Analysis of genetic variability in grapevine leafroll-associated virus 13 and the characteristics of the p23

Grapevine leafroll-associated virus 13 (GLRaV-13) is a newly discovered virus that infects grapevines; however, prior research on its sequence features and gene functions has been minimal. In this study, GLRaV-13 was detected in 34 of 234 grapevine samples, and the complete genome sequences of three Chinese isolates of GLRaV-13 were successfully obtained through high-throughput sequencing (HTS), overlapping RT-PCR, and RACE. Additionally, the interactions among GLRaV-13 encoded proteins were revealed by yeast two-hybrid (Y2H) and bimolecular fluorescence complementation (BiFC) assays. There are nine pairs of proteins that interact, and p23 has self-interaction. Furthermore, using Agrobacterium-mediated RNA silencing, we confirmed that p23 protein of GLRaV-13 acts as an RNA silencing suppressor (RSS). Further experiments demonstrated that the expression of p23 protein from the pGD or potato virus X (PVX) vectors can induce a strong hypersensitive response in Nicotiana benthamiana plants, indicating that this protein plays a crucial role in the pathogenic mechanism of the virus. Deletion analysis revealed that the N-terminal 1-178 amino acids region of the p23 protein is the critical domain for its self-interaction and RSS activity. Further mutational analysis demonstrated that the lysine (K) residue at position 99 plays a decisive role in p23 dimerization, specific interactions with other proteins, RSS function, and pathogenicity. In conclusion, the p23 protein exerts its symptom exacerbation function through a dimerization-dependent mechanism, which is closely associated with its RSS activity.