CRISPR/Cas9-Mediated Targeted Mutagenesis of GmUGT Enhanced Soybean Resistance Against Leaf-Chewing Insects Through Flavonoids Biosynthesis

Abstract

Leaf-chewing insects are important pests that cause yield loss and reduce seed quality in soybeans (<i>Glycine max</i>). Breeding soybean varieties that are resistant to leaf-chewing insects can minimize the need for insecticide use and reduce yield loss. The marker gene for QTL-M, <i>Glyma.07g110300</i> (LOC100775351) that encodes a UDP-glycosyltransferase (UGT) is the major determinant of resistance against leaf-chewing insects in soybean; it exhibits a loss of function in insect-resistant soybean germplasms. In this study, <i>Agrobacterium</i>-mediated transformation introduced the CRISPR/Cas9 expression vector into the soybean cultivar Tianlong No. 1 to generate <i>Glyma.07g110300-</i>gene mutants. We obtained two novel types of mutations, a 33-bp deletion and a single-bp insertion in the <i>GmUGT</i> coding region, which resulted in an enhanced resistance to <i>Helicoverpa armigera</i> and <i>Spodoptera litura</i>. Additionally, overexpressing <i>GmUGT</i> produced soybean varieties that were more sensitive to <i>H</i>. <i>armigera</i> and <i>S. litura</i>. Both mutant and overexpressing lines exhibited no obvious phenotypic changes. The difference in metabolites and gene expression suggested that <i>GmUGT</i> is involved in imparting resistance to leaf-chewing insects by altering the flavonoid content and expression patterns of genes related to flavonoid biosynthesis and defense. Furthermore, ectopic expression of the <i>GmUGT</i> gene in the <i>ugt72b1</i> mutant of <i>Arabidopsis</i> substantially rescued the phenotype of <i>H. armigera</i> resistance in the <i>atugt72b1</i> mutant. Our study presents a strategy for increasing resistance against leaf-chewing insects in soybean through CRISPR/Cas9-mediated targeted mutagenesis of the <i>UGT</i> genes.