Armadillo repeat only gene VlARO confers resistance to Botrytis cinerea by promoting guaijaverin accumulation in grapevine.

The ARM repeat only gene VlARO positively regulates grape resistance to gray mold by promoting the guaijaverin accumulation, which directly suppresses the mycelial growth of Botrytis cinerea. Gray mold, caused by the necrotrophic fungus Botrytis cinerea, severely threatens global grapevine production. Here, we identified VlARO, a nuclear-localized gene from the highly resistant grapevine variety 'Beta', as a positive regulator of gray mold resistance. Although VlARO contains Armadillo repeats, it lacks a canonical U-box domain, thus distinguishing it from typical plant U-box E3 ubiquitin ligases. Overexpression of VlARO in grape leaves significantly enhanced resistance to B. cinerea, whereas its silencing weakened this resistance. Stable overexpression in grape calli and transgenic Arabidopsis consistently conferred enhanced resistance, as evidenced by markedly reduced lesion formation. Mechanistically, VlARO significantly activated antioxidant enzymes, resulting in efficient ROS scavenging and reduced oxidative damage. Metabolomic analysis revealed that the overexpression of VlARO profoundly reshaped the metabolic landscape following B. cinerea inoculation, with the flavonoid biosynthesis pathway most prominently enriched. Notably, the accumulation of guaijaverin, a flavonoid glycoside, showed strong positive correlation with VlARO transcript levels. Further results demonstrated that guaijaverin directly inhibits the mycelial growth of B. cinerea in vitro and that its exogenous application significantly reduced disease severity in grape calli. Our findings establish the mechanism for VlARO in grapevine immunity by promoting guaijaverin accumulation. This study provides a promising genetic target for disease-resistant grapevine breeding and lays a theoretical foundation for developing eco-friendly fungicides based on guaijaverin.