Tomato Natural Insecticides: Elucidation of the Complex Pathway of Methylketone Biosynthesis

Plant species synthesize a multitude of specialized compounds 10 help ward off pests. and these in turn may well serve as an alternative to synthetic pesticides to reduce environmental damage and health risks to humans. The general goal of this research was to perform a genetic and biochemical dissection of the natural-insecticides methylketone pathway that is specific to the glandular trichomes of the wild species of tomato, Solanumhabrochaites f. glabratum (accession PI126449). Previous study conducted by us have demonstrated that these compounds are synthesized de novo as a derivate pathway of the fatty acid biosynthesis, and that a key enzyme. designated MethylketoneSynthase 1 (MKS 1). catalyzes conversion of the intermediate B-ketoacyl- ACPs to the corresponding Cn-1 methylketones. The approach taken in this proposed project was to use an interspecific F2 population. derived from the cross between the cultivated lV182 and the wild species PIl26449. for three objectives: (i) Analyze the association between allelic status of candidate genes from the fatty acid biosynthesis pathway with the methylketone content in the leaves (ii) Perform bulk segregant analysis of genetic markers along the tomato genome for identifying genomic regions that harbor QTLs for 2TD content (iii) Apply differential gene expression analysis using the isolated glands of bulk segregant for identifying new genes that are involved in the pathway. The genetic mapping in the interspecific F2 population included app. 60 genetic markers, including the candidate genes from the FAS pathway and SSR markers spread evenly across the genome. This initial; screening identified 5 loci associated with MK content including the candidate genes MKS1, ACC and MaCoA:ACP trans. Interesting observation in this genetic analysis was the connection between shape and content of the glands, i.e. the globularity of the four cells, typical to the wild species. was associated with increased MK in the segregating population. In the next step of the research transcriptomic analysis of trichomes from high- and 10w-MK plants was conducted. This analysis identified a new gene, Methy1ketone synthase 2 (MKS2), whose protein product share sequence similarity to the thioesterase super family of hot-dog enzymes. Genetic analysis in the segregating population confirmed its association with MK content, as well as its overexpression in E. coli that led to formation of MK in the media. There are several conclusions drawn from this research project: (i) the genetic control of MK accumulation in the trichomes is composed of biochemical components in the FAS pathway and its vicinity (MKS 1 and MKS2). as well as genetic factors that mediate the morphology of these specialized cells. (ii) the biochemical pathway is now realized different from what was hypothesized before with MKS2 working upstream to I\1KS 1 and serves as the interface between primary (fatty acids) and secondary (MK) metabolism. We are currently testing the possible physical interactions between these two proteins in vitro after the genetic analysis showed clear epistatic interactions. (iii) the regulation of the pathway that lead to specialized metabolism in the wild species is largely mediated by transcription and one of the achievements of this project is that we were able to isolate and verify the specificity of the MKS1 promoter to the trichomes which allows manipulation of the pathways in these cells (currently in progress). The scientific implications of this research project is the advancement in our knowledge of hitherto unknown biochemical pathway in plants and new leads for studying a new family in plants (hot dog thioesterase). The agricultural and biotechnological implication are : (i) generation of new genetic markers that could assist in importing this pathway to cultivated tomato hence enhancing its natural resistance to insecticides, (ii) the discovery of MKS2 adds a new gene for genetic engineering of plants for making new fatty acid derived compounds. This could be assisted with the use of the isolated and verified MKS1 promoter. The results of this research were summarized to a manuscript that was published in Plant Physiology (cover paper). to a chapter in a proceeding book. and one patent was submitted in the US.