Prepration of genetic constructs to modify the genomes of horticultural plants

Cell-to-cell signaling in plant depends also on peptide hormones. Hundreds of genes in plant genomes encode these small peptide signals, but only a tiny fraction of these genes have been functionally characterized. Complete identification of these peptide-encoding genes is challenging due to their small size and significant sequence diversity. The CLAVATA (CLV)/EMBRYO SURROUNDING REGION-RELATED (CLE) gene family encodes short peptides involved in plant meristem maintenance, cell differentiation, the defense from pathogens, and environmental responses. Recent findings brought the tomato (Solanum Lycopersicum) CLE gene family from 15 to 52 members, including 37 newly identified genes that had not been annotated before. Although we now know 52 CLE peptide genes in S. lycopersicum, their functions and roles remain unknown for most of them. We have identified CLE peptides that are highly expressed in flower, suggesting they may be involved in the flowering process. The overall goal of the project is understanding how these peptides influence flowering. Therefore, we have employed a multiplexing strategy using CRISPR/Cas9-mediated mutagenesis to knockout specific combinations of those genes. The genes that are involved in flowering are (CLE6, CLE19, CLE30, CLE31, CLE34) and for the ripening is CLE11. To assemble the vectors for the generation of the KO-lines we took advantage of pDIRECT_22C, a vector system based on Golden Gate Assembly techniques. CRISPR/Cas9-designed constructs will be delivered into S. lycopersicum with a transformation technique based on the infection of young cotyledon sections by Agrobacterium tumefaciens.