Interaction Between RSV NS2,NS3 Proteins And Hosts

Rice stripe virus (RSV), the type member of the genus Tenuivirus, was the causative agent of rice stripe disease which was one of the most economically important pathogens of rice and is repeatedly epidemic in China. The biological functions of virus-encoded proteins, pathogenicity differentiation, genetic diversity of virus, virus-plant host interactions and management of virus were studied during the past years. But RSV genomes, movement, and virus-host interactions, transmission were little known. In order to control the disease better and promote the sustainable development of rice production, it was essential to study the virus-rice interaction, virus-vector interaction in detail.Yeast two-hybrid system was used to detect the interaction between RSV and rice. Some positive clones interacted with RSV NS2 or NS3 were obtained. In order to study whether the full-length clones interacted with NS2 or NS3, five full-length clones interacted with NS2, three full-length clones interacted with NS3 were amplified inspectively and constructed into vector pGADT7. Then, these eight full-length clones were co-transformed into yeast strain AH109 with pGBKT7-NS2 or pGBKT7-NS3 respectively. Different synthetic dropout nutrient medium were detected. The results showed that full-length proteins pectinesterase, ras-related protein, cholinephosphate cytidylyltransferase, suppressor of gene silencing 3 (SGS3) and a function unknown protein interacted with NS2 respectively, and full-length proteins UBA/TS-N domain containing protein, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and SGS3 interacted with NS3. Full-length proteins O-methyltransferase did not interact with NS3.Rice (Oryza sativa L. ) SGS3 was homologous to Arabidopsis thaliana SGS3 (ATSGS3) protein and was designated as OsSGS3. OsSGS3 possessed the three domains typical of AtSGS3: zinc finger, XS and coiled coil. Three OsSGS3 deletion mutants: OsSGS3-1, which contained the znic finger domain, OsSGS3-2, which contained the XS domain, and OsSGS3-3, which contained the coiled coil domain were constructed. The coding sequences of the mutants were subcloned individually into pGADT7 and cotransformed with pGBKT7-NS2 or pGBKT7-NS3 into yeast strain AH109. All of the transformants were able to grow on SD/-His/-Trp medium and on SD medium lacking Ade, His, Leu, and Trp (SD/-Ade/-His/-Leu/-Trp), but the transformants of NS2 and SGS3-3 interacted weakly.The interaction between NS2 and Oryza sativa L. GAPDH (OsGAPDH) was verified by Bimolecular fluorescence complementation (BiFC). Cellular localization studies showed that OsGAPDH accumulated in cytoplasmic, OsSGS3 accumulated in cytoplasmic foci that perfectly resemble those of AtSGS3 and NS2 located in nucleus and punctate structures in association with cell membrane. We confirmed the interaction between OsSGS3 and NS2 by using co-IP, BiFC, and colocalization. The results of colocalization revealed that a considerable proportion of OsSGS3 was redistributed to nucleus by NS2. We speculate that the redistribution of OsSGS3 by NS2 might disrupt the normal function of OsSGS3. AtSGS3 has been demonstrated to be involved in small RNA production. To explore the possible biological implications of the interaction of RSV NS2 with OsSGS3, we first detected the expression patterns of 5 auxin responsive factors (ARFs) that had been domonstrated to be potential targets of trans acting siRNAs. The results indicated that the overall accumulation levels of the 5 genes increased in RSV infected rice. This suggested that the trans acting siRNA might indeed be affected by RSV infection. Further, we detected TAS3 derived ta-siRNAs of rice. The result indicated that the accumulation level of this small RNA was more than 2 fold higher in RSV infected rice compared with healthy control. One explanation for this observation was that a feedback regulation system of the trans acting small RNA pathway may have compensated for the loss of SGS3 activity in RSV infected rice. To test this possibility, we detected the accumulation of several other genes involved in the generation of trans acting small RNAs including the TAS3 transcript precursor and OsSGS3. The results indicated that the accumulation level of OsSGS3 and the accumulation level of TAS3 was more than 2.7 and 3 fold higher in RSV infected rice respectively compared to healthy control.A yeast two-hybrid cDNA library was constructed using CloneMiner? cDNA Library Construction Kit. The results showed that unamplified library consisted of 1.85×107 independent clones; the titer of amplified cDNA library was 7.7×108 cfu/mL, and the recombinant rate was about 97%. The size of most inserts were 1000-1500 bp. Random sequence analysis of ten clones showed that seven clones had homologous sequences in GenBank. Clones L2 and L9 were the same as the published Laodelphax striatellus. Construction of this yeast two-hybrid cDNA library of Laodelphax striatellus laid the foundation for cloning of functional genes in Laodelphax striatellus and carrying out the research on Laodelphax striatellus-Virus interaction. Bait plasmids pDB-NS3 was constructed and used to screen the yeast two-hybrid cDNA library of Laodelphax striatellus. The transformants were plated on different dropout nutrient mediums andβ-galactosidase activity were detected. One positive clone homologous to ribosomal protein L4 of some insects was obtained. The interaction between NS3 and ribosomal protein L4 of rice was also detected, the results showed that NS3 could either interact with ribosomal protein L4 of rice or ribosomal protein L4 of Laodelphax striatellus in yeast.