Proteomic Analysis And Identification Of Interacted Proteins Between Southern Rice Black-streaked Dwarf Virus-P10 And The Insect Vector Sogatella Furcifera

Southern rice black-streaked dwarf virus(SRBSDV) was first discovered in 2001 in Yangjiang city, Guangdong province of Southern China. SRBSDV is belong to genus Fijivirus, family Reoviridae, transmitted efficiently only by white-backed planthopper(WBPH, Sogatella furcifera Horvath, Hemiptera: Delphacidae) in a persistent propagative manner. During the last few years, SRBSDV has rapidly spread throughout southern China, northern Vietnam, Japan and Thailand, becoming one of the most important rice pathogens in East and Southeast Asia due to the migration of the viruliferous WBPH vectors. As a typical long-distance migratory pest carried by wind currents, WBPH is distributed in the most Asian counties, including China, Pakistan, Japan, Korea, Saudi Arabia, Siberia, Micronesia, Philippines, Laos, Cambodia, Myanmar, Nepal, Vietnam, Thailand, India, Indonesia, Fiji, Bangladesh and Seychelles. The dispersal of viruliferous WBPH leads to secondary infections of the virus and severe outbreaks of the viral diseases.The efficient and successful transmission of the virus requires specific interactions between the virus and unknown components of the vector to allow transport of the virus in and out of insect tissues and to overcome the various types of insect immune reactions. The molecular mechanism and involvement of proteins in the process of virus entry, transmission, movement and replication inside the insect are largely unknown. Thus, the investigation and identification of specific protein–protein interactions are essential for understanding and studying various aspects of cell biology. In this study, we examined protein interactions between SRBSDV-P10 as the bait and cDNA library of WBPH as the prey by using yeast two-hybrid system, and 130 protein–protein interactions between SRBSDV and WBPH were identified. Based on the classification of GO annotation analysis result, 130 proteins were categorized into 12 categories of molecular function, 9 categories of biological process and 8 categories of cellular components. The species distribution of the best match results for each sequence were 12.3% of the prey protein sequences had a maximum match with Zootermopsis nevadensis, followed by 9.2% with Tribolium castanetum, 4.6% with S. furcifera(WBPH) and Culex quinquefasciatu, and 3.8% with Diaphorina citri, Drosophila melanogaster, Bombus terrestris, and Megachile rotundata.We selected 28 candidate proteins according to their abundant clones, molecular function, biological process and identity percentage of blast result. Then performed retransformation analysis and β-galactosidase assay to find out the positive interactor to determine the strength of the interaction and to select the strongest candidates. Among them, 25 proteins expressed interaction and 15 of 25 showed as strong positive interactors. Moreover, we tried to conduct additional confirmation process as GST pull-dawn assay to examine the independently interaction between SRBSDV-P10 and each of 15 positive proteins. However, it is necessary to have a full-length sequence of genes to perform pull-down assay. Therefore, we tried to get the full-length sequence by using 5’ RACE system for Rapid Amplification of cDNA Ends(RACE). Among 15 proteins, full length sequence of 5 proteins: Vesicleassociated membrane protein 7(VAMP7), Vesicle Transport V-Snare Protein Vti1a(Vti1a), Growth hormone-inducible transmembrane protein(Ghitm), Nascent polypeptide-associated complex subunit alpha, and ATP synthase lipid-binding protein(mitochondrial) could be amplified and carried out for pull-down assay. According to the pull-down assay result, we could confirm that three proteins(VAMP7, Vti1 a, Ghitm) were interacted with SRBSDV-P10.As a next step, we also conducted gene expression analysis in different growth stages(nymphs, adult males and adult females) and organs(haemolymph, salivary gland, gut, fat body, ovary and malpighian tubule) of WBPH by RT-qPCR. The gene expression result showed that mRNA level of VAMP7 and Ghitm were highly expressed in male adult stage. Vti1 a was found abundantly in female adult stage. The relative expression result in 6 different organs of WBPH(haemolymph, salivary gland, gut, malpighian tubule, fat body, ovary) showed that mRNA transctipt level of VAMP7 was mainly found in the gut, both Vti1 a and Ghitm were highly expressed in malpighian tubule. The differential expression levels of genes in different developmental stages and different organs suggested that different functions might be involved in different stage and different organs.The result revealed that three different proteins of WBPH were interacted with SRBSDV and they were expected to be involved somehow in sophisticated molecular process for the virus transmission and movement in the different part of tissue, cell and organs of the insect body.