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Investigation Of The Interconnected Roles Of Cmal And Hopaa1-1 In The Virulence Of Pseudomonas Syringae Pv.Tomato Dc3000 In Nicotiana Benthamiana
Pseudomonas syringae pv. tomato DC3000 (DC3000) is a model plant pathogenic bacterium that infects tomato and Arabidopsis thaliana. It requires the phytotoxin coronatine and the delivery of type III effector proteins (T3Es) into the host cell cytoplasm for defense suppression and virulence. CmaL is a small protein found to be necessary for coronatine production. Coronatine is a potent molecular mimic of jasmonoyl-isoleucine, a plant hormone conjugate involved in regulating plant defenses. Coronatine is constructed of two amide bond-linked moieties, coronafacic acid and coronamic acid. CmaL was shown to be required for the production of L-allo-isoleucine, a precursor for coronamic acid biosynthesis. DC3000 mutants lacking both cmaL and the T3E gene hopAA1-1 are reduced in speck formation in tomato. hopAA1-1 is member of the conserved effector locus, a group of effector genes located adjacent to the genes encoding the type three secretion apparatus that are widespread among P. syringae strains. HopAA1-1 is toxic to both plants and yeast upon expression within them. To gain insight into the basis for its toxicity in eukaryotic cells, the subcellular localization of HopAA1-1 was investigated. HopAA1-1 was found to colocalize with plant peroxisomes. Truncated derivatives of HopAA1-1 that are not cytotoxic and cannot promote symptom formation do not localize with peroxisomes. Additionally, other truncated derivatives of HopAA1-1 colocalize with the endoplasmic reticulum in addition to peroxisomes, suggesting that HopAA1 -1 interacts with the endomembrane system. A DC3000 mutant with 28 T3E genes deleted (DC3000D28E) is a recently developed tool for investigating effector functions. DC3000D28E derivatives with small sets of effector genes progressively restored show increasing virulence wh en inoculated by infiltration with a blunt syringe into the model plant Nicotiana benthamiana. Because of its location in a cluster of effector genes, cmaL was inadvertently deleted in the construction of DC3000D28E. The importance of coronatine and its partial redundancy with HopAA1-1 in promoting an early stage of pathogenesis was revealed by restoring cmaL and hopAA1-1 to selected DC3000D28E derivatives and assaying the strains by dip inoculation of N. benthamiana leaves, which requires bacteria to follow a natural infection route through stomata.
Bacteriology; Pseudomonas syringae; Plant Pathology
Collmer, Alan Raymond
Martin, Gregory B; Hanson, Maureen R
Ph.D. of Microbiology
Doctor of Philosophy
dissertation or thesis