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Recently, we reported that a recombinant Tepary bean ( Phaseolus acutifolius ) lectin (rTBL-1) induces apoptosis in colon cancer cell lines and that cytotoxicity was related to differential recognition of β1-6 branched N -glycans. Sequencing analysis and resolution of the rTBL-1 3D structure suggest that glycan specificity could be strongly influenced by two arginine residues, R103 and R130, located in the carbohydrate binding pocket. The aim of this work was to determine the contribution of these residues towards cytotoxic activity. Two rTBL-1 mutants were produced in Pichia pastoris , biochemically characterized, and cytotoxic effects were evaluated on human colorectal cancer cells (HT-29). Substitution of either of the arginine residues with glutamines resulted in significant reductions in cytotoxic activity, with losses of 1.5 and 3 times for R103 and R130, respectively. Docking analysis showed that the mutations decreased lectin affinity binding to some Epidermal Growth Factor Receptor (EGFR)-related N -glycans. Together, these findings confirm that both of the selected arginine residues (R103 and R130) play a key role in the recognition of tumor cell glycoconjugates by rTBL-1.

A major cause of yield loss in wheat worldwide is the fungal pathogen Zymoseptoria tritici , a hemibiotrophic fungus which causes Septoria leaf blotch, the most destructive wheat disease in Europe. Resistance in commercial wheat varieties is poor, however, a link between reduced nitrogen availability and increased Septoria tolerance has been observed. We have shown that Septoria load is not affected by nitrogen, whilst the fungus is in its first, symptomless stage of growth. This suggests that a link between nitrogen and Septoria is only present during the necrotrophic phase of Septoria infection. Quantitative real-time PCR data demonstrated that WRKYs, a superfamily of plant-specific transcription factors, are differentially expressed in response to both reduced nitrogen and Septoria. WRKY39 was downregulated over 30-fold in response to necrotrophic stage Septoria, whilst changes in the expression of WRKY68a during the late biotrophic phase were dependent on the concentration of nitrogen under which wheat is grown. WRKY68a may therefore mediate a link between nitrogen and Septoria. The potential remains to identify key regulators in the link between nitrogen and Septoria, and as such, elucidate molecular markers for wheat breeding, or targets for molecular-based breeding approaches.

Herein, we report the production of a recombinant Tepary bean lectin ( r TBL-1), its three-dimensional (3D) structure, and its differential recognition for cancer-type glycoconjugates. r TBL-1 was expressed in Pichia pastoris, yielding 316 mg per liter of culture, and was purified by nickel affinity chromatography. Characterization of the protein showed that r TBL-1 is a stable 120 kDa homo-tetramer folded as a canonical leguminous lectin with two divalent cations (Ca 2+ and Mn 2+ ) attached to each subunit, confirmed in its 3D structure solved by X-ray diffraction at 1.9 Å resolution. Monomers also presented a ~2.5 kDa N -linked glycan located on the opposite face of the binding pocket. It does not participate in carbohydrate recognition but contributes to the stabilization of the interfaces between protomers. Screening for potential r TBL-1 targets by glycan array identified 14 positive binders, all of which correspond to β1-6 branched N -glycans' characteristics of cancer cells. The presence of α1-6 core fucose, also tumor-associated, improved carbohydrate recognition. r TBL-1 affinity for a broad spectrum of mono- and disaccharides was evaluated by isothermal titration calorimetry (ITC); however, no interaction was detected, corroborating that carbohydrate recognition is highly specific and requires larger ligands for binding. This would explain the differential recognition between healthy and cancer cells by Tepary bean lectins.

The parasitic small hive beetle ( Aethina tumida ) feeds on pollen, honey and brood of the European honey bee ( Apis mellifera ); establishment in North America and Australia has resulted in severe economic damage to the apiculture industry. We report potential for the "in-hive" use of a novel biopesticide that is toxic to this invasive beetle pest but harmless to honeybees. Constructs encoding the spider venom neurotoxin ω-hexatoxin-Hv1a (Hv1a) linked to the N - or C -terminus of snowdrop lectin (GNA) were used to produce recombinant Hv1a/GNA and GNA/Hv1a fusion proteins. Both were similarly toxic to beetles by injection (respective LD 50 s 1.5 and 0.9 nmoles/g larvae), whereas no effects on adult honeybee survival were observed at injection doses of > 200 nmoles/g insect. When fed to A. tumida larvae, GNA/Hv1a was significantly more effective than Hv1a/GNA (LC 50 s of 0.52 and 1.14 mg/ml diet, respectively), whereas both proteins were similarly toxic to adults. Results suggested that the reduced efficacy of Hv1a/GNA against larvae was attributable to differences in the susceptibility of the fusion proteins to cleavage by gut serine proteases. In laboratory assays, A. tumida larval survival was significantly reduced when brood, inoculated with eggs, was treated with GNA/Hv1a., Competing Interests: Conflict of interestThe authors declare they have no conflict of interest., (© The Author(s) 2019.)

RNA interference (RNAi) effects in insects are highly variable and may be largely dependent upon the stability of introduced double-stranded RNAs to digestion by nucleases. Here, we report a systematic comparison of RNAi effects in susceptible red flour beetle ( Tribolium castaneum ) and recalcitrant pea aphid ( Acyrthosiphon pisum ) following delivery of dsRNAs of identical length targeting expression of V-type ATPase subunit E ( VTE ) and inhibitor of apoptosis ( IAP ) genes. Injection and ingestion of VTE and IAP dsRNAs resulted in up to 100% mortality of T. castaneum larvae and sustained suppression (>80%) of transcript levels. In A. pisum , injection of VTE but not IAP dsRNA resulted in up to 65% mortality and transient suppression (ca. 40%) of VTE transcript levels. Feeding aphids on VTE dsRNA reduced growth and fecundity although no evidence for gene suppression was obtained. Rapid degradation of dsRNAs by aphid salivary, haemolymph and gut nucleases contrasted with stability in T. castaneum larvae where it appears that exo-nuclease activity is responsible for relatively slow digestion of dsRNAs. This is the first study to directly compare RNAi effects and dsRNA stability in receptive and refractory insect species and provides further evidence that dsRNA susceptibility to nucleases is a key factor in determining RNAi efficiency.

The Drosophila melanogaster (fruit fly) gene Diap1 encodes a protein referred to as DIAP1 (D rosophila Inhibitor 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.

Background: Aethina tumida is a serious pest of the European honey bee (Apis mellifera) in North America and Australia. Here we investigate whether Laccase 2, the phenoloxidase gene essential for cuticle sclerotisation and pigmentation in many insects, and vacuolar-ATPase V-type subunit A, vital for the generation of proton gradients used to drive a range of transport processes, could be potential targets for RNAi-mediated control of A. tumida., Results: Injection of V-ATPase subunit A (5 ng) and Laccase 2 (12.5 ng) dsRNAs resulted in 100% larval mortality, and qPCR confirmed significant decreases and enhanced suppression of transcript levels over time. Oral delivery of V-ATPase subunit A dsRNA in solutions resulted in 50% mortality; however, gene suppression could not be verified. We suggest that the inconsistent RNAi effect was a consequence of dsRNA degradation within the gut owing to the presence of extracellular nucleases. Target specificity was confirmed by a lack of effect on survival or gene expression in honey bees injected with A. tumida dsRNAs., Conclusion: This is the first study to show evidence for systemic RNAi in A. tumida in response to injected dsRNA, but further research is required to develop methods to induce RNAi effects via ingestion. © 2016 Crown copyright. Pest Management Science © 2016 Society of Chemical Industry., (© 2016 Crown copyright. Pest Management Science © 2016 Society of Chemical Industry.)

Background: The neurotoxin peptide ω-ACTX-Hv1a, fused to the carrier molecule GNA, presents potential for insect control as a biopesticide, being orally toxic to insect pests from different orders. However, thorough evaluation is required to assure its safety towards non-target invertebrates. Effects of this novel biopesticide on the parasitoid Eulophus pennicornis via its host Lacanobia oleracea are presented., Results: Hv1a/GNA did not cause mortality when injected or fed to fifth-stage L. oleracea, but caused up to 39% reduction in mean larval weight (P < 0.05) and increased developmental time when injected. When fed, GNA, but not Hv1a/GNA, caused ∼35% reduction in larval weight, indicating that host quality was not affected by the fusion protein. Although GNA and Hv1a/GNA were internalised by the hosts following ingestion, and thus were available to higher trophic levels, no significant changes in the rate of E. pennicornis parasitism occurred. Number of parasitoid pupae per host, adult emergence and sex ratio were unaffected by GNA- or Hv1a/GNA-treated hosts (P > 0.05). The fusion protein was degraded by parasitoid larvae, rendering it non-toxic., Conclusion: Hv1a/GNA has negligible effects on the parasitoid, even under worst-case scenarios. This low toxicity to these insects is of interest in terms of biopesticide specificity and safety to non-target organisms., (© 2015 Society of Chemical Industry.)

Background: The recombinant fusion proteins Pl1a/GNA and Hv1a/GNA contain the spider venom peptides δ-amaurobitoxin-PI1a or ω-hexatoxin-Hv1a respectively, linked to snowdrop lectin (GNA). Pl1a targets receptor site 4 of insect voltage-gated sodium channels (NaCh), while Hv1a targets voltage-gated calcium channels. Insecticide-resistant strains of peach-potato aphid (Myzus persicae) contain mutations in NaCh. The pyrethroid-resistant kdr (794J) and super-kdr (UKO) strains contain mutations at residues L1014 and M918 in the channel α-subunit respectively, while the kdr + super-kdr strain (4824J), insensitive to pyrethroids, contains mutations at both L1014 and M918., Results: Pl1a/GNA and Hv1a/GNA fusion proteins have estimated LC50 values of 0.35 and 0.19 mg mL(-1) when fed to wild-type M. persicae. For insecticide-resistant aphids, LC50 for the Pl1a/GNA fusion protein increased by 2-6-fold, correlating with pyrethroid resistance (wild type < kdr < super-kdr < kdr + super-kdr strains). In contrast, LC50 for the Hv1a/GNA fusion protein showed limited correlation with pyrethroid resistance., Conclusion: Mutations in the sodium channel in pyrethroid-resistant aphids also protect against a fusion protein containing a sodium-channel-specific toxin, in spite of differences in ligand-channel interactions, but do not confer resistance to a fusion protein targeting calcium channels. The use of fusion proteins with differing targets could play a role in managing pesticide resistance., (© 2014 Society of Chemical Industry.)

The grain aphid Sitobion avenae (F.) is a major pest of wheat, acting as a virus vector as well as causing direct plant damage. Commonly grown wheat varieties in the UK have only limited resistance to this pest. The present study was carried out to investigate the potential of a diploid wheat line (ACC20 PGR1755), reported as exhibiting resistance to S. avenae, to serve as a source of resistance genes. The diploid wheat line was confirmed as partially resistant, substantially reducing the fecundity, longevity and growth rate of the aphid. Proteomic analysis showed that approximately 200 protein spots were reproducibly detected in leaf extracts from both the resistant line and a comparable susceptible line (ACC5 PGR1735) using two-dimensional gel electrophoresis and image comparison software. Twenty-four spots were significantly up-regulated (>2-fold) in the resistant line after 24 h of aphid feeding (13 and 11 involved in local and systemic responses, respectively). Approximately 50 % of all differentially expressed protein spots were identified by a combination of database searching with MS and MS/MS data, revealing that the majority of proteins up-regulated by aphid infestation were involved in metabolic processes (including photosynthesis) and transcriptional regulation. However, in the resistant line only, several stress response proteins (including NBS-LRR-like proteins) and oxidative stress response proteins were identified as up-regulated in response to aphid feeding, as well as proteins involved in DNA synthesis/replication/repair. This study indicates that the resistant diploid line ACC20 PGR1755 may provide a valuable resource in breeding wheat for resistance to aphids.

Recombinant fusion proteins containing arthropod toxins have been developed as a new class of biopesticides. The recombinant fusion protein Hv1a/GNA, containing the spider venom toxin ω-ACTX-Hv1a linked to snowdrop lectin (GNA) was shown to reduce survival of the peach-potato aphid Myzus persicae when delivered in artificial diet, with survival <10% after 8 days exposure to fusion protein at 1 mg/ml. Although the fusion protein was rapidly degraded by proteases in the insect, Hv1a/GNA oral toxicity to M. persicae was significantly greater than GNA alone. A construct encoding the fusion protein, including the GNA leader sequence, under control of the constitutive CaMV 35S promoter was transformed into Arabidopsis; the resulting plants contained intact fusion protein in leaf tissues at an estimated level of 25.6 ± 4.1 ng/mg FW. Transgenic Arabidopsis expressing Hv1a/GNA induced up to 40% mortality of M. persicae after 7 days exposure in detached leaf bioassays, demonstrating that transgenic plants can deliver fusion proteins to aphids. Grain aphids (Sitobion avenae) were more susceptible than M. persicae to the Hv1a/GNA fusion protein in artificial diet bioassays (LC50 = 0.73 mg/ml after 2 days against LC50 = 1.81 mg/ml for M. persicae), as they were not able to hydrolyze the fusion protein as readily as M. persicae. Expression of this fusion protein in suitable host plants for the grain aphid is likely to confer higher levels of resistance than that shown with the M. persicae/Arabidopsis model system.

Background: Phloem feeding insects, such as aphids, feed almost continuously on plant phloem sap, a liquid diet that contains high concentrations of sucrose (a disaccharide comprising of glucose and fructose). To access the available carbon, aphids hydrolyze sucrose in the gut lumen and transport its constituent monosaccharides, glucose and fructose. Although sugar transport plays a critical role in aphid nutrition, the molecular basis of sugar transport in aphids, and more generally across all insects, remains poorly characterized. Here, using the latest release of the pea aphid, Acyrthosiphon pisum, genome we provide an updated gene annotation and expression profile of putative sugar transporters. Finally, gut expressed sugar transporters are functionally expressed in yeast and screened for glucose and fructose transport activity., Results: In this study, using a de novo approach, we identified 19 sugar porter (SP) family transporters in the A. pisum genome. Gene expression analysis, based on 214, 834 A. pisum expressed sequence tags, supports 17 sugar porter family transporters being actively expressed in adult female aphids. Further analysis, using quantitative PCR identifies 4 transporters, A. pisum sugar transporter 1, 3, 4 and 9 (ApST1, ApST3, ApST4 and ApST9) as highly expressed and/or enriched in gut tissue. When expressed in a Saccharomyces cerevisiae hexose transporter deletion mutant (strain EBY.VW4000), only ApST3 (previously characterized) and ApST4 (reported here) transport glucose and fructose resulting in functional rescue of the yeast mutant. Here we characterize ApST4, a 491 amino acid protein, with 12 predicted transmembrane regions, as a facilitative glucose/fructose transporter. Finally, phylogenetic reconstruction reveals that ApST4, and related, as yet uncharacterized insect transporters are phylogenetically closely related to human GLUT (SLC2A) class I facilitative glucose/fructose transporters., Conclusions: The gut enhanced expression of ApST4, and the transport specificity of its product is consistent with ApST4 functioning as a gut glucose/fructose transporter. Here, we hypothesize that both ApST3 (reported previously) and ApST4 (reported here) function at the gut interface to import glucose and fructose from the gut lumen.

Production of recombinant protein bio-insecticides on a commercial scale can only be cost effective if host strains with very high expression levels are available. A recombinant fusion protein containing an arthropod toxin, ω-hexatoxin-Hv1a, (from funnel web spider Hadronyche versuta) linked to snowdrop lectin (Galanthus nivalis agglutinin; GNA) is an effective oral insecticide and candidate biopesticide. However, the fusion protein was vulnerable to proteolysis during production in the yeast Pichia pastoris. To prevent proteolysis, the Hv1a/GNA fusion expression construct was modified by site-directed mutagenesis to remove a potential Kex2 cleavage site at the C-terminus of the Hv1a peptide. To obtain a high expressing clone of P. pastoris to produce recombinant Hv1a/GNA, a straightforward method was used to produce multi-copy expression plasmids, which does not require multiple integrations to give clones of P. pastoris containing high copy numbers of the introduced gene. Removal of the Kex2 site resulted in increased levels of intact fusion protein expressed in wild-type P. pastoris strains, improving levels of intact recombinant protein recoverable. Incorporation of a C-terminal (His)6 tag enabled single step purification of the fusion protein. These modifications did not affect the insecticidal activity of the recombinant toxin towards lepidopteran larvae. Introduction of multiple expression cassettes increased the amount of secreted recombinant fusion protein in a laboratory scale fermentation by almost tenfold on a per litre of culture basis. Simple modifications in the expression construct can be advantageous for the generation of high expressing P. pastoris strains for production of a recombinant protein, without altering its functional properties.

Evidence is accumulating that commonly used pesticides are linked to decline of pollinator populations; adverse effects of three neonicotinoids on bees have led to bans on their use across the European Union. Developing insecticides that pose negligible risks to beneficial organisms such as honeybees is desirable and timely. One strategy is to use recombinant fusion proteins containing neuroactive peptides/proteins linked to a 'carrier' protein that confers oral toxicity. Hv1a/GNA (Galanthus nivalis agglutinin), containing an insect-specific spider venom calcium channel blocker (ω-hexatoxin-Hv1a) linked to snowdrop lectin (GNA) as a 'carrier', is an effective oral biopesticide towards various insect pests. Effects of Hv1a/GNA towards a non-target species, Apis mellifera, were assessed through a thorough early-tier risk assessment. Following feeding, honeybees internalized Hv1a/GNA, which reached the brain within 1 h after exposure. However, survival was only slightly affected by ingestion (LD50>100 µg bee(-1)) or injection of fusion protein. Bees fed acute (100 µg bee(-1)) or chronic (0.35 mg ml(-1)) doses of Hv1a/GNA and trained in an olfactory learning task had similar rates of learning and memory to no-pesticide controls. Larvae were unaffected, being able to degrade Hv1a/GNA. These tests suggest that Hv1a/GNA is unlikely to cause detrimental effects on honeybees, indicating that atracotoxins targeting calcium channels are potential alternatives to conventional pesticides.

Recombinant fusion protein technology allows specific insecticidal protein and peptide toxins to display activity in orally-delivered biopesticides. The spider venom peptide δ-amaurobitoxin-PI1a, which targets insect voltage-gated sodium channels, was fused to the "carrier" snowdrop lectin (GNA) to confer oral toxicity. The toxin itself (PI1a) and an amaurobitoxin/GNA fusion protein (PI1a/GNA) were produced using the yeast Pichia pastoris as expression host. Although both proteins caused mortality when injected into cabbage moth (Mamestra brassicae) larvae, the PI1a/GNA fusion was approximately 6 times as effective as recombinant PI1a on a molar basis. PI1a alone was not orally active against cabbage moth larvae, but a single 30 μg dose of the PI1a/GNA fusion protein caused 100% larval mortality within 6 days when fed to 3rd instar larvae, and caused significant reductions in survival, growth and feeding in 4th - 6th instar larvae. Transport of fusion protein from gut contents to the haemolymph of cabbage moth larvae, and binding to the nerve chord, was shown by Western blotting. The PI1a/GNA fusion protein also caused mortality when delivered orally to dipteran (Musca domestica; housefly) and hemipteran (Acyrthosiphon pisum; pea aphid) insects, making it a promising candidate for development as a biopesticide., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

The interaction between Hessian fly (Mayetiola destructor) and wheat (Triticum aestivum) involves a gene-for-gene resistance mechanism. The incompatible interaction leading to resistance involves up-regulation of several Hfr (Hessian fly responsive) genes encoding proteins with potential insecticidal activity. The encoded proteins HFR-1, HFR-2 and HFR-3 all possess lectin-like domains. HFR-1 and HFR-3 were produced as recombinant proteins using Escherichia coli and Pichia pastoris, respectively as expression hosts. Purified recombinant proteins were assayed for insecticidal effects towards cereal aphid (Sitobion avenae), an insect to which wheat shows only tolerance. Both HFR-1 and HFR-3 were found to be insecticidal towards S. avenae when fed in artificial diet. Although HFR-3 has sequence similarity and similar chitin-binding activity to wheat germ agglutinin (WGA), the latter protein was almost non-toxic to S. avenae. HFR-3 binds strongly to aphid midguts after ingestion, whereas WGA binds but does not persist over a feed-chase period. Quantitative PCR showed that Hfr-3 mRNA does not increase in level after cereal aphid infestation. The results suggest that the lack of effective resistance to cereal aphid in wheat is not due to an absence of genes encoding suitable insecticidal proteins, but results from a failure to up-regulate gene expression in response to aphid attack., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Background: The spider-venom peptide ω-hexatoxin-Hv1a (Hv1a) targets insect voltage-gated calcium channels, acting directly at sites within the central nervous system. It is potently insecticidal when injected into a wide variety of insect pests, but it has limited oral toxicity. We examined the ability of snowdrop lectin (GNA), which is capable of traversing the insect gut epithelium, to act as a "carrier" in order to enhance the oral activity of Hv1a., Methodology/principal Findings: A synthetic Hv1a/GNA fusion protein was produced by recombinant expression in the yeast Pichia pastoris. When injected into Mamestra brassicae larvae, the insecticidal activity of the Hv1a/GNA fusion protein was similar to that of recombinant Hv1a. However, when proteins were delivered orally via droplet feeding assays, Hv1a/GNA, but not Hv1a alone, caused a significant reduction in growth and survival of fifth stadium Mamestra brassicae (cabbage moth) larvae. Feeding second stadium larvae on leaf discs coated with Hv1a/GNA (0.1-0.2% w/v) caused ≥ 80% larval mortality within 10 days, whereas leaf discs coated with GNA (0.2% w/v) showed no acute effects. Intact Hv1a/GNA fusion protein was delivered to insect haemolymph following ingestion, as shown by Western blotting. Immunoblotting of nerve chords dissected from larvae following injection of GNA or Hv1a/GNA showed high levels of bound proteins. When insects were injected with, or fed on, fluorescently labelled GNA or HV1a/GNA, fluorescence was detected specifically associated with the central nerve chord., Conclusions/significance: In addition to mediating transport of Hv1a across the gut epithelium in lepidopteran larvae, GNA is also capable of delivering Hv1a to sites of action within the insect central nervous system. We propose that fusion to GNA provides a general mechanism for dramatically enhancing the oral activity of insecticidal peptides and proteins.

Proteinase inhibitors which act on the digestive enzymes of insect herbivores are a basic mechanism of plant defence. Attempts to exploit this defence mechanism in plant genetic engineering have used over-expression of both endogenous and exogenous inhibitors. While significant protection against insect pests has been routinely achieved, the engineered plants do not show levels of resistance considered commercially viable. As a result of selective pressures, insect herbivores have developed multiple mechanisms of adaptation to overcome the defensive effects of plant proteinase inhibitors. Common polyphagous crop pests are well adapted to deal with a range of different inhibitors, which have only limited effects on fitness as a result. A range of strategies have been attempted to improve effectiveness of proteinase inhibitors as antimetabolites towards insects, including selection for inhibitory activity against insect digestive enzymes, mutagenesis for novel inhibitory activity, and engineering inhibitors with multiple functions. However, proteinase inhibitor genes have only been used in transgenic crops in combination with other insecticidal genes. In Chinese genetically engineered cotton varieties which express Bt toxins as an insecticidal protein against lepidopteran larvae, the CpTI (cowpea trypsin inhibitor) gene has been employed as a second transgene to improve protection. This gene combination represents the only commercial deployment of a proteinase inhibitor transgene to date, with Bt/CpTI cotton grown on over 0.5 million hectares in 2005. Future prospects for using proteinase inhibitor genes to enhance insect resistance in transgenic crops will require reassessment of their mechanisms of action, particularly in affecting processes other than digestion, as exemplified by effects on sap-feeding hemipteran pests.

Gut extracts from cereal aphids (Sitobion avenae) showed significant levels of proteolytic activity, which was inhibited by reagents specific for cysteine proteases and chymotrypsin-like proteases. Gut tissue contained cDNAs encoding cathepsin B-like cysteine proteinases, similar to those identified in the closely related pea aphid (Acyrthosiphon pisum). Analysis of honeydew (liquid excreta) from cereal aphids fed on diet containing ovalbumin showed that digestion of ingested proteins occurred in vivo. Protein could partially substitute for free amino acids in diet, although it could not support complete development. Recombinant wheat proteinase inhibitors (PIs) fed in diet were antimetabolic to cereal aphids, even when normal levels of free amino acids were present. PIs inhibited proteolysis by aphid gut extracts in vitro, and digestion of protein fed to aphids in vivo. Wheat subtilisin/chymotrypsin inhibitor, which was found to inhibit serine and cysteine proteinases, was more effective in both inhibitory and antimetabolic activity than wheat cystatin, which inhibited cysteine proteases only. Digestion of ingested protein is unlikely to contribute significantly to nutritional requirements when aphids are feeding on phloem, and the antimetabolic activity of dietary proteinase inhibitors is suggested to result from effects on proteinases involved in degradation of endogenous proteins., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Conotoxins are a diverse collection of more than 50,000 peptides produced by predatory marine snails of the genus Conus in order to immobilize their prey. Many conotoxins modulate the activity of ion channels, and show high specificity to their targets; as a result, some have valuable pharmaceutical applications. However, obtaining active peptide is difficult and to date has only been achieved though natural collection, chemical synthesis, or the use of prokaryotic expression systems, which often have the disadvantage of requiring subsequent steps to correctly fold the peptide. This paper reports the production of a conotoxin, TxVIA from Conus textile, as a biologically active recombinant protein, using the yeast Pichia pastoris as expression host. The presence of the pro-peptide was found to be necessary for the expression of biologically active conotoxin. We also show that TxVIA is not, as previously reported, mollusc-specific, but also shows insecticidal activity when injected into lepidopteran (cabbage moth) and dipteran (house fly) larvae. In contrast, recombinant TxVIA was not found to be molluscicidal to the grey field slug Deroceras reticulatum., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Aphids are major insect pests of cereal crops, acting as virus vectors as well as causing direct damage. The responses of wheat to infestation by cereal aphid (Sitobion avenae) were investigated in a proteomic analysis. Approximately, 500 protein spots were reproducibly detected in the extracts from leaves of wheat seedlings after extraction and 2-DE. Sixty-seven spots differed significantly between control and infested plants following 24 h of aphid feeding, with 27 and 11 up-regulated, and 8 and 21 down-regulated, in local or systemic tissues, respectively. After 8 days, 80 protein spots differed significantly between control and aphid treatments with 13 and 18 up-regulated and 27 and 22 down-regulated in local or systemic tissues, respectively. As positive controls, plants were treated with salicylic acid or methyl jasmonate; 81 and 37 differentially expressed protein spots, respectively, were identified for these treatments. Approximately, 50% of differentially expressed protein spots were identified by PMF, revealing that the majority of proteins altered by aphid infestation were involved in metabolic processes and photosynthesis. Other proteins identified were involved in signal transduction, stress and defence, antioxidant activity, regulatory processes, and hormone responses. Responses to aphid attack at the proteome level were broadly similar to basal non-specific defence and stress responses in wheat, with evidence of down-regulation of insect-specific defence mechanisms, in agreement with the observed lack of aphid resistance in commercial wheat lines., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Background: Asian rust (Phakopsora pachyrhizi) is a common disease in Brazilian soybean fields and it is difficult to control. To identify a biochemical candidate with potential to combat this disease, a new chitinase-like xylanase inhibitor protein (XIP) from coffee (Coffea arabica) (CaclXIP) leaves was cloned into the pGAPZα-B vector for expression in Pichia pastoris., Results: A cDNA encoding a chitinase-like xylanase inhibitor protein (XIP) from coffee (Coffea arabica) (CaclXIP), was isolated from leaves. The amino acid sequence predicts a (β/α)8 topology common to Class III Chitinases (glycoside hydrolase family 18 proteins; GH18), and shares similarity with other GH18 members, although it lacks the glutamic acid residue essential for catalysis, which is replaced by glutamine. CaclXIP was expressed as a recombinant protein in Pichia pastoris. Enzymatic assay showed that purified recombinant CaclXIP had only residual chitinolytic activity. However, it inhibited xylanases from Acrophialophora nainiana by approx. 60% when present at 12:1 (w/w) enzyme:inhibitor ratio. Additionally, CaclXIP at 1.5 μg/μL inhibited the germination of spores of Phakopsora pachyrhizi by 45%., Conclusions: Our data suggests that CaclXIP belongs to a class of naturally inactive chitinases that have evolved to act in plant cell defence as xylanase inhibitors. Its role on inhibiting germination of fungal spores makes it an eligible candidate gene for the control of Asian rust.

Background: Insect hosts have evolved immunity against invasion by parasitoids, and in co-evolutionary response parasitoids have also developed strategies to overcome host immune systems. The mechanisms through which parasitoid venoms disrupt the promotion of host immunity are still unclear. We report here a new mechanism evolved by parasitoid Pteromalus puparum, whose venom inhibited the promotion of immunity in its host Pieris rapae (cabbage white butterfly)., Methodology/principal Findings: A full-length cDNA encoding a C-type lectin (Pr-CTL) was isolated from P. rapae. Quantitative PCR and immunoblotting showed that injection of bacterial and inert beads induced expression of Pr-CTL, with peaks of mRNA and Pr-CTL protein levels at 4 and 8 h post beads challenge, respectively. In contrast, parasitoid venom suppressed Pr-CTL expression when co-injected with beads, in a time and dose-dependent manner. Immunolocalization and immunoblotting results showed that Pr-CTL was first detectable in vesicles present in cytoplasm of granulocytes in host hemolymph, and was then secreted from cells into circulatory fluid. Finally, the secreted Pr-CTL bound to cellular membranes of both granulocytes and plasmatocytes. Injection of double-stranded RNA specific for target gene decreased expression of Pr-CTL, and a few other host immune-related genes. Suppression of Pr-CTL expression also down-regulated antimicrobial and phenoloxidase activities, and reducing phagocytotic and encapsulation rates in host. The inhibitory effect of parasitoid venom on host encapsulation is consistent with its effect in suppressing Pr-CTL expression. Binding assay results showed that recombinant Pr-CTL directly attached to the surface of P. puparum egges. We infer that Pr-CTL may serve as an immune signalling co-effector, first binding to parasitoid eggs, regulating expression of a set of immune-related genes and promoting host immunity., Conclusions/significance: P. puparum venom inhibits promotion of host immune responses by silencing expression of host C-type lectin gene Pr-CTL, whose expression affected transcription of other host immune-related genes.

The ability of phytophagous insects to utilise the relatively low nitrogen content of plant tissues is typically the limiting factor in their nutritional uptake. In the larval stage, the vine weevil feeds predominantly on root tissues of plants. The root tissue as a whole has low levels of free amino acids, and thus effective hydrolysis of dietary proteins is essential for survival. In contrast to previous reports the present study demonstrates through both molecular and biochemical studies the presence of proteolytic enzymes from two mechanistic classes, cysteine and serine proteases, in the gut of larval vine weevil; with the latter being the predominant form. cDNA clones encoding cathepsin B-like and serine-like sequences were isolated from a gut specific cDNA library; the cathepsin B-like clone has the Cys-His-Asn catalytic triad. However, the sequence showed the replacement of the conserved His-His sequence in the "occluding loop" region of the enzyme with Asp-His. This may result in a change to the substrate specificity. Two trypsin precursors contained evidence of a signal peptide, activation peptide, and conserved N-termini (IVGG). Other structural features included typical His, Asp, and Ser residues of the catalytic amino acid triad indicative of serine proteases, characteristic residues in the substrate-binding pocket, and four pairs of cysteine residues for disulfide bridges. The apparent abundance of the trypsin-like cDNA clones compared to the cathepsin B clones suggests that serine proteases are the predominant form, thus supporting data from the biochemical studies., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

An expression construct encoding chicken (Gallus gallus) avidin was assembled from amplified fragments of genomic DNA. Recombinant, functional avidin was produced in Pichia pastoris, with yields of up to 80 mg/l of culture supernatant. The recombinant avidin had similar insecticidal activity to egg white avidin when assayed against larvae of a lepidopteran crop pest, cabbage moth (Mamestra brassicae), causing >90% reduction in growth and 100% mortality when fed in optimised diets at levels of 1.5 microM and 15 microM (100 ppm and 1000 ppm wet weight of recombinant protein). The recombinant protein was also highly toxic to a hemipteran pest, the pea aphid (Acyrthosiphon pisum), when fed in liquid artificial diet, causing 100% mortality after 4 days when present at concentrations > or = 3.8 microM (0.25 mg/ml, 250 ppm). Mortality was dose-dependent, with an estimated LC(50) of 2.1 microM. Toxicity to A. pisum was prevented by biotin supplementation of diet. In contrast, avidin had no significant effects on the survival of cereal aphid (Sitobion avenae) at concentrations up to 30 microM in liquid diet. Analysis of genomic DNA showed that symbionts from both aphid species lack the ability to synthesise biotin de novo. Cereal aphids appear to be less sensitive to recombinant avidin in the diet through proteolysis of the ingested protein, which would allow recovery of bound biotin., (Crown Copyright 2010. Published by Elsevier Ltd. All rights reserved.)

Analysis of the pea aphid (Acyrthosiphon pisum) genome using signatures specific to the Major Facilitator Superfamily (Pfam Clan CL0015) and the Sugar&#95;tr family (Pfam Family PF00083) has identified 54 genes encoding potential sugar transporters, of which 38 have corresponding ESTs. Twenty-nine genes contain the InterPro IPR003663 hexose transporter signature. The protein encoded by Ap&#95;ST3, the most abundantly expressed sugar transporter gene, was functionally characterized by expression as a recombinant protein. Ap&#95;ST3 acts as a low-affinity uniporter for fructose and glucose that does not depend on Na(+) or H(+) for activity. Ap&#95;ST3 was expressed at elevated levels in distal gut tissue, consistent with a role in gut sugar transport. The A. pisum genome shows evidence of duplications of sugar transporter genes.

Background: The toxicity of a fusion protein, ButalT/GNA, comprising a venom toxin (ButaIT) derived from the red scorpion, Mesobuthus tamulus (F.), and Galanthus nivalis agglutinin (GNA), was evaluated under laboratory conditions against several pest insects. Insecticidal activity was compared with SFI1/GNA, a fusion comprising a venom toxin (SFI1) derived from the European spider Segestria florentina (Rossi) and GNA, which has been previously demonstrated to be effective against lepidopteran and hemipteran pests, and to GNA itself., Results: Injection assays demonstrated that both fusion proteins were toxic to lepidopteran larvae, dipteran adults, coleopteran adults and larvae and dictyopteran nymphs. ButalT/GNA was more toxic than SFI1/GNA in all cases. GNA itself made a minor contribution to toxicity. Oral toxicity of ButalT/GNA towards lepidopteran pests was confirmed against neonate Spodoptera littoralis (Boisd.), where incorporation at 2% dietary protein resulted in 50% mortality and > 85% reduction in growth compared with controls. ButaIT/GNA was orally toxic to Musca domestica L. adults, causing 75% mortality at 1 mg mL(-1) in aqueous diets and, at 2 mg g(-1) it was orally toxic to Tribolium castaneum (Herbst.), causing 60% mortality and a 90% reduction in growth., Conclusions: Toxicity of the ButaIT/GNA recombinant fusion protein towards a range of insect pests from different orders was demonstrated by injection bioassays. Feeding bioassays demonstrated the potential use of the ButaIT/GNA fusion protein as an orally active insecticide against lepidopteran, dipteran and coleopteran pests. These experiments provide further evidence that the development of fusion protein technology for the generation of new, biorational, anti-insect molecules holds significant promise.

A cDNA encoding a cathepsin L-like cysteine proteinase (DcCathL) was prepared from gut tissue of larvae of wheat bulb fly (Delia coarctata: Diptera). The predicted protein is a homologue of the product of Drosophila melanogaster gene Cp-1 (CG6692), and is similar to a sub-family of cysteine proteinases found in other insects which have roles in tissue remodelling during development, and moulting. Recombinant DcCathL was produced using the yeast Pichia pastoris as expression host, and showed hydrolytic activity in vitro towards the synthetic substrate Z-Phe-Arg-AMC with a pH optimum of 4.5. DcCathL was insecticidal to lepidopteran larvae when injected into haemolymph, causing mortality that was accompanied by systemic melanisation, suggesting that DcCathL was affecting the immune-related proteolytic activation cascade leading to production of active phenoloxidase. This process is normally negatively regulated by serpins in the haemolymph. Recombinant serpins from cabbage moth (Mamestra brassicae) did not inhibit DcCathL, and were susceptible to degradation by the enzyme in vitro in buffer and extracted haemolymph. When M. brassicae larvae were co-injected with a lethal dose of DcCathL and exogenous recombinant serpins, no mortality or systemic melanisation was observed, suggesting that the insecticidal effects of DcCathL in vivo result from degradation of endogenous serpins.

Three genes encoding clan MB metalloproteinases (EpMP1-3) were identified from venom glands of the ectoparasitic wasp Eulophus pennicornis. The derived amino acid sequences predict mature proteins of approximately 46 kDa, with a novel two-domain structure comprising a C-terminal reprolysin domain, and an N-terminal domain of unknown function. EpMP3 expressed as a recombinant protein in Pichia pastoris had gelatinase activity, which was inhibited by EDTA. Injection of recombinant EpMP3 into fifth instar Lacanobia oleracea (host) larvae resulted in partial insect mortality associated with the moult to sixth instar, with surviving insects showing retarded development and growth. EpMP3 is expressed specifically in venom glands. These results suggest that EpMP3 is a functional component of Eulophus venom, which is able to manipulate host development.

The heterodimeric and homodimeric garlic lectins ASAI and ASAII were produced as recombinant proteins in the yeast Pichia pastoris. The proteins were purified as functional dimeric lectins, but underwent post-translational proteolysis. Recombinant ASAII was a single homogenous polypeptide which had undergone C-terminal processing similar to that occurring in planta. The recombinant ASAI was glycosylated and subject to variable and heterogenous proteolysis. Both lectins showed insecticidal effects when fed to pea aphids (Acyrthosiphon pisum) in artificial diet, ASAII being more toxic than ASAI at the same concentration. Acute toxicity (mortality at < or =48 h exposure; similar timescale to starvation) was only apparent at the highest lectin concentrations tested (2.0 mg ml(-)1), but dose-dependent chronic toxicity (mortality at >3d exposure) was observed over the concentration range 0.125-2.0 mg ml(-1). The recombinant lectins caused mortality in both symbiotic and antibiotic-treated aphids, showing that toxicity is not dependent on the presence of the bacterial symbiont (Buchnera aphidicola), or on interaction with symbiont proteins, such as the previously identified lectin "receptor" symbionin. A pull-down assay coupled with peptide mass fingerprinting identified two abundant membrane-associated aphid gut proteins, alanyl aminopeptidase N and sucrase, as "receptors" for lectin binding.

Downregulation of the expression of specific genes through RNA interference (RNAi), has been widely used for genetic research in insects. The method has relied on the injection of double-stranded RNA (dsRNA), which is not possible for practical applications in crop protection. By contrast, specific suppression of gene expression in nematodes is possible through feeding with dsRNA. This approach was thought to be unfeasible in insects, but recent results have shown that dsRNA fed as a diet component can be effective in downregulating targeted genes. More significantly, expression of dsRNA directed against suitable insect target genes in transgenic plants has been shown to give protection against pests, opening the way for a new generation of insect-resistant crops.

ScathL is a cathepsin L-like cysteine protease derived from the flesh fly Sarcophaga peregrina that functions in basement membrane (BM) remodeling during insect development. A recombinant baculovirus expressing ScathL (AcMLF9.ScathL) kills larvae of the tobacco budworm, Heliothis virescens, significantly faster than the wild-type virus. Here, we show that the occurrence of larval melanization prior to death was closely associated with the onset of high cysteine protease activity of ScathL in the hemolymph of fifth instars infected with AcMLF9.ScathL, but not with AcMLF9.ScathL.C146A, a recombinant baculovirus expressing a catalytic site mutant of ScathL. Fragmented fat body, ruptured gut and malpighian tubules, and melanized tracheae were observed in AcMLF9.ScathL-infected larvae. Phenoloxidase activity in hemolymph was unchanged, but the pool of prophenoloxidase was significantly reduced in virus-infected larvae and further reduced in AcMLF9.ScathL-infected larvae. The median lethal dose (LD(50)) for purified ScathL injected into fifth-instar H. virescens was 11.0 microg/larva. ScathL was also lethal to adult pea aphids, Acyrthosiphon pisum with a similar loss of integrity of the gut and fat body. Injection with purified ScathL.C146A or bovine trypsin at 20 microg/larva did not produce any effect in either insect. These results illustrate the potent insecticidal effects of ScathL cysteine protease activity and the potential for use of ScathL in development of insect resistant transgenic plants when combined with an appropriate delivery system.

Proteolytic activities in soluble protein extracts from Mamestra brassicae (cabbage moth) larval midgut were analysed using specific peptide substrates and proteinase inhibitors. Serine proteinases were the major activities detected, with chymotrypsin-like and trypsin-like activities being responsible for approximately 62% and 19% of the total proteolytic activity towards a non-specific protein substrate. Only small amounts of elastase-like activities could be detected. The serine proteinases were active across the pH range 7-12.5, with both trypsin-like and chymotrypsin-like activities maximal at pH 11.5. The digestive proteinases were stable to the alkaline environment of the lepidopteran gut over the timescale of passage of food through the gut, with 50% of trypsin and 40% of chymotrypsin activity remaining after 6h at pH 12, 37 degrees C. Soybean Kunitz trypsin inhibitor (SKTI) ingestion by the larvae had a growth-inhibitory effect, and induced inhibitor-insensitive trypsin-like activity. Qualitative and quantitative changes in proteinase activity bands after gel electrophoresis of gut extracts were evident in SKTI-fed larvae when compared with controls, with increases in levels of most bands, appearance of new bands, and a decrease in the major proteinase band present in extracts from control insects.

Phloem-sap feeding Hemipteran insects have access to a sucrose-rich diet but are dependent on sucrose hydrolysis and hexose transport for carbon nutrition. A cDNA library from Nilaparvata lugens (rice brown planthopper) was screened for clones encoding potential transmembrane transporters. A selected cDNA, NlHT1, encodes a 53kDa polypeptide with sequence similarity to facilitative hexose transporters of eukaryotes and prokaryotes, including GLUT1, the human erythrocyte hexose transporter. NlHT1 was expressed as a recombinant protein in the methylotropic yeast Pichia pastoris, and was identified in a membrane fraction isolated from transformed yeast cells. Transport experiments using membrane vesicles containing NlHT1 showed that the protein is a saturable, sodium independent transporter, with a relatively low affinity for glucose (K(m) 3.0mM), which can be inhibited by cytochalasin B. Competition experiments with fructose demonstrate NlHT1 is glucose specific. In situ localisation studies revealed that NlHT1 mRNA is expressed in N. lugens gut tissue, mainly in midgut regions, and that expression is absent in hindgut and Malpighian tubules. NlHT1 is therefore likely to play an important role in glucose transport from the gut, and in carbon nutrition in vivo. This is the first report of a facilitative glucose transporter from a phloem-feeding insect pest.

Glycoprotein folding is mediated by lectin-like chaperones and protein disulfide isomerases (PDIs) in the endoplasmic reticulum. Calnexin and the PDI homologue ERp57 work together to help fold nascent polypeptides with glycans located toward the N-terminus of a protein, whereas PDI and BiP may engage proteins that lack glycans or have sugars toward the C-terminus. In this study, we show that the PDI homologue PDILT is expressed exclusively in postmeiotic male germ cells, in contrast to the ubiquitous expression of many other PDI family members in the testis. PDILT is induced during puberty and represents the first example of a PDI family member under developmental control. We find that PDILT is not active as an oxido-reductase, but interacts with the model peptide Delta-somatostatin and nonnative bovine pancreatic trypsin inhibitor in vitro, indicative of chaperone activity. In vivo, PDILT forms a tissue-specific chaperone complex with the calnexin homologue calmegin. The identification of a redox-inactive chaperone partnership defines a new system of testis-specific protein folding with implications for male fertility.

ScathL is a cathepsin L-like cysteine proteinase from Sarcophaga peregrina (flesh fly), which is involved in differentiation of imaginal discs, through proteolysis of components of basement membranes. An expression system based on the methylotrophic yeast Pichia pastoris was used to produce recombinant ScathL. Although the expression construct contained the full proprotein coding sequence for ScathL, the proprotein was only detected in culture supernatant at early stages of expression by Western blotting. The purified recombinant protein contained only a polypeptide similar to mature ScathL, as a result of autocatalytic processing. After activation by reducing agents, the enzyme hydrolysed the cathepsin L substrate Z-Phe-Arg-AMC, with optimal activity at pH 5.5. ScathL showed decreasing activity with increasing ionic strength above 0.3M NaCl, and lost activity irreversibly at pH > or = 7.5. The enzyme showed limited activity towards protein substrates, digesting only to large fragments. ScathL was insecticidal towards larvae of the tomato moth, Lacanobia oleracera, following injection into the haemolymph. It caused melanisation, although no evidence of extensive proteolysis in haemolymph or gut was observed. Production of a inactive mutant form of ScathL showed that enzyme activity was necessary for the complete proprotein processing observed during production as a recombinant protein, and for insecticidal activity.

The hydrolysis of sucrose, the principal dietary source of carbon for aphids, is catalysed by a gut alpha-glucosidase/transglucosidase activity. An alpha-glucosidase, referred to as APS1, was identified in both a gut-specific cDNA library and a sucrase-enriched membrane preparation from guts of the pea aphid Acyrthosiphon pisum by a combination of genomic and proteomic techniques. APS1 contains a predicted signal peptide, and has a predicted molecular mass of 68 kDa (unprocessed) or 66.4 kDa (mature protein). It has amino acid sequence similarity to alpha-glucosidases (EC 3.2.1.20) of glycoside hydrolase family 13 in other insects. The predicted APS1 protein contains two domains: an N-terminal catalytic domain, and a C-terminal hydrophobic domain. In situ localisation and RT-PCR studies revealed that APS1 mRNA was expressed in the gut distal to the stomach, the same localisation as sucrase activity. When expressed heterologously in Xenopus embryos, APS1 was membrane-bound and had sucrase activity. It is concluded that APS1 is a dominant, and possibly sole, protein mediating sucrase activity in the aphid gut.

The adoption of insect-resistant transgenic crops has been increasing annually at double-digit rates since the commercial release of first-generation maize and cotton expressing a single modified Bacillus thuringiensis toxin (Bt) nine years ago. Studies have shown that these Bt crops can be successfully deployed in agriculture, which has led to a decrease in pesticide usage, and that they are environmentally benign. However, the sustainability and durability of pest resistance continues to be discussed. In this review, we focus on the science that underpins second- and third-generation insect-resistant transgenic plants and examine the appropriateness and relevance of models that are currently being used to determine deployment strategies to maximize sustainability and durability. We also review strategies that are being developed for novel approaches to transgenic insect pest control.

We report that protein phosphorylation is involved in the control of starch metabolism in Arabidopsis leaves at night. sex4 (starch excess 4) mutants, which have strongly reduced rates of starch metabolism, lack a protein predicted to be a dual specificity protein phosphatase. We have shown that this protein is chloroplastic and can bind to glucans and have presented evidence that it acts to regulate the initial steps of starch degradation at the granule surface. Remarkably, the most closely related protein to SEX4 outside the plant kingdom is laforin, a glucan-binding protein phosphatase required for the metabolism of the mammalian storage carbohydrate glycogen and implicated in a severe form of epilepsy (Lafora disease) in humans.

Background: Despite evidence suggesting a role in plant defence, the use of plant lectins in crop protection has been hindered by their low and species-specific insecticidal activity. Snowdrop lectin (Galanthus nivalis agglutinin; GNA) is transported to the haemolymph of insects after oral ingestion, and can be used as a basis for novel insecticides. Recombinant proteins containing GNA expressed as a fusion with a peptide or protein, normally only toxic when injected into the insect haemolymph, have the potential to show oral toxicity as a result of GNA-mediated uptake., Results: A gene encoding a toxin, ButaIT, from the red scorpion (Mesobuthus tamulus) was synthesised and assembled into expression constructs. One construct contained ButaIT alone, whereas the other contained ButaIT fused N-terminally to a GNA polypeptide (ButaIT/GNA). Both recombinant proteins were produced using the yeast Pichia pastoris as an expression host, and purified. Recombinant ButaIT and ButaIT/GNA were acutely toxic when injected into larvae of tomato moth (Lacanobia oleracea), causing slow paralysis, leading to mortality or decreased growth. ButaIT/GNA was chronically toxic when fed to L. oleracea larvae, causing decreased survival and weight gain under conditions where GNA alone was effectively non-toxic. Intact ButaIT/GNA was detected in larval haemolymph from insects fed the fusion protein orally, demonstrating transport of the linked polypeptide across the gut. Proteolysis of the fusion protein was also observed. ButaIT/GNA was significantly more toxic that GNA alone when fed to the homopteran Nilaparvata lugens (rice brown planthopper) in liquid artificial diet., Conclusion: The ButaIT/GNA recombinant fusion protein is toxic to lepidopteran larvae both when injected and when fed orally, showing the utility of GNA as a carrier to transport potentially toxic peptides and proteins across the insect gut. Although ButaIT has been claimed to be lepidopteran-specific, the fusion protein has more wide-ranging insecticidal activity. Fusion proteins based on plant lectins have potential applications in crop protection, both as exogenously applied treatments and as endogenous products in transgenic plants.

The SFI1/GNA fusion protein, comprising of snowdrop lectin (Galanthus nivalis agglutinin, GNA) fused to an insecticidal spider venom neurotoxin (Segestria florentina toxin 1, SFI1) was tested for toxicity against the rice brown planthopper Nilaparvata lugens (Stål) and the peach-potato aphid Myzus persicae (Sulzer) by incorporation into artificial diets. Significant effects on the mortality of N. lugens were observed, with 100% of the insects fed on the SFI1/GNA fusion protein diet dead by day 7. The survival of the aphid M. persicae was also reduced when fed on the SFI1/GNA fusion protein. After 14 days, only 49% of the aphids that were fed on the fusion protein were still alive compared with approximately 90% of the aphids fed on the control diet or on diet containing GNA only. The SFI1/GNA fusion protein also slowed the development of M. persicae, and the reproductive capacity of the aphids fed on the SFI1/GNA fusion protein was severely reduced. The ability of GNA to act as a carrier protein, and deliver the SFI1 neurotoxin to the haemolymph of N. lugens, following oral ingestion, was investigated. The successful delivery of intact SFI1/GNA fusion protein to the haemolymph of these insects was shown by western blotting. Haemolymph taken from the insects that were fed on the fusion protein contained two GNA-immunoreactive proteins of molecular weights corresponding to GNA and to the SFI1/GNA fusion protein., (Copyright 2005 British Crown Copyright. Published for SCI by John Wiley & Sons, Ltd.)

We investigated the response of Helicoverpa armigera larvae towards ingestion of Cicer arietinum Kunitz proteinase inhibitor (CaKPI), which caused antagonistic effects on developing H. armigera larvae. CaKPI-degrading proteinases were not detectable in either control or sensitized larvae. There were negligible increases in total proteinase activity, as well as in trypsin-like and chymotrypsin-like activities of H. armigera gut proteinases (HGPs). Decrease in sensitivity of HGPs to inhibition by CaKPI was not observed when the inhibitor was fed suggesting that the insect had not shown a specific adaptive response to dietary CaKPI. Semi-quantitative reverse transcriptase polymerase chain reaction (Q RT-PCR) analysis showed a general up-regulation of proteases in larvae that ingested CaKPI and a specific regulation of individual transcripts was not observed. CaKPI had maximum inhibitory activity against HGP derived from fourth instar larvae. CaKPI was equally potent in inhibition of HGPs derived from larvae fed on different host plants, as well as various proteinase inhibitors (PIs) to which larval adaptation was previously reported. The lack of larval response to CaKPI was attributable to the atypical active site sequence and inhibitory activity of CaKPI and/or to the pre-adaptation of H. armigera larvae due to the constant exposure to basal levels of CaKPI in chickpea seeds or a chickpea seed-based diet.

Bacillus thuringiensis (Bt) crystal protein genes encode insecticidal delta-endotoxins that are widely used for the development of insect-resistant crops. In this article, we describe an alternative transgenic strategy that has the potential to generate broader and more sustainable levels of resistance against insect pests. Our strategy involves engineering plants with a fusion protein combining the delta-endotoxin Cry1Ac with the galactose-binding domain of the nontoxic ricin B-chain (RB). This fusion, designated BtRB, provides the toxin with additional, binding domains, thus increasing the potential number of interactions at the molecular level in target insects. Transgenic rice and maize plants engineered to express the fusion protein were significantly more toxic in insect bioassays than those containing the Bt gene alone. They were also resistant to a wider range of insects, including important pests that are not normally susceptible to Bt toxins. The potential impact of fusion genes such as BtRB in terms of crop improvement, resistance sustainability, and biosafety is discussed.

Two residues that have been implicated in determining the substrate specificity of the thermophilic beta-glycosidase from the archaeon Sulfolobus solfataricus (SsbetaG), a member of the glycosyl hydrolase family 1, have been mutated by site-directed mutagenesis so as to create more versatile catalysts for carbohydrate chemistry. The wild-type and mutated sequences were expressed in E. coli with a His(7)-tag to allow one-step chromatographic purification. The E432C and W433C mutations removed key interactions with the OH-4 and OH-3 of the sugar substrates, thus reducing the discrimination of glucose, galactose and fucose with respect to other glycosides. This resulted in two glycosidases with greatly broadened substrate specificities. Observed changes include a 24-fold increase in Man:Gal activity and an 18-fold increase in GalA:Gal activity. This promiscuous substrate tolerance was further illustrated by the parallel synthesis of a beta-glycoside library of glucose, galactose, xylose and mannose in one pot at 50 degrees C, in organic solvent. The synthetic potential of the catalysts was further evaluated through alkyl glycoside transglycosylation yields, including the first examples of synthesis of beta-mannosides and beta-xylosides with SsbetaG.

Chickpea (Cicer arietinum L.) seeds contain Bowman-Birk proteinase inhibitors, which are ineffective against the digestive proteinases of larvae of the insect pest Helicoverpa armigera. We have identified and purified a low expressing proteinase inhibitor (PI), distinct from the Bowman-Birk Inhibitors and active against H. armigera gut proteinases (HGP), from chickpea seeds. N-terminal sequencing of this HGP inhibitor revealed a sequence similar to reported pea (Pisum sativum) and chickpea alpha-l-fucosidases and also homologous to legume Kunitz inhibitors. The identity was confirmed by matrix assisted laser desorption ionization - time of flight analysis of tryptic peptides and isolation of DNA sequence coding for the mature protein. Available sequence data showed that this protein forms a distinct phylogenetic cluster with Kunitz inhibitors from Glycine max, Medicago truncatula, P. sativum and Canavalia lineata. The isolated coding sequence was cloned into a yeast expression vector and produced as a recombinant protein in Pichia pastoris. alpha-l-fucosidase activity was not detectable in purified or recombinant protein, by solution assays. The recombinant protein did not inhibit chymotrypsin or subtilisin activity but did exhibit stoichiometric inhibition of trypsin, comparable to soybean Kunitz trypsin inhibitor. The recombinant protein exhibited higher inhibition of total HGP activity as compared to soybean kunitz inhibitor, even though it preferentially inhibited HGP-trypsins. H. armigera larvae fed on inhibitor-incorporated artificial diet showed significant reduction in average larval weight after 18 days of feeding demonstrating potent antimetabolic activity. The over-expression of this gene in chickpea could act as an endogenous source of resistance to H. armigera.

Abstract Insect-resistant transgenic plants have been suggested to have deleterious effects on beneficial predators feeding on crop pests, through transmission of the transgene product by the pest to the predator. To test this hypothesis, effects of oilseed rape expressing the serine protease inhibitor, mustard trypsin inhibitor -2 (MTI-2), on the predatory ground beetle Pterostichus madidus were investigated, using diamondback moth, Plutella xylostella as the intermediary pest species. As expected, oilseed rape expressing MTI-2 had a deleterious effect on the development and survival of the pest. However, incomplete pest mortality resulted in survivors being available to predators at the next trophic level, and inhibition studies confirmed the presence of biologically active transgene product in pest larvae. Characterization of proteolytic digestive enzymes of P. madidus demonstrated that adults utilize serine proteases with trypsin-like and chymotrypsin-like specificities; the former activity was completely inhibited by MTI-2 in vitro. When P. madidus consumed prey reared on MTI-2 expressing plants over the reproductive period in their life cycle, no significant effects upon survival were observed as a result of exposure to the inhibitor. However, there was a short-term significant inhibition of weight gain in female beetles fed unlimited prey containing MTI-2, with a concomitant reduction of prey consumption. Biochemical analyses showed that the inhibitory effects of MTI-2 delivered via prey on gut proteolysis in the carabid decreased with time of exposure, possibly resulting from up-regulation of inhibitor-insensitive proteases. Of ecological significance, consumption of MTI-2 dosed prey had no detrimental effects on reproductive fitness of adult P. madidus.

RNA extracted from guts of rice brown planthopper, Nilaparvata lugens, was used to clone cDNA predicted to encode a diuretic hormone receptor (DHR). The DHR, a member of the calcitonin/secretin/corticotropin-releasing factor family of G-protein-coupled receptors, contains seven transmembrane domains and a large N-terminal extracellular domain potentially involved in hormone binding. The N-terminal domain was expressed as a recombinant protein, purified and used to raise antibodies. Anti-DHR IgG bound specifically to Malpighian tubules in immunolocalization experiments using dissected guts, and to a putative DHR polypeptide from N. lugens gut on Western blots. Anti-DHR IgG delivered orally to insects was not detected in the haemolymph, and showed no binding to gut or tubules, confirming that DHR N-terminal hormone-binding domain is not exposed to the gut lumen.

Chitinases are vital to moulting in insects, and may also affect gut physiology through their involvement in peritrophic membrane turnover. A cDNA encoding chitinase was cloned from larvae of tomato moth (Lacanobia oleracea), a Lepidopteran pest of crops. The predicted protein contains 553 amino acid residues, with a signal peptide of 20 a.a. Sequence comparison showed 75-80% identity with other Lepidopteran chitinases. L. oleracea chitinase was produced as a functional recombinant enzyme in the yeast Pichia pastoris. A fusion protein containing chitinase joined to the N-terminus of snowdrop lectin (GNA) was also produced, to determine whether GNA could deliver chitinase to the haemolymph of Lepidopteran larvae after oral ingestion. The purified recombinant proteins exhibited similar levels of chitinase activity in vitro. Both proteins were highly toxic to L. oleracea larvae on injection, causing 100% mortality at low dose (2.5 microg/g insect). Injection of chitinase prior to the moult resulted in decreased cuticle thickness. The recombinant proteins caused chronic effects when fed, causing reductions in larval growth and food consumption by up to 60%. The oral toxicity of chitinase was not increased by attaching GNA in the fusion protein, due to degradation in the larval gut, preventing GNA acting as a "carrier".