RIN4-like proteins mediate resistance protein-derived soybean defense against Pseudomonas syringae

Abstract

Resistance (R) protein mediated recognition of pathogen avirulence effectors triggers signaling that induces a very robust form of species-specific immunity in plants. The soybean Rpg1-b protein mediates this form of resistance against the bacterial blight pathogen, Pseudomonas syringae expressing AvrB Pgyrace4. Likewise, the Arabidopsis RPM1 protein also mediates species-specific resistance against AvrB expressing bacteria. RPM1 and Rpg1-b are non-orthologous and differ in their requirements for downstream signaling components. We recently showed that the activation of Rpg1-b derived resistance signaling requires two host proteins that directly interact with AvrB. These proteins share high sequence similarity with the Arabidopsis RPM1 interacting protein 4 (RIN4), which is essential for RPM1-derived resistance. The two soybean RIN4-like proteins (GmRIN4a and b) differ in their abilities to interact with Rpg1-b as well as to complement the Arabidopsis rin4 mutation. Because the two GmRIN4 proteins interact with each other, we proposed that they might function as a heteromeric complex in mediating Rpg1-b-derived resistance. Absence of GmRIN4a or b enhanced basal resistance against bacterial and oomycete pathogens in soybean. Lack of GmRIN4a also enhanced the virulence of avrB bacteria in plants lacking Rpg1-b. Our studies suggest that multiple RIN4-like proteins proteins mediate R-mediated signaling, in soybean.

Figure 1

GmRIN4 proteins co-immunoprecipitate with AvrB. Agrobacterium cells expressing MYC-tagged GmRIN4a, b, c or d were expressed individually or together with FLAG (3X)-tagged AvrB in Nicotiana benthamiana. Proteins were immunoprecipitated (IP) from total extracts (T) using anti-FLAG antibodies, electrophoresed on SDS-PAGE and visualized using tagspecific antibodies (α-MYC for the various GmRIN4 proteins, α-FLAG for AvrB). Part showing AvrB is from the AvrB-GmRIN4a co-immunoprecipitation (Co-IP) experiment and is representative of Co-IPs with GmRIN4b, c and d.

Figure 2

GmRIN4 proteins co-immunoprecipitate with RPM1 (A) and AvrRpm1 (B). Agrobacterium cells expressing MYC tagged GmRIN4a, b, c or d were expressed individually (GmRIN4) or together with 3XFLAG tagged AvrRpm1 (A) or RPM1 (B) in Nicotiana benthamiana. Proteins were immunoprecipitated (IP) from total extracts (T) using anti-FLAG antibodies. Proteins were visualized on western blots using tag-specific antibodies. Parts showing RPM1 and AvrRpm1 are from co-immunoprecipitation (Co-IP) experiments with GmRIN4a and are representative of Co-IPs with GmRIN4b, c and d.

Figure 3

Silencing GmRIN4a or b does not enhance resistance to virulent Pseudomonas syrinage in Rpg1-b (cv. Harosoy) plants. Bacterial counts in plants silenced for GmRIN4a (S_4a) or GmRIN4b (S_4b) as compared to vector-inoculated (V) plants. LOG values of colony forming units (CFU) per unit leaf area from infected leaves at 0 or 4 days post-inoculation (dpi) are presented. Error bars indicate standard deviation (n = 5).

Figure 4

Role of soybean RIN4-like proteins (GmRIN4a & b) in Rpg1-b-derived resistance signaling. (A) GmRIN4a & b interact with AvrB and with each other. GmRIN4b, but not GmRIN4a, interacts with Rpg1-b. Binding of AvrB to GmRIN4a and/or b may be detected by Rpg1-b to induce resistance signaling. Whether activation of Rpg1-b requires binding of AvrB to GmRIN4a and/or b has not been demonstrated. (B) Absence of either GmRIN4a (right part) or b (left part) abrogates the induction of a resistance response.