Genomic Approaches to Identifying Acylsugar Metabolism Genes in Solanum pennellii

Acylsugars are specialized metabolites secreted by plants of the nightshade family. These chemicals have documented insecticidal characteristics. To make acylsugars, the plants attach branched-chain and straight-chain fatty acyl groups to sugar backbones. While many steps of acylsugar synthesis pathways have been identified, several acylsugar metabolic genes remain unknown. In this study, we used comparative genomic approaches in two independent experiments: a differential gene expression analysis between high- and low-acylsugar-producing S. pennellii accessions, and another differential gene expression analysis between 10 high- and 10 low-acylsugar-producing F2 plants of S. pennellii LA 0716 and S. lycopersicum cv. VF36 cross, to further investigate the biosynthesis of acylsugar in S. pennellii. Differential gene expression analysis tools were used to identify known and novel candidate genes, including genes putatively encoding fatty acid synthases, acyl-activating enzymes, ATP binding cassette (ABC) transporters, and CO2 fixation proteins, that were positively correlated with acylsugar accumulation. When S. pennellii leaves were treated with an inhibitor of BCAA biosynthesis, expression of known and candidate genes was repressed in response to inhibitor in a concentration-dependent manner. We identified four genes, Sopen05g009610, Sopen07g006810, Sopen05g032580 and Sopen05g034770 that were common in two differentially expressed gene (DEG) sets collected from the two independent comparative transcriptomics experiments and were under positive selection. I confirmed two candidate genes, Sopen05g009610, encoding a component of a fatty acid synthase, and Sopen07g006810, encoding a small subunit of Rubisco, through virus-induced gene silencing (VIGS), to be involved in medium-chain fatty acids synthesis pathway and carbon fixation associated with acylsugar production, respectively. These results provide strong support for the involvement of novel candidate genes we identified in acylsugar biosynthesis, and validate our approach of combining differential gene expression and evolutionary analysis