RNA interference (RNAi) has been considered as an alternative strategy to control agricultural pests whereby double-strandedRNA triggers a potent and specific inhibition of its homologous mRNA. Since small double-stranded RNAs are requiredfor various RNAi applications, there is a need for cost-effective methods for producing large quantities of high-qualitydsRNA. Bacillus thuringiensis produces much insecticidal proteins with expression of their encoding genes being drivenby sporulation-dependent promoters. To develop dsRNA mass-production platform utilizing Bt, the pHT1K-EGFP plasmidvector which has cyt1Aa sporulation-dependent promoter was constructed. The transcriptional level of target gene (EGFP)is higher 113 times than Bt reference gene (ssPE). It was applied to protect honeybee from Sacbrood virus, so targetgene was replaced to SBV-vp1. By ingestion of Bt-derived dsRNA to honeybee shows positive effect on SBV suppression.

이중나선형 RNA (dsRNA)를 이용한 유전자 발현 억제기술이 다양한 생명체에서 기능 유전체학을 연구하는 데 이용되고 있다. 이 기술의 원리는 전사후 단계에서 유전자 발현을 조절하는 RNA 간섭에 기인된다. dsRNA를 이용하여 특정 유전자의 발현 억제는 심각한 치사효과를 줄 수 있다. 이러한 분자기작을 해충 방제에 적용하여 특정 dsRNA를 이용한 새로운 살충제를 개발하고 있다. 본 종설은 dsRNA를 이용한 RNA 간섭 원리를 설명하고 이를 이용한 해충 방제를 구현한 여러 예를 살펴본다. 그리고 해충방제를 실현시키기 위해 현재 이 기술이 담고 있는 한계 를 고찰하고 이에 대한 대응 방안을 본 종설에서 제공하고자 한다.

RNA interference (RNAi) is an universal gene-knockdown mechanism in eukaryotic organisms including insects. RNAi has been considered as an alternative strategy to control agricultural pests whereby double-stranded RNA triggers a potent and specific inhibition of its homologous mRNA. Bacillus thuringiensis (Bt) is a spore-forming bacterium that produces a copious amount of crystal proteins δ-endotoxins under the control of sporulation-dependent promoter. In order to develop dsRNA mass-production platform utilizing recombinant Bt, the pHT1K-EGFP which expresses dsRNA against EGFP under the control of Cyt1-Aa sporulation-dependent promoter was constructed and the expression level of transgene (EGFP) was confirmed by qPCR analysis. These results suggested that Bt’s potential of becoming a new platform in dsRNA production.

RNAi (RNA interference) is a tool for silencing of target genes through sequence-specific manner. Spodoptera exigua belongs to Noctuidae family of Lepidoptera and is serious threat to crops of economic importance. One of S. exigua chymotrypsin gene (SeCHY2) was cloned into the L4440 vector to produce sequence specific dsRNAs (double-stranded RNAs). Recombinant L4440 vectors were transformed into Escherichia coli strain HT115 (DE3). Oral delivery of bacterially expressed dsRNA gave significant larval mortality. Quantitative real-time PCR results showed that expression level of target SeCHY2 gene in the larval gut tissue was significantly down-regulated. Pretreatment with an ultra-sonication and heating to disrupt bacterial cell wall/membrane significantly increased the insecticidal activity of the transformed bacteria

큰28점박이무당벌레(Henosepilachna vigintioctomaculata)는 감자, 토마토, 구기자 등 가지과 식물을 가해하는 저작형 해충이다. 한편, 최근에 RNA interference(RNAi)를 이용해 해충을 방제하려는 연구들이 많이 진행되고 있다. 따라서 이 실험은 RNAi를 이용하여 큰28점박이무당벌레 방제 및 유전자의 기능을 알아보고자 수행되었다. 먼저, RNAi에 이용할 target 유전자를 선발하기 위해 cDNA library를 제작하였다. 그 결과, 최종 titer 3.15×105의 cDNA library를 완성하였다. 완성한 cDNA library는 제한효소를 이용해 LITMUS 28i transcription vector에 cloning하였고, 이 후 insert의 크기와 중복성을 확인하였다. 그 결과, RNAi에 적합한 약 100~500bp 크기의 insert가 확인되었고, NCBI Blast search 결과, insert 사이에 중복성 없이 다양한 유전자가 cloning되었고, 대부분이 곤충 유전자임을 확인하였다. 확인된 유전자들은 T7 promoter를 이용해 dsRNA를 합성한 후, 0~2일된 큰28점박이무당벌레 4령 유충에 주입하였다. 앞으로의 실험에서는 RNAi 효과를 나타낸 유전자에 대해 Real-time PCR을 통해 큰28점박이무당벌레 체내 유전자 발현량 변화를 확인할 예정이다.

이중가닥 RNA (double-stranded RNA, dsRNA)는 표적 유전자의 발현을 억제하는 기능으로 해충방제에 응용되었다. 인테그린은 α와 β 단위체로 구성된 이량체 막 단백질이다. 진핵생명체에서 인테그린은 세포-세포 및 세포-세포외기질의 상호연결에 중요한 역할을 담당한다. 인 테그린 β 단위체 발현을 억제하는 특정한 dsRNA (= dsINT)는 해당 곤충에 뚜렷한 치사효과를 유발한다. 또한, dsINT를 발현시키는 형질전환 된 대장균도 해당 곤충에 뚜렷한 살충력을 가진다. 그러나 이 세균 살충제의 야외 적용을 위해서는 제형화 기술이 필요했다. 본 연구는 dsINT를 발현하는 재조합 세균을 동결 건조시켜 대상 곤충에 대해 살충효능을 검정하였다. 동결 건조된 세균은 파밤나방(Spodoptera exigua) 종령 유충에 높은 섭식독을 일으켰다. 파밤나방에 대해서 Bacillus thuringiensis 상용 살충제 처리는 불과 60%의 살충력을 보이는 반면, 동결 건조된 dsINT 발현 세균과 혼합 처리할 때 살충력은 크게 증가하였다. dsINT 발현 세균은 해당 인테그린 염기서열 유사성에 따라 차이를 보이는 해충 종에 선 택적 독성을 나타냈다. 이 결과는 인테그린에 특이적 dsRNA를 발현하는 세균이 동결 건조 제형화 조건하에서도 살충력을 유지한다는 것을 나타 냈다.

Tetranychus urticae is extremely hard to control by conventional acaricides due to its rapid development of resistance to nearly all arrays of acaricide. As an alternative control measure of acaricide-resistant mites, RNA interference (RNAi)-based method has recently been suggested. A double-stranded RNA (dsRNA) delivery method using multi-unit chambers was established and employed to screen the RNAi toxicity of 42 T. urticae genes. Among them, the dsRNA treatment of coatomer I (COPI) genes, such as coatomer subunit epsilon (COPE) and beta 2 (COPB2), resulted in high mortality [median lethal time (LT50) = 89.7 and 120.3 h, respectively]. The transcript level of the COPE gene was significantly (F3,9 = 16.2, P = 0.001) reduced up to 24% following dsRNA treatment, suggesting that the toxicity was likely mediated by the RNAi of the target gene. To identify the deferentially expressed gene upon dsRNA ingestion, RNA-seq was employed to compare the transcriptional profiles between mites fed dsEGFP and dsCOPB2. Approximately 928 of genes were up- or down-regulated significantly (P < 0.05) compared to control and 182 genes were commonly responded to the treatment of both dsRNAs. Those dsRNA-responsible genes were mainly categorized into metabolic enzymes, transporters and secretory proteins. Further study would be necessary to elucidate the roles of dsRNA-responsible genes in mite’s dsRNA uptake and defense.

Even though plant protections using chemical pesticides have several advantages, non-specific toxicities to other beneficial insects and humans and rapid development of tolerance and/or resistance of target pests to chemicals are major disadvantages. Recent researches suggested that using double stranded RNA (dsRNA) could be species specific and environmental friendly pest management protocols. However, efficiency of dsRNA treatments are known to be variable according to its application methods. For example, injections of dsRNAs to pests were known to be effective in all species. However, efficiencies of oral application of dsRNAs were known to be dependent on species. Thus, development of tools that could enhance the efficacy of orally treated dsRNAs are utmost important for widening usages of dsRNAs in plant protection. Recently, we found that the efficacy of oral treated dsRNAs to target pests could be enhanced by nano-technologies. I will show how applying nano-technology to dsRNAs enhance the efficiency of dsRNAs. (This work was carried out with the support of the Cooperative Research Program for Agriculture Science & Technology Development (Project title: Studies for biological characteristics of and control methods against the migratory locust, Locusta migratoria, Project No: PJ011630042016), Rural Development Administration).

Oral toxicity of double-stranded RNA (dsRNA) specific to integrin β1 subunit (SeINT) was known in a polyphagous insect pest, Spodoptera exigua. For an application of the dsRNA to control the insect pest, this study prepared a recombinant Escherichia coli expressing dsRNA specific to SeINT. The dsRNA expression was driven by T7 RNA polymerase overexpressed by an inducer in the transformed E. coli. The produced dsRNA amount was proportional to the number of the cultured bacteria. The bacteria gave a significant oral feeding mortality to S. exigua larvae with a significant reduction of the SeINT expression. The resulting insect mortality increased with the fed number of the bacteria. Pretreatment with a sonication to disrupt bacteria cell wall membrane significantly increased the insecticidal activity of the transformed bacteria. Compared to the control bacteria transformed by non-recombinant vector, the larvae fed the bacteria expressing dsRNA specific to SeINT suffered tissue damage in the midgut epithelium, which was characterized by a loose cell-cell contact and a significant cell death. The dsRNA-treated larvae were significantly more susceptible to a Cry toxin derived from Bacillus thuringinesis (Bt) than the larvae treated only with Cry toxin. This study demonstrates that a transformed bacterium expressing dsRNA specific to SeINT has a significant insecticidal activity by oral application against S. exigua and makes the target insects to be highly susceptible to Bt toxin.

The sweetpotato whitefly, Bemisia tabaci (Hemiptera: Aleyrodidae) is a species complex, including at least 24 biotypes which express different ecological and physiological characteristics. B. tabaci is one of the most serious pests in the horticultural crops in worldwide level. B. tabaci infests over 600 plant species and also indirectly damages plants by the honeydew excretion. Moreover, whiteflies transmit more than 100 plant viruses, especially begomoviruses. Tomato yellow leaf curl virus (TYLCV) which infested severely in tomato cultivars is transmitted by the vector insect, B. tabaci. Here, we demonstrated whether gene expression of B. tabaci is regulated by the oral ingestion of dsRNA. Double strand RNA (dsRNA) of heat shock protein 70 gene (hsp70) was produced from cDNA by using the specific primer. Whitefly adults were allowed to ingest sugar solution containing dsRNA for 3 hr in the two-layered parafilm feeding tube. Quantitative realtime PCR showed that whiteflies which ingest hsp70 dsRNA completely knockdown its transcript expression. Whiteflies ingestded dsRNA increased mortality by heat shock but not by cold shock. Further research will focus the role of hsp70 in various environmental stresses against insects.

Double-stranded RNA(dsRNA) had been used to specitically suppress target gene expression at post-tanscription level. Injection of dsRNA to hemocoel is the most efficient to knockdown target mRNA. However, some insects have shown to be susceptible to feeding dsRNA. Spodoptera exigua was susceptible to dsRNA at oral treatment. Especially dsRNA specific to β-integrin was potent to survival of S.exigua larvae. This study advanced our dsRNA application technology by generating recombinant E.coli expressing dsRNA specific the β-integrin. A recombinant vector L4440 was constructed with a partial β-integrin gene under T7 RNA polymerase promoter. The recombinant vector was used to transform HT115 competent cells of E.coli. The transformed E.coli expressed the dsRNA. The production of dsRNA was proportional to the bacterial number. By feeding the recombinant E.coli, S.exigua underwent significant mortality. By adding E.coli expressing Cry1Ca Bt toxin to E.coli expressing dsRNA, S.exigua exhibited highly enhanced mortality. This study suggests a possibility to use a recombinant E.coli expressing dsRNA to control S.exigua.

Mycoviruses have been found in many fungal species including mushrooms. Double-stranded (ds) RNA genomes were common type in mycoviruses, but single-stranded (ss) RNA mycoviruses were also reported in some fungal species. Sequencing analysis using cDNA cloning experiments revealed that mycoviruses can be classified into several different virus families such as Totiviridae, Hypoviridae, Partitiviridae and Barnaviridae etc. Because the nucleotide sequence data that are available in these days are very limited in a number of mycoviruses, the existence of more diverse viral groups in fungi are currently expected. In this review, we selected four different fungal groups, which were considered as the model systems for mycovirus related studies in both plant pathogenic fungi and edible mushroom species, and discussed about their molecular characteristics of diverse mycoviruses. The plant pathogenic fungi introduced here were Cryphonectria parasitica and Helminthosporium victoriae and the edible mushroom species were Agaricus bisporus and Pleurotus ostreatus.