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Recombinant fungal entomopathogen RNAi target insect gene.

ABSTRACT: RNA interference (RNAi) technology is considered as an alternative for control of pests. However, RNAi has not been used in field conditions yet, since delivering exogenous ds/siRNA to target pests is very difficult. The laboratory methods of introducing the ds/siRNA into insects through feeding, micro feeding / dripping and injecting cannot be used in fields. Transgenic crop is perhaps the most effective application of RNAi for pest control, but it needs long-time basic researches in order to reduce the cost and evaluate the safety. Therefore, transgenic microbe is maybe a better choice. Entomopathogenic fungi generally invade the host insects through cuticle like chemical insecticides contact insect to control sucking sap pests. Isaria fumosorosea is a common fungal entomopathogen in whitefly, Bemisia tabaci. We constructed a recombinant strain of I. fumosorosea expressing specific dsRNA of whitefly's TLR7 gene. It could silence the TLR7 gene and improve the virulence against whitefly. Transgenic fungal entomopathogen has shown great potential to attain the application of RNAi technology for pests control in fields. In the future, the research interests should be focused on the selection of susceptible target pests and their vital genes, and optimizing the methods for screening genes and recombinants as well.

SUBMITTER: Hu Q

PROVIDER: S-EPMC5153893 | biostudies-other | 2016 Nov

REPOSITORIES: biostudies-other

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Project description:BackgroundFungi are key dietary resources for many animals. Fungi, in consequence, have evolved sophisticated physical and chemical defences for repelling and impairing fungivores. Expression of such defences may entail costs, requiring diversion of energy and nutrients away from fungal growth and reproduction. Inducible resistance that is mounted after attack by fungivores may allow fungi to circumvent the potential costs of defence when not needed. However, no information exists on whether fungi display inducible resistance. We combined organism and fungal gene expression approaches to investigate whether fungivory induces resistance in fungi.Methodology/principal findingsHere we show that grazing by larval fruit flies, Drosophila melanogaster, induces resistance in the filamentous mould, Aspergillus nidulans, to subsequent feeding by larvae of the same insect. Larval grazing triggered the expression of various putative fungal resistance genes, including the secondary metabolite master regulator gene laeA. Compared to the severe pathological effects of wild type A. nidulans, which led to 100% insect mortality, larval feeding on a laeA loss-of-function mutant resulted in normal insect development. Whereas the wild type fungus recovered from larval grazing, larvae eradicated the chemically deficient mutant. In contrast, mutualistic dietary yeast, Saccharomyces cerevisiae, reached higher population densities when exposed to Drosophila larval feeding.Conclusions/significanceOur study presents novel evidence that insect grazing is capable of inducing resistance to further grazing in a filamentous fungus. This phenotypic shift in resistance to fungivory is accompanied by changes in the expression of genes involved in signal transduction, epigenetic regulation and secondary metabolite biosynthesis pathways. Depending on reciprocal insect-fungus fitness consequences, fungi may be selected for inducible resistance to maintain high fitness in fungivore-rich habitats. Induced fungal defence responses thus need to be included if we wish to have a complete conception of animal-fungus co-evolution, fungal gene regulation, and multitrophic interactions.

Project description:BackgroundHeterorhabditis bacteriophora is applied throughout the world for the biological control of insects and is an animal model to study interspecies interactions, e.g. mutualism, parasitism and vector-borne disease. H. bacteriophora nematodes are mutually associated with the insect pathogen, Photorhabdus luminescens. The developmentally arrested infective juvenile (IJ) stage nematode (vector) specifically transmits Photorhabdus luminescens bacteria (pathogen) in its gut mucosa to the haemocoel of insects (host). The nematode vector and pathogen alone are not known to cause insect disease. RNA interference is an excellent reverse genetic tool to study gene function in C. elegans, and it would be useful in H. bacteriophora to exploit the H. bacteriophora genome project, currently in progress.ResultsSoaking L1 stage H. bacteriophora with seven dsRNAs of genes whose C. elegans orthologs had severe RNAi phenotypes resulted in highly penetrant and obvious developmental and reproductive abnormalities. The efficacy of postembryonic double strand RNA interference (RNAi) was evident by abnormal gonad morphology and sterility of adult H. bacteriophora and C. elegans presumable due to defects in germ cell proliferation and gonad development. The penetrance of RNAi phenotypes in H. bacteriophora was high for five genes (87-100%; Hba-cct-2, Hba-daf-21, Hba-icd-1; Hba-nol-5, and Hba-W01G7.3) and moderate for two genes (usually 30-50%; Hba-rack-1 and Hba-arf-1). RNAi of three additional C. elegans orthologs for which RNAi phenotypes were not previously detected in C. elegans, also did not result in any apparent phenotypes in H. bacteriophora. Specific and severe reduction in transcript levels in RNAi treated L1s was determined by quantitative real-time RT-PCR. These results suggest that postembryonic RNAi by soaking is potent and specific.ConclusionAlthough RNAi is conserved in animals and plants, RNAi using long dsRNA is not. These results demonstrate that RNAi can be used effectively in H. bacteriophora and can be applied for analyses of nematode genes involved in symbiosis and parasitism. It is likely that RNAi will be an important tool for functional genomics utilizing the high quality draft H. bacteriophora genome sequence.

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Recombinant fungal entomopathogen RNAi target insect gene.

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