Molecular approaches to increasing resistance of wheat (Triticum aestivum L.) towards two insect pests; Cereal aphid (Sitobion avenae F.) and Wheat bulb fly (Delia coarctata Fallen).

Cereal aphid (Sitobion avenae) and wheat bulb fly (Delia coarctata) are serious pests of wheat in the UK. At the present, chemical pesticides are used to control these insects, but they are limited in effectiveness, and have undersirable ecological impacts. There is a need to improve wheat genetically to be resistant to such inset pests. The objectives of this work were to investigate digestive biochemistry in the selected insect pests of wheat, and to determine effects of potential endogenous resistance factors in wheat on digestion, nutrition and other insect metabolic processes. The aim was to develop new strategies for crop protection. Digestive biochemistry in S. avenae and D. coarctata was studied to characterise gut proteases and their inhibition by host plant proteinase inhibitors (PIs). Investigation of proteolytic digestion in S. avenae gut showed that in spite of being a phloem-feeding insect, cereal aphid could digest ingested protein, using cysteine proteases. D. coarctata larvae contained mainly serine protease activity. A serine protease (DcSP) and a cysteine protease (DcCathL) from D. coarctata gut tissue were expressed as recombinant proteins. Only DcCathL was recovered in active form. DcCathL was insecticidal to Mamestra brassicae when injected into hemolymph, causing systemic and extensive melanisation. DcCathL selectively degraded recombinant serpins from M. brassicae in in vitro assays, and is suggested to interfere with regulation of the proteolytic cascade leading to phenoloxidase activation and melanin production in vivo. DcCathL has potential as a biopesticide if it could be made effective when orally delivered. A cationic amino acid transporter from D. coarctata gut (DcCAAT) was also cloned as a target for RNA interference. Potential resistance factors in wheat were characterised by expression as recombinant proteins. Two PIs from wheat (subtilisin/chymotrypsin inhibitor; WSCI, and cysteine proteinase inhibitor; WCPI) were expressed in the yeast Pichia pastoris, and purified. WSCI inhibited gut protease activity of both insects in in vitro and in vivo assays, whereas WCPI only inhibited S. avenae gut extract activity. On feeding, WSCI was antimetabolic to both insects, affecting both survival and growth, whereas WCPI was antimetabolic to S. avenae only. Wheat Hessian fly responsive (Hfr) genes are up-regulated in response to herbivory by Hessian fly (Mayetiola destructor). The protein product Hfr-3 was expressed and purified, and showed antimetabolic effects on survival and growth of both S. avenae and D. coarctata. Both accumulated and induced defence proteins, like WSCI, WCPI and Hfr-3, have the potential to act as endogenous resistance factors in wheat towards a range of insect pests. Developing a wheat variety constitutively expressing these defence proteins by using traditional breeding methods and/or modern biotechnological tools is discussed

Heterologous production of the insecticidal pea seed albumin PA1 protein by Pichia pastoris and protein engineering to potentiate aphicidal activity via fusion to snowdrop lectin Galanthus nivalis agglutinin; GNA)

BackgroundNew bioinsecticides with novel modes of action are urgently needed to minimise the environmental and safety hazards associated with the use of synthetic chemical pesticides and to combat growing levels of pesticide resistance. The pea seed albumin PA1b knottin peptide is the only known proteinaceous inhibitor of insect vacuolar adenosine triphosphatase (V-ATPase) rotary proton pumps. Oral toxicity towards insect pests and an absence of activity towards mammals makes Pa1b an attractive candidate for development as a bioinsecticide. The purpose of this study was to investigate if Pichia pastoris could be used to express a functional PA1b peptide and if it’s insecticidal activity could be enhanced via engineering to produce a fusion protein comprising the pea albumin protein fused to the mannose-specific snowdrop lectin (Galanthus nivalis agglutinin; GNA).ResultsWe report the production of a recombinant full-length pea albumin protein (designated PAF) and a fusion protein (PAF/GNA) comprised of PAF fused to the N-terminus of GNA in the yeast Pichia pastoris. PAF was orally toxic to pea (Acyrthosiphon pisum) and peach potato (Myzus persicae) aphids with respective, Day 5 LC50 values of 54 µM and 105 µM derived from dose–response assays. PAF/GNA was significantly more orally toxic as compared to PAF, with LC50 values tenfold (5 µM) and 3.3-fold (32 µM) lower for pea and peach potato aphids, respectively. By contrast, no phenotypic effects were observed for worker bumble bees (Bombus terristrus) fed PAF, GNA or PAF/GNA in acute toxicity assays. Confocal microscopy of pea aphid guts after pulse-chase feeding fluorescently labelled proteins provides evidence that enhanced efficacy of the fusion protein is attributable to localisation and retention of PAF/GNA to the gut epithelium. In contact assays the fusion protein was also found to be significantly more toxic towards A. pisum as compared to PAF, GNA or a combination of the two proteins.ConclusionsOur results suggest that GNA mediated binding to V-type ATPase pumps acts to potentiate the oral and contact aphicidal activity of PAF. This work highlights potential for the future commercial development of plant protein-based bioinsecticides that offer enhanced target specificity as compared to chemical pesticides, and compatibility with integrated pest management strategies

Insecticidal effects of dsRNA targeting the Diap1 gene in dipteran pests

The Drosophila melanogaster (fruit fly) gene Diap1 encodes a protein referred to as DIAP1 (DrosophilaInhibitor of Apoptosis Protein 1) that acts to supress apoptosis in “normal” cells in the fly. In this study we investigate the use of RNA interference (RNAi) to control two dipteran pests, Musca domestica and Delia radicum, by disrupting the control of apoptosis. Larval injections of 125–500 ng of Diap1 dsRNA resulted in dose-dependent mortality which was shown to be attributable to down-regulation of target mRNA. Insects injected with Diap1 dsRNA have approx. 1.5-2-fold higher levels of caspase activity than controls 24 hours post injection, providing biochemical evidence that inhibition of apoptotic activity by the Diap1 gene product has been decreased. By contrast adults were insensitive to injected dsRNA. Oral delivery failed to induce RNAi effects and we suggest this is attributable to degradation of ingested dsRNA by intra and extracellular RNAses. Non-target effects were demonstrated via mortality and down-regulation of Diap1 mRNA levels in M. domestica larvae injected with D. radicum Diap1 dsRNA, despite the absence of 21 bp identical sequence regions in the dsRNA. Here we show that identical 15 bp regions in dsRNA are sufficient to trigger non-target RNAi effects

Analysis of haemolymph and nerve chords.

<p>Immunoblot analysis using anti-GNA antibodies of (A) haemolymph samples extracted from <i>M. brassicae</i> larvae 48 h after feeding on diet containing Hv1a/GNA (2 mg/5 g diet). “C” denotes control haemolymph (larvae fed on diet with no added protein). Lanes 1 and 2 are replicates of pooled samples (3 larvae per sample); 15 µl of haemolymph was loaded in all cases. (B) Nerve chord samples dissected from sixth stadium larvae that had been injected with 25 µg GNA (lanes 1–4) or Hv1a/GNA (lanes 5–8). Pooled samples were extracted 3 h (lanes 1, 2, 5, and 6) or 5 h (lanes 3, 4, 7, and 8) post injection. Pooled samples (4 nerve chords per sample) were extracted directly in 40 µl SDS-sample buffer and 20 µl was loaded per lane. “C” denotes control nerve chord sample. In panels (A) and (B), S1 and S2 are 50 ng standards of GNA and Hv1a/GNA respectively.</p

Droplet feeding assays.

<p>(A) Mean weight of fifth stadium <i>M. brassicae</i> larvae fed daily sucrose droplets containing either 9.2 µg Hv1a, 40 µg Hv1a/GNA, or 40 µg bovine serum albumin (BSA, control). Significant differences between Hv1a/GNA and control or Hv1a treatments were observed at days 1–4 (ANOVA Tukey post hoc; day 1, P = 0.0003; days 2–4, P<0.0001). (B) Image depicts larvae assayed in (A); control larvae on the left (BSA- and Hv1a-fed larvae) and Hv1a/GNA-fed larvae on the right. (C) Mean weight of fifth stadium <i>M. brassicae</i> larvae fed a single sucrose droplet containing either 40 µg Hv1a/GNA or control 40 µg BSA. Differences in mean weights between control and fusion protein treatments were significant from day 1 to day 6 of the assay (<i>t</i>-test; P<0.05).</p

Mortality recorded for fifth stadium <i>M. brassicae</i> larvae 72 h after injection of different concentrations of recombinant Hv1a and Hv1a/GNA.

<p>Doses of injected Hv1a/GNA are expressed as Hv1a equivalents to allow a direct comparison with the Hv1a treatment and are based on a mean larval weight at injection of 50 mg. Asterisks denotes significant difference in survival between control and toxin treatment (P<0.0001).</p

Binding of GNA to nerve chords.

<p>(A) Intact nerve chord dissected from sixth stadium <i>M. brassicae</i> larvae. (B–F) Composite of partial images of nerve tracts dissected from larvae injected with, or fed on, FITC-labelled proteins. Images were visualised with a fluorescent microscope under FITC filter and captured in OpenLab. B: FITC-GNA; C: FITC-Hv1a/GNA; D: control FITC; E: FITC-ovalbumin; F: FITC-GNA; G: FITC-Hv1a/GNA. Scale bar = 2 mm in (A) and 200 µM in (B–E).</p

Oligonucleotide sequences used for assembly and amplification of a synthetic gene encoding for the mature Hv1a toxin.

<p>Underlined bases depict restriction sites (<i>Pst</i>I and <i>Not</i> I) used for ligation of the full-length fragment into the yeast expression vector pGAPZαB.</p

Protein production and purification.

<p>(A) Schematic of construct encoding Hv1a/GNA showing predicted molecular masses of Hv1a and GNA as well as the total mass of the Hv1a/GNA fusion protein including the tri-alanine linker region and the additional two alanine residues at the N-terminus. (B) Coomassie blue stained SDS-PAGE gel (17.5% acrylamide) of recombinant Hv1a/GNA and GNA following purification by hydrophobic interaction and gel filtration chromatography. The approximate loading of protein (µg) is indicated above each lane, while the lane marked “M” contains molecular weight standards (Sigma SDS-7). (C) Composite of Western blots of recombinant proteins using (i) anti-GNA and (ii) anti-Hv1a antibodies. The approximate protein loading (ng) is denoted above each lane. FP denotes Hv1a/GNA fusion protein.</p