Identification of crucial residues or domain(s) of rice stripe virus NS3 protein required for self-interaction and for silencing suppressor activity

Abstract

Rice stripe virus (RSV), a member of the genus Tenuivirus, is one of the most harmful virus in rice cultivation. The genome of RSV consists of four single-stranded RNAs and encodes seven viral proteins. NS3, which is encoded from the RNA segment 3, was previously reported as a gene silencing suppressor for RNA silencing and self-interaction of NS3 is necessary for maintaining suppressor activity. To identify the crucial amino acid residues or domains(s) required for self-interaction of NS3, I used protein structure prediction program and constructed eleven NS3 mutant clones including four alpha-helix deletion and substitution mutants. With these mutated clones, yeast-two hybrid (Y2H) and bimolecular fluorescence complementation (BiFC) assays were conducted for interaction study. Y2H and BiFC results showed that the N-terminal region of NS3 is essential for self-interaction. All of alpha-helix deletion mutants and substitution mutants lost its self-interaction ability in Y2H and BiFC assays. To identify the relationship between NS3 self-interaction and silencing suppressor activity, we used GFP silencing system in Nicotiana benthamiana with Agrobacterium-mediated transient overexpression of each mutated NS3 protein. All the deletion and four alpha-helix substitution mutations resulted in the loss of the silencing suppressor ability except lysine77 substitution mutation which maintained self-interaction capacity. Altogether, these results suggest that the NS3-NS3 self-interaction might be necessary for maintaining suppressor activity as a counter defense to RNA silencing system of the host plant.