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 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.

A Systematic Study of RNAi Effects and dsRNA Stability in Tribolium castaneum and Acyrthosiphon pisum, 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 (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.

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. 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.

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. 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.

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. 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.

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. 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.

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. The amino acid sequence predicts a (ß/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.

A developmentally regulated chaperone complex for the endoplasmic reticulum of male haploid germ cells.

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.

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

Herein, we report the production of a recombinant Tepary bean lectin (rTBL-1), its three-dimensional (3D) structure, and its differential recognition for cancer-type glycoconjugates. rTBL-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 rTBL-1 is a stable 120 kDa homo-tetramer folded as a canonical leguminous lectin with two divalent cations (Ca2+ and Mn2+) 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 rTBL-1 targets by glycan array identified 14 positive binders, all of which correspond to ß1-6 branched N-glycans' characteristics of cancer cells. The presence of α1-6 core fucose, also tumor-associated, improved carbohydrate recognition. rTBL-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.

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 (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 LD50s 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 (LC50s 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.

RNAi-mediated crop protection against insects.

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.

The insecticidal activity of recombinant garlic lectins towards aphids.

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.

Biochemical characterization of midgut digestive proteases from Mamestra brassicae (cabbage moth; Lepidoptera: Noctuidae) and effect of soybean Kunitz inhibitor (SKTI) in feeding assays.

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.

Insecticidal activity of a basement membrane-degrading protease against Heliothis virescens (Fabricius) and Acyrthosiphon pisum (Harris).

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.

Recent developments and future prospects in insect pest control in transgenic crops.

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.

Functional expression and characterisation of a gut facilitative glucose transporter, NlHT1, from the phloem-feeding insect Nilaparvata lugens (rice brown planthopper).

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.

Characterisation of functional and insecticidal properties of a recombinant cathepsin L-like proteinase from flesh fly (Sarcophaga peregrina), which plays a role in differentiation of imaginal discs.

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.

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. 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.

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. 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.

A fusion protein containing a lepidopteran-specific toxin from the South Indian red scorpion (Mesobuthus tamulus) and snowdrop lectin shows oral toxicity to target insects.

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.