Lacanobia oleracea were reared continuously on artificial diet (Bown et al., 1997) at 25°C under a 16 h:8 h light:dark regime. Nilaparvata lugens were kept on 20 day old rice plants (Oryza sativa variety 'TN1'), at 28°C, 90% RH, under a 16 h:8 h light:dark regime.
Materials and recombinant DNA techniques
A cDNA containing the GNA coding sequence has been described previously . Sub-cloning was carried out using the TOPO cloning kit (pCR2.1 TOPO vector) purchased from Invitrogen . P. pastoris X33 strain and SMD1168H (Protease A deficient) strain, the expression vector pGAPZαB, and Easycomp Pichia transformation kit were also from Invitrogen. Oligonucleotide primers were synthesised by Sigma-Genosys Ltd. . Restriction endonucleases, T4 polynucleotide kinase, T4 DNA ligase, and Pfu DNA polymerase, were supplied by Promega . Plasmid DNA was prepared using Promega Wizard miniprep kits. GNA was obtained from Vector Laboratories Inc. or was produced as a recombinant protein in yeast . Anti-GNA antibodies, raised in rabbits, were prepared by Genosys Biotechnologies, Cambridge, UK, and anti-(His)6 (C-terminal) antibodies were from Invitrogen.
General molecular biology protocols were as described by Sambrook and Russell  except where otherwise noted. All DNA sequencing was carried out using dideoxynucleotide chain termination protocols on Applied Biosystems automated DNA sequencers by the DNA Sequencing Service, School of Biological and Biomedical Sciences, University of Durham, UK. Sequences were checked and assembled using Sequencher software  running on Mac OS computers.
Assembly of expression constructs for recombinant proteins
The ButaIT amino acid sequence (Genbank [AF481881]) was used as the basis for the assembly of a synthetic ButaIT gene. Each strand (i.e. coding and complementary strands) of the sequence encoding the mature ButaIT chain (114 nucleotides) was subdivided into 5 fragments, in such a way that each fragment overlapped neighbouring fragments on the complementary strand by 10–15 bases. Ten oligonucleotide primers based on these fragments were synthesised, and used in an assembly reaction of the full mature ButaIT coding sequence.
The oligonucleotides used were as follows:
The primers at the 5' and 3' ends of the coding sequence contained Pst I, and Sal I restriction sites (underlined), respectively, which were used to allow subsequent cloning of the ButaIT gene into the expression vector pGAPZαB. All primers were individually 5'-phosphorylated using enzyme T4 polynucleotide kinase. An equimolar solution of phosphorylated primers in standard T4-DNA ligase buffer (without adenosine triphosphate; ATP or dithiothreitol; DTT), was prepared in a final volume of 20 μl. The mix was boiled for 10 min, to denature secondary structures, and slowly cooled to room temperature to allow primers to anneal. After addition of ATP, DTT and DNA ligase, a ligation reaction was carried out for 24 h at 16°C. A PCR reaction was then performed, using sense and antisense primers at the 5' and 3' ends of the ButaIT gene, to obtain sufficient DNA for cloning into the intermediate vector PCR 2.1. The resulting clones were verified by sequencing. Subsequently, the ButaIT coding sequence was excised as a Pst I/Sal I fragment from PCR2.1, and cloned into the expression vector pGAPZαB to create ButaIT- pGAPZαB.
To create a construct encoding the ButaIT/GNA fusion protein, the sequence encoding the mature GNA peptide (105 residues), derived from LECGNA2 cDNA  (Genbank [A18023]), was excised from a previously generated construct (SFI1/GNA-pGAPZαA, ) by restriction digestion (5' Not I/3' Xba I) and ligated into ButaIT-pGAPZαB, which had been digested with the same enzymes. The expressed protein derived from this construct is described in fig. 1.
The construct for expressing recombinant GNA as a control also contained amino acids 1–105 of the mature GNA polypeptide (previously shown to produce a fully active lectin) inserted as an EcoR I – Xba I fragment in the vector pGAPZαA; the expressed protein, after post-translational cleavage of the vector-encoded α-factor prepro-sequence was predicted to contain two extra residues at the N-terminus (EF...) and one at the C-terminus (...D); these extra residues have been shown not to affect activity.
Expression and purification of ButaIT and ButaIT/GNA
Constructs for expressing recombinant proteins were transformed into P. pastoris (SMD1168H strain; ButaIT and ButaIT/GNA or X33 strain; GNA) according to the protocols supplied by Invitrogen . Transformants were selected by plating on media containing zeocin (100 μg/ml). Selected colonies were picked off and confirmed as positive transformants by colony PCR using gene-specific primers. Clones expressing recombinant proteins were identified by immuno-dot blot or Western analysis of supernatants from small-scale cultures, using an anti-(His)6 antibody (Invitrogen) for ButaIT, and anti-GNA antibodies for ButaIT/GNA.
For protein production, P. pastoris cells containing the ButaIT, ButaIT/GNA or GNA constructs were grown in shake flasks (30°C with shaking), or a BioFlo 110 laboratory fermenter  as previously described , except that the pH of the growing culture was maintained at 4.0. Recombinant protein samples were purified using hydrophobic interaction chromatography on a phenyl-Sepharose (Amersham-Pharmacia) column, as previously described . Recombinant ButaIT, ButaIT/GNA and GNA eluted at low salt concentration, or in water, and were analysed for purity by SDS-PAGE. Lyophilised ButaIT/GNA and GNA were further purified using gel filtration on a Sephacryl S-200 column (1.6 cm diameter, 90 cm length, 0.3 ml/min), equilibrated in PBS buffer. Fractions containing purified ButaIT/GNA or GNA were pooled, analysed for purity and concentration (absorbance 280 nm; SDS-PAGE), prior to use in injection and diet bioassays. Purified proteins were de-salted by dialysis and freeze-dried, or de-salted and concentrated using Microsep TM centrifugal concentrators (VivaScience AG, Hannover, Germany).
Proteins were analysed routinely by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Samples were prepared by adding 4× SDS sample buffer (containing 10% 2-mercaptoethanol) and boiled for 5–10 min prior to loading. For separation of low molecular weight polypeptides, SDS-PAGE was carried out using a Tris-Tricine buffer system according to the protocol of Schagger and von Jagow . Gels were either stained with Coomassie Blue, or transferred to nitrocellulose using a Biorad Trans-blot SD semi-dry transfer cell, according to the manufacturers recommendations.
Concentrations of purified recombinant proteins were estimated by comparison with known amounts of standard GNA after SDS-PAGE, or Western blotting using anti-GNA antibodies (1:3300 dilution) as previously described in detail . Haemagglutination assays were carried out as described previously .
Partially purified ButaIT and purified ButaIT/GNA and GNA were tested for biological activity by injecting 5–10 μl of aqueous samples (freeze-dried protein re-suspended in water or PBS) into fifth stadium L. oleracea larvae of approx. 50–70 mg in weight. For each concentration tested 10–15 larvae were injected and toxic effects monitored over the next 7 days. PBS or water were injected as negative controls; no difference was observed between these two treatments, and survival over 2–3 days after the injection was routinely >90%.
A potato leaf-based artificial diet  was used in assays of the toxicity of recombinant ButaIT/GNA on oral delivery to lepidopteran larvae. For each treatment 20 newly moulted third stadium L. oleracea larvae were maintained in clear plastic pots containing moist filter paper to prevent diet desiccation. Survival was monitored daily. Total larval weights (± 0.1 mg) per treatment were recorded daily for the first 4 days of the assay and subsequently, individual larval wet weights (± 0.1 mg) were recorded daily, and diet consumption (per replicate) was estimated on a wet weight basis. The amount of recombinant ButaIT/GNA and GNA added to diets was estimated as described in section 2.4; control diets contained no added protein.
Toxicity of proteins when fed to rice brown planthopper was assayed using a liquid diet. An artificial diet (MMD-1), suitable for the short-term maintenance of N. lugens, was prepared according to Mitsuhashi . Assays were set up by placing five second instar N. lugens nymphs in each feeding chamber, consisting of the base of a 35 mm petri dish lined with moist filter paper. Two layers of Parafilm M® were stretched over the top, with 100 μl of artificial diet sandwiched between the two layers . Feeding chambers were kept at 28°C, 90 % RH, 16 h:8 h light:dark regime, and the diet sachets were replaced every 2 days to avoid contamination. A total of 10 replicates (50 insects) were used per treatment, and survival was recorded daily over 8 days. The amounts of recombinant ButaIT/GNA and GNA added to diets was estimated as described in section 2.4; diet containing no added protein was used as negative control, and feeding chambers in which no diet was available, but insects were kept moist ("water only") were used as a positive control.
Analysis of haemolymph and gut tissue in L. oleracea larvae exposed to recombinant proteins
Haemolymph samples were extracted from L. oleracea larvae injected with recombinant proteins, or exposed to artificial diet containing ButaIT/GNA or GNA, as previously described . Protein concentrations were estimated by a microtitre-based Bradford assay (Biorad) using BSA as the standard protein. Aliquots of haemolymph were analysed for the presence of GNA immunoreactivity by Western blotting as described previously. Crude gut extracts were also prepared from larvae exposed to ButaIT/GNA diets to verify binding of the fusion protein to the gut epithelium. Whole guts, dissected over ice, were flushed with PBS to remove contents. Following homogenisation, samples were centrifuged (12 000 × rpm, 4°C for 20 mins) and the supernatant assayed for protein concentration prior to analysis by Western blotting. Diet samples were also prepared (as described for gut samples) for analysis by Western blotting to confirm that fusion protein incorporated into artificial diet remained intact in 2–3 day old diet.
All data analysis was carried out using the Statview (v. 5.0; SAS Inc., Carey, NC, USA) software packages on Apple Macintosh computers. Unpaired t-tests, ANOVA analysis (Bonferroni-Dunn), Mann-Whitney non-parametric tests and survival analyses were carried out to determine any significant differences between treatments in the parameters measured.