Directed Mutagenesis for Arginine Substitution of a Recombinant Lectin Disrupts Its Cytotoxic Activity.

Recently, we reported that a recombinant Tepary bean () lectin (rTBL-1) induces apoptosis in colon cancer cell lines and that cytotoxicity was related to differential recognition of β1-6 branched -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.

Septoria Leaf Blotch and Reduced Nitrogen Availability Alter WRKY Transcription Factor Expression in a Codependent Manner.

A major cause of yield loss in wheat worldwide is the fungal pathogen , 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.

Recombinant Lectin from Tepary Bean () with Specific Recognition for Cancer-Associated Glycans: Production, Structural Characterization, and Target Identification.

Herein, we report the production of a recombinant Tepary bean lectin (TBL-1), its three-dimensional (3D) structure, and its differential recognition for cancer-type glycoconjugates. TBL-1 was expressed in yielding 316 mg per liter of culture, and was purified by nickel affinity chromatography. Characterization of the protein showed that TBL-1 is a stable 120 kDa homo-tetramer folded as a canonical leguminous lectin with two divalent cations (Ca and Mn) attached to each subunit, confirmed in its 3D structure solved by X-ray diffraction at 1.

Demonstrating the potential of a novel spider venom-based biopesticide for target-specific control of the small hive beetle, a serious pest of the European honeybee.

The parasitic small hive beetle () feeds on pollen, honey and brood of the European honey bee (); 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 - or -terminus of snowdrop lectin (GNA) were used to produce recombinant Hv1a/GNA and GNA/Hv1a fusion proteins.

A Systematic Study of RNAi Effects and dsRNA Stability in and , Following Injection and Ingestion of Analogous dsRNAs.

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 () and recalcitrant pea aphid () following delivery of dsRNAs of identical length targeting expression of V-type ATPase subunit E () and inhibitor of apoptosis () genes. Injection and ingestion of and dsRNAs resulted in up to 100% mortality of larvae and sustained suppression (>80%) of transcript levels.

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 (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.

Systemic RNAi in the small hive beetle Aethina tumida Murray (Coleoptera: Nitidulidae), a serious pest of the European honey bee Apis mellifera.

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.

Sublethal effects of the insecticidal fusion protein ω-ACTX-Hv1a/GNA on the parasitoid Eulophus pennicornis via its host Lacanobia oleracea.

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.

Proteomic analysis shows that stress response proteins are significantly up-regulated in resistant diploid wheat () in response to attack by the grain aphid ().

The grain aphid (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.

Transgenic plants expressing ω-ACTX-Hv1a and snowdrop lectin (GNA) fusion protein show enhanced 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.

Genome-wide annotation and functional identification of aphid GLUT-like sugar transporters.

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.

Effect of insecticidal fusion proteins containing spider toxins targeting sodium and calcium ion channels on pyrethroid-resistant strains of peach-potato aphid (Myzus persicae).

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.

Novel biopesticide based on a spider venom peptide shows no adverse effects on honeybees.

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.

Optimising expression of the recombinant fusion protein biopesticide ω-hexatoxin-Hv1a/GNA in Pichia pastoris: sequence modifications and a simple method for the generation of multi-copy strains.

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.

A recombinant fusion protein containing a spider toxin specific for the insect voltage-gated sodium ion channel shows oral toxicity towards insects of different orders.

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.

Fusion to snowdrop lectin magnifies the oral activity of insecticidal ω-Hexatoxin-Hv1a peptide by enabling its delivery to the central nervous system.

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.

Insecticidal activity of wheat Hessian fly responsive proteins HFR-1 and HFR-3 towards a non-target wheat pest, cereal aphid (Sitobion avenae F.).

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.

Venom of parasitoid, Pteromalus puparum, suppresses host, Pieris rapae, immune promotion by decreasing host C-type lectin gene expression.

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).

Molecular interactions between wheat and cereal aphid (Sitobion avenae): analysis of changes to the wheat proteome.

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.

Protein digestion in cereal aphids (Sitobion avenae) as a target for plant defence by endogenous proteinase inhibitors.

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.

Prospects for using proteinase inhibitors to protect transgenic plants against attack by herbivorous insects.

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.

A new chitinase-like xylanase inhibitor protein (XIP) from coffee (Coffea arabica) affects Soybean Asian rust (Phakopsora pachyrhizi) spore germination.

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.

Molecular and biochemical characterisation of a dual proteolytic system in vine weevil larvae (Otiorhynchus sulcatus Coleoptera: Curculionidae).

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.

Insecticidal activity of recombinant avidin produced in yeast.

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.

Insecticidal activity of scorpion toxin (ButaIT) and snowdrop lectin (GNA) containing fusion proteins towards pest species of different orders.

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.