Sugar transporters of the major facilitator superfamily in aphids; from gene prediction to functional characterization.

Analysis of the pea aphid (Acyrthosiphon pisum) genome using signatures specific to the Major Facilitator Superfamily (Pfam Clan CL0015) and the Sugar_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_ST3, the most abundantly expressed sugar transporter gene, was functionally characterized by expression as a recombinant protein. Ap_ST3 acts as a low-affinity uniporter for fructose and glucose that does not depend on Na(+) or H(+) for activity. Ap_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.

A venom metalloproteinase from the parasitic wasp Eulophus pennicornis is toxic towards its host, tomato moth (Lacanobia oleracae).

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.

Molecular characterisation of a candidate gut sucrase in the pea aphid, Acyrthosiphon pisum.

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.

Crystal structure of a novel mid-gut procarboxypeptidase from the cotton pest Helicoverpa armigera.

The cotton bollworm Helicoverpa armigera (Hubner) (Lepidoptera: Noctuidae) is one of the most serious insect pests in Australia, India and China. The larva causes substantial economical losses to legume, fibre, cereal oilseed and vegetable crops. This pest has proven to be difficult to control by conventional means, mainly due to the development of pesticide resistance. We present here the 2.5 A crystal structure from the novel procarboxypeptidase (PCPAHa) found in the gut extracts from H. armigera larvae, the first one reported for an insect. This metalloprotease is synthesized as a zymogen of 46.6 kDa which, upon in vitro activation with Lys-C endoproteinase, yields a pro-segment of 91 residues and an active carboxypeptidase moiety of 318 residues. Both regions show a three-dimensional structure quite similar to the corresponding structures in mammalian digestive carboxypeptidases, the most relevant structural differences being located in the loops between conserved secondary structure elements, including the primary activation site. This activation site contains the motif (Ala)(5)Lys at the C terminus of the helix connecting the pro- and the carboxypeptidase domains. A remarkable feature of PCPAHa is the occurrence of the same (Ala)(6)Lys near the C terminus of the active enzyme. The presence of Ser255 in PCPAHa instead of Ile and Asp found in the pancreatic A and B forms, respectively, enlarges the S1' specificity pocket and influences the substrate preferences of the enzyme. The C-terminal tail of the leech carboxypeptidase inhibitor has been modelled into the PCPAHa active site to explore the substrate preferences and the enzymatic mechanism of this enzyme.

Characterisation of potato (Solanum tuberosum L.) extensins: a novel extensin-like cDNA from dormant tubers.

A cDNA library from dormant potato tubers was screened for extensin (Ext)-like cDNAs (PTEL). The cDNAs recovered were partially sequenced and could be divided into two classes. One class, containing the repeating amino acid (aa) motifs, SPPPPSPKYVYK and SPPPPSPSPPPPYYYK, constitutes the potato counterpart of the tomato Ext-encoding gene previously isolated by Zhou et al. [Plant Mol. Biol. 20 (1992) 5-17]. A full-length cDNA from the second class was fully sequenced. The product of this cDNA has the repetitive sequence and proline-rich composition typical of Ext. Features worthy of note are the signal peptide sequence, which shares close homology with signal sequences of a number of Ext of diverse origin, and the presence of a novel, histidine-rich motif (SPPPHHHHPVYK) repeated within the sequence. Wounding of the potato tuber causes a marked increase in Ext-like mRNAs, although the species induced are not those present in dormant tissue.

A gene from pea (Pisum sativum L.) with homology to metallothionein genes.

While searching for 'organ-specific' genes in pea (Pisum sativum L.) we have isolated a gene (designated PsMTA) which has an ORF encoding a predicted protein with some similarity to metallothioneins (MTs). The PsMTA transcript is abundant in roots which have not been exposed to elevated concentrations of trace metals.

Tailoring the substrate specificity of the beta-glycosidase from the thermophilic archaeon Sulfolobus solfataricus.

The substrate specificity of the thermophilic beta-glycosidase (lacS) from the archaeon Sulfolobus solfataricus (SSbetaG), a member of the glycohydrolase family 1, has been analysed at a molecular level using predictions from known protein sequences and structures and through site-directed mutagenesis. Three critical residues were identified and mutated to create catalysts with altered and broadened specificities for use in glycoside synthesis. The wild-type (WT) and mutated sequences were expressed as recombinant fusion proteins in Escherichia coli, with an added His(6)-tag to allow one-step chromatographic purification. Consistent with side-chain orientation towards OH-6, the single Met439-->Cys mutation enhances D-xylosidase specificity 4.7-fold and decreases D-fucosidase activity 2-fold without greatly altering its activity towards other D-glycoside substrates. Glu432-->Cys and Trp433-->Cys mutations directed towards OH-4 and -3, respectively, more dramatically impair glucose (Glc), galactose (Gal), fucose specificity than for other glycosides, resulting in two glycosidases with greatly broadened substrate specificities. These include the first examples of stereospecificity tailoring in glycosidases (e.g. WT-->W433C, k(cat)/K(M) (Gal):k(cat)/K(M) (mannose (Man))=29.4:1-->1.2:1). The robustness and high utility of these broad specificity SSbetaG mutants in parallel synthesis were demonstrated by the formation of libraries of beta-glycosides of Glc, Gal, xylose, Man in one-pot preparations at 50 degrees C in the presence of organic solvents, that could not be performed by SSbetaG-WT.

Transgenic indica rice resistant to sap-sucking insects.

Agrobacterium-mediated genetic transformation has been optimized in indica rice susceptible to sap-sucking insects, viz., brown planthopper (BPH) and green leafhopper (GLH). Snowdrop lectin gene (gna) from Galanthus nivalis, driven by phloem-specific rice-sucrose-synthase promoter, along with herbicide resistance gene (bar) driven by CaMV 35S promoter, was employed for genetic transformation. Embryogenic calli--after co-cultivation with Agrobacterium strain LBA4404 harbouring Ti plasmid pSB111-bar-gna--were selected on the medium containing phosphinothricin. PCR and Southern blot analyses confirmed the stable integration of both the genes into genomes of transgenic (T0) rice plants. Northern and Western blot analyses revealed the expression of gna in the transgenic plants. In the T1 and T2 generations, the gna and bar transgenes showed co-segregation at a ratio of 3 : 1. Plant progenies expressing gna, in T1 and T2, exhibited substantial resistance against BPH and GLH pests. This is the first report dealing with transgenic indica rice exhibiting high resistance to both insects.

Midgut carboxypeptidase from Helicoverpa armigera (Lepidoptera: Noctuidae) larvae: enzyme characterisation, cDNA cloning and expression.

Using synthetic substrates we have characterised carboxypeptidase activity in gut extracts from Helicoverpa armigera larvae. Carboxypeptidase A activity predominates, with only low levels of carboxypeptidase B activity present. Maximum carboxypeptidase A activity occurs over a broad pH range and is inhibited by phenanthroline and potato carboxypeptidase inhibitor. A cDNA clone encoding carboxypeptidase (the first such sequence from a lepidopteran insect) was isolated from a larval gut library. The sequence predicts a secreted polypeptide of Mr 46.6 k with homology to metallocarboxypeptidases from mammalian and invertebrate species. The presence of a serine residue at the active site suggests carboxypeptidase A activity. To further characterise the gene product, the complete cDNA sequence was expressed in insect cells using the baculovirus system. Culture supernatant from these cells contained carboxypeptidase A activity, with no activity against a carboxypeptidase B substrate; the carboxypeptidase B activity in gut extracts must thus be due to a separate enzyme. In agreement with this conclusion, the expressed carboxypeptidase cDNA is a member of a small multigene family. Chronic ingestion of soybean Kunitz trypsin inhibitor by H. armigera larvae results in increased accumulation of carboxypeptidase mRNA in the midgut cells, and an increase in carboxypeptidase A activity detected in gut extract.

Differentially regulated inhibitor-sensitive and insensitive protease genes from the phytophagous insect pest, Helicoverpa armigera, are members of complex multigene families.

Ingestion of soybean Kunitz trypsin inhibitor (SKTI) by larvae of the phytophagous insect pest Helicoverpa armigera induced production of inhibitor-insensitive protease activity. The induced activity was not due to proteolytic enzymes of different mechanistic classes, but rather to variants of the existing enzymes. Characterization of cDNAs showed that sequences encoding proteins of the serine protease family were abundant in gut tissue of both control and SKTI-fed insects. The majority of serine protease family cDNAs encode enzymes closely homologous to trypsin and chymotrypsin; comparison of these sequences shows variation in amino acid residues within the region which would be in contact with a protein protease inhibitor. More diverged sequences which may not encode active proteases are also present. All the cDNAs examined were found to derive from multigene families; at least 28 different genes are present in the serine protease family. Chronic ingestion of SKTI results in some serine protease-encoding mRNA species increasing in level, whereas others decrease. Chymotrypsin-encoding mRNAs tend to increase in level as a result of SKTI ingestion, but no clear trend is shown by trypsin-encoding mRNAs. It is suggested that multiple, varying protease-encoding genes are an adaptive mechanism for reducing the deleterious effects of plant protease inhibitors.

Ferritin acts as the most abundant binding protein for snowdrop lectin in the midgut of rice brown planthoppers (Nilaparvata lugens).

The mannose-specific snowdrop lectin [Galanthus nivalis agglutinin (GNA)] displays toxicity to the rice brown planthopper Nilaparvata lugens. A 26kDa GNA-binding polypeptide from N. lugens midgut was identified by lectin blotting and affinity chromatography, and characterized by N-terminal sequencing. This polypeptide is the most abundant binding protein for GNA in the N. lugens midgut. A cDNA (fersub2) encoding this protein was isolated from an N. lugens cDNA library. The deduced amino acid sequence shows significant homology to ferritin subunits from Manduca sexta and other arthropods, plants and vertebrates, and contains a putative N-glycosylation site. Native ferritin was purified from whole insects as a protein of more than 400kDa in size and characterized biochemically. Three subunits of 20, 26 and 27kDa were released from the native complex. The 26kDa subunit binds GNA, and its N-terminal sequence was identical to that of fersub2. A second cDNA (fersub1), exhibiting strong homology with dipteran ferritin, was identified as an abundant cDNA in an N. lugens midgut-specific cDNA library, and could encode the larger ferritin subunit. The fersub1 cDNA carries a stem-loop structure (iron-responsive element) upstream from the start codon, similar to structures that have been shown to play a role in the control of ferritin synthesis in other insects.

Putative protein digestion in a sap-sucking homopteran plant pest (rice brown plant hopper; Nilaparvata lugens: Delphacidae)--identification of trypsin-like and cathepsin B-like proteases.

Sap-sucking phytophagous insect species of the order Hemiptera have been assumed not to carry out digestive proteolysis, but instead to rely on free amino acids in the phloem and xylem saps for their nutritional requirements. Extracts prepared from isolated guts of rice brown planthopper (Nilaparvata lugens), a homopteran crop pest, were shown to contain protease activity, with hydrolysis of both protein and synthetic peptide substrates being observed. Assays with specific inhibitors suggested that a trypsin-like serine protease was responsible for most of hydrolytic activity against synthetic substrates. A cDNA library was prepared from RNA extracted from N. lugens gut tissue, and screened for protease-encoding sequences. cDNAs for a cathepsin B-like protease and a trypsin-like protease were isolated and fully characterised; the latter exhibits a novel C-terminal region and an unusual activation mechanism, and represents a small gene family. Soya bean Kunitz trypsin inhibitor (SKTI) is an effective inhibitor of protein hydrolysis by N. lugens gut extracts in vitro, explaining why transgenic rice plants expressing this protein are partially resistant to the insect (Mol. Breed. 5 (1999) 1). It is suggested that digestive proteolysis may be widespread in sap-sucking homoptera, and can make a significant contribution to nutrition.

Effect of dietary cowpea trypsin inhibitor (CpTI) on the growth and development of the tomato moth Lacanobia oleracea (Lepidoptera: Noctuidae) and on the success of the gregarious ectoparasitoid Eulophus pennicornis (Hymenoptera: Eulophidae).

Cowpea trypsin inhibitor (CpTI) was shown to have a deleterious effect on the growth and development of larvae of the tomato moth, Lacanobia oleracea, when incorporated in artificial diet (2.0% of soluble protein) and expressed in transgenic potato leaf (up to 1.0% of soluble protein). The effect of CpTI on parasitism of L oleracea by the ectoparasitoid Eulophus pennicornis was investigated. The parasitic success of the wasp was reduced by the presence of CpTI in the diet of the host and, in the case of transgenic potato leaves expressing the transgene protein, was collated with the length of time the host fed on the diet prior to parasitism. In all cases the proportion of hosts parasitised when fed CpTI-containing diets was reduced when compared with controls, although these differences were only significant when hosts were fed from the third instar on the transgenic potato leaves. Parasitoid progeny that developed on L oleracea reared on CpTI-containing diets, however, were not adversely affected. These results show that, whilst expression of CpTI in transgenic potato plants confers resistance to the lepidopterous pest L oleracea, adverse effects on the ability of the ectoparasitoid E pennicornis to parasitise this moth species successfully may also occur. These results are discussed in relation to the potential impact of transgenic crops on beneficial biological control agents.

Recombinant conotoxin, TxVIA, produced in yeast has insecticidal activity.

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.

Impact of oilseed rape expressing the insecticidal serine protease inhibitor, mustard trypsin inhibitor-2 on the beneficial predator Pterostichus madidus.

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.

Interaction of seed nuclear proteins with transcriptionally-enhancing regions of the pea (Pisum sativum L.) legA gene promoter.

An 873-basepairs promoter fragment (-833 to +40), of the legA (legumin seed storage protein) gene of pea is known to be fully functional in transgenic plants. This fragment has been enzymically cleaved, and the products examined for the ability to interact specifically with nuclear proteins. Use of DNA-binding and mobility-shift assays has shown that promoter sequences between -316 and +40 do not form stable complexes with seed nuclear extracts. Fragments from -549 to -316 and -833 to -582, however, did interact strongly with seed, but not leaf, nuclear proteins. Each probe reacted similarly to form three distinct and stable complexes, although only the complex with least mobility appeared to be specific when challenged with competitor DNA fragments. Competitor studies also indicate that a single factor (designated LABF1) forms these specific low-mobility complexes with both probes. Fractionation of seed nuclear proteins by sodium dodecyl sulphate polyacrylamide gel electrophoresis, followed by elution and renaturation, shows that LABF1 activity resides in the 84 000- to 116 000-Mr size range of polypeptides. The tissue-specific activity of LABF1 is temporally correlated with legumin gene expression, a relationship consistent with suggestions that this factor may act as a transcriptional enhancer.

A 38 bp repeat sequence within the pea seed storage protein promoter of legA is a binding site for a nuclear DNA-binding protein.

This paper describes a target sequence for a pea seed nuclear DNA-binding protein (of Pisum sativum) present at the cell expansion phase. Electromobility shift assays as well as in situ copper-phenanthroline footprinting have shown that this protein binds to an imperfect repeat sequence of the legA promoter located -404 tot -367 relative to the transcriptional start site, a region designated USR1 (-549 tot -316). Competition assays showed that the same or a related protein binds to USR2 located at -833 to -582. Another protein that does not recognize the repeat sequence as a target site binds adjacent but upstream to the repeat sequence in USR1. It is suggested that these protein-DNA interactions are initial events in the assembly of a transcriptional activation complex.

A comparison of the short and long term effects of insecticidal lectins on the activities of soluble and brush border enzymes of tomato moth larvae (Lacanobia oleracea).

When fed in semi-artificial diet in short- and long-term bioassays, the lectins from snowdrop (Galanthus nivalis; GNA) and jackbean (Canavalia ensiformis; Con A) affected the activities of soluble and brush border membrane (BBM) enzymes in the midgut of Lacanobia oleracea larvae. In the short term both lectins increased gut protein levels and BBM aminopeptidase activity. The lectins also increased trypsin activity, both in the gut (Con A) and in the faeces (GNA). GNA also increased the activity of alpha-glucosidase, but neither lectin had a significant effect on alkaline phosphatase activity. Trypsin mRNA levels were similar in lectin-fed and control larvae in the short term, showing that there is no direct effect on expression of the encoding genes. Larvae chronically exposed to GNA and Con A showed reductions in weight of 50-60%, and exhibited a significant reduction in alpha-glucosidase activity, but little change in other enzyme activities. Con A bound to many BBM and peritrophic matrix (PM) proteins in vitro, whereas GNA showed more specific binding, with strongest binding to a 94kDa uncharacterised BBM protein. Both lectins accumulated in gut tissues of insects after chronic exposure in vivo, but Con A was present at higher levels than GNA.

Sequence of a novel plant ras-related cDNA from Pisum sativum.

A clone isolated from a purple podded pea (Pisum sativum L.) cDNA library was shown to contain the complete coding sequence of a polypeptide with considerable homology to various members of the ras superfamily. The ras superfamily are a group of monomeric GTP-binding proteins of 21-25 kDa found in eukaryotic cells. Conserved sequences in the isolated clone include the GTP-binding site, GDP/GTP hydrolysis domain and C-terminal Cys residues involved in membrane attachment. Comparisons of the predicted amino acid sequence with those of other ras proteins show significantly higher homologies (ca. 70%) to two mammalian gene products, those of the BRL-ras oncogene, and the canine rab7 gene, than to any of the plant ras gene products so far identified (< 40% homology). The high percentage of amino acid identity suggests that this cDNA may be the product of a gene, designated Psa-rab, which is the plant counterpart of rab7. Rab/ypt proteins are a subfamily of the ras superfamily thought to be involved in intracellular transport from the endoplasmic reticulum to the Golgi apparatus and in vesicular transport. Northern blot hybridisation analysis of total RNA from green and purple podded pea revealed a mRNA species of approximately the same size as the isolated cDNAs.

Characterization and sequencing of cDNA clone encoding the phloem protein PP2 of Cucurbita pepo.

Direct N-terminal amino acid sequencing of the phloem protein 2 (PP2) from 3-month old Cucurbita pepo L. (pumpkin), purified by SDS-PAGE and blotted onto PVDF membrane, showed that the protein had a blocked N-terminus. However, after in situ cleavage of the polypeptide in a gel slice by cyanogen bromide, 75 residues of sequences on two cyanogen bromide fragments were determined. An oligonucleotide probe based on this amino acid sequence was used to screen a cDNA library, constructed from mRNA of 3-5-day old seedling hypocotyls, in lambda ZAP II. A cDNA clone (p11A) predicted an amino acid sequence of 218 residues, in full agreement with the sequences determined for two CNBr fragments of PP2, and suggests that the N-terminus of the protein is a blocked methionine residue which is cleaved off by CNBr. Two additional cDNA clones were sequenced but no heterogeneity in the PP2 sequence was found. The deduced amino acid sequence of C. pepo differs in nine residues from the recently published sequence of Cucurbita maxima (Bostwick et al., Plant Cell 4 (1992) 1539-1548). Southern blot showed that PP2 is encoded by a gene family with a relatively large number of members (estimated as 7-15 per haploid genome).