Plant & Animal Genomes XIV Conference

January 14-18, 2006
Town & Country Convention Center
San Diego, CA

Pseudomonas syringae Type III Effector AvrPtoB Acts As An E3 Ubiquitin Ligase To Suppress Plant Cell Death And Immunity

Robert B. Abramovitch ^1,2 , Radmila Janjusevic³ , C. Erec Stebbins³ , Gregory B. Martin^1,2

¹  Boyce Thompson Institute for Plant Research, Tower Road, Ithaca, New York, 14853, USA

²  Department of Plant Pathology, Cornell University, Ithaca, New York, 14853, USA

³  Laboratory of Structural Microbiology, Rockefeller University, New York, New York, USA

Pseudomonas syringae pv. tomato DC3000 is a model pathogen for studying bacterial pathogenesis of plants. DC3000 uses a type III secretion system to inject virulence effector proteins into the plant cell. Sequencing of the DC3000 genome revealed this pathogen carries over twenty type III effectors, and characterization of these effectors is an important step in understanding the mechanisms of DC3000 pathogenesis. Previously, we have shown that the AvrPtoB type III effector promotes DC3000 virulence by suppressing programmed cell death (PCD) associated with plant immunity. The goal of the current study was to identify the mechanism of AvrPtoB anti-PCD activity. In a yeast two-hybrid screen we isolated tomato Ubiquitin (Ub) as an AvrPtoB interactor. AvrPtoB is strongly ubiquitinated in vitro when incubated with rabbit reticulocyte lysates, and is observed as a smear when expressed in vivo. Together, these data suggested AvrPtoB might exhibit E3 ubiquitin ligase activity. Indeed, AvrPtoB E3 activity is observed in the presence of recombinant E1 activating enzyme and specific E2 ubiquitin conjugating enzymes. The C-terminus of AvrPtoB is necessary for both anti-PCD and E3 ubiquitin ligase activities, suggesting the two functions are associated. Mutation of AvrPtoB lysine residues in the C-terminus, between K512 and K529, disrupts AvrPtoB-ubiquitin interactions, decreases AvrPtoB-mediated anti-PCD activity and abrogates DC3000 pathogenesis of susceptible tomato plants. Remarkably, quantitative decreases in AvrPtoB anti-PCD activity are correlated with decreases in AvrPtoB ubiquitination, demonstrating that anti-PCD activity is dependent on AvrPtoB E3 ubiquitin ligase activity.