Multiplexed profiling of transcriptional regulators in plant cells

Transcriptional regulatory domains play key roles in plant growth, development, and environmental responses. Massively parallel reporter assays in yeast and animal cells have enabled large-scale studies of libraries of transcriptional regulators and other classes of proteins, however, similar technologies are limited in plants. Here, we developed ENTRAP-seq (Enrichment of Nuclear Trans -elements Reporter Assay in Plants), a high-throughput method that introduces protein-coding libraries into plant cells to drive a nuclear magnetic sorting-based reporter, enabling multiplexed measurement of regulatory activity from thousands of protein variants. Using ENTRAP-seq and machine learning, we screened 1,495 plant viruses and identified hundreds of novel transcriptional regulatory domains found in structural proteins and enzymes not previously associated with gene regulation. We further combined this approach with machine-guided design to provide the framework for semi-rational engineering of the regulatory activity of an Arabidopsis transcription factor. Our findings demonstrate how the development of scalable functional genomic platforms deployed in planta will enable the characterization of natural and synthetic coding diversity in plants.