PT  - JOURNAL ARTICLE
AU  - Stevens, Danielle M.
AU  - Tang, Andrea
AU  - Coaker, Gitta
TI  - A genetic toolkit for investigating <em>Clavibacter</em>: markerless deletion, permissive site identification and an integrative plasmid
AID  - 10.1101/2021.07.13.452269
DP  - 2021 Jan 01
TA  - bioRxiv
PG  - 2021.07.13.452269
4099  - http://biorxiv.org/content/early/2021/07/13/2021.07.13.452269.short
4100  - http://biorxiv.org/content/early/2021/07/13/2021.07.13.452269.full
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.