Identification of Bacterial Operons Required During The Plant Immune Response using a RB-TnSeq Approach

Today, the majority of agrochemicals used to increase agricultural output have adverse effects on the environment, and the developments of more sustainable approaches that utilize plant-growth promoting bacteria are needed 1. In order to benefit the plants, these microbes must be able to colonize the plant root in mixed environmental microbial communities that induce immune responses from the plant. Toward understanding the genetic basis of root colonization by plant-growth promoting microbes, a randomly barcoded transposon insertion library sequencing method (Bar-Seq) was utilized to identify genes that protect bacteria from the plant-immune response. Arabidopsis thaliana ecotype Col-0 and two mutants deficient in immune responses were grown for 5 weeks in liquid media and then an immune response was induced for 1 day using flg22 flagellin peptide. Exudates containing anti-microbial chemicals produced by the plants were collected. Bar-Seq libraries of four different plant-associated bacteria were grown in each exudate and the transposon barcodes corresponding to interrupted genes were sequenced. First, the genes are assigned fitness scores by comparing barcode abundance to input. Then, the fitness scores are compared (mutant vs. wild-type, + flg22 vs. – flg22) to identify genes required for growth during the immune response. These results will assist in the efforts to achieve higher crop yields in a more sustainable way, and provide insight into the genetic interactions between bacteria and their hosts.Bachelor of Scienc