David P. Bown

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.

Regulation of expression of genes encoding digestive proteases in the gut of a polyphagous lepidopteran larva in response to dietary protease inhibitors

Abstract.  Larvae of Helicoverpa armigera (Hübner), a polyphagous lepidopteran crop pest, adapt to the presence of protease inhibitors in their diet by differential regulation of multiple genes encoding digestive proteases. The time‐course of their response to dietary soybean Kunitz trypsin inhibitor (SKTI) involves several stages; an initial up‐regulation of all protease genes assayed (up to 12 h after exposure to inhibitor) is succeeded by a longer‐term down‐regulation of expression of specific genes that encode proteases most sensitive to the inhibitor, whereas genes encoding putative inhibitor‐insensitive proteases continue to be up‐regulated (after 24 h of exposure). Consequently, insect protease activity changes from being sensitive to the inhibitor, to being largely insensitive. The insect response is comparable in its timescale with that of the synthesis of protease inhibitors in the plant wounding response. SKTl causes similar effects on protease gene expression and gut protease activity when fed in diets containing casein or hydrolysed casein as sources of amino acid, suggesting that the insect response is not mediated through inhibition of digestive proteolysis. Soybean Bowman–Birk inhibitor, which has a broader range of inhibitory activity against gut proteases in H. armigera, but is a less effective inhibitor on an I50 basis, proves to be a more effective antimetabolite than SKTI, but does not induce inhibitor‐insensitive protease activity because it causes a general up‐regulation of protease‐encoding genes. A possible mechanism to account for these different responses is discussed.

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.

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.

Transcription of a legumin gene from pea (Pisum sativum L.) in vitro.

The transcriptional activity of the pea legumin gene leg A in the HeLa cell lysate in-vitro transcription system has been assayed. Labelled transcripts were examined by hybridisation to restriction fragments of Leg A, and by glyoxalation followed by agarose gel electrophoresis. Although the legumin gene was not transcribed efficiently, transcripts were shown to be produced from the correct region of Leg A, and analysis by S1 nuclease mapping was used to show that the transcription start was the same as in vivo. The transcripts produced in vitro did not extent over the whole coding sequence of the gene; termination of transcripts occurred in a semi-random fashion. Transcription of templates truncated at their 3′ ends (i.e. in coding sequence) showed that discrete transcripts were produced from the start to restriction sites at approx. +520 and +900 bases. Transcription of templates truncated at their 5′ ends showed that sequences upstream of-97 bases relative to the transcription start had no appreciable effect on transcription in vitro.

Differential Expression of Seed Storage Protein Genes in the Pea legJ Subfamily; Sequence of Gene legK

Summary The legJ subfamily of genes in garden pea, Pisum sativum L., encodes “minor” legumin seed storage protein polypeptides. Data on the differential expression of the 3 genes (legJ,K,L) within this subfamily is reported. The expression of one gene (legJ) is specifically upregulated during the desiccation phase of cotyledon development, when other storage protein genes are downregulated. The complete sequence of a second gene in the subfamily, legK, shows that the failure to observe any expression ofthis gene is due to the mutation of its initiator ATG (methionine) codon to a GTG (valine) codon. The third gene in the subfamily, legL, shows maximal expression during the cotyledon expansion phase of seed development, i.e. like other storage protein genes. Evidence for the use of alternative polyadenylation addition signal sequences in these genes is also presented.

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 1.5 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.