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<p>Elucidating gene function in highly redundant genetic programs such as signaling pathways is challenging in model and nonmodel plants with current whole-plant genetic screening tools. Many of these challenges could be overcome if screens were instead carried out using individual cells harboring genetic perturbations. Here we report a single-cell screening platform, PIVOT (protoplast isolation after virus overexpression <i>in planta</i>), to accelerate identification and functional characterization of plant genes. We use <i>Nicotiana</i> <i>benthamiana</i> as a heterologous host to test gene libraries arrayed in a single leaf. PIVOT harnesses viral superinfection exclusion to ensure single multiplicity of infection per cell during pooled library delivery. Additionally, we engineer a cell-surface protein as a phenotypic marker for isolating cells of interest from a heterogeneous population. Using this system, we recover regulators of cytokinin signaling from an <i>Arabidopsis</i> open reading frame library. We anticipate PIVOT will be broadly applicable for high-throughput, single-cell functional genetic screening across the plant kingdom.</p>

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A single-cell screening platform accelerates functional genetics in plants

  • 期刊论文

  • 发表日期：

    2026年5月5日

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  • Tara N. Lowensohn,
  • Will B. Cody,
  • Chun Tsai,
  • Alexander E. Vlahos,
  • Connor C. Call,
  • Xiaojing J. Gao,
  • Elizabeth S. Sattely

摘要

Elucidating gene function in highly redundant genetic programs such as signaling pathways is challenging in model and nonmodel plants with current whole-plant genetic screening tools. Many of these challenges could be overcome if screens were instead carried out using individual cells harboring genetic perturbations. Here we report a single-cell screening platform, PIVOT (protoplast isolation after virus overexpression in planta), to accelerate identification and functional characterization of plant genes. We use Nicotiana benthamiana as a heterologous host to test gene libraries arrayed in a single leaf. PIVOT harnesses viral superinfection exclusion to ensure single multiplicity of infection per cell during pooled library delivery. Additionally, we engineer a cell-surface protein as a phenotypic marker for isolating cells of interest from a heterogeneous population. Using this system, we recover regulators of cytokinin signaling from an Arabidopsis open reading frame library. We anticipate PIVOT will be broadly applicable for high-throughput, single-cell functional genetic screening across the plant kingdom.