GmRIN4 protein family members function nonredundantly in soybean race-specific resistance against Pseudomonas syringae

The Pseudomonas syringae effector AvrB interacts with four related soybean (Glycine max) proteins (GmRIN4ad), three (GmRIN4b, c, d) of which also interact with the cognate resistance (R) protein, Rpg1-b. Here, we investigated the specific requirements for the GmRIN4 proteins in R-mediated resistance and examined the mechanism of Rpg1-b activation. Using virus-induced gene silencing, we show that only GmRIN4a and b are required for Rpg1-b-mediated resistance. In planta binding assays show that GmRIN4a can associate with Rpg1-b indirectly via GmRIN4b. Pathogen-delivered AvrB induces the phosphorylation of GmRIN4b alone, and prevents interactions between GmRIN4b and Rpg1-b or GmRIN4a. Consistent with this result, a phosphomimic derivative of GmRIN4b (pm4b) fails to bind Rpg1-b and GmRIN4a. Conversely, a phosphodeficient derivative of GmRIN4b (pd4b) continues to bind the R protein and GmRIN4a, in the presence of AvrB. Coexpression of Rpg1-b with pm4b, but not GmRIN4b or pd4b, induces cell death and ion leakage in the heterologous Nicotiana benthamiana. Our data suggest that the AvrB-induced phosphorylation of GmRIN4b, and the subsequent inhibition of interaction among GmRIN4b, GmRIN4a and Rpg1-b, might activate the R protein. Furthermore, even though GmRIN4c and d are not required for Rpg1-b-derived resistance, they do function in resistance derived from other R loci.