To date, researchers studying proteins that travel the type III (Hrp) protein secretion system of bacterial plant pathogens do not know any of the virulence targets of these proteins - the bacterial-intended target that presumably contributes to the ability of these bacteria to be pathogenic. The experiments described in this proposal are designed to reveal the virulence target of HopPsyA (formally known as HrmA). HopPsyA is a protein that travels the type III (Hrp) secretion pathway of the plant pathogen Pseudomonas syringae and, based on indirect evidence, appears to be delivered into plant cells. On some plants, HopPsyA functions as an avirulence (Avr) protein and induces a rapid defense response. By focussing on plants that do not recognize HopPsyA by mounting a defense response, the researchers hope to identify HopPsyA's plant target to begin to unravel how Hrp-delivered proteins allow pathogenic bacteria to multiply and cause disease in plants. Yeast 2-hybrid data presented in this proposal suggest that HopPsyA targets the Mad2 protein in Arabidopsis. Mad2 is a conserved protein in eucaryotes that plays an important role in the cell cycle. When eucaryotic cells have damaged spindles during metaphase, a checkpoint, called the spindle checkpoint, stalls the cell cycle so the cells do not continue into anaphase. This helps prevent daughter cells from receiving too many or too few chromosomes during segregation. Mad2 plays a central role in the spindle checkpoint. Intriguingly, if HopPsyA does indeed interact with Mad2, this suggests that bacterial plant pathogens may be capable of modifying the plant cell cycle to improve the likelihood of successful pathogenesis. The proposed work will encompass many different molecular and cell biological techniques including standard light microscopy, yeast 2-hybrid screens, PCR mutagenesis, protein separation and immunoblot analysis, experiments with transgenic plants, coimmunoprecipitation and other methods to detect protein-protein interactions. Many components between the type III systems in pathogens of animal and plants are conserved. The research described in this proposal should provide important information about type III secretion systems and be of broad interest to researchers studying host-microbe interactions.
