Functional Characterization of RNA Silencing Suppressor Encoded by Cotton Leafroll Dwarf Virus

The P0 proteins encoded by Cotton leafroll dwarf virus (CLRDV) serve as viral suppressors of RNA silencing (VSR). CLRDV P0 proteins share a VSR-associated F-box-like motif. This study investigated the role of specific amino acid (aa) residues within the F-box-like motif, elucidating their implications for VSR potency, pathogenicity, and intracellular localization. Analysis of various single aa substitution mutants within the F-box-like motif to understand their effect on P0 proteins' VSR potency revealed that specific mutations significantly affect P0's ability to suppress RNA silencing, emphasizing the crucial role of the F-box-like motif. Subcellular localization examinations indicated that the P0 proteins associate with the nucleus and endoplasmic reticulum, which may be linked to their VSR function. Also, the induction of hypersensitive response (HR)-like symptoms and the accumulation of reactive oxygen species by P0 proteins was observed, suggesting their role as pathogenicity factors. These results indicated that specific mutations significantly affect the functions of multifaceted P0 proteins, highlighting the F-box-like motif's crucial role. Our study highlights the importance of further molecular investigations to elucidate how different CLRDV strains manifest various disease symptoms and severity. This is crucial considering the global economic importance of cotton and the potential emergence of more threatening CLRDV isolates.