Acylsugars protect Nicotiana benthamiana against insect herbivory and desiccation.

KEY MESSAGE: Nicotiana benthamiana acylsugar acyltransferase (ASAT) is required for protection against desiccation and insect herbivory. Knockout mutations provide a new resource for investigation of plant-aphid and plant-whitefly interactions. Nicotiana benthamiana is used extensively as a transient expression platform for functional analysis of genes from other species. Acylsugars, which are produced in the trichomes, are a hypothesized cause of the relatively high insect resistance that is observed in N. benthamiana. We characterized the N. benthamiana acylsugar profile, bioinformatically identified two acylsugar acyltransferase genes, ASAT1 and ASAT2, and used CRISPR/Cas9 mutagenesis to produce acylsugar-deficient plants for investigation of insect resistance and foliar water loss. Whereas asat1 mutations reduced accumulation, asat2 mutations caused almost complete depletion of foliar acylsucroses. Three hemipteran and three lepidopteran herbivores survived, gained weight, and/or reproduced significantly better on asat2 mutants than on wildtype N. benthamiana. Both asat1 and asat2 mutations reduced the water content and increased leaf temperature. Our results demonstrate the specific function of two ASAT proteins in N. benthamiana acylsugar biosynthesis, insect resistance, and desiccation tolerance. The improved growth of aphids and whiteflies on asat2 mutants will facilitate the use of N. benthamiana as a transient expression platform for the functional analysis of insect effectors and resistance genes from other plant species. Similarly, the absence of acylsugars in asat2 mutants will enable analysis of acylsugar biosynthesis genes from other Solanaceae by transient expression.