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Cysteine protease enhances plant-mediated bollworm RNA interference.

ABSTRACT: Oral ingestion of plant-expressed double stranded RNA (dsRNA) triggers target gene suppression in insect. An important step of this process is the transmission of dsRNA from plant to midgut cells. Insect peritrophic matrix (PM) presents a barrier that prevents large molecules from entering midgut cells. Here, we show that uptake of plant cysteine proteases, such as GhCP1 from cotton (Gossypium hirsutum) and AtCP2 from Arabidopsis, by cotton bollworm (Helicoverpa armigera) larvae resulted in attenuating the PM. When GhCP1 or AtCP2 pre-fed larvae were transferred to gossypol-containing diet, the bollworm accumulated higher content of gossypol in midgut. Larvae previously ingested GhCP1 or AtCP2 were more susceptible to infection by Dendrolimus punctatus cytoplasmic polyhedrosis virus (DpCPV), a dsRNA virus. Furthermore, the pre-fed larvae exhibited enhanced RNAi effects after ingestion of the dsRNA-expressing plant. The bollworm P450 gene CYP6AE14 is involved in the larval tolerance to gossypol; cotton plants producing dsRNA of CYP6AE14 (dsCYP6AE14) were more resistant to bollworm feeding (Mao et al. in Transgenic Res 20:665-673, 2011). We found that cotton plants harboring both 35S:dsCYP6AE14 and 35S:GhCP1 were better protected from bollworm than either of the single-transgene lines. Our results demonstrate that plant cysteine proteases, which have the activity of increasing PM permeability, can be used to improve the plant-mediated RNAi against herbivorous insects.

SUBMITTER: Mao YB

PROVIDER: S-EPMC3755213 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Functional analysis of the cathepsin-like cysteine protease genes in adult Brugia malayi using RNA interference.

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Symbiont-mediated RNA interference in insects.

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Ipomoea batatas: papain propeptide inhibits cysteine protease in main plant parasites and enhances resistance of transgenic tomato to parasites.

Project description:Different parasites cause severe lose in quantity and quality of crops. Many parasites develop haustorial cells and stylets that penetrate the host using secreted enzymes and mechanical pressure. Cysteine proteases are pre-pro-enzyme produced by parasites that are essential for normal parasitism. Papain is also a kind of cysteine proteases such that its propeptide segment has inhibitory properties and limits the protease activity of papain. To investigate the inhibitory effects of papain propeptide on some parasite proteases, we cloned inhibitory propeptide of papain of Ipomoea batatas, and enzymatic fragments of Diabrotica virgifera cathepsin L-like protease-1, Meloidogyne incognita cathepsin L-like protease 1, Heterodera glycines cysteine protease-1, Cuscuta chinesis cysteine protease and Orobanche cernua cysteine protease. After purification of recombinant inhibitory propeptide and enzymatic fragments, the inhibition activity of propeptide on cysteine proteases was measured. Finally inhibitory propeptide was transformed into tomato and transgenic plants resistance to parasites (bioassay) were examined. We demonstrated papain-propeptide inhibits cysteine protease of mentioned parasites. In transgenic tomato plants, papain-inhibitory propeptide effectively interrupted haustoria development. Haustoria-digitate cells of dodder could not differentiate and develop into the phloem and xylem hyphae on transgenic tomatoes. Parasites grown on transgenic tomatoes showed reduction in vigor and productivity due to defective connection of haustoria. Lower ratio of female nematodes and a decrease of nematode egg mass per transgenic line indicated biocontrol of nematode. The changes in growth factors of parasite challenged transgenic lines relative to controls, indicates the efficacy of papain propeptide in control of parasitism.

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Project description:In this study, we developed transgenic Arabidopsis thaliana (AT1703) expressing double-stranded (ds)RNA to silence S. sclerotiorum ABHYDROLASE-3 and slow infection through host induced gene silencing (HIGS). Leaf infection assays show reduced S. sclerotiorum lesion size, fungal load, and ABHYDROLASE-3 transcript abundance in AT1703 lines compared to wild-type Col-0. To better understand how HIGS influences host-pathogen interactions, we performed global RNA sequencing on AT1703 and wild-type Col-0 lines directly at the site of S. sclerotiorum infection. Together, these results demonstrate the utility of HIGS technology in slowing S. sclerotiorum infection and provides insight into the role of ABHYDROLASE-3 gene activity in the A. thaliana – S. sclerotiorum pathosystem.

2022-11-11 | GSE217513 | GEO

Knockdown of midgut genes by dsRNA-transgenic plant-mediated RNA interference in the hemipteran insect Nilaparvata lugens.

Project description:BackgroundRNA interference (RNAi) is a powerful technique for functional genomics research in insects. Transgenic plants producing double-stranded RNA (dsRNA) directed against insect genes have been reported for lepidopteran and coleopteran insects, showing potential for field-level control of insect pests, but this has not been reported for other insect orders.Methodology/principal findingsThe Hemipteran insect brown planthopper (Nilaparvata lugens Stål) is a typical phloem sap feeder specific to rice (Oryza sativa L.). To analyze the potential of exploiting RNAi-mediated effects in this insect, we identified genes (Nlsid-1 and Nlaub) encoding proteins that might be involved in the RNAi pathway in N. lugens. Both genes are expressed ubiquitously in nymphs and adult insects. Three genes (the hexose transporter gene NlHT1, the carboxypeptidase gene Nlcar and the trypsin-like serine protease gene Nltry) that are highly expressed in the N. lugens midgut were isolated and used to develop dsRNA constructs for transforming rice. RNA blot analysis showed that the dsRNAs were transcribed and some of them were processed to siRNAs in the transgenic lines. When nymphs were fed on rice plants expressing dsRNA, levels of transcripts of the targeted genes in the midgut were reduced; however, lethal phenotypic effects after dsRNA feeding were not observed.ConclusionsOur study shows that genes for the RNAi pathway (Nlsid-1 and Nlaub) are present in N. lugens. When insects were fed on rice plant materials expressing dsRNAs, RNA interference was triggered and the target genes transcript levels were suppressed. The gene knockdown technique described here may prove to be a valuable tool for further investigations in N. lugens. The results demonstrate the potential of dsRNA-mediated RNAi for field-level control of planthoppers, but appropriate target genes must be selected when designing the dsRNA-transgenic plants.

| S-EPMC3105074 | biostudies-literature

RNA Interference-Mediated Knockdown of Bombyx mori Haemocyte-Specific Cathepsin L (Cat L)-Like Cysteine Protease Gene Increases Bacillus thuringiensis kurstaki Toxicity and Reproduction in Insect Cadavers.

Project description:The silkworm's Cat L-like gene, which encodes a lysosomal cathepsin L-like cysteine protease, is thought to be part of the insect's innate immunity via an as-yet-undetermined mechanism. Assuming that the primary function of Cat L-like is microbial degradation in mature phagosomes, we hypothesise that the suppression of the Cat L-like gene expression would increase Bacillus thuringiensis (Bt) bacteraemia and toxicity in knockdown insects. Here, we performed a functional analysis of Cat L-like in larvae that were fed mulberry leaves contaminated with a commercial biopesticide formulation based on Bt kurstaki (Btk) (i.e., Dipel) to investigate its role in insect defence against a known entomopathogen. Exposure to sublethal doses of Dipel resulted in overexpression of the Cat L-like gene in insect haemolymph 24 and 48 h after exposure. RNA interference (RNAi)-mediated suppression of Cat L-like expression significantly increased the toxicity of Dipel to exposed larvae. Moreover, Btk replication was higher in RNAi insects, suggesting that Cat L-like cathepsin may be involved in a bacterial killing mechanism of haemocytes. Finally, our results confirm that Cat L-like protease is part of the antimicrobial defence of insects and suggest that it could be used as a target to increase the insecticidal efficacy of Bt-based biopesticides.

| S-EPMC9230843 | biostudies-literature

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