Begomoviral pre-coat protein boosts potato virus X in mixed infection through interfering with antiviral RNAi response

Plant-infecting viruses cause severe crop losses throughout the world. The frequent occurrence of mixed infection (co-existence of multiple viruses in a single infected plant) in the field makes the situation more alarming. Mixed infection of two viruses often boosts the fitness of either of the viruses and thus increases disease severity. Maize lethal necrosis disease and rice tungro disease are some devastating examples. This study focused on the mixed viral infection between begomovirus, a ssDNA virus, and potato virus X (PVX), a ssRNA virus. V2 protein encoded by a monopartite begomovirus, pepper leaf curl Bangladesh virus (PepLCBV), promotes the accumulation of PVX. When Nicotiana benthamiana plants were infected with both PVX and PepLCBV, aggravated PVX-induced symptoms and increased PVX titre were detected compared to plants infected with PVX alone. This observation suggests that PepLCBV promotes the viral titre of PVX during mixed infection. Next, when PepLCBVΔV2 infectious clone was co-infiltrated with PVX, no increase in PVX-associated symptoms was observed in the plants, which confirmed that the V2 protein plays a pivotal role in mediating the interaction between these two viruses. PepLCBV-V2 was experimentally identified as a potent suppressor of post-transcriptional gene silencing. Its interaction with suppressor of gene silencing 3 (NbSGS3) was confirmed through yeast two-hybrid and BiFC. Silencing of NbSGS3 facilitated PVX pathogenesis. Next, specific amino acids responsible for the suppressor activity of the V2 protein were identified, and the silencing suppressor mutant V2 failed to boost the PVX titre. A similar observation was recorded when the association of PVX with a bipartite begomovirus, tomato leaf curl New Delhi virus, was checked in both N. benthamiana and tomato. The present study, for the first time, reports that begomoviruses promote the pathogenesis of potato virus X during mixed infection and identifies begomoviral pre-coat protein (V2/AV2) as a key player in mediating this virus-virus interaction. We further demonstrated that the silencing suppression activity of V2 is important for regulating PVX titre in mixed-infected plants.