RESUMO
In situ hybridization (ISH) is a methodology by which nucleic acids are detected within fixed tissue samples. Recent advances in detection technology and target recovery have greatly enhanced the technique's ability to detect single mRNA molecules. Here we detail the fixation, paraffin embedding, sectioning, target recovery, and chromogenic detection of an mRNA (DvSSJ1), encoding for a membrane protein associated with the smooth septate junction (SSJ) in Western corn rootworm [Diabrotica virgifera (Dv)]. Further, we demonstrate, the expression of dsRNA of DvSSJ1 in maize root tissues using signal amplification and background suppression technology.
Assuntos
Zea mays , Animais , Besouros/genética , Hibridização In Situ , Larva , Plantas Geneticamente Modificadas , Interferência de RNA , RNA de Cadeia Dupla , RNA Mensageiro/genética , Zea mays/genéticaRESUMO
The western corn rootworm (WCR, Diabrotica virgifera virgifera) gene, dvssj1, is a putative homolog of the Drosophila melanogaster gene, snakeskin (ssk). This gene encodes a membrane protein associated with the smooth septate junction (SSJ) which is required for the proper barrier function of the epithelial lining of insect intestines. Disruption of DVSSJ integrity by RNAi technique has been shown previously to be an effective approach for corn rootworm control, by apparent suppression of production of DVSSJ1 protein leading to growth inhibition and mortality. To understand the mechanism that leads to the death of WCR larvae by dvssj1 double-stranded RNA, we examined the molecular characteristics associated with SSJ functions during larval development. Dvssj1 dsRNA diet feeding results in dose-dependent suppression of mRNA and protein; this impairs SSJ formation and barrier function of the midgut and results in larval mortality. These findings suggest that the malfunctioning of the SSJ complex in midgut triggered by dvssj1 silencing is the principal cause of WCR death. This study also illustrates that dvssj1 is a midgut-specific gene in WCR and its functions are consistent with biological functions described for ssk.
Assuntos
Besouros/efeitos dos fármacos , Besouros/genética , Controle de Insetos/métodos , RNA de Cadeia Dupla/farmacologia , Zea mays/parasitologia , Animais , Besouros/crescimento & desenvolvimento , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Genes de Insetos/efeitos dos fármacos , Proteínas de Insetos/genética , Inseticidas/farmacologia , Larva/efeitos dos fármacos , Larva/genética , Larva/crescimento & desenvolvimento , Proteínas de Membrana/genética , Controle Biológico de Vetores/métodos , Interferência de RNA , RNA Mensageiro/genéticaRESUMO
RNA interference (RNAi) in transgenic maize has recently emerged as an alternative mode of action for western corn rootworm (Diabrotica virgifera virgifera) control which can be combined with protein-based rootworm control options for improved root protection and resistance management. Currently, transgenic RNAi-based control has focused on suppression of genes that when silenced lead to larval mortality. We investigated control of western corn rootworm reproduction through RNAi by targeting two reproductive genes, dvvgr and dvbol, with the goal of reducing insect fecundity as a new tool for pest management. The results demonstrated that exposure of adult beetles, as well as larvae to dvvgr or dvbol dsRNA in artificial diet, caused reduction of fecundity. Furthermore, western corn rootworm beetles that emerged from larval feeding on transgenic maize roots expressing dvbol dsRNA also showed significant fecundity reduction. This is the first report of reduction of insect reproductive fitness through plant-mediated RNAi, demonstrating the feasibility of reproductive RNAi as a management tool for western corn rootworm.
Assuntos
Controle Biológico de Vetores , Doenças das Plantas/genética , Interferência de RNA , Reprodução/genética , Animais , Besouros/genética , Besouros/patogenicidade , Fertilidade/genética , Proteínas de Insetos/genética , Larva/genética , Larva/patogenicidade , Doenças das Plantas/microbiologia , Plantas Geneticamente Modificadas/genética , RNA de Cadeia Dupla/genética , RNA de Plantas/genética , Zea mays/genética , Zea mays/crescimento & desenvolvimento , Zea mays/microbiologiaRESUMO
RNA interference (RNAi) is a promising new technology for corn rootworm control. This paper presents the discovery of new gene targets - dvssj1 and dvssj2, in western corn rootworm (WCR). Dvssj1 and dvssj2 are orthologs of the Drosophila genes snakeskin (ssk) and mesh, respectively. These genes encode membrane proteins associated with smooth septate junctions (SSJ) which are required for intestinal barrier function. Based on bioinformatics analysis, dvssj1 appears to be an arthropod-specific gene. Diet based insect feeding assays using double-stranded RNA (dsRNA) targeting dvssj1 and dvssj2 demonstrate targeted mRNA suppression, larval growth inhibition, and mortality. In RNAi treated WCR, injury to the midgut was manifested by "blebbing" of the midgut epithelium into the gut lumen. Ultrastructural examination of midgut epithelial cells revealed apoptosis and regenerative activities. Transgenic plants expressing dsRNA targeting dvssj1 show insecticidal activity and significant plant protection from WCR damage. The data indicate that dvssj1 and dvssj2 are effective gene targets for the control of WCR using RNAi technology, by apparent suppression of production of their respective smooth septate junction membrane proteins located within the intestinal lining, leading to growth inhibition and mortality.