An improved Nicotiana benthamiana strain for aphid and whitefly research

Nicotiana benthamiana is used extensively as a platform for transient gene expression and as a model system for studying plant-virus interactions. However, many tobacco-feeding generalist herbivores, including Myzus persicae (green peach aphid), Bemisia tabaci (whitefly), Macrosiphum euphorbiae (potato aphid), Heliothis virescens (tobacco budworm), Trichoplusia ni (cabbage looper), and Helicoverpa zea (corn earworm), grow poorly on N. benthamiana, limiting its utility for research on plant-insect interactions. Using CRISPR/Cas9, we generated knockout mutations in two N. benthamiana acylsugar acyltransferases, ASAT1 and ASAT2, which contribute to the biosynthesis of insect-deterrent acylsucroses. Whereas asat1 mutations reduced the abundance of two predominant acylsucroses, asat2 mutations caused almost complete depletion of foliar acylsucroses. The tested hemipteran and lepidopteran species survived, gained weight, and/or reproduced significantly better on asat2 mutant plants than on wildtype N. benthamiana. Furthermore, both asat1 and asat2 mutations reduced the water content and increased the temperature of leaves, indicating that foliar acylsucroses can protect against desiccation. Two experiments demonstrated the utility of the N. benthamiana asat2 mutant line for insect bioassays. Transmission of turnip mosaic virus by M. persicae was significantly improved by an asat2 mutation. Tobacco rattle virus constructs were used for virus-induced gene silencing of acetylcholinesterase, squalene synthase, toll-like receptor 7, and tubulin-specific chaperon D genes in B. tabaci, an experiment that would have been difficult with wild-type N. benthamiana due to high insect mortality. Additionally, the absence of acylsugars in asat2 mutant lines will simplify transient expression assays for the functional analysis of acylsugar biosynthesis genes from other Solanaceae.