A single-cell screening platform accelerates functional genetics in plants.

Area of Science:

• Plant Biology
• Genetics
• Molecular Biology

Background:

• Studying gene function in complex plant genetic pathways, like signaling, is difficult with current whole-plant screening.
• Redundant genetic programs pose significant challenges for traditional genetic screens in both model and nonmodel plants.

Purpose of the Study:

• To develop a novel single-cell screening platform, PIVOT, for accelerated plant gene identification and functional characterization.
• To overcome limitations of whole-plant screening by enabling genetic perturbation analysis at the cellular level.

Main Methods:

• Developed PIVOT (protoplast isolation after virus overexpression in planta) for high-throughput, single-cell genetic screening.
• Utilized Nicotiana benthamiana as a heterologous host for testing gene libraries in single leaves.
• Employed viral superinfection exclusion for single multiplicity of infection and engineered a cell-surface marker for cell isolation.

Main Results:

• Successfully recovered regulators of cytokinin signaling from an Arabidopsis open reading frame library using the PIVOT system.
• Demonstrated the platform's capability to handle pooled library delivery and isolate specific cells from heterogeneous populations.

Conclusions:

• PIVOT platform enables efficient functional genetic screening at the single-cell level in plants.
• Anticipate broad applicability of PIVOT for high-throughput plant gene discovery across diverse species.