RT Journal Article SR Electronic T1 A genetic toolkit for investigating Clavibacter: markerless deletion, permissive site identification and an integrative plasmid JF bioRxiv FD Cold Spring Harbor Laboratory SP 2021.07.13.452269 DO 10.1101/2021.07.13.452269 A1 Stevens, Danielle M. A1 Tang, Andrea A1 Coaker, Gitta YR 2021 UL http://biorxiv.org/content/early/2021/07/13/2021.07.13.452269.abstract AB The development of knockout mutants and expression variants are critical for understanding genotype-phenotype relationships. However, advancements of these techniques in Gram-positive actinobacteria have stagnated over the last decade. Actinobacteria in the Clavibacter genus are composed of diverse crop pathogens which cause a variety of wilt and cankering diseases. Here, we present a suite of tools for genetic manipulation in the tomato pathogen C. michiganensis including a markerless deletion system, an integrative plasmid, and an R package for identification of permissive sites for plasmid integration. The vector pSelAct-KO is a recombination based, markerless knockout system that uses dual selection to engineer seamless deletions of a region of interest, providing opportunities for repeated higher-order genetic knockouts. The efficacy of pSelAct-KO was demonstrated in C. michiganensis and confirmed using whole genome sequencing. We developed permissR, an R package to identify permissive sites for chromosomal integration, which can be used in conjunction with pSelAct-Express, a non-replicating integrative plasmid that enables recombination into a permissive genomic location. Expression of eGFP by pSelAct-Express was verified in two candidate permissive regions predicted by permissR in C. michiganensis. These molecular tools are essential advancements for investigating Gram-positive actinobacteria, particularly for important pathogens in the Clavibacter genus.Competing Interest StatementThe authors have declared no competing interest.