UBC Theses and Dissertations
Pseudomonas biofilm regulation in the Arabidopsis rhizosphere as a plant-immune evasion strategy Liu, Zhexian
Plant root-associated (“rhizosphere”) bacteria provide diverse benefits to their plant hosts including growth promotion and protection from pathogens. Pseudomonas fluorescens is a model bacterium that robustly colonizes the roots of the model plant Arabidopsis. To identify bacterial genes required for P. fluorescens to colonize the plant rhizosphere, we performed a forward genetic screen using transposon mutagenesis coupled with next generation sequencing (Tn-seq). Using this approach, we identified bacterial genes required for P. fluorescens rhizosphere fitness and plant immune evasion. We found that P. fluorescens requires MorA, a c-di-GMP phosphodiesterase, and SpuC, a putrescine aminotransferase, to avoid triggering plant immunity. Deletion of morA or spuC leads to increased biofilm formation in vitro. Furthermore, we found that exogenous putrescine promotes biofilm formation. These findings suggest that P. fluorescens attenuates biofilm formation in the rhizosphere to avoid triggering a plant immune response. To dissect the role of polyamine biosynthesis and metabolism in promoting biofilm in Pseudomonas, I constructed markerless deletions in genes required for polyamine metabolism in P. aeruginosa, a model organism for biofilm research and a relative of P. fluorescens. I found that deletion of spuC and speD, genes involved in converting putrescine to succinate and spermidine, respectively, significantly increased biofilm formation in P. aeruginosa. Additionally, using a GFP-based c-di-GMP biosensor, I measured the intracellular levels of c-di-GMP in P. aeruginosa in response to exogenous polyamines and polyamine precursors such as L-arginine. I found that exogenous putrescine, spermidine, and arginine increase the c-di-GMP levels in P. aeruginosa as indicated by increased GFP fluorescence signal. Finally, I found that exogenous putrescine promotes P. aeruginosa and P. fluorescens biofilm formation. We postulate that putrescine may serve as a plant-derived signal that triggers lifestyle switching in rhizosphere bacterial commensal and pathogen.
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