Potential Application of Virus-Induced Gene Silencing in Cucurbit Plants Using Cucumber Fruit Mottle Mosaic Virus Vector

Approach of target gene silencing for ascertaining its function in plants can be surely well-established by transgenic technique such as RNAi strategy. However, producing transgenic cucurbit plants is recalcitrant and also the success rate is usually very low. Virus-induced gene silencing (VIGS) for functional genomics has been developed constantly and reported successful results in representative plants such as Arabidopsis, tomato and rice so on. VIGS is believed to be one of possible ways to solve the bottleneck in loss-of-function in plant genomic studies. Prior to VIGS application, a virus vector should be preceded. To perform functional genomic analysis in cucurbits, we isolated, cloned, and sequenced Cucumber fruit mottle mosaic virus from melon (CFMMV-Cm), which belongs to cucurbit-infecting tobamovirus genus. We constructed a CaMV 35S promoter-driven CFMMV vector by mapping coat protein (CP) subgenomic promoter and inserting multiple cloning site in front of the CP gene. To estimate efficiency of the subgenomic promoter activities of CFMMV vector, EGFP was used as a reporter gene which is possible to observe visually in vivo. We constructed a CFMMV-pds vector carrying the gene coding phytoene desaturase (PDS) that is one of the key enzymes in the beta-carotene biosynthesis pathway and located upstream of lycopene. When the virus vector was inoculated to cotyledons of watermelon, melon, and cucumber, photobleaching phenomena were observed in upper true leaves. Furthermore, the inoculated watermelon fruits (45 days after inoculation to cotyledons) showed white color instead of red in flesh of the fruit accompanied by low copy numbers of PDS gene and high accumulation of the virus vector. The lycopene contents found to be significantly low levels in three different cultigens of watermelon.