Identification And Functional Study On Proteins Of Vector Sogatella Furcifera (Horvath) Interacted With Southern Rice Black-streaked Dwarf Virus P7-1

A rice viral disease caused by Southern rice black-streaked dwarf virus (SRBSDV), a tentative member of the genus Fijivirus in the family Reoviridae, was first recorded in2001in Yangjiang city, Guangdong province of Southern China and has caused serious losses in rice production in recent year. In the past decade, SRBSDV has rapidly spread throughout Southern China, Northern Vietnam, South Korea, Japan and Thailand, becoming one of the most important rice pathogens in East and Southeast Asia. SRBSDV is effectively transmitted by white-backed planthopper (WBPH; Sogatella furcifera Horvath) in a persistent-propagative manner. As a typical long-distance migratory pest carried by wind currents, the dispersal of viruliferous WBPH leads to secondary infections of the virus and severe outbreaks of the viral disease.In the present study, the protein interaction analysis was done between SRBSDV P7-1as bait protein and a cDNA library of S. furciferaas prey protein. In the preliminary yeast two hybrid system,153potential protein-protein interactions were identified by Blast2go database. The species distribution of the best match results for each sequence were:12%of the prey protein with Riptortus pedestris (bean bug) sequences, followed by each of7%with Tribolium castanetum (red flour beetles) and S. furcifera (WBPH) sequences and6%with Pediculus humanus corporis (body louse). In the classification of GO annotation analysis,153putative proteins were well distributed into10categories of biological process,6categories of cellular component and17categories of molecular function. To determine the positive interactors, we selected24candidate prey proteins based on their molecular activities and cellular components of which18proteins were confirmed as true positive interactors by two confirmation assays in yeast:elimination of false positives byretransformation analysis and selection of the strength of interaction by β-galactosidase assay.To confirm the yeast two-hybrid results, we independently examined the interactions of P7-1with18positive prey proteins in mammalian cells by Chemiluminescent Co-IP assay. The relative strengths of the interactions between P7-1and seven prey proteins:neuroglian, myosin light chain2(MLC2), chitin bind4(cuticular-RR2), E3ubiquitin-protein ligase MARCH5, polyubiquitin, ribophorin ii and profilin, were13,12,7,6.8,6.6,6.3and5.6times higher than that of the respective control and other11prey proteins were2-4times higher than that of control. According to these results, the seven candidates:neuroglian, MLC2, chitin bind4(cuticular-RR2), ribophorin ii, E3ubiquitin-protein ligase MARCH5, polyubiquitin and profilin, are highly interacted with P7-1. The most abundant subcategories of differentially highly expressed proteins in Co-IP assay were proteins involved in binding, posttranslational modification, protein turnover and folding, lipid transport, and metabolism, energy production and conversion, protein biosynthesis, cytoskeleton maintenance and biogenesis.Among seven candidates, six proteins of S. furcifera:neuroglian, MLC2, ribophorin ii, E3ubiquitin protein ligase, polyubiquitin, and profilin were studied for gene expression analysis by qRT-PCR. mRNA expression levels of neuroglian, MLC2, polyubiqutin, E3ubiquitin-protein ligase and profilin were highly expressed in male adult stage than that of female adult stage and nymph stages. However, ribophorin ii was mainly expressed in female adult stage with highest expression level compared with the nymph and male adult stages. For mRNA level in five different tissue organs neuroglian, MLC2, polyubiquitin and profilin showed the highest expression in gut, ribophorin ii was predominantly expressed in the salivary glands and the highest mRNA level of E3ubiquitin-protein ligase was found in the haemolymph.The level of four candidate proteins:neuroglian, MLC2, E3ligase and profilin in three different organs of WBPH were analyzed by Indirect ELISA. The result showed that neuroglian can be detected in gut and haemolymph, but not in salivary glands. MLC2and profilin were highly detectable in gut followed by salivary glands, but not in haemolymph. The highest protein expression level of E3ligase can be detected in haemolymph than salivary glands and gut. This ELISA result was consistent with the qRT-PCR result and supporting that neuroglian, MLC2, profilin and E3ligase might involve in the virus movement process to successful pass through the different tissue tropism in insect vector.In order to clarify the interaction between SRBSDV P7-1and WBPH prey proteins, MLC2was chosen as a model to determine the involvement of MLC2in the virus movement process in insect vector using sub-cellular localization analysis by immunofluorescence microscopy. The results showed that virus containing tubular structure P7-1proteins were attached to MLC2proteins of the actomyosin complex along the midgut at4days and8days post acquisition period (PADP), whereas P7-1attached with MLC2in accessary salivary glands of WBPH at the8days PADP and not at4days PADP, supporting that MLC2protein involved in the trafficking process of virus in insect vector.To elucidate the functional role of MLC2of S. furcifera, the full-length gene of MLC2was amplified and characterized. The amplified cDNA sequence is1030bp in length with an open reading frame of624bp encoding a polypeptide of207amino acids. Alignment analysis revealed that the deduced protein sequence has67%identity to myosin light chain2of Hemiptera species,54-59%similarity with myosin regulatory light chain from Diptera group insects. MLC2protein contained two "EF-hand" domains. The MLC2protein sequence was used to construct phylogenetic trees with other known vertebrate and invertebrate and this result was consistent with the traditional classification of phylogenetic. These findings suggested that the obtained protein was myosin regulatory light chain (MLC2) of S. furcifera.Present study revealed that six candidate proteins are involved in the complex reaction to SRBSDV for successful virus transmission by insect vector and provide new insights toward unraveling the molecular basis of virus and insect vector relationship.