Plant Synthetic Biology Approaches to Decipher Solanaceae Acylsugar Structural Effects on Insect Herbivory

Plants use their remarkable specialized metabolic capacity to interact with the biotic environment, including deterring and attracting insects and microbes. Solanaceae trichome-synthesized acylsugars confer defense against herbivores and offer the possibility of engineering plants with environmentally safe pesticides. Acylsugars show striking structural variety within Solanaceae family, with mono- and di-saccharide cores and acylchains of C2-C12 found across the family. We ask whether the repeated structure diversification bears the fingerprint of an evolutionary arms race. An alternative hypothesis is that the diversity is simply the result of genetic drift. Deciphering effects of structurally diverse acylsugars faces the challenge of interference of other defense traits (for example, different levels of alkaloids and terpenes in plants). To critically assess the impact of acylsugars on herbivory, we used CRISPR/Cas9 gene editing of domesticated Solanum lycopersicum and Solanum pennellii (LA0716) pathway enzymes to modify the amounts and types of metabolites produced. We generated isogenic S. pennellii lines differing in glucose- or sucrose-based esters to critically assess the impact of sugar core. Meanwhile, knockout lines of the first three acylsugar acyltransferase (ASAT) genes in both backgrounds accumulate no detectable acylsugars, without altering plant growth or trichome development. These lines provide a good 'null mutant' background to test the efficacy of acylsugars. Finally, cultivated tomato slasat4 mutants lacking acetylated acylsucroses permit us to evaluate the impact of acetyl groups in insect protection. Our results suggest that even the performance of the specialist, Manduca sexta, is hindered by the presence of acylsugars, while we observe larger protective effects of acylsucroses than acylglucoses. The CRISPR mutated plants are suitable for investigating the importance of acylsugars in the field. Moreover, these plants serve as a promising molecular engineering platform that allows for the investigation of the mechanisms underpinning specialized metabolic evolution.