AvrRpm1 Functions as an ADP-Ribosyl Transferase to Modify NOI-domain Containing Proteins, Including Arabidopsis and Soybean RPM1-interacting Protein 4.

The Pseudomonas syringae effector protein AvrRpm1 activates the Arabidopsis intracellular innate immune receptor protein RPM1 via modification of a second Arabidopsis protein, RIN4. Prior work has shown that AvrRpm1 induces phosphorylation of AtRIN4, but homology modeling indicated that AvrRpm1 may be an ADP-ribosyl transferase. Here we show that AvrRpm1 induces ADP-ribosylation of RIN4 proteins from both Arabidopsis and soybean within two highly conserved nitrate-induced (NOI) domains. It also ADP-ribosylates at least ten additional Arabidopsis NOI domain-containing proteins. The ADP-ribosylation activity of AvrRpm1 is required for subsequent phosphorylation on threonine 166 of Arabidopsis RIN4, an event that is necessary and sufficient for RPM1 activation. We also show that the C-terminal NOI domain of AtRIN4 interacts with the exocyst subunits EXO70B1, EXO70E1, EXO70E2 and EXO70F1. Mutation of either EXO70B1 or EXO70E2 inhibited secretion of callose induced by the bacterial flagellin-derived peptide flg22. Substitution of RIN4 threonine 166 with aspartate enhanced the association of AtRIN4 with EXO70E2, which we posit inhibits its callose deposition function. Collectively, these data indicate that AvrRpm1 ADP-ribosyl transferase activity contributes to virulence by promoting phosphorylation of RIN4 threonine 166, which inhibits the secretion of defense compounds by promoting the inhibitory association of RIN4 with EXO70 proteins.