Effective removal of a range of Ti/Ri plasmids using a pBBR1-type vector having a repABC operon and a lux reporter system
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Ti and Ri plasmids of pathogenic Agrobacterium strains are stably maintained by the function of a repABC operon and have been classified into four incompatibility groups, namely, incRh1, incRh2, incRh3, and incRh4. Removal of these plasmids from their bacterial cells is an important step in determining strain-specific virulence characteristics and to construct strains useful for transformation. Here, we developed two powerful tools to improve this process. We first established a reporter system to detect the presence and absence of Ti/Ri plasmids in cells by using an acetosyringone (AS)-inducible promoter of the Ti2 small RNA and luxAB from Vibrio harveyi. This system distinguished a Ti/Ri plasmid-free cell colony among plasmid-harboring cell colonies by causing the latter colonies to emit light in response to AS. We then constructed new “Ti/Ri eviction plasmids,” each of which carries a repABC from one of four Ti/Ri plasmids that belonged to incRh1, incRh2, incRh3, and incRh4 groups in the suicidal plasmid pK18mobsacB and in a broad-host-range pBBR1 vector. Introduction of the new eviction plasmids into Agrobacterium cells harboring the corresponding Ti/Ri plasmids led to Ti/Ri plasmid-free cells in every incRh group. The Ti/Ri eviction was more effective by plasmids with the pBBR1 backbone than by those with the pK18mobsacB backbone. Furthermore, the highly stable cryptic plasmid pAtC58 in A. tumefaciens C58 was effectively evicted by the introduction of a pBBR1 vector containing the repABC of pAtC58. These results indicate that the set of pBBR1-repABC plasmids is a powerful tool for the removal of stable rhizobial plasmids.
KeywordsTi plasmid Plasmid curing Agrobacterium Plasmid incompatibility Luciferase reporter
This work was supported by the Japan Society for the Promotion of Science (JSPS) KAKENHI Grant Numbers 25850003, 24580115, and 15H04479. We thank Dr. E. Szegedi for providing bacterial strains and Dr. T. Oyama (Kyoto University) for the luxAB plasmid. Pathogenic strains isolated outside of Japan were imported with permissions issued by the Japanese Plant Quarantine Office and were handled under directions given by the same organization.
This study was funded by the JSPS (25850003, 24580115, and 15H04479).
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflict of interest.
This article does not contain any studies with human participants or animals performed by any of the authors.
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