Degree Type

Dissertation

Date of Award

2021

Degree Name

Doctor of Philosophy

Department

Plant Pathology and Microbiology

Major

Genetics

First Advisor

Gwyn A Beattie

Abstract

Light is a diel signal that is prevalent in many terrestrial environments. Light availability occurs in a cycle which temporally links light to a range of environmental fluctuations, such as temperature and ambient humidity. Therefore, light may be a powerful cue that photosensing organisms can exploit in an anticipatory manner to prepare for subsequent stresses. Photosensory proteins are widespread among plant-associated microbes, which is consistent with the requirement of light for plant growth. The leaf colonist and foliar pathogen Pseudomonas syringae pv. syringae (Pss) B728a contains genes for three photosensory proteins, one blue light-sensing LOV protein, and two red/far-red light-sensing bacteriophytochromes, BphP1 and BphP2. Previous work has determined roles for LOV and BphP1 in influencing virulence, leaf colonization, and swarming motility in Pss B728a. Although LOV, BphP1, and BphP2 are histidine kinases, the target proteins that they phosphorylate have not yet been identified. To begin to identify cellular components that are influenced by these photosensory proteins, we conducted global transcriptome analyses characterizing the effects of individual wavelengths of light (far-red, red, and blue light) on wild-type Pss B728a and mutants lacking one or more photosensory proteins. We found that light is a global signal, initiating transcriptional changes in 31% of the genes in Pss B728a. While all tested wavelengths of light promoted changes in gene expression, far-red light influenced the most genes, suggesting a heightened sensitivity to far-red light. BphP1 was required for the majority of the transcriptional responses to all three wavelengths tested, consistent with the activation of purified BphP1 by all three wavelengths. We demonstrated that light intensity, not just wavelength, impacts gene expression using algD as an indicator gene. Based on light- and BphP1-regulation of genes involved in conjugation, we performed functional tests and found that far-red light and BphP1 negatively regulate conjugation in Pss B728a. Light had an especially noticeable impact on genes involved in the response to osmotic stress, with these genes among those most strongly induced by light. A comparison of the light-responsive, BphP1-induced regulon with the previously characterized osmotic stress-responsive, AlgU-induced regulon showed a large, but not complete, overlap, highlighting that these two regulated pathways are integrated. We explored the possibility that light functions to help cells anticipate osmotic stress, finding that pre-exposure to far-red or red light enhances Pss B728a osmotolerance and that this light enhancement of osmotolerance requires BphP1. This anticipatory light-enhanced osmotolerance requires the extracytoplasmic function (ECF) sigma factor AlgU, which is a key regulator of osmotic stress response genes, as well as the AlgU-regulated functions of osmoprotectant transport (for experiments performed in the presence of the osmoprotectant choline) and compatible solute synthesis. Integration of the light- and osmotic stress-regulated pathways is further highlighted by BphP1 regulation of algU expression and AlgU regulation of expression of the photosensory gene lov. By monitoring the onset of light, dew accumulation, and surface water evaporation on sunlight-exposed surfaces at dawn, we provide evidence that exposure of terrestrial surfaces to light can precede rapid evaporative water loss during a natural diel cycle. This finding illustrates the potential biological benefit of anticipatory light-enhanced osmotolerance to leaf-surface bacteria. Lastly, we demonstrate that this anticipatory light-enhanced osmotolerance is likely a widespread phenomenon in plant-associated pseudomonads, and potentially within terrestrial microbes, although these have not yet been examined. Collectively, this work contains the most thorough investigation of light and photosensory proteins in a non-photosynthetic bacterium to date, and provides evidence of global regulation by a bacteriophytochrome, integration of responses to light and water stress, and a novel phytochrome-driven anticipatory stress response that modulates cellular fitness.

DOI

https://doi.org/10.31274/etd-20210609-67

Copyright Owner

Bridget M Hatfield

Language

en

File Format

application/pdf

File Size

118 pages

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