RepB C-terminus mutation of a pRi-repABC binary vector affects plasmid copy number in Agrobacterium and transgene copy number in plants.

Zarir Vaghchhipawala,Stanton B Gelvin,Larry A Gilbertson,Mary L Russell,Ervin Nagy,Xudong Ye,Sharon Radke,Susan Johnson,Erh-Min Lai

doi:10.1371/journal.pone.0200972

Zarir Vaghchhipawala, Stanton B Gelvin + Show 7 more

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https://doi.org/10.1371/journal.pone.0200972

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Abstract

A native repABC replication origin from pRiA4b was previously reported as a single copy plasmid in Agrobacterium tumefaciens and can improve the production of transgenic plants with a single copy insertion of transgenes when it is used in binary vectors for Agrobacterium-mediated transformation. A high copy pRi-repABC variant plasmid, pTF::Ri, which does not improve the frequency of single copy transgenic plants, has been reported in the literature. Sequencing the high copy pTF::Ri repABC operon revealed the presence of two mutations: one silent mutation and one missense mutation that changes a tyrosine to a histidine (Y299H) in a highly conserved area of the C-terminus of the RepB protein (RepBY299H). Reproducing these mutations in the wild-type pRi-repABC binary vector showed that Agrobacterium cells with the RepBY299H mutation grow faster on both solidified and in liquid medium, and have higher plasmid copy number as determined by ddPCR. In order to investigate the impact of the RepBY299H mutation on transformation and quality plant production, the RepBY299H mutated pRi-repABC binary vector was compared with the original wild-type pRi-repABC binary vector and a multi-copy oriV binary vector in canola transformation. Molecular analyses of the canola transgenic plants demonstrated that the multi-copy pRi-repABC with the RepBY299H mutation provides no advantage in generating high frequency single copy, backbone-free transgenic plants in comparison with the single copy wild-type pRi-repABC binary vector.

Highlights

Vector backbone-free transgene events are required by government regulatory agencies for commercial transgenic product deployment, and are preferred for many other biotechnology applications
These two plasmids were designated pMON83937-repBT486C and pMON83937-RepBY299H, respectively. Both plasmids and the control pMON83937 were separately introduced into the nopaline-type strain Agrobacterium tumefaciens ABI [9] by electroporation, and the cultures were spread on LB medium containing 50 mg/L spectinomycin and 10 mg/L gentamicin
The faster growth could result from a gene dose effect of the elevated plasmid copy number, providing a growth advantage from the multiple copies of the aadA gene as increased plasmid copy number [26], which is consistent with our observation that replacing the original aadA promoter with a strong prokaryotic promoter can drastically increase the spectinomycin resistance in Agrobacterium with the native oriRi binary vector backbone [27]

Summary

Introduction

Vector backbone-free transgene events are required by government regulatory agencies for commercial transgenic product deployment, and are preferred for many other biotechnology applications. The specific roles of these authors are articulated in the "author contributions" section

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