(1) Background: is a dioecious plant, whose sex is determined by XY-type sex chromosomes. is a smut fungus that infects plants and causes masculinization in female flowers, as if were acting as a sex-determining gene. Recent large-scale sequencing efforts have promised to provide candidate genes that are involved in the sex determination machinery in plants. These candidate genes are to be analyzed for functional characterization. A virus vector can be a tool for functional gene analyses; (2) Methods: To develop a viral vector system in plants, we selected (ALSV) as an appropriate virus vector that has a wide host range; (3) Results: Following the optimization of the ALSV inoculation method, plants were infected with ALSV at high rates in the upper leaves. In situ hybridization analysis revealed that ALSV can migrate into the flower meristems in plants. Successful VIGS (virus-induced gene silencing) in plants was demonstrated with knockdown of the phytoene desaturase gene. Finally, the developed method was applied to floral organ genes to evaluate its usability in flowers; (4) Conclusion: The developed system enables functional gene analyses in plants, which can unveil gene functions and networks of plants, such as the mechanisms of sex determination and fungal-induced masculinization.