explain why Pto has an functional kinase activity, and the kinase activity is responsible for a Prf-independent resistance. The second question is why avirulence target was evolved? A Decoy Model was put forward and explained that avirulence targets originated to function as decoy proteins to compete pathogen virulence effectors with the virulence targets.42 However, this kind of evolution manner is not completely feasible, because generated decoys does not fully mask the virulence function of pathogen effectors. Here, we suggested that decoy is not a periodical intention but only a phenomenon for evolution of plant immunity. When some one signal component was targeted by pathogen effector, plants inclined to duplicate the gene of the virulence target, and to relieve this opposing selection pressure through increasing the dosage of the virulence target. Amplified homology proteins generally diversified in structure and function. Some lost the ability on PTI mediation but enhanced its binding affinity with pathogen effector, and these kinds of proteins just were so-called “Decoy.” Because the virulence activity of pathogen had not been abolished until this stage, plant subsequently evolved a suicide defense system through generating R protein. As mentioned above that Pto is an active kinase, and the kinase activity of Pto was preserved during evolution possibly because it is benefit for plant immunity, because the AvrPto-Pto interaction was confirmed just to be mediated by the phosphorylation-stabilized P+1 loop in Pto.20 Although the various possibilities discussed above seem to be reasonable, there are also some questions are difficult to understand based on our opinions. For example, what are the virulence targets of AvrRpt2 and AvrRpm1, and whether RIN4 indeed


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structurally mimics the virulence targets of the two effectors? As a specific protein, RIN4 was evolved for interaction with pathogen effectors and ETI activation, why it is so conserved in plants? Except for AvrRpt2, AvrRpm1 and AvrB, at least five pathogen References 1.

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