Abstract
Gram-negative bacterial plant pathogens inject effectors into their hosts to hijack and manipulate metabolism, eluding surveillance at the battle frontier on the cell surface. The effector AvrRpm1Pma from Pseudomonas syringae pv. maculicola functions as an ADP-ribosyl transferase that modifies RESISTANCE TO P. SYRINGAE PV MACULICOLA1 (RPM1)-INTERACTING PROTEIN4 (RIN4), leading to the activation of Arabidopsis thaliana (Arabidopsis) resistance protein RPM1. Here we confirmed the ADP-ribosyl transferase activity of another bacterial effector, AvrRpm2Psa from P. syringae pv. actinidiae, via sequential inoculation of Pseudomonas strain Pto DC3000 harboring avrRpm2Psa following Agrobacterium-mediated transient expression of RIN4 in Nicotiana benthamiana. We conducted mutational analysis in combination with mass spectrometry to locate the target site in RIN4. A conserved glutamate residue (Glu156) is the most likely target for AvrRpm2Psa, as only Glu156 could be ADP-ribosylated to activate RPM1 among candidate target residues identified from the MS/MS fragmentation spectra. Soybean (Glycine max) and snap bean (Phaseolus vulgaris) RIN4 homologs without glutamate at the positions corresponding to Glu156 of Arabidopsis RIN4 are not ADP-ribosylated by bacterial AvrRpm2Psa. In contrast to the effector AvrB, AvrRpm2Psa does not require the phosphorylation of Thr166 in RIN4 to activate RPM1. Therefore, separate biochemical reactions by different pathogen effectors may trigger the activation of the same resistance protein via distinct modifications of RIN4.