Development of a Proteomics Platform for Analysis of the Plant-Pathogen Interface

Plants possess an elaborate tiered immune system to detect the presence of pathogens. Once triggered, immune responses are sufficient to abrogate pathogen growth. As a result, effective phytopathogens must either evade immune detection or interdict plant immune signalling and responses. The Gram negative bacteria Pseudomonas syringae causes diseases of economically important crops and can also infect the model plant Arabidopsis thaliana. The type III secretion system (T3SS) represents a critical virulence strategy employed by P. syringae to cause disease in hosts. The T3SS functions as a molecular syringe to inject proteinaceous type III secreted effectors (T3SE) into the plant cytosol. After injection, T3SE inhibit plant immunity through direct interaction with host proteins. Because T3SE interact with plant immune components, T3SE can be used as molecular probes for defining novel components of plant immunity. In this role, T3SE have been exploited to discover crucial components of plant immune systems using classical genetics and yeast two-hybrid analyses. However, there have been limited attempts to leverage T3SE as proteomic probes for isolating protein complexes involved in plant immunity. This thesis presents the first successful proteomic identification of a T3SE/Arabidopsis protein complex. The T3SE HopF2 was purified from transgenic Arabidopsis using a combination of gel filtration chromatography and immunoaffinity purification. During gel filtration, HopF2 existed as both a member of a high molecular weight complex and a monomer. Immunoaffinity purification of high molecular weight complexes containing HopF2, and identification of complex members LC-MS/MS, suggested HopF2 complexes contain plasma membrane H+-ATPases. This lead to the hypothesis that HopF2 may manipulate stomatal apertures during infection because plasma membrane H+-ATPases play a role in regulating stomatal aperture. Consistent with this, transgenic expression of HopF2 inhibits stomatal immunity and increases the virulence of surface inoculated P. syringae. Intriguingly, HopF2's ability to inhibit stomatal aperture is independent of HopF2's characterized ADP-ribosyltransferase activity, suggesting HopF2 is a multifunctional T3SE. Together this implies a novel virulence function of HopF2 through the manipulation of stomatal immunity.