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<p>The <i>Agrobacterium tumefaciens</i> mediated transformation is the prevailing methodology for plant genetic manipulation; however, <i>A. tumefaciens</i> overgrowth is a common constraint in the process. Exploring auxotrophic <i>A. tumefaciens</i> could reduce overgrowth and enhance plant transformation efficiency. The <i>ILVC</i> gene, which encodes the ketol-acid isomeroreductase, is critical for Valine (Val) and isoleucine (Ile) biosynthesis in some microorganisms. However, its function in <i>A. tumefaciens</i> is unclear. To ascertain the function of <i>ILVC</i> and generate an auxotrophic <i>A. tumefaciens</i>, this study employed an allelic exchange to disrupt the <i>ILVC</i> in <i>A. tumefaciens</i> strain GV3101. This resulted in the loss of ketol-acid isomeroreductase activity and the prevention of Val and Ile biosynthesis, creating a dual-auxotrophic GV3101<sub>∆ILVC</sub>. Transient expression assays in <i>Nicotiana benthamiana</i> transformation demonstrated that the GV3101<sub>∆ILVC</sub> was capable of T-DNA transfer. Moreover, stable genetic transformation analysis in <i>N. benthamiana</i> indicated that the introduction of GV3101<sub>∆ILVC</sub> led to a reduction in overgrowth within infected plant tissues. Additionally, an enhancement in transformation efficiency was observed with the prolongation of the co-cultivation time of the explant-infected strain. This study revealed the function of <i>ILVC</i> and explored a dual-auxotrophic <i>A. tumefaciens</i> for Val and Ile, potentially broadening the utilization of auxotrophic strains in plant genetic transformation.</p>

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Knockout of the Agrobacterium ILVC gene generates a valine-isoleucine auxotrophic strain for plant transformation

  • 期刊论文

  • 发表日期：

    2025年5月2日

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  • Yanhui Yang,
  • Jun Lu,
  • Tianrui Xue,
  • Zhenli Cao,
  • Mingjie Li,
  • Zhongyi Zhang,
  • Rong Wang,
  • Tongyu Wu

摘要

The Agrobacterium tumefaciens mediated transformation is the prevailing methodology for plant genetic manipulation; however, A. tumefaciens overgrowth is a common constraint in the process. Exploring auxotrophic A. tumefaciens could reduce overgrowth and enhance plant transformation efficiency. The ILVC gene, which encodes the ketol-acid isomeroreductase, is critical for Valine (Val) and isoleucine (Ile) biosynthesis in some microorganisms. However, its function in A. tumefaciens is unclear. To ascertain the function of ILVC and generate an auxotrophic A. tumefaciens, this study employed an allelic exchange to disrupt the ILVC in A. tumefaciens strain GV3101. This resulted in the loss of ketol-acid isomeroreductase activity and the prevention of Val and Ile biosynthesis, creating a dual-auxotrophic GV3101∆ILVC. Transient expression assays in Nicotiana benthamiana transformation demonstrated that the GV3101∆ILVC was capable of T-DNA transfer. Moreover, stable genetic transformation analysis in N. benthamiana indicated that the introduction of GV3101∆ILVC led to a reduction in overgrowth within infected plant tissues. Additionally, an enhancement in transformation efficiency was observed with the prolongation of the co-cultivation time of the explant-infected strain. This study revealed the function of ILVC and explored a dual-auxotrophic A. tumefaciens for Val and Ile, potentially broadening the utilization of auxotrophic strains in plant genetic transformation.