Document Type

Thesis

Degree

Master of Science

Major

Biology

Date of Defense

12-17-2015

Graduate Advisor

Lisa Schechter, PhD.

Committee

Wang, Xuemin

Zolman, Bethany

Schechter, Lisa

Abstract

Pseudomonas syringae pathovar tomato strain DC3000 is a model bacterial plant-pathogen that utilizes a dedicated protein export apparatus, the type III secretion system (T3SS), to translocate virulence proteins called effectors directly into host cells. Because effectors suppress plant immune responses, activation of the T3SS is critical upon entry into the host. The P. syringae T3SS is controlled by the hrpRS-hrpL regulatory cascade, and is activated quickly by specific conditions. Different environmental stimuli have been reported to modulate T3SS gene expression in culture, however it is unclear how each signal affects hrpRS or hrpL. My objective was to identify how environmental variables activate or repress hrpRS or hrpL in Pst DC3000. To this aim, I created three T3SS::gusA transcriptional reporter strains by fusing a promoterless gusA after hrpRS, hrpL, and a downstream effector gene, avrPto in the chromosome of Pst DC3000. I then analyzed GUS activity of each reporter strain cultured under variable conditions. I verified that repression of Pst DC3000 T3SS genes in KB acts upstream of the hrpRS operon, and that this repression is relieved by overexpression of either hrpR or hrpS. Furthermore, I demonstrated that hrpRS, hrpL, and avrPto, are differentially regulated by pH and carbon sources, although all carbon source tested (including sugars, a sugar alcohol, glycerol, and organic acids) initially induced T3SS gene expression. Results of several assays suggest that quorum sensing may be involved in regulation of the T3SS in Pst DC3000. First, T3SS genes were optimally expressed when growth media contained carbon sources that promoted slower growth, and when bacteria were cultured at low cell densities. In addition, I show that a T3SS repressive signal accumulated in high cell density Pst DC3000 cultures. However, density-dependent repression of T3SS genes was independent of psyRI, which mediates quorum sensing by acyl homoserine lactones (AHLs), and T3SS gene expression was unaffected by addition of 3-oxo-C6 or C6 AHLs. In contrast, T3SS genes were repressed when another small molecule produced by P. syringae, the auxin IAA, is added to Pst DC3000 cultures. However the biological relevance of IAA as a T3SS repressing signal remains to be explored.

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