Functional analysis of Dicer-2 gene in Bombyx mori resistance to BmNPV virus

Bombyx mori nucleopolyhedrovirus (BmNPV) is a primary pathogen in silkworm, and the molecular mechanism of B. mori defense to BmNPV infection is still unclear. RNA interference (RNAi) is well-known as an intracellular conserved mechanism that is critical in gene regulation and cell defense. The antiviral RNAi pathway processes viral double-stranded RNA (dsRNA) into viral small interfering RNAs that guide the recognition and cleavage of complementary viral target RNAs. In this study, a Dicer-2 (Dcr2) gene was identified in B. mori and its antiviral function was explored. Dcr2 messenger RNA (mRNA) expression was the highest in hemocytes and expressed in all stages of silkworm growth. After infection with BmNPV, the expression of Dcr2 mRNA was significantly increased after infection in midgut and hemocytes. The expression of Dcr2 was significantly upregulated by injecting dsRNA (dsBmSPH-1) into silkworm after 48 hr. Knocking down the expression level of Dcr2 using specific dsRNA in silkworm, which modestly enhanced the production of viral genomic DNA. Our results suggested that the Dcr2 gene in B. mori plays an important role in against BmNPV invasion.     

The OST‐complex as target for RNAi‐based pest control in Nilaparvata lugens

RNAi‐based pest control strategies are emerging as environment friendly and species‐specific alternatives for the use of conventional pesticides. Because N‐glycosylation is important for many biological processes, such as growth and development, the early steps of protein N‐glycosylation are promising targets for an RNAi‐based pest control strategy. Through injection of dsRNAs, the expression of the catalytic subunits of the oligosaccharyl transferase complex was efficiently silenced in nymphs of the notorious rice pest insect Nilaparvata lugens. Silencing of both STT3 isoforms resulted in a high mortality of the N. lugens nymphs. However, our data reveals the occurrence of a functional redundancy between the two isoforms when silencing only one of the isoforms. These observations confirm the potential to use the early genes in the N‐glycosylation pathway as targets for an RNAi‐based pest control strategy. In addition, the existence of a functional redundancy between the two STT3 isoforms presents a factor which one must take into account when designing RNAi‐based approaches.     

The Insect Ecdysone Receptor is a Good Potential Target for RNAi-based Pest Control

RNA interference (RNAi) has great potential for use in insect pest control. However, some significant challenges must be overcome before RNAi-based pest control can become a reality. One challenge is the proper selection of a good target gene for RNAi. Here, we report that the insect ecdysone receptor (EcR) is a good potential target for RNAi-based pest control in the brown planthopper Nilaparvata lugens, a serious insect pest of rice plants. We demonstrated that the use of a 360 bp fragment (NlEcR-c) that is common between NlEcR-A and NlEcR-B for feeding RNAi experiments significantly decreased the relative mRNA expression levels of NlEcR compared with those in the dsGFP control. Feeding RNAi also resulted in a significant reduction in the number of offspring per pair of N. lugens. Consequently, a transgenic rice line expressing NlEcR dsRNA was constructed by Agrobacterium- mediated transformation. The results of qRT-PCR showed that the total copy number of the target gene in all transgenic rice lines was 2. Northern blot analysis showed that the small RNA of the hairpin dsNlEcR-c was successfully expressed in the transgenic rice lines. After newly hatched nymphs of N. lugens fed on the transgenic rice lines, effective RNAi was observed. The NlEcR expression levels in all lines examined were decreased significantly compared with the control. In all lines, the survival rate of the nymphs was nearly 90%, and the average number of offspring per pair in the treated groups was significantly less than that observed in the control, with a decrease of 44.18-66.27%. These findings support an RNAi-based pest control strategy and are also important for the management of rice insect pests.     

New insights into an RNAi approach for plant defence against piercing‐sucking and stem‐borer insect pests

Insect double‐stranded (ds)RNA expression in transgenic crops can increase plant resistance to biotic stress; however, creating transgenic crops to defend against every insect pest is impractical. Arabidopsis Mob1A is required for organ growth and reproduction. When Arabidopsis roots were soaked in dsMob1A, the root lengths and numbers were significantly suppressed and plants could not bolt or flower. Twenty‐four hours after rice roots were immersed in fluorescent‐labelled dsEYFP (enhanced yellow fluorescent protein), fluorescence was observed in the rice sheath and stem and in planthoppers feeding on the rice. The expression levels of Ago and Dicer in rice and planthoppers were induced by dsEYFP. When rice roots were soaked in dsActin, their growth was also significantly suppressed. When planthoppers or Asian corn borers fed on rice or maize that had been irrigated with a solution containing the dsRNA of an insect target gene, the insect's mortality rate increased significantly. Our results demonstrate that dsRNAs can be absorbed by crop roots, trigger plant and insect RNAi and enhance piercing‐sucking and stem‐borer insect mortality rates. We also confirmed that dsRNA was stable under outdoor conditions. These results indicate that the root dsRNA soaking can be used as a bioinsecticide strategy during crop irrigation.     

Characterization and functional study of juvenile hormone diol kinase gene in Nilaparvata lugens (Stål) (Hemiptera: Delphacidae)

The brown planthopper, Nilaparvata lugens (Stål) is an important pest in rice. It has been widely recognized that the juvenile hormone (JH) is regulated by its hydrolase, which includes juvenile hormone esterase (JHE), juvenile hormone epoxide hydrolase (JHEH) and juvenile hormone diol kinase (JHDK). In this paper, we cloned the gene of Jhdk and the gene expression at different stages of N. lugens was analysed, and the relationship with Jhe and Jheh was studied after silencing the jhdk gene of N. lugens (Nljhdk) through double-stranded RNA (dsRNA) feeding. We also explored the expression of the three JH hydrolase after indoxacarb treatments. RT-PCR was used to amplify the full length Jhdk cDNA, and the Nljhdk gene was expressed throughout all the development periods tested and showed the lowest level at the 4th instar and the highest in the 5th instar. The expression level of Nljhdk in male adults was higher than that of female adults. Through feeding, dsRNA against Nljhdk successfully knocked down the target gene, which had no significant effect on the expression of the jhe gene of N. lugens (Nljhe), while the expression of Nljheh was upregulated. Indoxacarb could inhibit N. lugens reproduction, and the expression level of Nljhe and Nljhdk increased with the increasing of indoxacarb concentration, but the expression of the jheh gene of N. lugens (Nljheh) was reduced. These studies provide a line of experimental evidence in N. lugens to support that Nljhdk encodes the functional protein involved in JH degradation and further showed the relationship of the three hydrolases and the mechanism of indoxacarb inhibition of the fecundity of N. lugens.     

dsRNA with 5′ overhangs contributes to endogenous and antiviral RNA silencing pathways in plants

In plants, SGS3 and RNA‐dependent RNA polymerase 6 (RDR6) are required to convert single‐ to double‐stranded RNA (dsRNA) in the innate RNAi‐based antiviral response and to produce both exogenous and endogenous short‐interfering RNAs. Although a role for RDR6‐catalysed RNA‐dependent RNA polymerisation in these processes seems clear, the function of SGS3 is unknown. Here, we show that SGS3 is a dsRNA‐binding protein with unexpected substrate selectivity favouring 5′‐overhang‐containing dsRNA. The conserved XS and coiled‐coil domains are responsible for RNA‐binding activity. Furthermore, we find that the V2 protein from tomato yellow leaf curl virus, which suppresses the RNAi‐based host immune response, is a dsRNA‐binding protein with similar specificity to SGS3. In competition‐binding experiments, V2 outcompetes SGS3 for substrate dsRNA recognition, whereas a V2 point mutant lacking the suppressor function in vivo cannot efficiently overcome SGS3 binding. These findings suggest that SGS3 recognition of dsRNA containing a 5′ overhang is required for subsequent steps in RNA‐mediated gene silencing in plants, and that V2 functions as a viral suppressor by preventing SGS3 from accessing substrate RNAs.     

Impacts of nitrogen fertilizer on major insect pests and their predators in transgenic Bt rice lines T2A‐1 and T1C‐19

Nitrogen is a critical factor for plant development and nitrogen input is one of the important tactics to enhance the development and yield of crops. Nevertheless, nitrogen input could influence the occurrence of insects positively or negatively. Nitrogen is also one of the main elements composing the insecticidal crystal (Cry) protein. Cry protein production could affect nitrogen partitioning in Bt plants and as such nitrogen input may influence insect pest management in transgenic Bt rice, Oryza sativa L. (Poaceae). To test this possibility, we evaluated the impacts of nitrogen regimes on the main insect pests and their predators on two Bt rice lines, T2A‐1 and T1C‐19, expressing Cry2A and Cry1C, respectively, and their non‐transgenic parental counterpart MH63. The results showed that Cry proteins with different nitrogen regimes have enough insecticidal activity on rice leaffolder, Cnaphalocrocis medinalis Guenée (Lepidoptera: Crambidae), in both laboratory and field experiments. Laboratory studies indicated that relevant parameters of ecological fitness in brown planthopper, Nilaparvata lugens (Stål) (Hemiptera: Delphacidae), a non‐target insect pest, were significantly affected by nitrogen input both on Bt and MH63 rice lines. Nymphal survival, female adult longevity, and egg hatchability in N. lugens differed significantly among rice varieties. The experiments conducted in rice fields also demonstrated that nitrogen was positively correlated with the abundance of N. lugens on Bt rice, similar to that on MH63 rice. The abundances of two predators – the wolf spider Pirata subpiraticus (Boesenberg & Strand) (Araneae: Lycosidae) and the bug Cyrthorhinus lividipennis Reuter (Hemiptera: Miridae) – were significantly affected by rice growth stages but not by nitrogen input and rice varieties. In conclusion, the above results indicate that high nitrogen regimes for Bt rice (T2A‐1 and T1C‐19) and non‐Bt rice (MH63) cannot facilitate the management of insect pests.     

Transport of orally delivered dsRNA in southern green stink bug,Nezara viridula

The southern green stink bug (SGSB, Nezara viridula) is an emerging polyphagous pest in many regions of the world. RNA interference (RNAi) is a valuable method for understanding gene function and holds great potential for pest management. However, RNAi efficiency is variable among insects and the differences in transport of double-stranded RNA (dsRNA) are one of the major factors that contribute to this variability. In this study, Cy3 labeled dsRNA was used to track the transport of dsRNA in SGSB tissues. Cy3_dsRNA was detected in the hemocytes, fat body (FB), epidermis, and midgut tissues at 24–72 hr after injection. Orally delivered Cy3_dsRNA or Cypher-5E labeled dsRNA was mostly detected in the midgut and a few signals were detected in parts of the FB and epidermis. Both injected and fed Cy3_dsRNA showed stronger signals in SGSB tissues when compared to Cy3_siRNA (small interfering RNA) or Cy3_shRNA (short hairpin RNA). dsRNA targeting the gene for a vacuolar-sorting protein, SNF7, induced higher knockdown of the target gene and greater SGSB mortality compared to siRNA or shRNA targeting this gene. ³²P-labeled dsRNA injected into SGSB was processed into siRNA, but fed ³²P-labeled dsRNA was not efficiently processed into siRNA. These data suggest that transport of orally delivered dsRNA across the midgut epithelium is not efficient in SGSB which may contribute to variable RNAi efficiency. Targeting genes expressed in the midgut rather than other tissues and using dsRNA instead of siRNA or shRNA would be more effective for RNAi-mediated control of this pest.     

The role of chemosensory protein 10 in the detection of behaviorally active compounds in brown planthopper,Nilaparvata lugens

Chemosensory proteins (CSPs) play important roles in insects’ chemoreception, although their specific functional roles have not been fully elucidated. In this study, we conducted the developmental expression patterns and competitive binding assay as well as knock‐down assay by RNA interference both in vitro and in vivo to reveal the function of NlugCSP10 from the brown planthopper (BPH), Nilaparvata lugens (Stål), a major pest in rice plants. The results showed that NlugCSP10 messenger RNA was significantly higher in males than in females and correlated to gender, development and wing forms. The fluorescence binding assays revealed that NlugCSP10 exhibited the highest binding affinity with cis‐3‐hexenyl acetate, eicosane, and (+)‐β‐pinene. Behavioral assay revealed that eicosane displayed attractant activity, while cis‐3‐hexenyl acetate, similar to (+)‐β‐pinene significantly repelled N. lugens adults. Silencing of NlugCSP10, which is responsible for cis‐3‐hexenyl acetate binding, significantly disrupted cis‐3‐hexenyl acetate communication. Overall, findings of the present study showed that NlugCSP10 could selectively interrelate with numerous volatiles emitted from host plants and these ligands could be designated to develop slow‐release mediators that attract/repel N. lugens and subsequently improve the exploration of plans to control this insect pest.