Type III effector AvrPtoB requires intrinsic E3 ubiquitin ligase activity to suppress plant cell death and immunity

Microbial pathogens of both plants and animals employ virulence factors that suppress the host immune response. The tomato pathogen Pseudomonas syringae injects the AvrPtoB type III effector protein into the plant cell to suppress programmed cell death (PCD) associated with plant immunity. AvrPtoB also inhibits PCD in yeast, indicating that AvrPtoB manipulates a conserved component of eukaryotic PCD. To identify host targets of AvrPtoB, we performed a yeast two-hybrid screen and identified tomato ubiquitin (Ub) as a strong AvrPtoB interactor. AvrPtoB is ubiquitinated in vitro and exhibits E3 Ub ligase activity in the presence of recombinant E1 activating enzyme and specific E2 Ub-conjugating enzymes. The C terminus of AvrPtoB is sufficient for both anti-PCD and E3 Ub ligase activities, suggesting the two functions are associated. Indeed, mutation of AvrPtoB lysine residues in the C terminus, between K512 and K529, disrupts AvrPtoB-Ub interactions, decreases AvrPtoB-mediated anti-PCD activity, and abrogates P. syringae pathogenesis of susceptible tomato plants. Remarkably, quantitative decreases in AvrPtoB anti-PCD activity are correlated with decreases in AvrPtoB ubiquitination and E3 Ub ligase activity. Overall, these data reveal a unique bacterial pathogenesis strategy, where AvrPtoB manipulates the host Ub system and requires intrinsic E3 Ub ligase activity to suppress plant immunity.