A conserved glutamate residue in RPM1-interacting protein4 is ADP-ribosylated by Pseudomonas effector AvrRpm2 to activate RPM1-mediated response

ABSTRACT Gram-negative bacterial plant pathogens directly inject effectors into their hosts to hijack and manipulate metabolism, eluding the frontier surveillance at the cell surface. The effector AvrRpm1Pma from Pseudomonas syringae pv. maculicola functions as an ADP-ribosyl transferase, modifying RPM1-interacting protein4 (RIN4), leading to the activation of Arabidopsis resistance protein RPM1. We identified the ADP-ribosyl transferase activity of another bacterial effector AvrRpm2Psa from Pseudomonas syringae pv. actinidiae via infection using a Pseudomonas syringae pv. tomato strain following Agrobacterium-mediated transient expression of RIN4 in N. benthamiana. We conducted mutational analysis in combination with mass spectrometry to genetically locate the modified residue. We show that a conserved glutamate residue (E156) of AtRIN4 is the target site for AvrRpm2Psa by demonstrating the modified AtRIN4 with E156A substitution is no longer ADP-ribosylated. Accordingly, naturally occurring soybean and snap bean RIN4 homologs with no glutamate at the positions corresponding to the E156 of AtRIN4 are not ADP-ribosylated by AvrRpm2Psa. In contrast with another effector AvrB, modifications of potential phosphorylation sites including T166 in AtRIN4 affected neither ADP-ribosylation nor RPM1 activation by AvrRpm2Psa. This study suggests that separate biochemical reactions by different pathogen effectors may trigger the activation of the same resistance protein through distinct modifications of RIN4. One sentence summaryA conserved glutamate residue (E156) in the C-NOI domain of RPM1-interacting protein4 is ADP-ribosylated by Pseudomonas effector AvrRpm2 to activate RPM1-mediated defence response, independently of phosphorylation at T166.