Elucidating the function of a novel cysteine-rich peptide during flower-to-fruit transition in tomato

During fruit initiation, secreted peptides mediate communication between pollen and the female gametophyte, as well as the embryo sac and the surrounding female tissues. The cysteine-rich peptides (CRPs), a diverse class of secreted peptides that contain an N-terminal secretion signal and a C-terminal cysteine-rich region, play an important role in cell-cell signaling during flower-to-fruit transition. However, their role in tomato fertilization, fruit initiation, and seed formation is still unknown. Previous tissue-specific transcriptomic studies in our lab identified OVULE-SECRETED PROTEIN (OSP), a CRP highly expressed in the tomato ovule integument at anthesis. Based on this expression profile, we hypothesized that OSP may be involved in processes during flower-to-fruit transition, such as micropylar pollen tube guidance, ovule maturation, or seed formation. In this project, we conducted studies aimed at the functional characterization of OSP, which included phylogenetic analysis, a study of OSP spatiotemporal expression patterns, and the phenotypic characterization of OSP-RNAi plants. In addition, we designed and created molecular tools to generate osp knock-out mutants using CRISPR technology. Multiple sequence alignment and phylogeny tools demonstrated that the cysteine motif in OSP is unique to the cultivated tomato and its wild relatives. Using gOSP::YFP transgenic plants and confocal microscopy, we have observed that OSP is expressed in ovaries and fruit at stages surrounding anthesis. Our results suggest that OSP expression in the ovule inner integument beginning prior to anthesis. OSP-RNAi plants with reduced levels of OSP expression showed no abnormal phenotype when we examined seed weight, seed number per fruit, external seed and ovule morphology, and pollen tube guidance. We have therefore assembled a CRISPR vector that includes guide RNAs targeting the OSP coding region. We anticipate that osp knockout mutants will show a stronger phenotype that will help elucidate the function of OSP.

My Experience

The Plant Genome Research Program at BTI has been an invaluable experience. I felt immediately welcomed by my lab and the community at BTI. In addition to learning a variety of microscopy and molecular biology techniques, I had the opportunity to make experimental decisions and develop confidence as a researcher. Through working independently and with my mentor, I am better prepared for a research career. This internship has reinforced my intention to pursue plant science during graduate school, and I look forward to a continued relationship with my lab and the interns I have met this summer.