| Rice stripe disease, which is caused by rice stripe virus, has brought huge loss of rice production in China. As a plant virus, it is transmitted by the small brown planthopper (Laodelphax striatellus Fallen, SBPH), and RSV can propagate and circulate in insect vector. Moreover, RSV can be transmitted from female adults to their progeny via eggs. At present, little is known about how the virus fights the RNAi antiviral immunity in insect vector. RSV-NS3 is the suppressor of gene silencing, which might play key roles in the process of RSV infecting SBPH. In this paper, RSV-NS3 was used as a bait to screen the yeast two-hybrid cDNA library of high viruliferous SBPH, and a series of special receptors interacted with RSV-NS3 were obtained. Afterward, the interactions between NS3 and a key factor RNaseⅢ (a domain of Dicer-2 protein) were further analyzed.1. Screening of RSV-NS3 interaction factors in SBPH using the yeast two-hybrid systemFirstly, the bait vector pGBKT7-NS3 was constructed and transformed into yeast AH 109 cells, and then the cDNA library vectors were transformed into AH 109 competent cells. The transformants were screened on SD/-Trp-Leu-His-Ade medium (plus X-a-gal). Plasmids in these colonies were extracted, and the cDNA inserts in pGAD vectors were detected using PCR. Vectors that contained above 500 bp inserts were transformed into E.coli DH5a and sequenced. Sequences of inserts in positive vectors were identified gene attribution using blast tool in Genbank database. The results, these positive cDNA inserts belonged to 10 different genes, including vitellogenin C2 domain-containing protein, lipid-recognition protein-like isoform 1, RNaseⅢ domain(Dicer-2-like), DUF1639 superfamily, DUF4497 superfamily, TweedleE (DUF243 superfamily), heat shock cognate protein 70, Icarapin-like protein, BTB/POZ domain-containing protein 2.2. The interaction between SBPH RNaseⅢ and RSV-NS3According to the results of screening cDNA library, RNaseⅢ (a key domain of Dicer-2 protein) was selected to further study. Dicer-2 protein structure was analyzed, and RNaseⅢ domain was determined and cloned. The interaction between RNaseⅢ and NS3 was verified using the yeast two-hybrid system.3. Cleavage activity analysis of RNaseⅢExpression vector of RNaseⅢ was constructed, and RNaseⅢ was expressed in E. coli strain BL21 (DE3). Fusion protein was purified with Ni-NTA resin affinity chromatography. The dsGFP was incubated with purified RNaseⅢ, the results showed that purified RNaseⅢ protein exhibited dsRNA cleavage activity. Titer concentration of RNase III was 6 ng/μL via doubling dilution detection, and the time gradient analysis showed that the cleavage activity of RNaseⅢ enhanced with the prolonging of reaction time.4. Effect of NS3 protein on the cleavage activity of RNaseⅢNS3 protein was expressed in E. coli strain BL21 (DE3) and was purified. In the cleavage system of RNaseⅢ, NS3 protein was added, and the results showed that NS3 promoted dsRNA cleavage activity of RNaseⅢ. A mediated-RNAi SBPH Embryo Lysate system was preliminary established, in which RNaseⅢ domain of SBPH Dicer-2 protein also had dsRNA cleavage activity. The dsRNA was incubated with NS3 in Embryo Lysate system, and the results showed that NS3 also promoted dsRNA cleavage activity of RNase III domain.Previous research reported that NS3 can bind siRNA, but not dsRNA, to suppress the assembly of RNA-induced silencing complex (RISC) for suppressing RNAi antiviral immunity in the host. Research of this paper found that NS3 could promote RNaseⅢ domain of Dicer-2 to cut dsRNA into its binding target siRNA, which will help NS3 exercise the function of suppression silencing. |
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