Validation of a Tobacco Rattle Virus (TRV)-Induced Gene Editing System in Physalis grisea (Groundcherry)

Abstract

Physalis grisea, known as groundcherry, is an orphan crop with agricultural potential due to its nutritional, medicinal, and economic value. Limited genetic tools for its improvement have harmed this crop’s wider adoption. This project validated a TRV-induced gene editing system in Physalis grisea. In this method, Agrobacterium infiltration introduced TRV constructs carrying gene-specific sgRNAs, including an FT motif to enhance systemic movement, into leaf tissue. The sgRNAs, combined with the Cas9 expressed in the plant, produced edits in the target gene: the PHYTOENE DESATURASE (PDS) gene. PDS modifications disrupted carotenoid production, leaving chlorophyll vulnerable to light-induced damage and producing photobleaching. This PDS-loss phenotype was observed in >94% surviving PDS-infiltrated Cas9-expressing plants, while control groups were unaffected (n>450 across 3 replicates). The degree of photobleaching varied and mosaic patterns were observed in leaves, husks and possibly fruits. Microscopy verified and quantified the degree of photobleaching (31%). Plant growth was unaffected by photobleaching, but was negatively influenced by agroinfiltration, which also caused mortality. These results validate TRV mediated PDS editing as a proof-of-concept in P. grisea by showing the efficacy and reproducibility of the system thereby supporting the use of the system for future gene studies and improvement strategies in the crop.