AbstractMany persistent transmitted plant viruses, includingRice stripe tenuivirus(RSV), cause serious damages to crop productions in China and worldwide. Although many reports have indicated that successful insect-mediated virus transmission depends on proper virus–insect vector interactions, the mechanism(s) controlling interactions between viruses and insect vectors for virus persistent transmission remained poorly understood. In this study, we used RSV and its small brown planthopper (SBPH) vector as a working model to elucidate the molecular mechanism controlling RSV virion entrance into SBPH midgut for persistent transmission. We have now demonstrated that this non-envelopedTenuivirususes its non-structural glycoprotein NSvc2 as a helper component to bridge the specific interaction between virion and SBPH midgut cells, leading to overcome SBPH midgut barriers for virus persistent transmission. In the absence of this glycoprotein, purified RSV virion is not capable of entering SBPH midgut cells. In RSV-infected cells, glycoprotein NSvc2 is processed into two mature proteins: an amino-terminal protein NSvc2-N and a carboxyl-terminal protein NSvc2-C. We determined that NSvc2-N interacted with RSV virion and bound directly to midgut lumen surface via its N-glycosylation sites. Upon recognition by midgut cells, the midgut cells underwent endocytosis followed by compartmentalizing RSV virion and NSvc2 into early and then late endosomes. The acidic condition inside the late endosome triggered conformation change of NSvc2-C and caused cell membrane fusion via its highly conserved fusion loop motifs, leading to the release of RSV virion from endosome into cytosol. In summary, our results showed for the first time that a riceTenuivirususes a molecular bridge strategy to ensure proper interactions between virus and insect midgut for successful persistent transmission.Author summaryOver 75% of the known plant viruses are insect transmitted. Understanding how plant viruses interacted with their insect vectors during virus transmission is one of the key steps to manage virus diseases worldwide. Both the direct and indirect virus–insect vector interaction models have been proposed for virus non-persistent and semi-persistent transmission. However, the indirect virus–vector interaction mechanism during virus persistent transmission has not been reported previously. In this study, we developed a new reverse genetics technology and demonstrated that the circulative and propagative transmittedRice stripe tenuivirusutilizes a glycoprotein NSvc2 as a helper component to ensure a specific interaction betweenTenuivirusvirion and midgut cells of small brown planthopper (SBPH), leading to conquering the midgut barrier of SBPH. This is the first report of a helper component mediated-molecular bridge mechanism for virus persistent transmission. These new findings and our new model on persistent transmission expand our understanding of molecular mechanism(s) controlling virus–insect vector interactions during virus transmission in nature.