2025/08/07 by Tara N. Lowensohn, Will B. Cody, Chun J. Tsai +4 · 1 voice
Agricultural and Biological Sciences · Biochemistry, Genetics and Molecular Biology · #CRISPR and Genetic Engineering #Plant Virus Research Studies #Transgenic Plants and Applications
paper · pdf · doi:10.1101/2025.08.06.668818
openalex publication_date 2025/08/07 · openalex created_date 2025/10/10 · openalex updated_date 2026/07/19
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.