Type III protein secretion and host cell death in plant-Pseudomonas syringae interaction

Hypersensitive response and pathogenicity (hrp) genes control the ability of major groups of plant pathogenic bacteria to elicit the hypersensitive response (HR) in resistant plants and to cause disease in susceptible plants. A number of Hrp proteins are components of type III secretion, which delivers bacterial virulence and avirulence (Avr) proteins into host cells. However, the molecular mechanism of type III protein secretion is poorly understood. Similarly, although plant programmed cell death (PCD) is an integral part of a plant's defense during pathogenic plant-bacteria interactions, little is known about the underlying mechanisms. This research is focused on (1) studying the proteins secreted via the type III secretion system and their roles in interactions between plants and Pseudomonas syringae pv. tomato (Pst) DC3000, and (2) investigating possible similarities between PCD in plant defense and animal apoptosis.;I showed that Pst DC3000 secreted multiple extracellular proteins under hrp-gene-inducing conditions. By creating hrp gene mutations that affect type III secretion, I demonstrated that the production of at least five extracellular proteins, including HrpW, HrpZ, and HrpA, is under the control of the Pst DC3000 type III secretion system. In collaboration with Dr. Martin Romantschuk's group at the University of Helsinki, I showed that the HrpA protein is associated with a novel filamentous appendage (named the Hrp pilus) on the bacterial surface, and that assembly of the Hrp pilus is controlled by the type III secretion system. These results provide the first evidence that the type III system is involved in the secretion of multiple proteins and pilus biogenesis. Furthermore, I provided evidence that HrpA is strongly associated with host cell walls.;Caspases are a family of cysteine proteases that promote apoptosis in animal systems. I found that N-benzyloxycarbonyl-Val-Ala-Asp-chloromethylketone (Z-YVAD-CMK), an inhibitor of the interleukin-1beta converting enzyme (ICE)-type caspases, prevented both the HR necrosis triggered by avirulent pathogens and the disease-associated necrosis caused by virulent pathogens on tobacco leaves. Caspase-like activity was detected in tobacco leaf tissues undergoing cell death. These results demonstrate that a key step in plant PCD involves caspase-like activities. Affinity labeling using biotinylated-YVAD identified three tobacco proteins that bind to the caspase inhibitor Z-YVAD-CMK.;Bcl-2 is a pro-survival protein that appears to inhibit apoptosis in several animal systems. However, instead of inhibiting cell death, I found that the transgenic plants expressing the bcl-2 gene exhibited spontaneous leaf necrosis and showed increased resistance to the virulent bacterial pathogen Pst DC3000. Leaf cells undergoing cell death in these transgenic plants showed nuclear condensation, a phenomenon often observed in animal apoptosis. However, two functional conserved domains, BH1 and BH2, of Bcl-2 were not found to be involved in the necrotic phenotype caused by Bcl-2 expression in transgenic plants. These results suggest that expression of the human bcl-2 gene can trigger plant cell death and disease resistance, but that this process is independent of the anti-apoptotic activity of Bcl-2 in animals.