New tools for faster and simpler site-directed mutagenesis in Xanthomonas

Authors: Li, Dandan; Schachterle, Jeffrey

Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: 2/27/2023
Publication Date: 3/2/2023
Citation: Li, D., Schachterle, J.K. 2023. New tools for faster and simpler site-directed mutagenesis in Xanthomonas [abstract]. Bacterial Leaf Streak Conference. 2:9.

Interpretive Summary:

Technical Abstract: The genus Xanthomonas includes several devastating diseases, including bacterial leaf streak caused by X. translucens. Despite the importance of these diseases, genetic tools to understand the molecular basis of disease and host-pathogen interactions remain limited. The implementation of high-throughput sequencing has facilitated discovery of many genetic elements related to virulence that are candidates for further molecular characterization. The functional characterization of candidate genes is limited by challenges to reverse genetics. One goal of our current work is to develop and improve genetic tools in Xanthomonas to enable functional characterization of genes in virulence processes. To this end, we have generated a series of plasmids with broad host-range that allow for inducible expression of phage lambda red-recombinase genes, which have been successfully used for recombineering in various bacterial species. With these plasmids we have developed protocols for implementation of recombinase-based mutagenesis in X. translucens, allowing replacement of a target DNA sequence in the chromosome with an antibiotic cassette. Using our approach, we have successfully generated gene knockouts for various proof-of-concept genes using PCR products for gene replacement in multiple pathovars and strains. We anticipate that these tools will be broadly applicable to the genus of Xanthomonas and will facilitate molecular studies in the genetic and physiological basis of host-pathogen interactions.