Genome editing-induced remodeling of allergen and glycoalkaloid composition in tomato

Tomato breeding has been mainly driven by aims to increase in fruit size and productivity and, only more recently, by consumer driven traits. Tomato is a highly consumed vegetable and contributes positively to human health thanks to its high contents in carotenoids, vitamins, flavonoids, folates, fibers and minerals; however, it can elicit allergic reactions in a small number of consumers. Moreover, tomato fruit can also accumulate a series of "antinutritional" compounds, so called for interfering with the normal absorption of nutrients or showing toxic effects in humans. Among those, glycoalkaloids are well-known metabolites which can exert negative effects on human health. Thus, the purpose of this work was to simultaneously eliminate and/or decrease some of these antinutrients, either at large (protein) and small (metabolite) component level, by using a new plant breeding technology (NPBT) approach. More in detail, we designed specific gRNAs for the two following target genes: Glycoalkaloid metabolism 4 (GAME4), key step in the biosynthetic pathway of the glycoalkaloids, and Sola l 4, a PR-10 protein which is among the major allergens in tomato. Genetically stable (homozygous and Cas9-free) plants carrying loss of function mutations in both GAME4 and Sola l 4 genes were obtained. LC-HRMS analyses, used to metabolically profiling the edited plants, highlighted a dramatic reduction (up to 99.99%) in the glycoalkaloids content, as well the complete absence of Sola l 4 protein. Furthermore, a great extent of additional metabolic changes was observed, partially explainable by the GAME4-KO induced flux diversion. Notably, and contrary to our expectations, increased fitness was noted in the edited lines as evaluated in biotic assays against fungi and insects.