Type III protein secretion in plant-bacterial pathogen interactions

Hypersensitive response and pathogenicity (hrp) genes control the ability of major groups of plant pathogenic bacteria to elicit the defense response in resistant plants and to cause disease in susceptible plants. A number of Hrp proteins share significant sequence similarities with components of type III secretion systems and flagellar assembly machineries in animal pathogenic bacteria. The hrp gene-encoded type III secretion system (the Hrp system) is believed to deliver bacterial virulence or avirulence (Avr) proteins into host cells upon infection. However, the molecular mechanism of type III protein secretion is poorly understood in both animal and plant pathogenic bacteria. My doctoral dissertation research is mainly focused on studying the mechanism of type III protein secretion and the function of secreted proteins in a model plant pathogenic bacterium, Pseudomonas syringae pv. tomato (Pst) DC3000.;I began by examining the function of the hrpA gene, which encodes an extracellular protein secreted via the Hrp system of Pst DC3000. A non-polar hrpA mutant was constructed by replacing the hrpA gene open reading frame (ORF) with the aph ORF. In collaboration with Dr. Martin Romantschuk's group I demonstrated that HrpA is a major structural protein of a novel filamentous appendage on the bacterial surface, called the Hrp pilus. The hrpA mutant could no longer cause disease in Arabidopsis thaliana or elicit a resistance response in the non-host tobacco or resistant tomato plants, providing genetic evidence for a critical role of the Hrp pilus in plant-Pst DC3000 interactions.;In the functional study of the Hrp pilus, I found that the hrpA gene was required for expression of all examined Pst DC3000 genes (hrc, hrp and avr) that are associated with the type III secretion. HrpA-mediated gene regulation was achieved through activation of two previously characterized regulatory genes (hrpR and hrpS) at the RNA level. Ectopic expression of the hrpRS operon restored gene expression in the hrpA mutant, but did not restore type III protein secretion. These results define a dual role of the Hrp pilus structural protein in global regulation of the Hrp system and control of type III protein secretion in Pst DC3000.;I also explored the possibility of using an Avr protein as a bait to identify host interacting proteins. Specifically, there is one unique Avr family of proteins, known as Avr/Pth proteins, that are presumably secreted via the Hrp system of the cotton pathogen Xanthomonas campestris pv. malvacearum (Xcm). Some members (e.g., Avrb6) of this family are also required for bacterial virulence in susceptible cotton cultivars. A yeast two-hybrid system was employed to identify cotton receptors of Avrb6. After screening over 1.8 x 108 cotton cDNA clones, I confirmed that over 152 clones encode proteins that specifically bind to the bait, the central repeat region of Avrb6. Sequencing analysis revealed three clones, a serine/threonine kinase homologous to the resistance protein Pto in tomato, a calcineurin B-like protein, and a putative transcription factor with a C2H2-type zinc-finger motif. The functional relevance of these cotton proteins to Xcm avirulence and virulence in plants remains to be determined.