Movement and nucleocapsid proteins coded by two tospovirus species interact through multiple binding regions in mixed infections

Negative-stranded tospoviruses (family: Bunyaviridae) are among the most agronomically important viruses. Some of the tospoviruses are known to exist as mixed infections in the same host plant. Iris yellow spot virus (IYSV) and Tomato spotted wilt virus (TSWV) were used to study virus-virus interaction in dually infected host plants. Viral genes of both viruses were separately cloned into binary pSITE-BiFC vectors. BiFC results showed that the N and NSm proteins of IYSV interact with their counterparts coded by TSWV in dually infected Nicotiana benthamiana plants. BiFC results were further confirmed by pull down and yeast-2-hybrid (Y2H) assays. Interacting regions of the N and NSm proteins were also identified by Y2H system and β-galactosidase activity. Several regions of the N and NSm were found interacting with each other. The regions involved in these interactions are presumed to be critical for the functioning of the tospovirus N and NSm proteins. This is the first report of in vivo protein interactions of distinct tospoviruses in mixed infection.