Changes in gene expression in the permissive larval host lightbrown apple moth (Epiphyas postvittana, Tortricidae) in response to EppoNPV (Baculoviridae) infection.

Host cell and virus gene expression were measured five days after per os inoculation of 3rd instar lightbrown apple moth (LBAM) larvae with the Epiphyas postvittana nucleopolyhedrovirus (EppoNPV). Microarray analysis identified 84 insect genes that were up-regulated and 18 genes that were down-regulated in virus-infected larvae compared with uninfected larvae. From the 134 viral open reading frames represented on the microarray, 81 genes showed strong expression. Of the 38 functionally identifiable regulated insect genes, 23 coded for proteins that have roles in one of five processes; regulation of transcription and translation, induction of apoptosis, and maintenance of both juvenility and actin cytoskeletal integrity. Of the 34 functionally identifiable viral genes that were most strongly expressed, 12 had functions associated with these five processes, as did a further seven viral genes which were expressed at slightly lower levels. A survey of the LBAM-expressed sequence tag library identified further genes involved in these processes. In total, 135 insect genes and 38 viral genes were analysed by quantitative polymerase chain reaction. Twenty-one insect genes were strongly up-regulated and 31 genes strongly down-regulated. All 38 viral genes examined were highly expressed. These data suggest that induction of apoptosis and regulation of juvenility are the major 'battlegrounds' between virus and insect, with the majority of changes observed representing viral control of insect gene expression. Transcription and translational effects seem to be exerted largely through modulation of mRNA and protein degradation. Examples of attempts by the insect to repel the infection via changes in gene expression within these same processes were, however, also noted. The data also showed the extent to which viral transcription dominated in the infected insects at five days post inoculation.

Serratia entomophila inoculation causes a defect in exocytosis in Costelytra zealandica larvae.

Rapid elimination of midgut luminal proteinase activity and gut clearance are the two major symptoms of amber disease in Costelytra zealandica larvae because of the three-subunit protein toxin complex produced in Serratia entomophila and Serratia proteamaculans. Quantitative PCR analysis of mRNA from the major serine proteinase gene families showed that loss of proteinase activity did not result from transcriptional downregulation. Unexpectedly, protein levels and rates of protein synthesis increased, rather than decreased, in the midgut of diseased insects. Proteomic analysis of midgut tissues showed marked differences between healthy and diseased midguts. Large increases in soluble forms of both actin and tubulin were identified from 2D-gels, together with concurrent decreases in the levels of polymeric actin-associated proteins: actin depolymerizing factor and cyclophilin. These results suggest that the Serratia toxin acts to cause degradation of the cytoskeletal network and prevent secretion of midgut gut digestive proteinases as both the actin cytoskeleton and microtubules are involved in exocytosis. Proteinases synthesized in the diseased midgut must be rapidly degraded because they do not accumulate in an inactive form.

Serine proteases identified from a Costelytra zealandica (White) (Coleoptera: Scarabaeidae) midgut EST library and their expression through insect development.

Costelytra zealandica larvae are pests of New Zealand pastures causing damage by feeding on the roots of grasses and clovers. The major larval protein digestive enzymes are serine proteases (SPs), which are targets for disruption in pest control. An expressed sequence tag (EST) library from healthy, third instar larval midgut tissue was constructed and analysed to determine the composition and regulation of proteases in the C. zealandica larval midgut. Gene mining identified three trypsin-like and 11 chymotrypsin-like SPs spread among four major subgroups. Representative SPs were examined by quantitative PCR and enzyme activity assayed across developmental stages. The serine protease genes examined were expressed throughout feeding stages and downregulated in nonfeeding stages. The study will improve targeting of protease inhibitors and bacterial disruptors of SP synthesis.

Expressed sequence tags from the midgut of Epiphyas postvittana (Walker) (Lepidoptera: Tortricidae).

The midgut is a key tissue in insect science. Physiological roles include digestion and peritrophic membrane function, as well as being an important target for insecticides. We used an expressed sequence tag (EST) approach to identify candidate genes and gene families involved in these processes in the light brown apple moth, Epiphyas postvittana (Walker) (Lepidoptera: Tortricidae). Two cDNA libraries were constructed from dissected midgut of third to fifth instar larvae. Clustering analysis of 6416 expressed sequence tags produced 1178 tentative unique genes comprising 725 tentative contigs and 453 singletons. The sequences show similar codon usage to sequences from other lepidopterans, a Kozak consensus sequence similar to Drosophila and single nucleotide polymorphisms (SNPs) were detected at a frequency of 1.35/kb. The identity of the most common Interpro families correlates well with major known functions of the midgut. Phylogenetic analysis was conducted on representative sequences from selected multigene families. Gene families include a broad range of digestive proteases, lipases and carbohydrases that appear to have degradative capacity against the major food components found in leaves, the diet of these larvae; and carboxylesterases, glutathione-S-transferases and cytochrome P450 monooxygenases, potentially involved in xenobiotic degradation. Two of the larger multigene families, serine proteases and lipases, expressed a high proportion of genes that are likely to be catalytically inactive.

Influence of plant development and environment on transgene expression in potato and consequences for insect resistance.

Clonal replicates of different transformed potato plants expressing transgene constructs containing the constitutive Cauliflower Mosaic Virus (CaMV) 35S promoter, and sequences encoding the plant defensive proteins snowdrop lectin (Galanthus nivalis agglutinin; GNA), and bean chitinase (BCH) were propagated in tissue culture. Plants were grown to maturity, at first under controlled environmental conditions, and later in the glasshouse. For a given transgene product, protein accumulation was found to vary between the different lines of clonal replicates (where each line was derived from a single primary transformant plant), as expected. However, variability was also found to exist within each line of clonal replicates, comparable to the variation of mean expression levels observed between the different clonal lines. Levels of GNA, accumulated in different parts of a transgenic potato plant, also showed variation but to a lesser extent than plant-plant variation in expression. With the majority of the clonal lines investigated, accumulation of the transgene product was found to increase as the potato plant developed, with maximum levels found in mature plants. The variation in accumulation of GNA among transgenic plants within a line of clonal replicates was exploited to demonstrate that the enhanced resistance towards larvae of the tomato moth, Lacanobia oleracea L., caused by expression of this protein in potato, was directly correlated with the level of GNA present in the plants, and that conditions under which the plants were grown affect the levels of GNA expression and subsequent levels of insect resistance.

The effects of Phaseolus vulgaris erythro- and leucoagglutinating isolectins (PHA-E and PHA-L) delivered via artificial diet and transgenic plants on the growth and development of tomato moth (Lacanobia oleracea) larvae; lectin binding to gut glycoproteins in vitro and in vivo.

Red kidney bean, Phaseolus vulgaris, contains a lectin phytohemagglutinin (PHA) with toxicity towards higher animals. PHA exists in the isoforms PHA-E and PHA-L, which agglutinate erythrocytes and lymphocytes, respectively. Lacanobia oleracea larvae were reared from hatch on artificial diets containing PHA-E or PHA-L at 2% (w/w) dietary protein, and on transgenic Arabidopsis plants expressing either lectin at 0.4-0.6% of total soluble proteins. In artificial diet bioassays neither lectin affected larval survival, development, growth nor consumption. In transgenic plant bioassays both PHA-E and PHA-L promoted larval growth and development. This effect was greatest for PHA-E. Mean larval biomass of insects fed on plants expressing PHA-E was significantly greater (up to two-fold) than controls during the final two instars and the insects developed at a significantly greater rate so that after 26 days 83% of PHA-E exposed insects were in the final instar compared to 44% for control insects. PHA-E and PHA-L were detected by Western blotting in haemolymph, sampled from insects fed diets or plant material containing the lectins. However, despite the demonstrated potential for both isolectins to bind to gut glycopolypeptides in vitro neither was found to accumulate in vivo in the guts of exposed insects. Since lectin binding to gut polypeptides is thought to be necessary for insecticidal activity the failure of PHA-E and PHA-L to bind in vivo may account for their lack of toxicity to L. oleracea.

Characterization of major midgut proteinase cDNAs from Helicoverpa armigera larvae and changes in gene expression in response to four proteinase inhibitors in the diet.

A Helicoverpa armigera larval midgut cDNA library from larvae raised on an artificial, protein-rich, inhibitor-free diet contained very large numbers of serine proteinase positive clones. DNA sequencing of six random positive cDNAs and 12 PCR derived products identified trypsin genes classifiable into three families, and chymotrypsin and elastase genes classifiable into a single family each. Genomic blots established that the most highly expressed of the trypsin families contained about 18 genes, and that the chymotrypsin and elastase families contained about 14 and 2 genes respectively. The levels of mRNA corresponding to the highly expressed trypsin and chymotrypsin families were determined following chronic ingestion of four proteinase inhibitors. Compared to insects on an inhibitor-free diet, chymotrypsin mRNA was increased by all inhibitors, and trypsin mRNA levels decreased. This occurred independent of whether the inhibitor was solely a trypsin inhibitor (aprotinin), an inhibitor of both trypsin and chymotrypsin (proteinase inhibitor II, soybean trypsin inhibitor) or predominantly a chymotrypsin inhibitor (proteinase inhibitor I). Changing the protein level of the diet did not affect trypsin mRNA levels, but chymotrypsin mRNA levels decreased with increasing dietary protein.

Expression of a Brassica napus extensin gene in the vascular system of transgenic tobacco and rape plants.

The organs and tissues where the Brassica napus extA extensin gene is expressed have been identified. The extA gene with 3.75 kb of 5' flanking sequence was transferred to tobacco via disarmed Agrobacterium tumefaciens vectors and transgenic plants regenerated. The gene was found to be inactive in transgenic tobacco leaf, but was active as measured by RNA transcript assays in both stem and root tissues. To determine the cell-specific expression pattern of the extA gene, a promoter-reporter gene fusion construct was made consisting of 1.0 kb of 5' extA sequence fused to the coding region of the glucuronidase (GUS) gene. This fusion construct was introduced into B. napus via Agrobacterium rhizogenes, and expression of GUS in transgenic rape hairy roots was examined. GUS activity was only seen in the vascular tissues of the rape root, and was found to be specifically localised in the phloem.

The extensin gene family in oilseed rape (Brassica napus L.): characterisation of sequences of representative members of the family.

A family of cross-hybridising cDNA clones has been isolated from a cDNA library produced with poly(A)+ RNA from the roots of oilseed-rape (Brassica napus L.). The clones were selected as abundantly expressed in root by differential screening of the root cDNA library with cDNA probes prepared from root, green leaf, etiolated leaf and developing seed. mRNA species corresponding to the selected abundant clones were expressed in roots at levels of at least 400 times those in other organs, as shown by Northern blot analysis and RNase protection assays. Complete nucleotide sequence determination of the cDNA clones showed that they encoded proteins homologous to carrot extensin and were the products of at least three different genes. An extensin gene, designated extA, was obtained from an oilseed rape (B. napus L.) genomic library screened with a cDNA species encoding a protein expressed abundantly in roots. The gene is a member of a multigene family, consisting of about 3 members per haploid genome with strong homology to the probe, and a further 20 or so members with weaker homology. The isolated gene, although not identical to the cDNA probe, was also found to be specifically expressed in roots, and was transcribed into a mRNA species approximately 1,300 nucleotides in size. A single transcription start was identified by S1 mapping. The complete nucleotide sequence of the extA gene and its flanking regions has been determined and shown to encode a protein homologous to carrot and tomato extensins.

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.