Construction of Host Plant Insect-Resistance Mutant Library by High-Throughput CRISPR/Cas9 System and Identification of A Broad-Spectrum Insect Resistance Gene

Insects pose significant challenges in cotton-producing regions. Here, they describe a high-throughput CRISPR/Cas9-mediated large-scale mutagenesis library targeting endogenous insect-resistance-related genes in cotton. This library targeted 502 previously identified genes using 968 sgRNAs, generated approximate to 2000 T0 plants and achieved 97.29% genome editing with efficient heredity, reaching upto 84.78%. Several potential resistance-related mutants (10% of 200 lines) their identified that may contribute to cotton-insect molecular interaction. Among these, they selected 139 and 144 lines showing decreased resistance to pest infestation and targeting major latex-like protein 423 (GhMLP423) for in-depth study. Overexpression of GhMLP423 enhanced insect resistance by activating the plant systemic acquired resistance (SAR) of salicylic acid (SA) and pathogenesis-related (PR) genes. This activation is induced by an elevation of cytosolic calcium [Ca2+]cyt flux eliciting reactive oxygen species (ROS), which their demoted in GhMLP423 knockout (CR) plants. Protein-protein interaction assays revealed that GhMLP423 interacted with a human epidermal growth factor receptor substrate15 (EPS15) protein at the cell membrane. Together, they regulated the systemically propagating waves of Ca2+ and ROS, which in turn induced SAR. Collectively, this large-scale mutagenesis library provides an efficient strategy for functional genomics research of polyploid plant species and serves as a solid platform for genetic engineering of insect resistance. A high-throughput screening strategy for cotton endogenous insect-resistance-related genes is established by a CRISPR/Cas9-mediated large-scale mutant library. It identifies promising genes related to plant innate insect-resistance, including major latex-like protein 423 (GhMLP423). GhMLP423 enhanced plant immunity to chewing and sap-sacking insects via the induction of GhEPS15 that initiates the systemic acquired resistance elicited by the systemically propagating waves of Ca2+-mediated ROS signaling.image