| Rice stripe disease has been reported to be epidemic and cause severe crop losses in rice fields in China in recent years, and the disease’s pathogen is Rice stripe virus (RSV). RSV is known to be transmitted mainly by the small brown planthopper (Laodelphax striatellus Fallen, SBPH) in a persistent, circulative-propagative manner. After invaded into SBPH, RSV can escape from midgut, salivary gland and ovary barriers and propagate in the body. Moreover, RSV can be transmitted from female adults to their progeny via eggs. The epidemic and outbreak of rice stripe disease have close relationship with the outbreak of viruliferous populations of SBPH. Therefore, it is crucial for disease control to research the mechanisms how RSV is transmitted specifically by SBPH. At present, the known interaction mechanisms between RSV and vector were still about traditional biology, and the molecular mechanisms remained unclear. To elucidate transmission mechanisms of RSV, author carried out research to search RSV specific interaction factors in SBPH in this paper.Analysis of rice stripe virus seven-gene expression in rice and in the small brown planthopper:Expression levels of RSV seven genes in RSV-infected rice tissues and in viruliferous SBPH were investigated by using a sensitive and reliable real-time quantitative PCR method. The results revealed that NS3gene exhibited the most abundant expression quantity both in rice plants and in SBPH. As a suppressor of gene silencing, the dominant expression of NS3gene suggested that after invading the host cells, RSV must suppress the immune responses of hosts for its replication. But silencing suppressor NS2gene did not exhibit a higher expression in plant and insect hosts. Therefore, the function coordination of NS3and NS2was important and worthy of further study. Disease-specific protein (SP) gene was only present in a higher abundance in rice, not in SBPH. Compared with its inapparent presence (0.2-fold of NCP) in SBPH, higher abundance (0.8-fold of NCP) of replicase RdRP mRNA in rice was considered that the replication and assembly of RSV was probably more intensive in rice plants than in the insect.Five proteins of small brown planthopper are potentially involved in the interactions between rice stripe virus and vector:High-affinity and non-viruliferous SBPH populations were screened in the laboratory for several generations and used as experimental materials. Total proteins of SBPH, separated by two-dimensional electrophoresis (2-DE), were screened as potential RSV binding molecules using a virus overlay assay of protein blots. The results, five SBPH proteins that bound to purified RSV particles in vitro were resolved and identified using Nano LC-ESI-CID-MS/MS mass spectrometry. Identified five proteins included the receptor for activated protein kinase C (RACK),60S ribosomal protein L5(RPL5), glyceraldehyde-3-phosphate dehydrogenase (GAPDH3),60S RPL7a and60S RPL8. The peptide sequences from LC-MS/MS were used to search matched proteins against the SBPH transcriptome database with the tBlastn tool. After obtaining partial contigs of virus-binding proteins, full-length ORFs of five proteins were cloned via RT-PCR. The virus-binding capacities of five proteins were further elucidated. In yeast two-hybrid experiment, RACK and GAPDH3did not interact with RSV nucleocapsid protein (NCP), and three ribosomal proteins (RPL5, RPL7a and RPL8) had specific interactions with RSV. Five interaction factors were expressed, and expressed proteins were used to perform virus-binding experiments (including far-Western blot and DIBA) in vitro. The results of far-Western blot were accord with the results of yeast two-hybrid assay, and in dot immunobinding assay (DIBA), all five proteins were able to bind to RSV particles. The five proteins’potential contributions to the interactions between RSV and SBPH were discussed. Author proposed that the interaction capacities with virus of RACK and GAPDH3(protein biomarkers for efficient virus transmission in aphid) might rely on their specific conformation in membrane systems, and RACK and GAPDH3might be involved in the epithelial transcytosis of virus particles. Three ribosomal proteins (RPL5, RPL7a and RPL8) probably played potential crucial roles in the infection and propagation of RSV in vector cells.Construction of yeast two-hybrid cDNA library of high-viruliferous (RSV-infected) SBPH populations and screening of NCP interaction factors:High-affinity and high-viruliferous (RSV-infected) SBPH populations (briefly referred to as high-viruliferous populations) were screened in the laboratory and used as experimental materials. The mRNA was isolated and purified from SBPH, and used as the template to synthesize the double-strand cDNAs. The cDNAs were ligated with three-frame adapter and purified. By using homologous recombination reaction, the cDNA entry library was prepared, and then entry library was transferred into compatible vector pGADT7-DEST to construct yeast two-hybrid cDNA library by using gateway strategy. Detection of the library indicated that it contained3.68×107independent clones, and the titer of the amplified library was2.62×1010cfu/mL. The recombination rate was above95%, and the average size of inserts was above1kb in the cDNA library. The results demonstrated that the library database met the requirements of the standard cDNA library. The yeast two-hybrid cDNA library of high-viruliferous SBPH will be useful for the future research on the interaction between insect vector and RSV. Afterwards, in order to search SBPH proteins that can interact with NCP, the cDNA library was screened by a GAL4-based yeast two-hybrid system using RSV NCP as the bait. A series of positive colonies were identified and sequence analysis, revealing that they might correspond to13independent proteins of SBPH. According to the literature reports, implications of these findings in the interactions with RSV were preliminarily discussed.Preliminary analysis of correlations between parasitic HiPV and transmission of RSV in SBPH:With parasitic (symbiotic) microorganisms as a breakthrough, the contribution of parasitic Himetobi P virus (HiPV) in SBPH to RSV transmission was analyzed. Capsid protein VP1of HiPV was expressed in E. coli strain BL21(DE3) pLys S using pET32a vector, and fusion proteins were purified with Ni-NTA resin affinity chromatography. The polyclonal antibody against VP1was obtained via immunizing rabbits. HiPV in SBPH could be specifically detected in Western-blot with polyclonal antibody. Detecting system of HiPV in single insect was established using RT-PCR and Western-blot methods as basis. Through detecting HiPV in various SBPH populations, the correlations between HiPV and transmission of RSV were preliminarily understand, and the results showed that there was widespread prevalence of HiPV in high-affinity SBPH populations, and certain corresponding relationship between RSV and HiPV infection was observed. In yeast two-hybrid assay, RSV-NCP had stronger interaction with capsid protein VP1of HiPV. According to present results, a research hypothesis was proposed that SBPH infecting HiPV in high-affinity populations was a main reason that populations exhibited a high affinity for RSV, which will offer a help to research RSV transmission mechanisms from point of view of SBPH’s parasitic (symbiotic) microorganisms.Predictive analysis and verification of RSV NSvc2protein structure:RSV has phylogenetic similarity to Tomato spotted wilt virus (TSWV), and RSV has genomic structure and function similarity to TSWV. NSvc2protein of RSV is corresponded to glycoprotein (determinants for thrips transmission) of TSWV, so author speculated that NSvc2was determinant for SBPH transmission of RSV. After predictive analysis of NSvc2structure using bio-software, schematic representation of protein topology structure was mapped. Author predicted that NSvc2existed potential cleavage in hosts, and might be cleaved to generate two individual proteins R-GN and R-Gc. Western-blot analysis in RSV-infected rice tissue and viruliferous SBPH were carried out. The results revealed that RSV NSvc2was cleaved into two proteins in RSV-infected rice tissue, which was accord with predictive analysis. But in SBPH, NSvc2produced a single protein (94kDa), which was full-length pattern of NSvc2without cleavage. This was the first report that RSV NSvc2protein existed in two different forms in rice and in SBPH, which was different from other RSV gene products. Research results laid the foundation for identifying NSvc2as determinant for SBPH transmission of RSV.Research of this paper offered new theoretical basis and thoughts for further understanding mechanisms of RSV transmission and control of rice stripe disease. |
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