Differential binding specificity of the bacterial effector protein AvrPto to plant receptor-like kinase virulence targets

Abstract

Pseudomonas syringae is a widespread and highly adaptable bacterial plant pathogen that can infect both economically relevant crop plants and Arabidopsis thaliana, making it a highly relevant model pathogen for investigating the origin of pathogenicity in the plant immunity field. P. syringae uses a large and diverse arsenal of effector proteins secreted into the host cell to suppress plant immune responses during the infection process and thereby increase its virulence. One of these effectors, AvrPto, has historically been studied in-depth as a part of a classical gene-for-gene resistance pair in tomato. Though it has been the target of scientific inquiry for more than two decades, the virulence mechanism of AvrPto remained largely elusive. AvrPto binds the intracellular kinase domains of multiple plant plasma membrane pattern recognition receptors, and previous qualitative investigations into these interactions led to inconclusive and conflicting hypotheses regarding AvrPto’s virulence target. This thesis provides insights into these unresolved questions by using quantitative methodology to investigate differences in binding specificity and interaction kinetics in AvrPto’s multi-faceted interactome. AvrPto was shown to bind the intracellular domain of the PRR-interacting co-receptor AtBAK1 with high affinity, while binding the equivalent domain of the PRR AtFLS2 with demonstrable, but significantly lower affinity. Quantitative investigation into the interaction of AvrPto with SlPto, the guarded effector target forming the molecular basis for R-gene mediated immunity in response to AvrPto recognition in tomato, show that the affinity of this interaction ranks between those of AvrPto with either BAK1 or FLS2. These data indicate that AvrPto likely evolved to bind BAK1 homologues in various host plant species, and in turn, SlPto evolved in tomato to bind AvrPto and mediate its recognition. In contrast, binding to FLS2 likely occurs due to its structural similarity to the primary target BAK1. Importantly, additional kinetic analysis of these interactions provided a conceptually different methodological approach to corroborate the affinity data, and added temporal information consistent with the described evolutionary model. Although the exact molecular determinants of AvrPto’s kinase binding specificity remain elusive, mutational analysis on BAK1’s intracellular domain indicate that the interaction likely occurs structurally similarly to that of AvrPto with Pto, an interaction which has already been structurally elucidated.