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. 2021 Aug 11;13(8):1591.
doi: 10.3390/v13081591.

Regulation of RNA Interference Pathways in the Insect Vector Laodelphax striatellus by Viral Proteins of Rice Stripe Virus

Yan Xiao  1   2 , Qiong Li  1   3 , Wei Wang  1 , Yumei Fu  4 , Feng Cui  1   3
Affiliations

Regulation of RNA Interference Pathways in the Insect Vector Laodelphax striatellus by Viral Proteins of Rice Stripe Virus

Yan Xiao et al. Viruses. .

Abstract

RNA interference (RNAi), especially the small interfering RNA (siRNA) and microRNA (miRNA) pathways, plays an important role in defending against viruses in plants and insects. However, how insect-transmitted phytoviruses regulate the RNAi-mediated antiviral response in vector insects has barely been uncovered. In this study, we explored the interaction between rice stripe virus (RSV) and the miRNA and siRNA pathways of the small brown planthopper, which is a vector insect. The transcript and protein levels of key genes in the two RNAi pathways did not change during the RSV infection process. When the expression of insect Ago1, Ago2, or Translin was silenced by the injection of double-stranded RNAs targeting these genes, viral replication was promoted with Ago2 silencing but inhibited with Translin silencing. Protein-protein binding assays showed that viral NS2 and RNA-dependent RNA polymerase interacted with insect Ago2 and Translin, respectively. When NS2 was knocked down, the transcript level of Ago2 increased and viral replication was inhibited. Therefore, viral NS2 behaved like an siRNA suppressor in vector insects. This protein-binding regulation of insect RNAi systems reflects a complicated and diverse coevolution of viruses with their vector insects.

Keywords: Ago2; miRNA; planthopper; rice stripe virus; siRNA; translin; viral replication.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Response of miRNA and siRNA pathways to RSV infection in planthoppers. (A) The relative RNA levels of RSV NP in the planthoppers after injection of RSV crude preparations from viruliferous planthoppers measured with quantitative real-time PCR (qPCR). Significant differences are indicated by different lowercase letters. (B–I) The relative transcript levels of Drosha, Pasha, Dicer1, Ago1, Dicer2, Ago2, Translin, and Trax in the miRNA and siRNA pathways during the infection process of RSV in the planthoppers were measured with qPCR. The crude preparations from nonviruliferous planthoppers were injected into the control groups (CK). The RNA or transcript levels of these genes were normalized to that of EF2. The values represent the means ± SEs. NS, no significant difference. **, p < 0.01.
Figure 2
Figure 2
Western blot assay showing the protein levels of NP, Ago1, Ago2, and Translin in the planthoppers at 6 d after injection of RSV crude preparations. The crude preparations from nonviruliferous planthoppers were injected into the control group (CK). Three biological replicates were shown for each group. The monoclonal antibodies for NP, Ago1 and Ago2, and polyclonal antibody for Translin were used. The protein level of planthopper tubulin was revealed using the monoclonal anti-β-tubulin antibody as an internal reference. The relative grayscales of Ago1, Ago2, and Translin to that of tubulin were quantified and compared between the RSV-infected and CK groups. The values represent the means ± SEs. NS, no significant difference.
Figure 3
Figure 3
Roles of miRNA and siRNA pathways in the control of RSV replication. (A–C) Effect of planthopper Ago1, Ago2, and Translin transcript levels, and viral NP and RNA3 levels at 6 d after injection of double-stranded RNAs (dsRNAs) with RSV crude preparations. The control group was injected with dsGFP-RNA and RSV crude preparations. (D) Effect of planthopper Translin transcript level and viral NP and RNA3 levels in the stable viruliferous strain at 6 d after injection of dsTranslin-RNA. The control group was injected with dsGFP-RNA. The relative RNA levels of NP and RNA3 to that of EF2 and relative transcript levels of the three planthopper genes to that of EF2 were quantified with quantitative real-time PCR. The protein levels of NP and tubulin were measured using monoclonal anti-NP and anti-β-tubulin antibodies by western blot. Four biological replicates were shown for each group. The relative grayscale of NP to that of tubulin was quantified and compared between the gene interference groups and dsGFP-RNA injection groups. The values represent the means ± SEs. *, p < 0.05. **, p < 0.01. ***, p < 0.001. NS, no significant difference.
Figure 4
Figure 4
Regulation of RNAi pathways through insect protein-viral protein interactions. (A) Coimmunoprecipitation (Co-IP) assay demonstrating that the recombinant expressed NS2-His pulled down Ago2 from the crude extracts of viruliferous planthoppers. (B) Co-IP assay demonstrating that the recombinant expressed RdRp2-His pulled down Translin from the crude extracts of viruliferous planthoppers. Monoclonal antibodies against Ago2 and His and polyclonal antibodies against Translin were applied. IgG was used as a negative control. (C) Effect of planthopper Ago1 and Ago2 transcript levels and viral NS2, NP, and RNA3 levels in the planthoppers at 6 d after injection of double-stranded RNAs (dsRNAs) of NS2 with RSV crude preparations. The control group was injected with dsGFP-RNA and RSV crude preparations. The relative transcript or RNA levels to that of EF2 were quantified with quantitative real-time PCR. The protein levels of NP and tubulin were measured using monoclonal anti-NP and anti-β-tubulin antibodies by western blot. Four biological replicates were shown for each group. The relative grayscale of NP to that of tubulin was quantified and compared between the gene interference group and dsGFP-RNA injection groups. The values represent the means ± SEs. *, p < 0.05. NS, no significant difference.
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