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<p>Virus-induced gene silencing (VIGS) is a powerful reverse genetics tool to rapidly and transiently knockdown gene expression in plants. In this study, we optimized a tobacco rattle virus (TRV)-based VIGS system for <i>Spinacia oleracea</i> to enhance its utility in functional genomics. Using <i>SoPDS</i> (phytoene desaturase gene) as a visual marker, we evaluated key factors that influence the silencing efficiency. Systematic analysis revealed that <i>Agrobacterium</i> strain GV2260, an optical density at 600&#xa0;nm of 1.0, and wound infiltration via sandpaper on a true leaf produced the most robust silencing, reducing the <i>SoPDS</i> transcript level by more than 80% and decreasing the total chlorophyll content by 69%. Cotyledon infiltration was also effective, achieving a greater than 60% reduction in the <i>SoPDS</i> transcript level and a comparable decrease in the total chlorophyll content. Furthermore, the optimized system successfully enabled dual-gene silencing of <i>SoPDS</i> and <i>SoTCM</i>, with expression of both genes suppressed by more than 65% at the transcript level. These results demonstrate that the optimized TRV-VIGS protocol is a reliable and efficient platform for functional genomics in spinach and can facilitate broader applications in leafy crops.</p>

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Advanced optimization of a TRV-based virus-induced gene silencing (VIGS) system for functional genomics in spinach

  • 期刊论文

  • 发表日期：

    2026年2月12日

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  • Jiwon Kim,
  • Surim Lee,
  • Eunsu Ko,
  • Kyung-Hwan Boo

摘要

Virus-induced gene silencing (VIGS) is a powerful reverse genetics tool to rapidly and transiently knockdown gene expression in plants. In this study, we optimized a tobacco rattle virus (TRV)-based VIGS system for Spinacia oleracea to enhance its utility in functional genomics. Using SoPDS (phytoene desaturase gene) as a visual marker, we evaluated key factors that influence the silencing efficiency. Systematic analysis revealed that Agrobacterium strain GV2260, an optical density at 600 nm of 1.0, and wound infiltration via sandpaper on a true leaf produced the most robust silencing, reducing the SoPDS transcript level by more than 80% and decreasing the total chlorophyll content by 69%. Cotyledon infiltration was also effective, achieving a greater than 60% reduction in the SoPDS transcript level and a comparable decrease in the total chlorophyll content. Furthermore, the optimized system successfully enabled dual-gene silencing of SoPDS and SoTCM, with expression of both genes suppressed by more than 65% at the transcript level. These results demonstrate that the optimized TRV-VIGS protocol is a reliable and efficient platform for functional genomics in spinach and can facilitate broader applications in leafy crops.