Abstract
Leaf-chewing insects are important pests that cause yield loss and reduce seed quality in soybeans (Glycine max). 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, Glyma.07g110300 (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, Agrobacterium-mediated transformation introduced the CRISPR/Cas9 expression vector into the soybean cultivar Tianlong No. 1 to generate Glyma.07g110300-gene mutants. We obtained two novel types of mutations, a 33-bp deletion and a single-bp insertion in the GmUGT coding region, which resulted in an enhanced resistance to Helicoverpa armigera and Spodoptera litura. Additionally, overexpressing GmUGT produced soybean varieties that were more sensitive to H. armigera and S. litura. Both mutant and overexpressing lines exhibited no obvious phenotypic changes. The difference in metabolites and gene expression suggested that GmUGT 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 GmUGT gene in the ugt72b1 mutant of Arabidopsis substantially rescued the phenotype of H. armigera resistance in the atugt72b1 mutant. Our study presents a strategy for increasing resistance against leaf-chewing insects in soybean through CRISPR/Cas9-mediated targeted mutagenesis of the UGT genes.
Highlights
Soybean (Glycine max) is an important industrial crop that provides edible oil, vegetable protein, and active compounds, such as flavonoids (Arifin et al, 2021)
Findings from this study present a strategy to increase the resistance to leaf-chewing insects in soybean and other crops through CRISPR/Cas9-mediated targeted mutagenesis of UGT genes
The target adaptor was designed using the CRISPR-P1 web tool; it was integrated into the single guide RNA expression cassettes driven by the GmU6 promoter, which was subsequently built into the pSC1-CRISPR/Cas9PGmUbi3-BK vector
Summary
Soybean (Glycine max) is an important industrial crop that provides edible oil, vegetable protein, and active compounds, such as flavonoids (Arifin et al, 2021). Breeding soybean varieties that are resistant to leaf-chewing insects can minimize the need for insecticides, improve soybean yields, and reduce concerns stemming from pesticide use (Ortega et al, 2016). Modified cultivars expressing insecticidal proteins have become vital to the effective management of leaf-chewing insect pests. Major insecticidal proteins include the crystal protein (CRY), the vegetative insecticidal protein (VIP), and the protease inhibitor (PI), whose genes have been used to develop insect-resistant genetically modified cultivars (Saikhedkar et al, 2019; Kumar et al, 2020). Many insect-resistant transgenic crops exist; there are six commercialized soybean cultivars with various insect-resistance (IR) genes (Ichim, 2019; Kumar et al, 2020). This genetic trait provides an effective control strategy for defoliators, including H. armigera. Transgenic crops that have foreign DNA randomly integrated into their genomes have always been controversial, owing to the durability of resistance, environmental safety, and potential adverse health effects on consumers, limiting such transgenic breeding (Domingo and Bordonaba, 2011; Raman, 2017; Schiemann et al, 2019; Pozebon et al, 2020; Gao, 2021)
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