The Pseudomonas syringae effector protein, AvrRPS4, requires in planta processing and the KRVY domain to function.

A Pseudomonas syringae pv. pisi effector protein, AvrRPS4, triggers RPS4-dependent immunity in Arabidopsis. We characterized biochemical and genetic aspects of AvrRPS4 function. Secretion of AvrRPS4 from Pst DC3000 is type III secretion-dependent, and AvrRPS4 is processed into a smaller form in plant cells but not in bacteria or yeast. Agrobacterium-mediated transient expression analysis of N-terminally truncated AvrRPS4 mutants revealed that the C-terminal 88 amino acids are sufficient to trigger the hypersensitive response in turnip. N-terminal sequencing of the processed AvrRPS4 showed that processing occurs between G133 and G134. The processing-deficient mutant, R112L, still triggers RPS4-dependent immunity, suggesting that the processing is not required for the AvrRPS4 avirulence function. AvrRPS4 enhances bacterial growth when delivered by Pta 6606 into Nicotiana benthamiana in which AvrRPS4 is not recognized. Transgenic expression of AvrRPS4 in the Arabidopsis rps4 mutant enhances the growth of Pst DC3000 and suppresses PTI (PAMP-triggered immunity), showing that AvrRPS4 promotes virulence in two distinct host plants. Furthermore, full virulence activity of AvrRPS4 requires both proteolytic processing and the KRVY motif at the N-terminus of processed AvrRPS4. XopO, an Xcv effector, shares the amino acids required for AvrRPS4 processing and the KRVY motif. XopO is also processed into a smaller form in N. benthamiana, similar to AvrRPS4, suggesting that a common mechanism is involved in activation of the virulence activities of both AvrRPS4 and XopO.