[논문]Suppression of Rice Stripe Virus Replication in Laodelphax striatellus Using Vector Insect-Derived Double-Stranded RNAs

Fang, Ying; Choi, Jae Young; Park, Dong Hwan; Park, Min Gu; Kim, Jun Young; Wang, Minghui; Kim, Hyun Ji; Kim, Woo Jin; Je, Yeon Ho

doi:10.5423/ppj.oa.03.2020.0052

Suppression of Rice Stripe Virus Replication in Laodelphax striatellus Using Vector Insect-Derived Double-Stranded RNAs 원문보기

The plant pathology journal, v.36 no.3, 2020년, pp.280 - 288

Fang, Ying (Department of Agricultural Biotechnology, College of Agriculture & Life Science, Seoul National University) , Choi, Jae Young (Department of Agricultural Biotechnology, College of Agriculture & Life Science, Seoul National University) , Park, Dong Hwan (Department of Agricultural Biotechnology, College of Agriculture & Life Science, Seoul National University) , Park, Min Gu (Department of Agricultural Biotechnology, College of Agriculture & Life Science, Seoul National University) , Kim, Jun Young (Department of Agricultural Biotechnology, College of Agriculture & Life Science, Seoul National University) , Wang, Minghui (Department of Agricultural Biotechnology, College of Agriculture & Life Science, Seoul National University) , Kim, Hyun Ji (Department of Agricultural Biotechnology, College of Agriculture & Life Science, Seoul National University) , Kim, Woo Jin (Department of Agricultural Biotechnology, College of Agriculture & Life Science, Seoul National University) , Je, Yeon Ho (Department of Agricultural Biotechnology, College of Agriculture & Life Science, Seoul National University)

Abstract ▼ AI-Helper

RNA interference (RNAi) has attracted attention as a promising approach to control plant viruses in their insect vectors. In the present study, to suppress replication of the rice stripe virus (RSV) in its vector, Laodelphax striatellus, using RNAi, dsRNAs against L. striatellus genes that are strongly upregulated upon RSV infection were delivered through a rice leaf-mediated method. RNAi-based silencing of peroxiredoxin, cathepsin B, and cytochrome P450 resulted in significant down regulation of the NS3 gene of RSV, achieving a transcriptional reduction greater than 73.6% at a concentration of 100 ng/μl and, possibly compromising viral replication. L. striatellus genes might play crucial roles in the transmission of RSV; transcriptional silencing of these genes could suppress viral replication in L. striatellus. These results suggest effective RNAi-based approaches for controlling RSV and provide insight into RSV-L. striatellus interactions.

주제어

표/그림 (6)

표 Table 1. Differentially expressed genes in RSV-viruliferous Laodelphax striatellus
그림 Fig. 1. Quantitative PCR (qPCR) analysis of the transcription of Laodelphax striatellus genes up-regulated in rice stripe virus (RSV)-viruliferous L. striatellus.
표 Table 2. List of genes selected for RNAi application to suppress RSV replication in Laodelphax striatellus
그림 Fig. 2. Silencing of RNA interference target gene transcription in rice stripe virus (RSV)-viruliferous Laodelphax striatellus nymphs that had ingested the corresponding double-stranded RNA (dsRNA).
그림 Fig. 3. Suppression of rice stripe virus (RSV) transmission in RSV-viruliferous Laodelphax striatellus nymphs that had ingested vector-derived double-stranded RNAs (dsRNAs).
그림 Fig. 4. Dose-dependent suppression of rice stripe virus (RSV) transmission in RSV-viruliferous Laodelphax striatellus nymphs that had ingested vector-derived double-stranded RNAs (dsRNAs).

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제안 방법

Synthesis of dsRNA. Candidate siRNA sites in target genes were predicted using BLOCK-iT RNAi Designer (https://rnaidesigner.thermofisher.com/rnaiexpress) and dsRNA sequences 500-700 bp in length and containing at least three candidate sites of siRNA were designed against each target gene. Single-strand cDNA of each target gene was synthesized from total RNA of L.
Single-strand cDNA of each target gene was synthesized from total RNA of L. striatellus using the QuantiTect Reverse Transcription Kit (Qiagen, Hilden, Germany) according to manufacturer’s instructions and the target gene was amplified using KOD Neo FX DNA polymerase (Toyobo, Osaka, Japan) and primers with a T7 promoter sequence (5'-TAATACGACTCACTATAG-3') at the 5'-end (Supplementary Table 1) as previously described (Fang et al., 2017).
Comparative transcriptome analysis. To investigate genes in L. striatellus that were differentially expressed upon RSV infection, total raw reads from the RSVviruliferous and non-viruliferous L. striatellus individuals were mapped to the 3,703 contig sequences that were functionally categorized in the gene ontology (GO) analysis to obtain the FPKM values. While 885 genes were over two-fold up-regulated in RSV-viruliferous L.
Selection and validation of target genes for RNAi application. To investigate whether RSV transmission in L. striatellus could be suppressed by silencing host genes related to RSV transmission, we selected 8 genes identified as up-regulated in the RSV-viruliferous L. striatellus based on the comparative transcriptome analysis and qPCR validation of relative transcription level (Table 2). Among these RNAi target genes, 6 genes (all except contig nos.
striatellus nymphs that ingested vector-derived dsRNAs. To investigate whether dsRNAs against L. striatellus genes up-regulated in RSV-viruliferous L. striatellus suppress replication of RSV in L. striatellus, qPCR analysis of the transcription of RSV genes in RSV-viruliferous L. striatellus individuals that had ingested dsRNA was performed. The transcription level of NS3, which is crucial for RSV replication and act as a viral suppressor of RNAi, was significantly reduced in L.

데이터처리

Statistical analysis. Statistical analysis was performed by one-way ANOVA using SPSS Statistics version 24 (IBM Corp., Armonk, NY, USA). Multiple comparisons of mean values were performed by post hoc Scheffé's tests.

이론/모형

The relative transcription levels are expressed as fold differences in RSV-viruliferous L. striatellus relative to non-viruliferous L. striatellus as determined by qPCR and calculated using the 2-ΔΔCt method.

성능/효과

In this study, exogenous application of dsRNA against these 4 genes resulted in significant suppression of RSV replication in L. striatellus (Fig. 3).
1). The other 2 target genes, contig nos. 78749 and 80690, were shown to be highly up-regulated in the RSV-viruliferous L. striatellus in the qPCR analysis (2.39- and 4.53-fold up-regulated, respectively) (Fig. 1), although they were found to be only slightly up-regulated in the transcriptome sequencing analysis (Table 2).
4). These results demonstrated that comparative transcriptome analysis can aid the identification of genes related to the transmission of plant viruses in vector insects and that vector insect-derived genes related to replication of plant viruses could be effectively exploited to control plant viruses.

후속연구

, 2012), relationships between cytochrome P450s and plant viruses have not been described. Further studies are required to explore the effects of cytochrome P450s on the replication of RSV in L. striatellus, which might provide further insights into RSV-L. striatellus interactions.

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