Cara Haney

Plants form commensal associations with soil microorganisms, creating a root microbiome that provides benefits to the host including protection against pathogens. While bacteria can inhibit pathogens through production of antimicrobial compounds in vitro, it is largely unknown how microbiota contribute to pathogen protection in planta. I used a gnotobiotic model system consisting of Arabidopsis thaliana, and an opportunistic pathogen Pseudomonas sp. N2C3, to identify mechanisms that determine the outcome of plant-pathogen-microbiome interactions in the rhizosphere. I screened 25 phylogenetically diverse Pseudomonas strains for their ability to protect against N2C3 and found that commensal strains closely related to N2C3 were more likely to protect against pathogenesis. I used a comparative genomics approach to identify unique genes in the protective strains that revealed no genes that correlate with protection, suggesting that variable regulation of components of the core Pseudomonas genome may contribute to pathogen protection. I found that commensal colonization level was highly predictive of protection and so tested deletions of genes previously shown to be required for Arabidopsis rhizosphere colonization. I identified a response regulator colR that is required for Pseudomonas protection against N2C3 and fitness in competition with N2C3 indicating that competitive exclusion may contribute to pathogen protection. I found that Pseudomonas sp. WCS365 also protects against the agricultural pathogen Pseudomonas fuscovaginae SE-1, the causal agent of bacterial sheath brown rot of rice. This work establishes a gnotobiotic model to uncover mechanisms by which members of the microbiome can protect hosts from pathogens and informs our understanding of the use of beneficial strains for microbiome engineering in dysbiotic soil systems.

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Pseudomonas aeruginosa is a versatile bacterium found ubiquitously in the environment and is an opportunistic pathogen of diverse organisms, including animals and plants. A key determinant of bacterial adaptability is the ability to sense and respond appropriately to environmental stimuli. ColRS is a two-component regulatory system previously shown to be required for P. fluorescens colonization of plant roots (the “rhizosphere”) and P. aeruginosa virulence in C. elegans; however, the mechanisms by which ColRS regulates fitness in a host environment are unknown. I found that colR and colS deletion mutants in P. aeruginosa were significantly impaired in their ability to colonize the rhizosphere of Arabidopsis. In addition, we showed that colR in the P. aeruginosa cystic fibrosis epidemic isolate, LESB58, is required for virulence in a mouse abscess model. Using RNA-seq, I found a total of 128 genes that were dysregulated in the colR mutant in the rhizosphere, many of which have products that are predicted to localize to the cytoplasmic or outer membranes, suggesting ColRS may function in maintaining membrane integrity. Using P. aeruginosa transposon insertion mutants in colR-dependent genes, I identified novel genes required for rhizosphere colonization, including the protein tyrosine phosphatase tpbA, diacylglycerol kinase dgkA, and a type 2 phosphatidic acid phosphatase. Lastly, I showed that P. aeruginosa colR is required for tolerance to high levels of iron, zinc, and manganese, and for growth at acidic pH. Because functional analysis of rhizosphere gene expression showed that high iron concentration and low pH are stresses that may be present in the rhizosphere, the ColRS two-component system likely promotes P. aeruginosa colonization of the Arabidopsis rhizosphere through regulation of genes required to protect against these stresses.

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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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