Viral Proteins as Biocontrol Agents: Mechanisms and Delivery of Mycovirus REP for Sustainable Plant Disease Management

ABSTRACT

Hypovirulent mycoviruses offer a promising and environmentally sustainable approach for managing plant pathogenic fungi, yet the specific viral factors responsible for suppressing fungal virulence and their potential for translational application remain poorly defined. This dissertation investigates the replication-associated protein (REP) encoded by Sclerotinia sclerotiorum hypovirulence-associated DNA virus 1 (SsHADV1) as a protein-based antifungal agent and establishes a comprehensive framework for characterizing its biological activity, molecular function, and delivery potential. REP application markedly suppressed Sclerotinia sclerotiorum growth, development, and aggressiveness across multiple heterologous systems, and site-directed mutagenesis identified a conserved 14-amino-acid motif essential for REP activity. This motif is conserved across REPs from other ssDNA mycoviruses and is important but not sufficient for hypovirulence, the REP structure may also be critical for this functional conservation. To enable practical deployment, Bacillus subtilis RIK1285 was engineered to express and secrete REP, and extensive optimization of protein expression, secretion, and quantification workflows significantly improved extracellular protein accumulation. In addition, standardized protocols were developed for REP production in bacterial cultures, fungal inhibition assays, and plant infection models, providing a reproducible pipeline for functional evaluation of secreted viral proteins. Secreted and purified REP inhibited fungal growth in vitro and reduced disease severity in a greenhouse sunflower infection model, demonstrating its translational potential as a protein-based biocontrol strategy. Mechanistic analyses revealed REP-induced alterations in fungal morphology, decreased oxalic acid production, and modulation of growth- and virulenceassociated gene expression. Taken together, this work elucidates the multifunctional antifungal iv activity of a mycoviral REP protein and provides foundational biological, mechanistic, and delivery frameworks to inform the development of viral factor-derived, sustainable strategies for controlling fungal diseases in agriculture.