In Silico Screening of Natural Bioactive Compounds Targeting RXLR Effector Protein 25 In Plasmopara Viticola, The Causal Agent of Grapefruit Downy Mildew

Abstract

Downy mildew caused by the obligate biotrophic oomycete Plasmopara viticola remains one of the most destructive diseases of grapevine globally, leading to significant yield losses and a heavy reliance on synthetic fungicides, which are increasingly compromised by resistance development. This study utilized structure-based in silico screening to identify natural bioactive compounds targeting RXLR Effector Protein 25 (PvRXLR25), a critical virulence factor that suppresses grapevine immune responses. The three-dimensional structure of PvRXLR25 was predicted using AlphaFold 3, followed by virtual screening of diverse natural compound libraries from specialized databases, including Seaweed Metabolites 2024, Phenol-Explorer 2024, Lichen Database 2024, and others. Molecular docking with PyRx/AutoDock Vina revealed Strychnine Acid (CNP0260135.6) as the most promising candidate with the highest binding affinity of -6.7 kcal/mol, followed by thelephoric Acid (-6.2 kcal/mol) and variolaric Acid (-6.1 kcal/mol). Detailed interaction analysis demonstrated stable binding via a strong conventional hydrogen bond to PHE242 (2.0191 Å), a π–π T-shaped interaction with PHE241, and multiple hydrophobic contacts. These results indicate that lichen-derived secondary metabolites, particularly Strychnine Acid, possess strong potential as inhibitors of PvRXLR25 and offer promising scaffolds for the development of novel, eco-friendly strategies to manage grape downy mildew.