A Genetic Toolkit for Investigating Clavibacter Species: Markerless Deletion, Permissive Site Identification, and an Integrative Plasmid
| Autor: | Danielle M. Stevens, Andrea Tang, Gitta Coaker |
|---|---|
| Jazyk: | angličtina |
| Rok vydání: | 2021 |
| Předmět: | |
| Zdroj: | Molecular Plant-Microbe Interactions, Vol 34, Iss 12, Pp 1336-1345 (2021) |
| Druh dokumentu: | article |
| ISSN: | 1943-7706 0894-0282 |
| DOI: | 10.1094/MPMI-07-21-0171-TA |
| Popis: | The development of knockout mutants and expression variants are critical for understanding genotype-phenotype relationships. However, advances in these techniques in gram-positive actinobacteria have stagnated over the last decade. Actinobacteria in the Clavibacter genus are composed of diverse crop pathogens that cause a variety of wilt and cankering diseases. Here, we present a suite of tools for genetic manipulation in the tomato pathogen Clavibacter 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 was 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 nonreplicating integrative plasmid that enables recombination into a permissive genomic location. Expression of enhanced green fluorescent protein by pSelAct-Express was verified in two candidate permissive regions predicted by permissR in C. michiganensis. These molecular tools are essential advances for investigating gram-positive actinobacteria, particularly for important pathogens in the Clavibacter genus.[Graphic: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license. |
| Databáze: | Directory of Open Access Journals |
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