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Plant defense to microbial pathogens involves pre-formed barriers as well as induced responses. A well-known form of induced defense involves the deployment of plant resistance (R) proteins, which confer protection against specific races of pathogens. R-mediated specificity is determined by the presence of cognate effector molecules in the pathogen, termed avirulence (Avr) proteins (Flor, 1971). Direct/indirect interactions between the R and Avr proteins are thought to activate one or more signal transduction pathways that eventually prevent pathogen colonization. Early advances in the understanding of Avr-dependent defense activation in a host genotype-specific manner were made from the analyses of soybean (Glycine max) responses to the bacterial blight pathogen Pseudomonas syringae pv. glycinea (Psg) (Staskawicz et al., 1987). Psg infection of susceptible soybean cultivars results in chlorotic and spreading lesions, whereas resistant cultivars exhibit localized cell death, resulting in necrotic lesions in the cells surrounding the infection site. The localized cell death phenotype is one of the earliest visible manifestations of induced defense reactions in resistant plants, and is termed hypersensitive response (HR; Holliday et al., 1981).
Studies with naturally occurring strains of Psg led to the identification and characterization of several functional bacterial Avr proteins (Keen & Holliday, 1982). These include AvrA1Pgyrace6 (Napoli & Staskawicz, 1987), AvrB1Pgyrace4 (commonly designated AvrB, Staskawicz et al., 1987; Lindeberg et al., 2005), and AvrB2Pgyrace4 (original designation AvrC, Tamaki et al., 1988; Lindeberg et al., 2005). In addition, AvrD1PtoPT23 from P. syringae pv. tomato (Pto), the causal agent of bacterial speck in tomato, has been shown to impart avirulence to Psg in a host cultivar-specific manner (Keen et al., 1990; Kobayashi et al., 1990a). Several Psg strains also express endogenous AvrD1 alleles; however, these do not elicit HR in soybean (Kobayashi et al., 1990b; Keith et al., 1997).
Of these different Avr proteins, resistance to AvrB has been extensively studied. These studies have been carried out in the heterologous host Arabidopsis thaliana using AvrB-expressing Pto (Innes et al., 1993; Bisgrove et al., 1994; Grant et al., 1995; Mackey et al., 2002, 2003; Chung et al., 2011; Liu et al., 2011). In Arabidopsis, the RPM1 (resistance to P. syringae pv. maculicola) gene provides specificity to Pto strains expressing AvrB or AvrRPM1 (Keen & Holliday, 1982; Debener et al., 1991; Innes et al., 1993; Bisgrove et al., 1994; Grant et al., 1995). AtRIN4 (RPM1-interacting 4) protein, which binds both RPM1 and AvrB, is essential for RPM1 resistance (Mackey et al., 2003). Extensive analysis has shown that RPM1 activation is associated with the AvrB-dependent phosphorylation of AtRIN4 (Mackey et al., 2003). This phosphorylation of AtRIN4 via a receptor-like cytoplasmic kinase (designated RIPK for RPM1-induced protein kinase) was recently shown to be essential for RPM1 activation (Mackey et al., 2002; Chung et al., 2011; Liu et al., 2011).
Although genetic resistance to AvrB-expressing bacteria was first identified in soybean, the corresponding R gene was identified more recently. In soybean, resistance to AvrB-expressing Psg (avrB Psg) is determined by the Rpg1 (resistance to Psg) locus (Staskawicz et al., 1987; Keen et al., 1990; Keen & Buzzell, 1991). The Rpg1-b gene on this locus encodes a protein that belongs to the coiled coil-nucleotide binding-leucine rich repeat (CC-NB-LRR) class of R proteins and provides specificity against Psg avrB (Bisgrove et al., 1994; Ashfield et al., 2003; Ashfield et al., 2004). Although Rpg1-b and RPM1 are nonorthologous, they share several signaling features. For example, both R proteins require RAR1 (required for Mla12-mediated resistance, Torp & Jorgensen, 1986) for resistance signaling (Tornero et al., 2002; Fu et al., 2009), and neither R protein directly interacts with AvrB (Grant et al., 1995; Ashfield et al., 2004; Selote & Kachroo, 2010a). Furthermore, like RPM1, Rpg1-b also interacts with RIN4-like proteins; of the four related soybean proteins, designated GmRIN4a–d for their high sequence similarities to AtRIN4, GmRIN4b, c and d interact with Rpg1-b (Selote & Kachroo, 2010a,b). Like AtRIN4, the GmRIN4 proteins all bind AvrB and at least GmRIN4a and b are required for resistance derived from Rpg1-b (Selote & Kachroo, 2010a,b). Here, we further investigated the role of the four GmRIN4 isoforms in resistance derived from Rpg1-b and other R loci in soybean and examined the changes in these proteins in response to the AvrB effector.
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The soybean GmRIN4 family comprises four related isoforms, which bind the P. syringae avirulence effector, AvrB. We show that only two (GmRIN4a and b) of these isoforms are essential for resistance derived from Rgp1-b, the soybean R protein that specifies resistance to Psg avrB. The Psg avrB-dependent phosphorylation of GmRIN4b not only inhibits its own association (and, thereby, that of GmRIN4a) with Rpg1-b, but also activates Rpg1-b. In addition to Rpg1-b, the four GmRIN4 proteins contribute to bacterial resistance derived from three other R loci, including Rpg2, 3, and 4.
Our previous work showing that GmRIN4b directly binds Rpg1-b, but that GmRIN4a does not, posed the dilemma of how the Rpg1-b noninteracting GmRIN4a might contribute to resistance derived from this R protein. The data presented here support the possibility of a GmRIN4a–GmRIN4b–Rpg1-b complex in planta, and inhibition of these associations by Psg avrB. This raised the possibility that the dissociation of the GmRIN4a-b complex from Rpg1-b may contribute to the activation of resistance signaling. Supporting this notion are the facts that GmRIN4b directly interacts with Rpg1-b and GmRIN4a (Selote & Kachroo, 2010a) and that the three proteins were detected in a single complex when expressed heterologously in planta. Interestingly, the presence of Psg avrB inhibited binding of GmRIN4b with GmRIN4a or Rpg1-b. Psg avrB also specifically induced the phosphorylation of GmRIN4b. Clearly, the AvrB-dependent phosphorylation of GmRIN4b was necessary and sufficient to inhibit its binding with GmRIN4a or Rpg1-b; a phosphomimic derivative of GmRIN4b was unable to bind either GmRIN4a or Rpg1-b. Moreover, a phosphodeficient GmRIN4b derivative continued to bind both GmRIN4a and Rpg1-b even in the presence of Psg avrB. This raises the possibility that the dissociation of Rpg1-b from GmRIN4a and GmRIN4b in the presence of Psg avrB might be associated with Rpg1-b activation. However, such a possibility can be discounted, unless N. benthamiana encodes functionally orthologous proteins that can associate with Rpg1-b, because expression of Rpg1-b alone did not induce cell death or ion leakage in N. benthamiana. Furthermore, coexpression of Rpg1-b with the phosphomimic GmRIN4b derivative induced ion leakage and cell death, in the absence of GmRIN4a. This suggests that the AvrB-dependent phosphorylation of GmRIN4b is sufficient to activate Rpg1-b, at least in the heterologous N. benthamiana. Thus, it is possible that, whereas GmRIN4b contributes to the activation of Rpg1-b, GmRIN4a might contribute to resistance signaling, downstream of Rpg1-b activation (Fig. 7). Another possibility is that N. benthamiana ortholog(s) of RIN4 facilitated the pm4b-mediated activation of Rpg1-b in the absence of GmRIN4a. Notably, BLAST analysis of the draft N. benthamiana genome (solgenomics.net, Gomez et al., 2012) identified at least three sequences with significant identities to the GmRIN4a protein (Fig. S10a,c). Furthermore, western blot analysis of total protein extracts from N. benthamiana leaves detected protein(s) of similar molecular weight as GmRIN4, which cross-react with GmRIN4a antibodies (Fig. S10b). Although these data support the possibility of such proteins facilitating pm4b-mediated activation of Rpg1-b in N. benthamiana, conclusive evidence will require the analyses of their binding affinities for Rpg1-b, AvrB, and GmRIN4b, and the effects of AvrB on such associations. Therefore, while our findings clarify the putative mechanism of Rpg1-b activation in the heterologous N. benthamiana, it is quite possible that activation of resistance signaling in the native soybean may involve additional unidentified events.
Figure 7. Model depicting possible AvrB-dependent changes in GmRIN4b and the activation of Rpg1-b-derived resistance. GmRIN4a (4a), GmRIN4b (4b), and Rpg1-b are present in a single complex. Presence of pathogen-delivered AvrB induces the phosphorylation of GmRIN4b and inhibits its binding with GmRIN4a or Rpg1-b. Phosphorylation of GmRIN4b activates Rpg1-b in Nicotiana benthamiana. Dissociation of GmRIN4a may contribute to Rpg1-b activation. Alternatively, GmRIN4a might regulate soybean resistance downstream of Rpg1-b activation.
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Coexpression of Rpg1-b with AvrB induced significant ion leakage in N. benthamiana, in the absence of GmRIN4b. Furthermore, presence of GmRIN4b or the other GmRIN4 isoforms did not significantly alter this response. This suggests that N. benthamiana-encoded RIN4 orthologs likely enable the activation of Rpg1b in the presence of AvrB. Apparently these factors do not interfere with the Avr-independent activation of Rpg1-b by pm4b. Notably, this Avr-independent activation of Rpg1-b required phosphomimic substitutions at both T22 and T198 residues of GmRIN4b, because unlike pm4b (T22/198D), the T22D or T198D derivatives were unable to induce significant ion leakage when coexpressed with Rpg1-b. By contrast, a phosphomimic substitution at T166 of AtRIN4 was sufficient to activate RPM1 (Chung et al., 2011). Furthermore, unlike the GmRIN4b–Rpg1-b interaction, phosphorylation of AtRIN4 does not inhibit its interaction with RPM1. Also, the AtRIN4–RPM1 interaction is thought to be essential for RPM1 activation in the absence of pathogen effector (Chung et al., 2010). Unlike phosphomimic AtRIN4, which did not bind AvrB in yeast (Liu et al., 2009), pm4b was able to associate with AvrB in IP assays. These differences could perhaps be attributed to the fact that multiple isoforms participate in Rpg1-b signaling in soybean, whereas RPM1 activation and derived signaling have been studied in the context of the single AtRIN4. Notably, Arabidopsis encodes several other AtRIN4-related proteins whose functions in RPM1 activation and/or signaling have not yet been examined.
The Psg avrB-dependent phosphorylation of GmRIN4b is not surprising, given the fact that this protein shares very high sequence similarity to AtRIN4, whose AvrB-dependent phosphorylation is well documented. Surprisingly, AvrB-dependent phosphorylation was not detected for GmRIN4a, c and d, even though these isoforms share equally high sequence similarities to AtRIN4. Like GmRIN4b, GmRIN4a, c and d also contain the T residues corresponding to the phosphorylated residues of AtRIN4 (Chung et al., 2011; Liu et al., 2011). Moreover, GmRIN4a, but not GmRIN4b, contains the corresponding S residue, which is phosphorylated in AtRIN4. One untested possibility is that only the GmRIN4b isoform may serve as a substrate for RIPK-like protein kinase(s). Testing the substrate specificities of RIPK-like proteins from soybean during pathogen presence might help to address this. The fact that the AvrB-dependent phosphorylation of GmRIN4b was detectable in the heterologous N. benthamiana suggests that N. benthamiana also encodes RIPK-like proteins, which phosphorylate AtRIN4. This is supported by the observation that related proteins can possibly phosphorylate AtRIN4 in Arabidopsis plants lacking RIPK (Liu et al., 2011). The AvrB-dependent phosphorylation of GmRIN4b, but not GmRIN4a, does correlate with our previous report demonstrating the ability of GmRIN4b, but not GmRIN4a, to complement RPM1 function in the atrin4 mutant (Selote & Kachroo, 2010a) as well as the fact that RPM1 activation requires the phosphorylation of AtRIN4 (Chung et al., 2011; Liu et al., 2011). Thus, it is possible that GmRIN4a was unable to complement RPM1 function because it was not phosphorylated in an AvrB-dependent manner.
Both GmRIN4c and d bind Rpg1-b and AvrB (Selote & Kachroo, 2010a), yet silencing the genes encoding these proteins did not alter Rpg1-b-derived resistance. One possibility is that GmRIN4c and d function redundantly in resistance derived from this R protein. Testing this would require silencing GmRIN4c and d simultaneously, without affecting the expression of GmRIN4a or b, which we were unable to achieve because of the high sequence similarities between these isoforms. However, the GmRIN4c and d isoforms do function independently in resistance derived from other R loci, as well as in basal defense to virulent pathogens. Interestingly, the absence of GmRIN4a, c, or d resulted in the activation of Rpg3 and/or other associated R loci in Flambeau plants. This is similar to the inappropriate activation of RPM1, and the ectopic induction of RPS2 in Arabidopsis plants lacking AtRIN4 (Mackey et al., 2002, 2003; Belkhadir et al., 2004). Interestingly, Rpg1-b does not appear to be activated by the absence of any of the GmRIN4 proteins. Understanding the precise functions of each of the GmRIN4 isoforms and how they regulate the activation and/or signaling derived from multiple R proteins could provide important insights into how plants perceive and activate defenses in response to diverse pathogen effectors.