Most plant viruses are transmitted by hemipteroid insects. Some viruses can be transmitted from female parent to offspring usually through eggs, but the mechanism of this transovarial transmission remains unclear. Rice stripe virus (RSV), a Tenuivirus, transmitted mainly by the small brown planthopper (Laodelphax striatellus), is also spread to the offspring through the eggs. Here, we used the RSV–planthopper system as a model to investigate the mechanism of transovarial transmission and demonstrated the central role of vitellogenin (Vg) of L. striatellus in the process of virus transmission into the eggs. Our data showed Vg can bind to pc3 in vivo and in vitro and colocalize in the germarium. RSV filamentous ribonucleoprotein particles (RNPs) only accumulated in the terminal filaments and pedicel areas prior to Vg expression and was not present in the germarium until Vg was expressed, where RSV RNPs and Vg had colocalized. Observations by immunoelectron microscopy (IEM) also indicated that these two proteins colocalized in nurse cells. Knockdown of Vg expression due to RNA interference resulted in inhibition of the invasion of ovarioles by RSV. Together, the data obtained indicated that RSV RNPs may enter the nurse cell of the germarium via endocytosis through binding with Vg. Finally, the virus enters the oocytes through nutritive cords, using the same route as for Vg transport. Our results show that the Vg of L. striatellus played a critical role in transovarial transmission of RSV and shows how viruses can use existing transovarial transportation systems in insect vectors for their own purposes. Numerous parasites including viruses, bacteria, and microsporidia can be maternally transmitted, with the parasite passing from mother to offspring, usually through eggs. However, the process of the parasites spreading into eggs from primarily infected tissues and the factors that mediate this process in live hosts or vectors are unknown due to the lack of useful tools. Here, we used several techniques to investigate the molecular mechanisms of transovarial transmission of Rice stripe virus (RSV), a plant virus belonging to the genus Tenuivirus, by its insect vector (Laodelphax striatellus). We found that the nucleocapsid protein of RSV bound to insect's vitellogenin (Vg) in vitro and in vivo. We also found that RSV invaded the egg tubes of the ovariole until Vg is highly expressed, then colocalized with Vg in the germarium. When Vg expression was knocked down due to RNA interference, the invasion of ovarioles by RSV decreased largely. Our study provides new insights into the transovarial transmission of an important viral pathogen that uses existing transovarial transportation systems in insect vectors to invade eggs.

Numerous viruses can be transmitted by their corresponding vector insects; however, the molecular mechanisms enabling virus transmission by vector insects have been poorly understood, especially the identity of vector components interacting with the virus. Here, we used the yeast two-hybrid system to study proteomic interactions of a plant virus (Rice stripe virus, RSV, genus Tenuivirus) with its vector insect, small brown planthopper (Laodelphax striatellus). Sixty-six proteins of L. striatellus that interacted with the nucleocapsid protein (pc3) of RSV were identified. A virus–insect interaction network, constructed for pc3 and 29 protein homologs of Drosophila melanogaster, suggested that nine proteins might directly interact with pc3. Of the 66 proteins, five (atlasin, a novel cuticular protein, jagunal, NAC domain protein, and vitellogenin) were most likely to be involved in viral movement, replication, and transovarial transmission. This work also provides evidence that the novel cuticular protein, CPR1, from L. striatellus is essential for RSV transmission by its vector insect. CPR1 binds the nucleocapsid protein (pc3) of RSV both in vivo and in vitro and colocalizes with RSV in the hemocytes of L. striatellus. Knockdown of CPR1 transcription using RNA interference resulted in a decrease in the concentration of RSV in the hemolymph, salivary glands and in viral transmission efficiency. These data suggest that CPR1 binds RSV in the insect and stabilizes the viral concentration in the hemolymph, perhaps to protect the virus or to help move the virus to the salivary tissues. Our studies provide direct experimental evidence that viruses can use existing vector proteins to aid their survival in the hemolymph. Identifying these putative vector molecules should lead to a better understanding of the interactions between viruses and vector insects.

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Insect transmission is obligatory for persistently transmitted viruses because the vector insect is the only means of virus spread in nature. The insect midgut is the first major barrier limiting virus acquisition, but the mechanisms by which viruses are able to cross the cell membrane and then infect the midgut epithelial cells of the insect have not been elucidated completely. Here, we found that the outer capsid or nucleocapsid protein (NP) of three viruses can interact and colocalize with sugar transporter 6 that is highly expressed in the midgut of Laodelphax striatellus (LsST6). In contrast, LsST6 did not interact with the NP of rice grassy stunt virus, which cannot be transmitted by the same planthopper. LsST6 not only altered the cellular location of viral proteins and then colocalized with them in the cell membrane, but also mediated the entry of rice stripe virus (RSV) particles into Spodoptera frugiperda 9 (Sf9) cells that expressed the heterologous gene LsST6. We further showed that RSV particles initially bound to the cell membrane of midgut epithelial cells where it colocalized with LsST6, and then invaded the cytoplasm. When LsST6 expression was knocked down, viral titre, acquisition percentage and transmission efficiency of the treated insect decreased significantly, but virus replication was not affected. This work thus uncovered a strategy by which LsST6 mediates viral entry into midgut epithelial cells and leads to successful transmission by the insect vector. Sap/blood-feeding arthropods are major vectors of viruses that infect plants and vertebrates. Studies on the insect midgut, the first barrier for virus transmission, and its interactions with viruses and parasites are fundamental to understanding the transmission mechanism in vector insects and the epidemics caused by the vectored pathogen. Some putative receptors in arthropods have been discovered by in vitro protein interactions, but in vivo evidence is still lacking. Here, we found that the specific interaction between viral nucleocapsid protein and vector sugar transporter 6 of Laodelphax striatellus (LsST6) determines whether the virus can invade midgut epithelial cells or not. These results provide direct evidence that LsST6 is an essential and key factor in crossing the midgut infection barrier for viruses, especially for RSV. This vector protein may be a promising target for blocking transmission of diverse plant viruses. Our discovery has important implications for better understanding the interaction among host–virus–insect vector and disease epidemics caused by plant and animal arboviruses.

Rice plants infected by Rice stripe virus (RSV) usually leads to chlorosis and death of newly emerged leaves. However, the mechanism of RSV-induced these symptoms was not clear. We used an iTRAQ approach for a quantitative proteomics comparison of non-infected and infected rice leaves. RT-qPCR and Northern blot analyses were performed for assessing the transcription of candidate genes. As a whole, 681 (65.8 % downregulated, 34.2 % upregulated infected vs. non-infected) differentially accumulated proteins were identified. A bioinformatics analysis indicated that ten of these regulated proteins are involved in chlorophyll biosynthesis and three in cell death processes. Subsequent RT-qPCR results showed that downregulation of magnesium chelatase was due to reduced expression levels of the genes encoding subunits CHLI and CHLD, which resulted in chlorophyll reduction involved in leaf chlorosis. Three aspartic proteases expressed higher in RSV-infected leaves than those in the control leaves, which were also implicated in RSV-induced cell death. Northern blot analyses of CHLI and p0026h03.19 confirmed the RT-qPCR results. The magnesium chelatase and aspartic proteases may be associated with RSV-induced leaf chlorosis and cell death, respectively. The findings may yield new insights into mechanisms underlying rice stripe disease symptom formation.