Assembly of viroplasms by viral nonstructural protein Pns9 is essential for persistent infection of rice gall dwarf virus in its insect vector

•Infection route of RGDV in its insect vector is revealed.•RGDV spread directly from the initially infected epithelial cells of insect intestine toward the visceral muscle tissues.•Assembly of viroplasms by nonstructural protein Pns9 is essential for persistent infection of RGDV in vector. Rice gall dwarf virus (RGDV), a plant reovirus, is transmitted by leafhopper vector Recilia dorsalis in a persistent-propagative manner. In a sequential study of RGDV infection of its insect vector, the virus initially infected the filter chamber epithelium, then directly crossed the basal lamina into the visceral muscles, from where it spread throughout the entire midgut and hindgut. Finally, RGDV spread into the salivary glands. During RGDV infection of the continuous cultured cells of R. dorsalis, viroplasm that was mainly comprised of viral nonstructural protein Pns9 was formed and acted as the site of viral replication and assembly of progeny virions. Knockdown of Pns9 expression in cultured insect vector cells using synthesized dsRNAs from the Pns9 gene strongly inhibited viroplasm formation and viral infection. The microinjection of dsRNAs from the Pns9 gene strongly abolished viroplasm formation in the initially infected filter chamber epithelium and prevented viral spread into leafhopper visceral muscles. These results indicated that the assembly of viroplasms was essential for the persistent infection and spread of RGDV in its insect vector.