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bioRxiv · 10.1101/2025.08.06.668818

An in planta single-cell screen to accelerate functional genetics

Lowensohn, T. N.·Cody, W. B.·Tsai, C.·Vlahos, A. E.·Call, C. C.·Gao, X. J.·Sattely, E. S.
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Abstract

Genetic screens in whole plants are a powerful tool for functional genetics. However, elucidating gene function in highly redundant genetic programs such as signaling pathways remains challenging in both model and non-model plants. 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 used Nicotiana benthamiana as a heterologous host to test gene libraries arrayed in a single leaf. Two elements of our system made pooled screens possible in planta: (1) we harnessed viral superinfection exclusion to ensure single multiplicity of infection per cell during pooled library delivery, and (2) we engineered a cell surface protein as a phenotypic marker for isolating cells of interest from a heterogeneous population. Using this system, we recovered known and new 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.

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Lowensohn, T. N., Cody, W. B., Tsai, C., Vlahos, A. E., Call, C. C., Gao, X. J., Sattely, E. S.. 2025-08-07. An in planta single-cell screen to accelerate functional genetics. https://doi.org/10.1101/2025.08.06.668818

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