Degree Type

Dissertation

Date of Award

2013

Degree Name

Doctor of Philosophy

Department

Plant Pathology and Microbiology

First Advisor

Gwyn A. Beattie

Abstract

Pseudomonas syringae is a model bacterial plant pathogen that is adapted for growth and survival on leaf surfaces and in the leaf interior of its host plants. P. syringae likely experiences distinct environmental conditions in different phases of its interaction with plants. In this study, we used global transcriptome profiling of P. syringae pv. syringae B728a to analyze genes and traits that were responsive to growth and survival on the leaf surface versus in the leaf apoplast. We found that the epiphytic environment specifically favors active relocation via flagellar motility, swarming motility, chemosensing, and chemotaxis, whereas the apoplastic environment favors traits contributing to virulence including the synthesis of two phytotoxins and syingolin A as well as the degradation of an alternative amino acid that suppresses virulence. Through comparing the transcriptomes of in planta cells to those of cells exposed to various stresses in culture media, we found that water limitation is a major stress that limits B728a growth and survival in these leaf habitats. P. syringae can adapt to water stress by the production of a potent compatible solute, trehalose. Distinct P. syringae strains vary in their ability to tolerate water stress, possibly due, in part, to differences in the production or regulation of trehalose. To investigate this possibility, we compared the relative contribution of the trehalose biosynthetic pathways to trehalose synthesis in two closely related P. syringae strains B728a and DC3000 and characterized an apparent interdependency between these pathways. Our data showed that, of the two trehalose biosynthesis pathways, only one was required for trehalose production in B728a whereas both were needed in DC3000, and moreover that differences in trehalose production may help explain differences in their water stress tolerance. Lastly, to understand the contribution of distinct regulators to fitness and pathogenicity of P. syringae, we performed a transcriptome analysis of B728a and mutants lacking each of nine regulators, including quorum-sensing regulators (AhlR and AefR), global regulators (GacS, SalA and RetS), and alternative sigma factors (RpoN, AlgU, RpoS, and HrpL), with cells recovered from the surface and interior of bean leaves as well as exposed to various environmental stresses. Our data showed that AhlR and AefR had negligible roles during B728a leaf colonization, whereas GacS and SalA had major roles. GacS/SalA formed a large regulatory network with both plant signal-dependent and plant signal-independent branches. RetS functioned almost exclusively to repress secondary metabolite genes when B728a cells were not in the leaf environment. Among the alternative sigma factors, RpoN influenced the majority of the genome whereas AlgU influenced a large number and RpoS a small number of genes, with plant signals strongly attenuating RpoN activation of the AlgU-regulated genes. Lastly, HrpL influenced very few genes in planta, due primarily to suppression by GacS and SalA. Collectively, our results highlight the role of these regulators during P. syringae colonization of leaves and the central importance of signals in the leaf environment on their regulation.

Copyright Owner

Xilan Yu

Language

en

File Format

application/pdf

File Size

203 pages

Included in

Microbiology Commons

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