Investigating auxin biosynthesis and its contribution to virulence by Pseudomonas syringae pv tomato DC3000

Auxin is a major hormone that regulates many processes in plant development and has been shown to be important in several plant-pathogen interactions. Many microbes, including multiple pathovars of Pseudomonas syringae produce indole-3-aceitc acid (IAA), a naturally occurring auxin. However IAA production and its biosynthesis pathway remains undetermined for many bacteria including P. syringae strain DC3000, the subject of this thesis. To investigate IAA biosynthesis by DC3000, I examined IAA production in cultures fed with different pathway intermediates. I show that DC3000 produces IAA in culture and cultures supplemented with Indole-3-acetaldehyde (IAAld) produced high levels of IAA, indicating that IAAld is an important intermediate for DC3000 IAA biosynthesis. Subsequently I investigated the IAA pathway(s) that utilize IAAld as an intermediate focusing on enzymes that convert IAAld to IAA. Through bioinformatics, I identified 6 putative DC3000 aldehyde dehydrogenase enzymes and showed that E. coli expressing genes encoding three of these, PSPTO_0092, PSPTO_2673, and PSPTO_3644, displayed IAAld dehydrogenase activity. Mutations in each of these aldehyde dehydrogenase genes, which we designated as aldA, aldB and aldC respectively, were made and tested for IAA production. The aldA and aldB mutants displayed reductions in IAA when grown in cultures supplemented with IAAld, however the aldC mutant did not. These results indicate that DC3000 utilizes IAAld as an important intermediate for IAA production and AldA and AldB function in DC3000 IAA biosynthesis as indole-3-acetaldehyde dehydrogenases that convert IAAld to IAA. Additionally I tested whether IAA derived from P. syringae DC3000, a bacterial speck causing bacteria, contributes to its pathogenesis. To investigate the role of DC3000 derived IAA during virulence, I utilized the DC3000 IAA biosynthesis mutants aldA and aldB. I showed that both ald mutants display reduced growth in Arabidopsis plants and that the severity of the reduced virulence is correlated with the degree of reduced IAA production. Additionally I showed that the ald mutants do not display reduced growth on a SA deficient mutant, sid2-2 suggesting that DC3000 derived auxin functions to suppress SA-mediated host defenses. Elevated endogenous IAA in transgenic plants overexpression the Arabidopsis YUCCA1 auxin biosynthesis gene did not rescue the ald mutant phenotype, indicating that plant derived auxin and pathogen derived auxin function to regulate DC3000 virulence in independent manners