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 ≈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.

Silencing of a LIM gene in cotton exhibits enhanced resistance against Apolygus lucorum.

Plant bugs (Miridae species) have become major agricultural pests that cause increasing and severe economic damage. Plant-mediated RNA interference (RNAi) is emerging as an eco-friendly, efficient, and reliable strategy for pest management. In this study, we isolated and characterized a lethal gene of Apolygus lucorum and named it Apolygus lucorum LIM (AlLIM), which produced A. lucorum mortality rates ranging from 38% to 81%. Downregulation of the AlLIM gene expression in A. lucorum by injection of a double-stranded RNA (dsRNA) led to muscle structural disorganization that resulted in metamorphosis deficiency and increased mortality. Then we constructed a plant expression vector that enabled transgenic cotton to highly and stably express dsRNA of AlLIM (dsAlLIM) by Agrobacterium-mediated genetic transformation. In the field bioassay, dsAlLIM transgenic cotton was protected from A. lucorum damage with high efficiency, with almost no detectable yield loss. Therefore, our study successfully provides a promising genetically modified strategy to overpower A. lucorum attack.