|Title of Invention||
"INSECT CHYMOTRYPSIN INHIBITOR, POT1A "
|Abstract||The present invention relates generally to a novel chymotrypsin that exhibits resistance to a plant serine proteinase inhibitor. More particularly, the present invention provides a chymotrypsin which is up-regulated in the gut of Helicoverpa armigera and Helicoverpa punctigera insect larvae when fed the serine proteinase inhibitors of Nicotiana alata. The novel chymotrypsin represents, therefore, a target for the identification of antagonists including inhibitors which are proposed to be useful in the control of Helicoverpa spp. populations that have become resistant to serine proteinase inhibitors produced in plants. The antagonists of the chymotrypsin may be topically applied to the plants or, when in proteinaceous form, may be produced by genetic means in plant cells. The antagonists may act at the level of gene expression or protein activity.|
|Full Text||FIELD OF THE INVENTION
The present invention relates generally to a novel chymotrypsin that exhibits resistance to a plant serine proteinase inhibitor. More particularly, the present invention provides a chymotrypsin which is up-regulated in the gut of Helicoverpa armigera and Helicoverpa punctigera insect larvae when fed the serine proteinase inhibitors of Nicotiana alata. The novel chymotrypsin represents, therefore, a target for the identification of antagonists including inhibitors which are proposed to be useful in the control of Helicoverpa spp. populations that have become resistant to serine proteinase inhibitors produced in plants. The antagonists of the chymotrypsin may be topically applied to the plants or, when in proteinaceous form, may be produced by genetic means in plant cells. The antagonists may act at the level of gene expression or protein activity.
DESCRIPTION OF THE PRIOR ART
Bibliographic details of the publications referred to in this specification are also collected at the end of the description.
Reference to any prior art in this specification is not, and should not be taken as, an acknowledgment or any form of suggestion that this prior art forms part of the common general knowledge in any country.
Female reproductive tissues and wounded leaves of the ornamental tobacco, Nicotiana alata amass high levels of serine proteinase inhibitors (PIs) for protection against pests and pathogens (Atkinson et al, The Plant Cell 5: 203-213, 1993). These 6 kDa PIs accumulate in the vacuole (Miller et al., Plant Cell 11: 1499-1508, 1999) and are derived in vivo from the post-translational modification of a 40.3kDa precursor protein. The precursor of the PI
protein (referred to as "NaPI") is composed of six repeated regions of high sequence identity (Figure 1) each with a potential PI reactive site. Processing of the six-repeat precursor protein unexpectedly occurs at sites located within, rather than between the repeated regions. Complete removal of the linker sequence (Glu-Glu-Lys-Lys-Asn) [SEQ ID NO:1] contained within each repeated region, generates five contiguous 6 kDa inhibitors (Cl and T1-T4) and a novel two-chain chyrnotrypsin inhibitor (C2) formed by disulphide bond linkage of N-terminal and C-terminal peptide fragments (Heath et al., European Journal of Biochemistry 230(1): 25-257, 1995; Lee et al., Nature Structural Biology 6(6): 526-530, 1999). The structures of Cl, T1-T4 and C2 have been solved using 1H-NMR techniques (Nielson et al., J. Mol. Biol 242: 231-243, 1994; Nielson et al, Biochemistry 34: 14304-14311,1995; Lee et al, 1999, supra).
Nicotiana alata also has a second gene related to NaPI that encodes a closely related precursor protein with four rather than six repeated domains (Miller et al, Plant Mol. Biol. 42: 329-333, 2000). This precursor is also processed in vivo resulting in the release of three contiguous 6 kDa inhibitors (Cl, T4 and T5) and the two-chain inhibitor C2 (Figure 1). Three of the inhibitors (Cl, C2 and T4) are identical to those released from the six-domain precursor. Related multidomain precursors have been described for other solanaceous plants including N. tabacum (Balandin et al, Plant Mol Biol 27: 1197-1204, 1995), N. glutinosa (Choi et al, Biochim. et Biophys. Acta 1492: 211-215, 2000), L esculentum (Taylor et al, Plant Mol Biol. 23: 1005-1014, 1993) and Capsicum annum (Moura and Ryan, Plant Physiol 126: 289-298, 2001; Antcheva et al., Protein Sci. 10: 2280-2290, 2001).
Several groups have reported on the affect of serine proteinase inhibitors on the activity of the digestive proteases of insects and have suggested that they are produced by plants for protection against the damaging affects of insect pests and microorganisms (Ryan, Annu. Rev. Phytopathol 28: 425-449, 1990; Gatehouse et al, In: Plant Genetic Manipulation for Crop Protection, Biotech, in Agriculture No. 7, Eds. Gatehouse, Hilder & Boulter, International U.K., pp. 155-181, 1992). Insects that are specialist feeders on a particular host plant are generally resistant to the serine Pis produced by that plant, but are sensitive
to PIs produced by non-hosts (Broadway and Villani, Entomol Expo. Appl. 76: 303-312, 1995; Broadway, J. Insect. Physiol. 41: 107-116, 1995). There is interest, therefore, in transferring genes encoding serine PIs from non-hosts into crop plants to enhance insect resistance and to decrease reliance on chemical pesticides. Recently, however, several groups have reported on the ability of certain insects to change the relative proportions of proteolytic enzymes in their midgut following ingestion of high levels of Pis (Broadway, 1995, supra; Jongsma et al, Proc. Natl. Acad Sci. USA 92(17): 8041-8045, 1995a). Broadway (1995, supra), for example, found that certain lepidopteran insects produce two broad classes of trypsin like proteases, one of which is insensitive to PIs from cabbage leaves. After ingestion of the cabbage PIs the insects increased production of the trypsin class not affected by the PIs and thus were able to grow and develop unhindered. Jongsma and coworkers (1995, supra) made a similar observation with Spodoptera exigua larvae fed on PIs from potato (PotII) and tobacco. The factors that regulate the secretion of these proteases under these conditions are not known.
These studies indicate that PNs specific for only one or two of the range of proteinases in an insect gut will be of limited use for long term plant protection. The gene encoding the N.alata PI has a potential advantage over other plant PNs for the enhancement of insect resistance in transgenie plants. Most plant serine PNs contain only one or two inhibitory domains, whereas the N. alata PI precursors have four or six (Figure 1). Thus, there is potential to engineer the individual domains of the N. alata PI to provide inhibitory activity against several proteinases in the insect gut.
The midgut proteases of several Lepidoptera, Coleoptera and Orthoptera have been partially characterized. In most Lepidopteran species the endoproteinase activity is due primarily to serine proteinases (trypsin, chymotrypsin and/or elastase) and cysteine and metalloproteinases are not detectable (Christeller et al, Insect Biochem. Molecul. Biol. 22: 735-746,1992; Terra and Ferreira, Comp. Biochem. Physiol 109: 1-62, 1994; Xu and Qin, J. Econ. Entomol. 87: 334-338, 1994; Lee and Anstee, Insect. Biochem. Molec. Biol. 25: 63-71, 1995a; Johnston et al, Insect Biochem. 21: 389-397, 1991; Johnston et al, Insect Biochem. Molec. Biol. 25(3): 375-383, 1995). Exopeptidase and leucineaminopeptidase
have also been identified (Christeller et al, 1992, supra; Lee and Anstee, Insect. Biochem. Moke. Bioll 25(1): 49-61,1995b).
The mechanism of action of PIs on insects is only partially understood. Three responses have been described:-
(i) Severe retardation of growth without a decrease in gut proteolytic activity. Broadway and Duffey (J. Insect Physiol. 32: 673-680, 1986a; Broadway and Duffey, J. Insect Physiol. 32: 827-833, 1986b) found that insects fed on PIs had remarkably reduced growth rates that were not associated with a decrease in the total proteolytic activity in the gut. Indeed the gut proteolytic activity often increased. They suggested that a feedback mechanism was operating that led to hyperproduction of proteases, that led in turn to a depletion of essential sulphur containing amino acids. This phenomenon has been recorded for other insects after chronic ingestion of PIs (Burgess et al, Entomol. Exp. App. 61: 123-130, 1991; De Leo et al., Plant Physiol. 118: 997-1004, 1998; Markwick et al., J. Economic Entomology 91 (6): 1265-76, 1998).
(ii) Severe retardation of growth with a decrease in gut proteolytic activity. Broadway (1995, supra) found that the lepidopteran species, Agrotis ipsilon (black cutworm) had reduced growth and delayed pupation after exposure to soybean trypsin inhibitor and did not respond by secreting Pi-insensitive proteases. These insects had up to a 70% reduction in total gut proteolytic activity. Codling moth larvae (Lepidoptera:Tortricidae) fed on 'elastase inhibitors' were also retarded in growth and development that was associated with diminished elastase activity in the gut (Markwick et al., Journal of Economic Entomology 88(1): 33-39, 1995).
(iii) No effect on growth - change in the complement of gut proteinases. Some insects can compensate for the inhibition of one group of proteinases by inducing a new proteinase activity. The genomes of lepidopteran insects contain genes for a range of serine proteases and insects can modify the expression of specific isozymes to suit dietary components (Bown, et al., Insect Biochem. Molec. Biol. 27: 625-638, 1997; Broadway, J. Insect. Physiol. 43(9): 855-874, 1997). Changes in the complement of gut trypsins and chymotrypsins have been detected using Northern blot analysis on RNA from H. armigera (Bown, et al, 1997, supra; Gatehouse, et al, Insect Biochem. Molecul. Biol. 27: 929-944, 1997), H. zea and Agrotis ipsilon (Magumdar-Leighton and Broadway, Insect Biochem. Mol. Biol. 31: 645-657, 2001a; Mazumdar-Leighton and Broadway, Insect Biochem. Mol Biol 31:633-644, 2001 b). Corresponding changes at the protein level have also been observed using electrophoretic separation of isozymes for H. armigera (Harsulkar, et al, Plant Physiol 121: 497-506, 1999; Patankar, et al, Insect Biochem. & Mol Biol 31: 453-464, 2001), Spodoptera frugiperda (Paulillo, et al, J. Econ. Entomol 95:892-896, 2000), H. zea and Trichoplusia ni (Broadway, Arch. Insect Biochem. Physiol 32(1): 39-53, 1996). Sometimes specific isozymes have been up-regulated, and occasionally proteases previously undetected have been observed.
Recently, Mazumdar-Leighton and Broadway (200la, supra) demonstrated that the production of Pi-insensitive trypsins in H. zea is regulated at the transcriptional level and can be abolished using the transcriptional regulator actinomycin. Broadway and colleagues examined changes in gut trypsin and chymotrypsin activity after H. zea and Trichoplusia ni larvae were fed for 48 h on artificial diet containing 1% SBTI (Broadway, 1996, supra). Trypsin activity increased after SBTI consumption and protease banding patterns on zymograms indicated a change in the relative complement of proteases. The researchers showed in vitro that SBTI could inhibit 74% of the trypsin activity in gut extracts from control larvae, but only 3% of the gut trypsin activity in larvae that had consumed SBTI. They suggested the new protease bands (one new band for H. zea and 6 new bands for T. ni) on the zymograms may be SBTI-insensitive trypsins or SBTI-insensitive chymotrypsins and concluded that the production of these new proteases was enhanced by the ingestion of SBTI. Further studies using Northern blot analysis showed that consumption of SBTI resulted in transcriptional induction of mRNAs encoding trypsins and chymotrypsins by H. zea and Agrotis ipsilon (Mazumdar-Leighton and Broadway, 200la, supra; Mazumdar-Leighton and Broadway, 2001b, supra), although it was not
determined if these proteases were SBTI-insensitive.
The novel trypsin transcript induced in H. zea after ingestion of SBTI was designated HzT15 (Mazumdar-Leighton and Broadway, 2001b, supra). Recently, the first insect digestive enzyme insensitive to several proteinase inhibitors was purified from the gut of H. zea and corresponds to the protein encoded by HzTI5 (Volpicella et al, Eur. J. Biochem. 270: 10-19, 2003). The authors identified several differences in charge distribution across the surface of the structural model of mis PI-insensitive trypsin relative to the PI inhibitable trypsins, but were unable to identify the structural changes that led to resistance.
Until recently, chymotrypsins were assumed to contribute relatively little to protein digestion in Lepidoptera and consequently most biochemical studies focused on characterization of the trypsins. This problem arose due to the initial use of synthetic substrates Ihat worked well with mammalian chymotrypsins, but not at all or poorly with the Lepidopteran enzymes. Lepidopteran chymotrypsins prefer synthetic substrates with at least four amino acids to occupy the S1-S4 binding subsites on the enzyme, whereas mammalian trypsins are active on shorter substrates with one amino acid that is specific for the S1 binding subsite. That is, the insect chymotrypsins appear to have an extended substrate binding site requiring at least four amino acids for efficient catalysis. Recent studies have shown that chymotrypsins do respond to PI ingestion and are worthy of more detailed investigation. When larvae from H. armigera were fed on diets consisting of either potato proteinase inhibitor II, soybean trypsin inhibitor, aprotin (trypsin inhibitor) or potato proteinase inhibitor I, levels of chymotrypsin mRNA increased in all cases while trypsin mRNA decreased (Gatehouse et al., 1997, supra). Other reports also mention upregulation of chymotrypsins in preference to trypsins (Bown et al., 1997, supra; Wu et al, Molecular Breeding 3: 371-380, 1997). Mazumdar-Leighton and Broadway (2001a, supra) assayed chymotrypsin activity in the gut of H. zea larvae and found that SBTI inhibited 95% of the chymotrypsin from the gut of control insects but only 35% of activity from the gut of insects that had prior exposure to SBTI in the diet.
Hence, consumption of proteinase inhibitors can lead to a drastic change in the complements of gut proteases which allows insects to adapt to the diet and survive. Changes in the complement of proteases after exposure to PIs have been detected in insects fed on both artificial diets and transgenic plants. The triggers that regulate these changes are still unknown and the responses vary with the species, the PI and its concentration, and the base diet. It is unclear why some inhibitors induce this response and others do not. It is clear, however, that some larvae are genetically pre-adapted to PIs, since prior exposure to a specific inhibitor is not necessary for an insect to be resistant (Broadway, 1996, supra),
There is a need to identify and investigate novel insect proteinases which are insensitive to PIs and to use these to screen for antagonists of the proteinases in order to develop agents useful in controlling insect growth, maintenance, development and/or survival.
SUMMARY OF THE INVENTION
Throughout this specification, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element or integer or group of elements or integers but not the exclusion of any other element or integer or group of elements or integers.
Nucleotide and amino acid sequences are referred to by a sequence identifier number (SEQ ID NO:). The SEQ ID NOs: correspond numerically to the sequence identifiers 1 (SEQ ID NO:1), 2 (SEQ ID NO:2), etc. A summary of the sequence identifiers is provided in Table 1. A sequence listing is provided at the end of the specification.
The present invention provides a novel chymotrypsin from Helicoverpa spp. referred to herein as "HpCh5". Reference to "HpCh5" includes all variants, derivatives, homologs and analogs as well as members of a HpCh5 family of chymotrypsins. Examples of variants of HpCh5 include proteinase inhibitor (PI) sensitive forms. Such sensitive forms may carry inter alia as substitution of the arginine at position 192 to an asparagine or glutamine. This substitution is referred to herein as "R192N/Q" using single amino acid nomenclature or "Arg 192 Asn/Gln" using three letter amino acid code. Other derivatives of HpF5 include the signal sequence of HpF5.
The HpCh5 chymotrypsin is encoded by a nucleotide sequence referred to as "HpF5". Again, reference to "HpF5" includes variants, homologs and analogs thereof. The term "HpF5" encompasses both a genomic sequence as well as a cDNA sequence. The amino acid sequence of HpCh5 is set forth in SEQ ID NO:2. The amino acid sequence of the N-terminal activation peptide is shown in SEQ ID NO:3. The nucleotide sequence of the coding region of HpF5 is set forth in SEQ ID N0:4 with the nucleotide sequence encoding the activation peptide is shown in SEQ ID NO:5 and its entire 5'-3' sequence shown in SEQ ID NO:6. HpCh5 is generally characterized by being substantially insensitive to inhibition by a PI from N. data.
Variants and homologs of HpCh5 include molecules having at least 75% amino acid identity to SEQ ID NO:2 after optimal alignment. Variants and homologs of HpF5 include nucleotide sequences having at least about 75% similarity to SEQ ID N0:4 or SEQ ID NO:6 after optimal alignment or a nucleotide sequence capable of hybridizing to SEQ ID NO:4 or SEQ ID NO:6 or its complementary form under low stringency conditions.
Accordingly, one aspect of the present invention provides an isolated nucleic acid molecule comprising a sequence of nucleotides encoding a chymotrypsin from Helicoverpa ssp. or a variant, derivative, homolog or analog of said chymotrypsin, wherein said chymotrypsin exhibits resistance to a PI from N. alata.
Another aspect of the present invention provides an isolated chymotrypsin from Helicoverpa ssp. wherein said chymotrypsin exhibits resistance to a PI from N. alata or a variant, derivative, homolog or analog of said chymotrypsin.
The present invention provides compounds which inhibit HpCh5 or its variants and homologs or members of the HpCh5 chymotrypsin family or which inhibit expression of the HpF5 gene or its variants and homologs.
The compounds may be chemical type compounds such as those sprayed or provided to plants or genetic type molecules which may be either topically applied or generated in plant cells. The HpCh5 or HpF5 antagonists may also be a modified form of an existing plant PI.
The present invention provides, therefore, methods for inhibiting insect infestation of a plant or for retarding insect growth and development by the application or dispersement of an antagonist of HpCh5 activity or HpF5 gene expression.
The antagonists include compounds which bind to and inhibit HpCh5 as well as antisense or sense nucleic acid molecules generated by a plant cell and then ingested by an insect.
Reference to an "antagonist" includes reference to an inhibitor.
The present invention further provides genetically modified plants which are engineered to produce a HpCh5 or HpF5 antagonist. Reference to a "plant" includes a monocotyledonous or dicotyledonous plant and may be a plant regenerated from genetically transformed callus or tissue or progeny of such a plant. The present invention further provides seeds and other reproductive material from the genetically modified plants of the present invention.
Plants contemplated herein include cotton, sweet corn, tomato, tobacco, piniento, potato, sunflower, citrus, plums, sorghum, leeks, soybean, alfalfa, beans, pidgeon peas, chick peas, artichokes, curcurbits, lettuce, Dianthus (an ornamental plant) and geraniums, cape gooseberry, maize, flax and linseed, alfalfa, lupins, broad beans, garden peas, peanuts, canola, snapdragons, cherry, sunflower, pot marigolds, Helichrysum (an ornamental plant), wheat, barley, oats, triticale, carrots, onions orchids, roses and/or petunias.
The present invention further provides nucleic acid molecules which encode potato-derived protenase inhibitors such as but not limited to PotlA and PotlB or their homologs or derivatives as well as transgenic plants comprising and capable of expressing same.
Yet another aspect of the present invention contemplates a method for screening for an inhibitor of an insect chymotrypsin which is insensitive to inhibition by NaPI such as HpCh5. Such a method generally involves testing for chymotryptic activity in the presence of potential inhibitors. The assay is conveniently contacted in vitro although the use of H, argmigera and/or H. punctiga is also encompassed by the present invention. An isolated inhibitor identified by the subject assay is also contemplated by the present invention.
A summary of sequence identifiers used throughout the subject specification is provided in Table 1.
Summary of sequence identifiers
BRIEF DESCRIPTION OF THE FIGURES
Figure 1 is a graphical representation showing temperature stability of baculovirus expressed H. punctigera chymotrypsin. Bovine chymotrypsin (♦) and HpF5 (▲) in 50 mM Na acetate were compared by incubating 100 µL aliquots at 5°C intervals between 40°C and 70°C. After chilling the heated samples on ice, duplicate activity assays were performed by incubating 10 µL of be or 50 µL of HpF5 with substrate at 30°C. Residual activity was presented as a percentage of the activity of the untreated control.
Figure 2 is a graphical representation showing growth of H. punctigera larvae fed on either low protein haricot bean artificial diet or cotton leaf artificial diet, in the presence or absence of 0.26% (w/v) NaPIs. Weight gain (mg) was monitored for 21 days. Larvae grew at a similar rate on both artificial diets and growth was retarded in the presence of NaPIs. (A) Growth of larvae. (B) Relative size of larvae at 21 days after feeding on cotton artificial diet, in the presence or absence of 0.26% NaPIs. Only five of the 20 larvae fed on NaPI survived and all five weighed less than control larvae after 21 days. (C) The effect of NaPIs on gut trypsin and chymotrypsin activity. When each H. punctigera larva reached the late fourth/ early fifth instar stage of development, the gut was removed and gut extract prepared prior to assays for trypsin and chymotrypsin activity. Extracts from individual larvae fed -on control cotton-leaf artificial diet (CC) are indicated in grey and larvae fed on the same diet containing 0.26% ((w/v)) NaPI (NC) are indicated in black. Only four test larvae survived to fifth instar, whereas most control larvae survived. All assays were performed in duplicate. Trypsin activity was determined using BApNA substrate and chymotrypsin activity with SAAPFpNA substrate. Units of activity are expressed as change in absorbance at 405 nm/min/mg gut extract protein (± standard deviation). Trypsin activity was almost abolished, relative to controls, in extracts from NaPI-fed larvae, while chymotrypsin activity was low in two of the four NaPI-fed larvae. (D) The effect of NaPIs on trypsin and chymotrypsin activity in the frass. Frass was collected from each larva fed on the cotton leaf artificial diet and faecal extracts were prepared. Trypsin and chymotrypsin activity in extracts from larvae fed on the control diet (CC) are indicated in grey and larvae fed on the same diet containing 0.26% ((w/v)) NaPI (NC) are indicated in
black. Note that frass of larvae CC16 and NCI3 were included in this analysis but excluded from Figure 2C because the gut were damaged during preparation of extracts. All assays were performed in duplicate. Trypsin activity was determined using BApNA substrate and chymotrypsin activity with SAAPFpNA substrate. Units of activity are expressed as change in absorbance at 405 nm/min/mg faecal extract protein (± standard deviation). Negligible trypsin activity was evident in extracts from NaPI-fed larvae, while chymotrypsin activity was elevated, relative to controls. (E) Levels of trypsin in the frass of control and NaPI fed larvae. Frass extracts were analysed on 15% ((w/v)) SDS-PAGE and transferred to nitrocellulose. The nitrocellulose filter was probed with rabbit anti-HpTRYl serum (1 in 2000 dilution) as the primary antibody, followed by the secondary antibody, donkey anti-rabbit IgG-horse radish peroxidase conjugate (1 in 2000 dilution). Immuno-reactive proteins were visualized using Enhanced Chemiluminescence (ECL) reagents and HyperfilmECL X-ray film. The trypsin in the frass of larvae that consumed NaPIs (NC) was significantly increased relative to controls (upper panel) but inactive (lower panel), presumably because it was in complex with the NaPIs. In comparison, trypsin was active in the frass of the control larvae (CC) even though the amount present was below the detection level of the antibody. (F) Production of NaPI-insensitive proteases in the gut of NaPI fed larvae. The gut extracts of the larvae were subjected to inhibition assays to identity NaPI-insensitive trypsins and chymotrypsins. Each extract was pre-incubated for 30 min at 30°C in the presence or absence of 80 nM NaPI inhibitor (Tl monomer for trypsin assays and Cl monomer for chymotrypsin assays), prior to the addition of substrate to initiate the reaction. Results from extracts assayed without inhibitor are indicated in grey(control larvae) and black (NaPI-fed larvae). Results from extracts assayed with inhibitor are stippled. All assays were performed in duplicate. Units of activity are expressed as substrate hydrolysis at 405 nm/min/mg extract protein (± standard deviation). (A) Inllibition of trypsin activity by Tl. Trypsin activity was determined using BApNA substrate. (B) Inllibition of chymotrypsin activity by Cl. Chymotrypsin activity was determined with SAAPFpNA substrate. Tl almost totally inhibited trypsin activity in the extracts of control larvae, indicating these larvae did not produce NaPI-insensitive trypsins. The extracts from NaPI-fed larvae contained negligible trypsin activity, but this activity could not be inhibited by Tl. Cl did not inhibit chymotrypsin activity in the extracts of six control larvae and only partially inhibited the gut chymotrypsins in the rest of the controls indicating the control insects contained a Cl-insensitive chymotrypsin. Most of the chymotrypsin activity in extracts from NaPI-fed larvae can be attributed to NaPI-insensitive chymotrypsins. When extracts from control and NaPI-fed larvae were pre-incubated with 80 nM chymostatin, all chymotrypsin activity was abolished.
Figure 3 is a graphical representation showing the effect of various proteinase inhibitors on the chymotrypsin activity in unfractionated gut extracts from H. punctigera. Proteinase inhibitors were mixed with 1 µg of protein from an unfractionated gut extract before incubation with the chymotrypsin substrate N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide. Inhibition is expressed as a percentage of the total activity in the control samples.
Figure 4 is a photographic representation showing purification and N-terminal sequence of an NaPI inhibitable chymotrypsin from H. punctigera gut. (A and B) Protein gel analysis of fractions at various stages of purification using an affinity column with immobilized Cl inhibitor (Figure 1). (A) 15% ((w/v)) SDS-polyacrylamide gel loaded with (a) unfractionated gut extract (b) and (c) unbound proteins. (B) 12.5% (w/v) SDS-polyacrylamide gel loaded with (d) wash fraction (e) protein bound to the Cl column. (C) N-terminal; sequence of the ~24 kDa protein in lane (e).
Figure 5 is a diagrammatic representation showing purification and N-terminal sequence of an NaPI-insensitive chymotrypsin from H. punctigera. (A) PVDF blot of chymotrypsin (i) eluted from potato inhibitor column. Potato Inhibitor II (ii) and potato inhibitor I (iii) both co-eluted from the matrix under denaturing conditions. (B) N-terminal amino acid sequence obtained from PVDF blot. Rechla was the most abundant of the four sequences obtained.
Figure 6 is a graphical representation showing the effect of pH and a range of substrates on the activity of the NaPI-insensitive chymotrypsin from H. punctigera midgut. SA2PF-pNA, N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide; SA2PL-pNA, N-succinyl-Ala-Ala-Pro-Leu-p-nitroanilide;MA2PM-pNA,N-methoxysuccinyl-Ala-Ala-Pro-Met-p-nitroanilide.
Figure 7 is a diagrammatic representation showing the design of oligonucleotide primers for amplification of chymotrypsins from H. pimctigera. An alignment of chymotrypsins from Helicoverpa armigera predicted from DNA sequences in the GenBank database. NCBI protein database accession numbers are left of the sequences. Regions corresponding to the oligonucleotide primers are boxed and the direction of amplification are indicated by arrows.
Figure 8 is a diagrammatic representation showing oligonucleotide sequences used in RT-PCR amplification of Helicoverpa chymotrypsins,
Figure 9 is a diagrammatic representation showing PCR products from amplification of cDNAs encoding H. punctigera chymotrypsins. PCR amplification of cDNA prepared from gut mRNA yielded partial sequence for five distinct chymotrypsins. The translated sequence is aligned and the region corresponding to the PCR primers is boxed.
Figure 10 is a diagrammatic representation showing alignment of predicted amino acid sequence of chymotrypsins from H. pimctigera. The catalytic residues are marked by a solid triangle (▼). The highly conserved active site motifs are highlighted with grey. The dipeptide R-I (↓↓), conserved among all chymotrypsins is the site for the proposed cleavage of the activation peptide by trypsin. The residues that lie in the substrate binding pocket and confer substrate specificity are indicated by the symbols ¥,§,#. The cysteine (●) residues are highly conserved among all chymotrypsins.
Figure 11 is a diagrammatic representation showing design of oligonucleotide primers for amplification of cDNA encoding the NaPI-insensitive chymotrypsins from H. punctigera. (A) Comparison of the N-terminal sequence of two NaPI-insensitive chymotrypsins with Helicoverpa chymotrypsins predicted from the cDNA clones. The unique regions Fl and F2 are shaded. (B) Oligonucleotide primers complementary to unique regions at the N-terminus of the insensitive chymotrypsin.
Figure 12 is a diagrammatic representation showing nucleotide sequence and deduced amino acid sequence from the cDNA encoding the insensitive chymotrypsin. The nucleotide sequence of the insensitive chymotrypsin cDNA and deduced amino acid sequence. The amino acid sequence obtained from N-terminal sequence of purified protein is shaded in grey. The putative site for endoproteolytic cleavage by trypsin is shown by the arrow. The double underlined regions in the nucleotide sequence refer to the positions of the degenerate primers used for PCR amplification. The polyadenylation signal sequence is single underlined and an asterisk marks the stop codon. The deduced amino acid sequence of the putative activation peptide is numbered -40 to -1 followed by the mature domain (+1). The three amino acids that correspond to the catalytic residues are marked by ths symbol #. The chymotrypsin substrate specificity residue, serine, located at the base of the primary substrate-binding pocket is marked with the symbol §.
Figure 13 is a representation showing alignment of H. punctigera chymotrypsin families showing sequence identity. ClustalW alignment of members from the five families of H. punctigera chymotrypsins. Protein sequence is given in single letter code. Identical amino acids are coloured black, similar amino acids are grey. Amino acids are numbered on the right and gaps have been introduced to maximize the alignment. The NaPI- insensitive chymotrypsins are members of family 5 and are characterized by a unique arginine residue (arrowed) at position 185. Human trypsin IV also contains an arginine residue (arrowed) in a similar position.
Figure 14 is a representation showing alignment of the H. punctigera NaPI-insensitive chymotrypsin with a liomolog from H. armigera that also has an arginine residue at position 185.
Figure 15 is a representation showing the deduced protein sequences for the insensitive (HpCh5) and sensitive (HpCh2A) chymotrypsins from H. punctigera aligned to the bovine chymotrypsin isoforms A and B. H. punctigera chymotrypsins HpCh2A and HpCh5 were aligned to the bovine chymotrypsin isoforms A and B using ClustalW. The numbering system (excluding gaps) is according to the nomenclature of Greer, Proteins 7: 317-34,
1990 used for bovine chymotrypsin. Dots throughout the sequences represent conserved residues. The regions shaded in grey designate residues that form surface loops that are involved in recognition and binding of substrates or inhibitors. The primary substrate-binding poctet is formed by the regions labeled S1 A, S1B and S1C. The S1 site is formed by loops 35 and 60. Black boxes mark residues in the HpF5 sequence that differ significantly to amino acids in the corresponding positions in other chymotrypsins.Using the Greer, 1990, supra nomenclature these residues are Asp36, Arg 63, Thr72, Pro 83, Gly 109, Ileu 120, Glu insertion between 129 and 130, Glu 134, Ser145, Arg 192 and Pro 207. The boxed amino acids are removed from bovine chymotrypsins by autocatalytic cleavage that results is the formation of α-chymotrypsin.
Figure 16 is a diagrammatic representation showing several surface loops in the structural model of H. punctigera chymotrypsin HpCh2A are larger than the cognate loops in bovine chymotrypsin. The structural model of H. punctigera F2A chymotrypsin (grey) was superimposed onto the structure of alpha-chymotrypsin from Bos taurus (black). Surface loops 60, 35, and 142 that are implicated in substrate recognition are larger in the insect chymotrypsin model. The chymotrypsin substrate specificity residue, serine 189, positioned at the base of the primary substrate-binding pocket is viewed as a space filled representation of the van der Waals radius.
Figure 17 is a representation showing glutamine 192 (Greer, 1990, supra nomenclature, Figure 15) of the sensitive chymotrypsin HpCh2A appears easily accommodated when modeled in complex with Cl. Structural model of the sensitive chymotrypsin HpCh2A (grey) in complex with the proteinase inhibitor Cl (black). The side chain of glutamine 192 is arrowed The residues of Cl in the vicinity of Gln 192 are represented in stick configuration (black).
Figure 18 is a diagrammatic representation showing comparison of the environment surrounding residue 192 of the sensitive and insensitive chymotrypsin complexed to Cl. Enlarged view of the boxed area shown in Figure 17. Arginine 192 of the insensitive chymotrypsin (B) appears to clash with residues of the Cl inhibitor, in contrast there is no apparent conflict with glutamine 192 of the sensitive chymotrypsin (A).
Figure 19 is a diagrammatic representation showing the environment surrounding arginine 192 when the insensitive chymotrypsin is complexed to the Type 1 potato proteinase inhibitor (PotIB, Fig24). (A) Structural model of the insensitive chymotrypsin HpCh5 (grey) in complex with the potato type I proteinase inhibitor PotI (black). (B) Enlarged view of the region around Arg 192 (boxed area in B). The side chain of arginine 192 is labeled. The residues of PotI in the vicinity of Arg 192 are represented in stick configuration (black).
Figure 20 is a photographic representation showing expression of the chymotrypsin clone HpF2B in E. coli cells for the production of a polyclonal antibody. (A) The separation of total cell lysates taken at time points 0-5 hr after induction of HpCh2B on a 12.5% (w/v) SDS-PAGE gel stained with Coomassie Blue. The lanes are marked by the number of hours after induction and the arrow indicates the position of the induced protein with the correct predicted molecular mass. (B) Panel 1: Bacterially expressed HpCh2B purified on Talon resin (BD Biosciences Clontech), separated on a 15% (w/v) SDS-PAGE gel and stained with Coomassie Blue. Panel 2: Identical sample to Panel 1 transferred to nitrocellulose and immunostained with anti-HpCh2B antibodies. (C) Decreasing amounts (200, 150, 100, 75, 50, 25, 20, 10, 0 ng) of bacterially expressed chymotrypsin HpCh2B separated by SDS-PAGE and stained with silver and a protein blot of an identical gel probed with anti-chymotrypsin HpCh2B antibodies (1/2500). The H. punctigera antibody had a detection limit of 20 ng of bacterially expressed protein.
Figure 21 is a photographic representation showing specificity of antibodies raised against bacterially expressed NaPI-insensitive (HpCh5) and sensitive (HpCh2B) chymotrypsins from H. punctigera. Bacterially expressed NaPI-insensitive (R) and sensitive (C) chymotrypsins were separated by SDS-PAGE on 12.5% (w/v) polyacrylamide gels and (A) stained with Coomassie Blue, (B) immunoblotted with an α-His tag antibody, (C) immunoblotted with the antibody to the HpCh2B chymotrypsin (α-RC), and (D) immunoblotted with the antibody to the HpCh5 chymotrypsin (α-SC).
Figure 22 are representations showing purification of PotI from potato tubers. PotI was purified from potato tubers (Russet Burbank) by acid extraction, ammonium sulphate precipitation and gel filtration. (A) SDS-PAGE stained with silver, lane 1: molecular size markers (kDa), lane 2: pooled PotI containing fractions from G-75 column, lane 3: immunoblot of lane 2 using an antibody raised in rabbits to a commercial preparation of PotI (Calbiochem) linked to keyhole limpet hemocyanin. PotI was identified as a single band with an approximate mass of 6 kDa. (B) RP-HPLC of pooled G-75 fractions from A. Peaks 1,2 and 3 are PotI isoforms, peak 4 is a contaminating protein.
Figure 23 is a graphical representation showing growth of H. armigera larvae on artificial diet containing NaPI and PotI. Growth of H. armigera, larvae fed on a cotton leaf artificial diet in the presence or absence of 0.26% (w/v) NaPI or 0.26% (w/v) NaPI plus 0.26% (w/v) PotI. The PotI was purified from potato tubers (var Russet Burbank), see Figure 22. Twenty five larvae were used on each diet. The weight of the larvae was measured at days 7, 10, 12, 14 and 17 post egg hatch. At day 17, larvae fed NaPI alone were 84% of the control and larvae fed NaPI and PotI were 34% of the control. Two larvae fed on the control diet died, seven larvae fed the NaPI diet died and six larvae fed on the NaPI plus PotI diet died.
Figure 24 is a representation showing the alignment of predicted amino acid sequence of StPotIA and StPotIB with members of the potato Inhibitor I family. ClustalW alignment of several members of the Potato Inhibitor I family. X67950: potato cDNA, (Beuning and Christeller, Plant Physiol 102: 1061, 1993), P01052: potato protein (Richardson and Cossins, FEBS Letters, 52: 161, 1975), M17108: potato genomic sequence (Cleveland et al, Plant Mol Biol, 8: 199-207, 1987), K03290: tomato (Graham et al, J. Biol. Chem., 260: 6555-6560, 1985) Z12619: tobacco (Lindhorst et al., Plant Mol. Biol. 21: 985-992, 1993), X78988: maize (Jose Cordero et al, Plant J. 6: 141-150, 1994), EILXCH: leech (See Mullet et al, Hoppe-Seyler's Z. Physiol Chem. 358: 1105-1117, 1977). * PI reactive site. Both StPotIA and StPotIB are similar to other family members from potato. However, StPotIA has an additional four amino acids at position 41 to 44 that are also found in a wound induced PotI from tomato (K03290). StPotIB has a methionine at the PI site which is common for potato isolates. StPotIA has an alanine at the PI site which has not been reported for PotI isolates from potato, but is present in a PotI isolate from maize (X78988).
Figure 25 is a graphical representation showing purification of E. coli expressed StPotIA and StPotIB. StPotIA and StPotIB were purified using the N-terminal HIS tag fused to StPotIA and StPotIB and a metal affinity resin followed by RP-HPLC. Profile after separation by RP-HPLC. Protein was eluted with a linear gradient of 0-100% Buffer B (80% (v/v) acetonitrile, 0.1% (v/v) TFA) at a flow-rate of 1 ml/min over 60 min. (A) StPotIA, (B) StPotIB. StPotIA eluted in one major peak at 36 min retention time (60% Buffer B) and StPotIB eluted in one major peak at 25 min retention time (42% Buffer B). The peaks were analyzed by SDS-PAGE and stained with silver (insert in A). Lane 1 is StPotIA (pkl), lane 2 is StPotIB (pk2) and lane 3 is purified PotI from potato tubers. StPotIA and StPotIB have a different apparent mobility (10 kDa) to a mix of PotI isoforms isolated from tubers (6 kDa), due to the additional HIS-tag epitope at the N terminus.
Figure 26 is a graphical representation showing inhibition of NaPI-insensitive chymotrypsin by bacterially expressed StPotIA and StPotIB. Inhibition of the NaPI-insensitive chymotrypsin from the gut of H. punctigera with purified PotI. (A) substrate SA2PFpNA, development time 30 min, (B) substrate SA2PLpNA, 30 min incubation. Mix of PotI isoforms from potato tuber (circle), StPotIA (square), StPotIB (triangle). StPotIA, StPotIB and the PotI from potato tubers were good inhibitors of the NaPI- insensitive chymotrypsin.
Figure 27 is a graphical representation showing growth of H. armigera larvae on transgenic cotton expressing NaPI and PotI. Transgenic cotton cv Coker 315 was used in bioassays with H. armigera,. Thirty larvae were fed leaves of either the control untransformed Coker 315, transgenic line 1 (NaPI), transgenic line 2 (StPotIA) or transgenicplant 3 (NaPI X StPotIA). The weight of the larvae was measured at day 7 post-
egg hatch. (A) Growth of larvae. At day 7, larvae fed leaves expressing NaPI were 86% of the weight of the control larvae fed untransformed leaves. Larvae fed leaves expressing StPotIA were 92% of the control and larvae fed leaves expressing both NaPI and StPotIA were 46% of the control. (B) The effect of ingestion of NaPI and PotI on gut trypsin and chymotrypsin activity. Gut from the larvae in each experiment were pooled and extracts prepared. All assays were performed in duplicate. Trypsin activity (black) was determined using BApNA substrate and chymotrypsin activity (grey) with SA2PFpNA substrate. Units of activity are expressed as change in absorbance at 405 nm/min/ug gut extract protein. Trypsin activity was reduced, relative to the control, in the extracts from larvae fed leaves expressing NaPI and NaPI+StPotlA. Chymotrypsin activity was elevated in extracts from larvae fed leaves expressing NaPI or NaPI + StPotIA and reduced in extracts from larvae fed StPotIA alone.
Figure 28 diagrammatic representation showing the nucleotide sequence and deduced amino acid sequence from the HpF5 cDNA encoding the NaPI-insensitive chymotrypsin and the location of the oligonucleotide primers used to add an endoplasmic reticulum sequence to HpCh5. The FwBacRECHl primer was used to add the first half of the ER signal sequence as well as a silent mutation, changing A to G to destroy the BamHI cut site. The FwBacRECH2 primer added the remainder of the coding sequence for the ER signal as well as a BamHI cut site to the 3' end of the sequence. The ER signal was added before the hexahistidine tag to enable purification of the expressed protein by metal affinity chromatography after cellular processing. The added amino acids are shaded in grey.
Figure 29 is a photographic representation showing expression of the chymotrypsin clone HpF5 in baculovirus infected insect cells. Expressed proteins were separated on 12.5% (w/v) SDS+PAGE gels and subjected to immunoblots with the α-HpCh5-antibody (A) RCDNA. Production of HpCh5 by HIGH FIVE (trademark) insect cells transfected with 20µl of bacmid DNA. Controls; (pFastBacVector) insect cells transfected with bacmid DNA transposed with pFastBac vector without the HpF5insert; (blue colony) insect cells transfected with untransposed bacmid DNA; (cells alone) untransfected insect cells
(Cellfectin). insect cells treated with CELFECTIN (registered trademark) alone; The positive control, + is bacterially expressed HpCh5 with a hexahistidine tag. (E) Production of HpCh5 by HIGH FIVE (trademark) insect cells infected with virus. RC 24, 48 and 72. Medium collected 24, 48 and 72 hours after infection with HpF5 recombinant virus. Controls 24, 48 and 72. Medium from cells treated with virus prepared from nontransfected bachmid (blue colony) for 24, 48 and 72 hours. Positive control is bacterially expressed HpCh5 with a hexahistidine tag.
DETAILED DESCRIPTION OF THE INVENTION
The present invention is predicated in part on the identification and cloning of a novel insect chymotrypsin molecule termed "HpCh5". cDNA encoding HpCh5 is referred to herein as "HpF5". The isolation of this molecule permits the identification and design of a range of products which are useful in controlling the growth, development and/or overall biological fitness of Helicoverpa spp. and other insects. These products generally act as antagonists of HpCh5 function or HpF5 gene expression and are useful as insect control agents.
The amino acid sequence of HpCh5 is set forth in SEQ ID N0:2. The nucleotide sequence of HpF5 is set forth in SEQ ID N0s:4 and 6.
Reference herein to "HpCh5" should be understood as a reference to all forms of HpCh5 including, for example, any peptide isoforms which arise from alternative splicing of HpF5 mRNA, mutants or polymorphic variants of HpCh5, any post-translation modified forms of HpCh5 or any non-post-translational modified forms of HpCh5 as well as any homolog in other insect species or strains. The term "HpCh5" also encompasses members in a HpCh5 family of chymotrypsin molecules. To the extent that it is not specified, reference herein to HpCh5 includes derivatives, homologs, analogs, chemical equivalents and mimetics thereof. Reference to HpCh5 also refers to any variant having at least 75% amino acid identity to SEQ ID NO:2 after optimal alignment. Examples of variants of HpCh5 include Pi-sensitive variants such as those inter alia having an Arg 192 Gln or Arg 192 Asn substitution. Other variants include the N-terminal signal sequence of HpCh5 as defined in SEQ ID NO:3 and which is encoded by the nucleotide sequence set forth in SEQ ID NO:5. Such variants include a signal sequence comprising an amino acid sequence having at least about 75% similarity to SEQ ID N0:3 after optimal alignment or encoded by a nucleotide sequence having at least about 75% identity to SEQ ID NO:5 or a nucleotide sequence capable of hybridizing to SEQ ID NO: 5 after optimal alignment.
Reference to "HpF5" should be understood as reference to all forms of HpF5 including any cDNA isoform, genomic forms, mutants and polymorphic variants of HpF5 as well as any homologs from other species or strains of insect. The term "HpF5" also encompasses members of a HpF5 family of genes which encode HpCh5 or HpCh5-type chymotrypsins. To the extent that it is not specified, reference herein to HpF5 includes derivatives of HpF5 as well as a nucleotide sequence having at least about 75% identity to SEQ ID N0:4 or SEQ ID N0:6 or a nucleotide sequence capable of hybridizing to SEQ ID NO:4 or SEQ ID N0:6 or its complementary form under low stringency conditions. The signal sequence of HpF5 as defined in SEQ ID NO:3 and encoded by SEQ ID NO:5 also encompasses variants thereof. As indicated above, such variants include a signal sequence having at least about 75% similarity to SEQ ID N0:3 after optimal alignment and/or being encoded by a nucleotide sequence capable of hybridizing to SEQ ID NO:5 under low stringency conditions and/or a nucleotide sequence having at least about 75% identity to SEQ ID N0:5 after optimal alignment.
Before describing the present invention detail, it is to be understood that unless otherwise indicated, the subject invention is not limited to specific formulations of components, manufacturing methods, administration regimens, or the like, as such may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.
It must be noted that, as used in the subject specification, the singular forms "a", "an" and "the" include plural aspects unless the context clearly dictates otherwise. Thus, for example, reference to an "agent" or "antagonist" includes a single agent or antagonist as well as two or more agents or antagonists and so forth.
In describing and claiming the present invention, the following terminology is used in accordance with the definitions set forth below.
The terms "compound", "agent", "active agent" and "active" are used interchangeably herein to refer to a chemical compound which inhibits the activity of HpChS or the expression of a genomic gene corresponding to HpF5. The terms also encompass
agriculturally or horticultural active ingredients of those active agents specifically mentioned herein including but not limited to salts, esters, amides, prodrugs, active metabolites, analogs and the like. When the term "compound", "agent", "active agent" or "active" is used, then it is to be understood that this includes the agent per se as well as agriculturally or horticulturally acceptable, physiologically active salts, esters, amides, prodrugs, metabolites, analogs, etc. The term "compound" is not to be construed as a chemical compound only but extends to peptides, polypeptides and proteins as well as genetic molecules such as RNA, DNA and chemical analogs thereof.
The present invention contemplates, therefore, compounds useful in down-regulating the activity of HpCh5 or down-regulating expression of a genomic gene corresponding to HpCh5. The term "down-regulating" encompasses inhibition of HpCh5 activity. The inhibition of HpCh5 or reduction in its levels has the effect of reducing or retarding the growth of the insect. The inhibition of HpCh5 activity or HpF5 gene expression may occur by producing an inhibitor in a plant which is then consumed by the insect or the inhibitor may be topically applied to a plant or sprayed or otherwise dispersed to insects or a source of insects. In this regard, the plant may produce a nucleic acid molecule that interferes with HpF5 expression when consumed by the insect. Alternatively, the plant may produce a PI capable of inhibiting HpCh5. Still in a further alternative, the HpCh5 inhibitor is a non-proteinaceouss chemical applied to the surface of a plant or taken up by the root system of a plant. Reference herein to a "plant" is not to exclude trees or cultured tissues (e.g. callus) from a plant (or tree).
Reference to compounds, agents and actives also includes combinations of compounds, agents or actives. Such combinations may be formulated in multi-part agricultural or horticultural compositions which are admixed together prior to dispersement or given sequentially.
The terms "effective amount", "insecticidal effective amount" and "insect-static effective amount" of an agent as used herein mean a sufficient amount of the agent to reduce or retard insect growth and development and/or to kill or inhibit the insect.
By "agriculturally acceptable" or "horticulturally acceptable" carrier, excipient or diluent is meant a vehicle comprised of a material that is not environmentally or otherwise undesirable to a plant or non-target insect. Carriers may include excipients and other additives such as diluents, detergents, coloring agents, wetting or emulsifying agents, pH buffering agents, preservatives, and the like.
The terms "treating" and "treatment" as used herein in relation to plants or eradication of insects refer to reduction in severity of symptoms of insect infestation of a plant or the application of the agents to a group of insects resulting in retardation of their growth, development or biological fitness or wellbeing.
The compounds of the present invention may be large or small molecules, nucleic acid molecules (including antisense or sense molecules), peptides, polypeptides or proteins or hybrid molecules such as RNAi- or siRNA-complexes, ribozymes or DNAzymes.
The term "nucleic acid molecule" is also encompassed by the expression "genetic molecule" and includes hairpin constructs such as those which include RNAi-mediated post-transcriptional gene silencing or methylation-mediated silencing.
Accordingly, one aspect of the present invention provides an isolated nucleic acid molecule or derivative, homolog or analog thereof comprising a nucleotide sequence encoding a novel chymotrypsin protein or a derivative, homolog or mimetic thereof wherein said chymotrypsin is insensitive to a PI of N. alata.
More particularly, the present invention is directed to a nucleic acid molecule or derivative, homolog or analog thereof comprising a nucleotide sequence encoding, or a nucleotide sequence complementary to a nucleotide sequence encoding, an amino acid sequence substantially as set forth in SEQ ID N0:2 or a derivative, homolog or mimetic thereof or having at least about 75% or greater identity to SEQ ID NO:2 after optimal alignment or a nucleotide sequence set forth in SEQ ID NO:4 or SEQ ID N0:6 or a nucleotide sequence
having at least about 75% similarity or greater to SEQ ID N0:4 or SEQ ID NO:6 or a nucleotide sequence capable of hybridizing to SEQ ID N0:4 or SEQ ID NO:6 or its complementary form under low stringency conditions.
Another aspect of the present invention provides an isolated chymotrypsin Helicoverpa ssp. wherein said chymotrypsin exhibits resistance to a PI from N. alata or a variant, derivative, homolog or analog of said chymotrypsin.
More particularly, the isolated chymotrypsin comprises an amino acid sequence set forth in SEQ ID N0:2 or an amino acid sequence having at least about 75% similarity to SEQ ID NO:2 after optimal alignment.
The term "similarity" as used herein includes exact identity between compared sequences at the nucleotide or amino acid level. Where there is non-identity at the nucleotide level, "similarity" includes differences between sequences which result in different amino acids that are nevertheless related to each other at the structural, functional, biochemical and/or conformational levels. Where there is non-identity at the amino acid level, "similarity" includes amino acids that are nevertheless related to each other at the structural, functional, biochemical and/or conformational levels. In a particularly preferred embodiment, nucleotide and amino acid sequence comparisons are made at the level of identity rather than similarity.
Terms used to describe sequence relationships between two or more polynucleotides or polypeptides include "reference sequence", "comparison window", "sequence similarity", "sequence identity", "percentage of sequence similarity", "percentage of sequence identity", "substantially similar" and "substantial identity". A "reference sequence" is at least 12 but frequently 15 to 18 and often at least 25 or above, such as 30 monomer units, inclusive of nucleotides and amino acid residues, in length. Because two polynucleotides may each comprise (1) a sequence (i.e. only a portion of the complete polynucleotide sequence) that is similar between the two polynucleotides, and (2) a sequence that is divergent between the two polynucleotides, sequence comparisons between two (or more)
polynucleotides are typically performed by comparing sequences of the two polynucleotides over a "comparison window" to identify and compare local regions of sequence similarity, A "comparison window" refers to a conceptual segment of typically 12 contiguous residues that is compared to a reference sequence. The comparison window may comprise additions or deletions (i.e. gaps) of about 20% or less as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. Optimal alignment of sequences for aligning a comparison window may be conducted by computerized implementations of algorithms (GAP, BESTF1T, FASTA, and TFASTA in the Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Drive Madison, WI, USA) or by inspection and the best alignment (i.e. resulting in the highest percentage homology over the comparison window) generated by any of the various methods selected. Reference also may be made to the BLAST family of programs as, for example, disclosed by Altschul et al. (Nucl. Acids Res. 25: 3389-3402, 1997). A detailed discussion of sequence analysis can be found in Unit 19.3 of Ausubel et al. ("Current Protocols in Molecular Biology" John Wiley & Sons Inc, 1994-1998, Chapter 15,1998).
The terms "sequence similarity" and "sequence identity" as used herein refers to the extent that sequences are identical or functionally or structurally similar on a nucleotide-by-nucleotide basis or an amino acid-by-amino acid basis over a window of comparison. Thus, a "percentage of sequence identity", for example, is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base (e.g. A, T, C, G, I) or the identical amino acid residue (e.g. Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gin, Cys and Met) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity. For the purposes of the present invention, "sequence identity" will be understood to mean the "match percentage" calculated by the DNASIS computer program (Version 2.5 for windows; available from Hitachi Software engineering Co., Ltd,, South San Francisco, California, USA) using standard defaults as used in the
reference manual accompanying the software. Similar comments apply in relation to sequence similarity. The term "similarity" is particularly useful to describe amino acid sequence comparisons. The term "identity" is particularly useful to describe nucleotide sequence comparisons.
Reference to at least about 75% identity or 75% similarity includes percentage identities and similarities greater than 75% such as 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,. 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% and 100%.
Reference herein to a low stringency means from at least about 0 to at least about 15% (v/v) formamide (including 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10% 11%, 12%, 13% and 14% (v/v) formamide) and from at least about 1 M to at least about 2 M salt for hybridization, and at least about 1 M to at least about 2 M salt for washing conditions. Generally, low stringency is at from about 25-30°C to about 42°C. The temperature may be altered and higher temperatures used to replace formamide and/or to give alternative stringency conditions. Alternative stringency conditions may be applied where necessary, such as medium stringency, which includes and encompasses from at least about 16% (v/v) to at least about 30% (v/v) formamide and from at least about 0.5 M to at least about 0.9 M salt for hybridization, and at least about 0.5 M to at least about 0.9 M salt for washing conditions, or high stringency, which includes and encompasses from at least about 31% (v/v) to at least about 50% (v/v) formamide and from at least about 0.01 M to at least about 0.15 M salt for hybridization, and at least about 0.01 M to at least about 0.15 M salt for washing conditions. In general, washing is carried out Tm = 69.3 + 0.41 (G+C)% (Marmur and Doty, J. Mol. Biol. 5: 109, 1962). However, the Tmof a duplex DNA decreases by 1°C with every increase of 1% in the number of mismatch base pairs (Bonner and Laskey, Eur. J. Biochem. 46: 83, 1974). Formamide is optional in these hybridization conditions. Accordingly, particularly preferred levels of stringency are defined as follows: low stringency is 6 x SSC buffer, 0.1% (w/v) SDS at 25-42°C; a moderate stringency is 2 x SSC buffer, 0.1% (w/v) SDS at a temperature in the range 20°C to 65°C; high stringency is 0.1 x SSC buffer, 0.1% (w/v) SDS at a temperature of at least 65°C.
A further aspect of the present invention contemplates a nucleic acid molecule or derivative, bomolog or analog thereof comprising a nucleotide sequence substantially as set forth in SEQ ID N0:4 or SEQ ID N0:6 or a nucleotide sequence having at least 75% or greater similarity to SEQ ID NO:4 or SEQ ID NO.6 or a derivative, homolog or analog thereof, or capable of hybridizing to SEQ ID NO:4 or SEQ ID NO:6 or its complementary form under low stringency conditions and which encodes an amino acid sequence substantially as set forth in SEQ ID NO:2 or a derivative, homolog or mimetic thereof or having at least 75% or greater identity to SEQ ID NO:2 after optimal alignment which nucleic acid molecule encodes a chymotrypsin which is insensitive to a proteinaceous inhibitor of N. alata.
Yet another aspect of the present invention contemplates nucleic acid molecule comprising a sequence of nucleotides substantially as set forth in SEQ ID NO:4 or SEQ ID NO:6. The nucleic acid molecule encoding HpCh5 is preferably a sequence of deoxyribonucleic acids such as a cDNA sequence or a genomic sequence. A genomic sequence may also comprise exons or introns. A genomic sequence may also include a promoter region or other regulatory regions. The present invention further contemplates isolated introns and exons of HpF5 such as those involved in genetic networking within a plant cell.
The nucleic acid molecule according to this aspect of the invention corresponds herein to HpF5. This cDNA has been determined, in accordance with the present invention, to encode a protein that defines a new family of chymotrypsins, family 5, within the group of chymotrypsin gene families, and this protein is referred to herein as HpCh5. Reference to "HpF5" also includes a genomic form of the gene. Within the Helicoverpa punctigera chymotrypsin gene families, there are varying levels of homology as shown in Table 2. Family 5 is exemplified by HpF5, and is most similar to family 2A at 73% and least similar to family 4 at
substitution is associated with one of the ß-strands that forms a wall of the primary substrate-binding pocket. The location of this substitution and conversion to an arginine, from glutamine, is highly unusual for the S1 domain that is predominantly lined with non-polar residues that define chymotrypsin specificity
Percentage protein sequence identity between members of the H. punctigera
chmotrypsin gene family
The present invention provides, therefore, an isolated protein having chymotrypsin activity which is not substantially inhibited by a PI from N. alata. Accordingly, another aspect of the present invention is directed to an isolated protein selected from the list consisting of:
(i) a novel chymotrypsin protein or a derivative, homolog or mimetic thereof wherein said chymotrypsin is insensitive to the proteinase inhibitors of N. alata;
(ii) a protein having an amino acid sequence substantially as set forth in SEQ ID N0:2 or a derivative, homolog or mimetic thereof or having at least 75% or greater identity to SEQ ID N0:2 after optimal alignment;
(iii) a protein encoded by a sequence of nucleotides substantially as set forth in SEQ ID NO:4 or SEQ ID NO:6 or a derivative, homolog or analog thereof or a nucleotide sequence having at least 75% similarity to SEQ ID N0:4 or SEQ ID NO:6 or a sequence encoding an ammo acid sequence substantially as set forth in SEQ ID NO:2 or a derivative, homolog or mimetic thereof or having at least 75% or greater similarity to SEQ ID NO:2 after optimal alignment;
(iv) a protein encoded by a nucleic acid molecule or derivative, homolog or analog thereof comprising a nucleotide sequence substantially as set forth in SEQ ID NO:4 or SEQ ID NO.6 or a derivative, homolog or analog thereof, or capable of hybridizing to SEQ ID NO:4 or SEQ ID NO:6 under low stringency conditions and which encodes an amino acid sequence substantially as set forth in SEQ ID N0:2 or a derivative, homolog or mimetic thereof or having at least 75% or greater similarity to SEQ ID N0:2 after optimal alignment.
(v) a novel chymotrypsin protein or a derivative, homolog or mimetic thereof that has an arginine substituted for an asparagine or glutamine in the primary substrate-binding pocket.
The present invention discloses the amino acid, and corresponding cDNA sequence of a novel chymotrypsin that is insensitive to the Type II serine proteinase inhibitors produced by solanaceous species such as N. alata. Therefore, this may be used as a target for agents to control insects carrying this insensitive proteinase. A number of compounds have been shown to inhibit the activity of HpCh5, and a list of these compounds as preferred embodiments is found in Table 3.
Effect of various proteinase inhibitors on the activity of the NaPI- Insensitive chymotrypsins from H. punctigera and bovine chymotrypsin
SBTI, soybean trypsin inhibitor; PMSF, phenylmethyl sulphonyl fluoride; Bowman Birk, soybean Bowman Birk inhibitor; lima bean, lima bean trypsin inhibitor (Sigma); Pot I, potato proteinase inhibitor Type I. Bovine chymotrypsin (BC).
Without limiting the mode of action of any of these compounds to any one activity, 3-D modeling is used to investigate the binding ability of PotI and the peptide encoded by the Cl domain of NaPI to both NaPI-insensitive and NaPI-sensitive insect chymotrypsins.
The deduced amino acid sequences from the cDNA clones HpF2A (NaPI-sensitive) and HpF5 (NaPI-insensitive) were modeled on the structures of the Solenopsis invicta (fire ant) and Bos taurus (cow) chymotrypsins obtained from the Research Collaboratory for Structural Bibinformatics (RCSB) Protein Data Bank.
The Helicoverpa chymotrypsins are predicted to adopt similar structures to those reported for all the chymotrypsin structures available in the data bank. The modeled structures have the classic serine protease fold consisting of two, six-stranded anti-parallel beta barrels with the catalytic triad located between the two domains. Certain surface loops are cleaved in the mammalian chymotrypsins (loop 142), but remain intact within insect chymotrypsins. Therefore, the fire ant chymotrypsin structure (Botos et al, J. Mol. Biol. 298: S95-901, 2000 ) was required to help refine the orientation of these surface loops in the Helicoverpa chymotrypsin models. Two surface loops, 60 and 142 are considerably larger in the H. punctigera chymotrypsins (Figures 15,16).
Cl was modeled in complex with NaPI-sensitive and NaPI-insensitive chymotrypsins to investigate what residues in the NaPI-insensitive chymotrypsin might be involved in the loss of inhibitor binding. The structure of the chymotrypsin inhibitor Cl was previously determined by 1H NMR (Nielson et al., 1994, supra) but has not been determined in a proteinase complex. Therefore the related proteinase inhibitor PCI-1 from Solanum tuberosum in complex with Proteinase B from Streptomyces griseus (Greenblatt et al, J. Mol Biol. 205: 201, 1989) provided an appropriate basis guiding the alignment of the complexes. Energy minimization of the Cl-chymotrypsin complexes revealed Argl92 (chymotrypsin numbering system) as the only likely candidate to cause such resistance from a passible 24 putative contact residues. The NaPI-insensitive chymotrypsin containing the Arg192 (Greer nomenclature, Greer, Proteins 7:317-34, 1990. Figure 15) residue in complex with Cl could not be properly energy minimized due to steric contacts between Algl92 and Cl whereas the Gln192 residue in the Na-PI sensitive chymotrypsin in complex with Cl caused no such problems due to its much smaller size. Figure 17 shows a close up view of the binding region surrounding Gin 192 in the Cl-HpF2A chymotrypsin complex. It is clear that Gin 192 is not in conflict with any regions on the inhibitor molecule. However, comparison to the cognate Arg residue in the Cl-HpCh5 chymotrypsin model demonstrates there is not enough space to accommodate this much larger residue (Figure 18) making contact with Thr5 and Ala9 in Cl. Furthermore, modeling the StPotlA inhibitor into HpCh5 revealed that the NaPI-insensitive chymotrypsin could accommodate the Argl92 residue consistent with the inhibition of this chymotrypsin by StPotlA (Figure 19).
In summary, the NaPI-insensitive chymotrypsin from Helicoverpa species has an arginine in place of an asparagine or glutamine at position 192 that extends into the S1 binding
pocket and appears to interfere with Cl binding. Furthermore, it is clear that this Arg residue does not interfere with PotI binding, consistent with the observation that PotI is a much more efficient inhibitor of insect chymotrypsins than the NaPI inhibitors. Large quantities of the PotI inhibitor were purified from potato tubers (Figure 22) to evaluate the combined effect of NaPI and PotI on the growth of H. armigera larvae (Figure 23). Bioassays confirmed that PotI significantly enhances the activity of the NaPI inhibitors. Caterpillars fed NaPI and PotI in combination (0.26 and 0.34% (w/v), respectively) were 34% the size of control larvae at the fifth instar stage of development whereas caterpillars feeding on NaPls alone were about 84% the size of the controls.
Therefore, another aspect of the present invention provides a method for modulating activity of the HpCh5 or a homolog or variant thereof in an insect, said method comprising contacting the HpCh5 protein or its homolog or variant with an effective amount of an agent for a time and under conditions sufficient to decrease or increase HpCh5 activity.
Yet another aspect of the present invention provides a method for modulating expression of HpF5 or homolog or variant in an insect, said method comprising contacting HpF5 or its homolog or variant with an effective amount of an agent for a time and under conditions sufficient to decrease or increase HpF5 expression.
The preferred insects targeted in accordance with these and other aspects are species of Helicoverpa and other Lepidopteran species. In addition, plants to be protected include those sensitive to H. armigera, H. punctigera, H, zea and H. virescens. Such plants include the H. armigera sensitive plants such as cotton, sweet corn, tomato, tobacco, piniento, potato, sunflower, citrus, plums, sorghum, leeks, soybean, alfalfa, beans, pidgeon peas, chick peas, artichokes, curcurbits, lettuce, Dianthus (an ornamental plant), geraniums, cape gooseberry, maize, flax and linseed, alfalfa, lupins, broad beans, garden peas, peanuts, canola, snapdragons, cherry, sunflower, pot marigolds and Helichiysum (an ornamental plant)
Other plants contemplated herein include cereals (such as wheat, barley, oats, triticale,
etc.), horticultural plants (e.g. apples, carrots, onions, etc.), ornamental plants (such as orchids, roses, petunias, etc.) and trees.
The present invention contemplates, therefore, methods of screening for compounds which inhibit or act as antagonists of HpCh5 activity or HpF5 gene expression. For example, one method involves contacting a candidate compound with HpCh5. The screening procedure includes assaying (i) for the presence of a complex between the compound and ITpCh5, or (ii) an alteration in the expression levels of HpF5 cDNA or genomic DNA. One form of assay involves competitive binding assays. In such competitive binding assays, HpCh5 is typically labeled. Free HpCh5 is separated from any putative complex and the amount of free (i.e. uncomplexed) label is a measure of the binding of the agent being tested to bind to HpCh5. One may also measure the amount of bound, rather than free, HpCh5. It is also possible to label the compound rather than HpCh5 and to measure the amount of compound binding to target in the presence and in the absence of the compound being tested. Such compounds may inhibit HpCh5. A similar approach may be adopted for compounds which bind to and inhibit HpF5 or mRNA transcripts thereof.
Another technique for agent screening provides high throughput screening for compounds having suitable binding affinity to HpCh5 and is described in detail in Geysen (International Patent Publication No. WO 84/03564). Briefly stated, large numbers of different small peptide test compounds are synthesized on a solid substrate, such as plastic pins or some other surface. The peptide test compounds are reacted with HpCh5 and washed. Bound HpCh5 molecules are then detected by methods well known in the art. This method may be adapted for screening for non-peptide, chemical entities. This aspect, therefore, extends to combinatorial approaches including phage display to screen for HpCh5 antagonists.
Purified HpCh5 can be coated directly onto plates for use in the aforementioned agent screening techniques. However, non-neutralizing antibodies to HpCh5 may also be used to immobilize HpCh5 on the solid phase.
Live animals such as H. armigera and/or H. punctigera may also be used in feeding trials to find potential inhibitors.
The present invention also contemplates the use of competitive agent screening assays in which neutralizing antibodies capable of specifically binding HpCh5 compete with a test compound for binding to HpCh5 or fragments thereof. In this manner, the antibodies can be used to detect the presence of any peptide which shares one or more antigenic determinants of HpCh5.
Yet another useful source of analogs of compounds which are chemically modified may be used to induce feed-back inhibition of biochemical or genetic pathways for generating authentic HpCh5.
Analogs of HpCh5 contemplated herein include but are not limited to modification to side chains, incorporating of unnatural amino acids and/or their derivatives during peptide, polypeptide or protein synthesis and the use of crosslinkers and other methods which impose conformational constraints on HpCh5.
Examples of side chain modifications of HpCh5 contemplated by the present invention include modifications of amino groups such as by reductive alkylation by reaction with an aldehyde followed by reduction with NaBH4; amidination with methylacetimidate; acylation with acetic anhydride; carbamoylation of amino groups with cyanate; trinitrobenzylation of amino groups with 2, 4, 6-trinitrobenzene sulphonic acid (TNBS); acylation of amino groups with succinic anhydride and tetrahydrophthalic anhydride; and pyridoxylation of lysine with pyridoxal-5-phosphate followed by reduction with NaBH4
The guanidine group of arginine residues may be modified by the formation of heterocyclic condensation products with reagents such as 2,3-butanedione, phenylglyoxal and glyoxal.
The carboxyl group may be modified by carbodiimide activation via O-acylisourea formation fallowed by subsequent derivitization, for example, to a corresponding amide.
Sulphydryl groups may be modified by methods such as carboxymethylation with iodoacetic acid or iodoacetamide; perfomiic acid oxidation to cysteic acid; formation of a mixed disulphides with other thiol compounds; reaction with maleimide, maleic anhydride or other substituted maleimide; formation of mercurial derivatives using 4-chloromercuribenzoate, 4-chloromercuriphenylsulphonic acid, phenylmercury chloride, 2-chloromercuri-4-nitrophenol and other mercurials; carbamoylation with cyanate at alkaline pH.
Tryptophan residues may be modified by, for example, oxidation with N-bromosuccinimide or alkylation of the indole ring with 2-hydroxy-5-nitrobenzyl bromide or sulphonyl halides. Tyrosine residues on the other hand, may be altered by nitration with tetranitromathane to form a 3-nitrotyrosine derivative.
Modification of the imidazole ring of a histidine residue may be accomplished by alkylation with iodoacetic acid derivatives or N-carbethoxylation with diethylpy roparbonate.
Examples of incorporating unnatural amino acids and derivatives during peptide synthesis include, but are not limited to, use of norleucine, 4-amino butyric acid, 4-amino-3-hydroxy-5-phenylpentanoic acid, 6-aminohexanoic acid, t-butylglycine, norvaline, phenylglycine, ornithine, sarcosine, 4-amino-3-hydroxy-6-methylheptanoic acid, 2-thienyl alanine and/or D-isomers of amino acids. A list of unnatural amino acid, contemplated herein is shown in Table 4.
TABLE 4 Codes for non-conventional amino acids
Crosslinkers can be used, for example, to stabilize 3D conformations, using homo-bifunctionat crosslinkers such as the bifunctional imido esters having (CH2)n spacer groups with n=l to n=6, glutaraldehyde, N-hydroxysuccinimide esters and hetero-bifunctional reagents which usually contain an amino-reactive moiety such as N-hydroxysuccinimide and another group specific-reactive moiety such as maleimido or dithio moiety (SH) or carbodiimide (COOH). In addition, peptides can be conformationally constrained by, for example, incorporation of Cα and Nα-methylamino acids, introduction of double bonds between Cα and Cß atoms of amino acids and the formation of cyclic peptides or analogs by introducing covalent bonds such as forming an amide bond between the N and C termini, between two side chains or between a side chain and the N or C terminus.
Such analogs, especially if they retain activity or even the HpCL5 molecule itself may have indistinct applications such as in washing powder or as in a stain removal formulation.
Another aspect of the present invention contemplates any compound which binds or otherwise interacts with HpCh5 or its derivatives or variants or which induces feed-back inhibition of HpCh5 synthesis resulting in down-regulation of HpCh5 activity or levels.
The present invention is also useful for screening for other compounds which reduce expression of HpF5. A variety of agent screening techniques may be employed such as those described herein and in International Publication No. WO 97/02048.
A compound antagonist includes a variant of HpCh5 such as a variant comprising an analog amino acid residue as indicated above. In one embodiment, the target is the HpCh5 polypeptide. The term "polypeptide" refers to a polymer of amino acids and its equivalent and does not refer to a specific length of the product, thus, peptides, oligopeptides and proteins are included within the definition of a polypeptide. This term also does not exclude modifications of the polypeptide, for example, glycosylations, aceyiations, phosphorylations and the like. Included within the definition are, for example, polypeptides containing one or more analogs of an amino acid (including, for example, unnatural amino acids such as those given in Table 4) or polypeptides with substituted linkages.
A substance identified as an antagonist of HpCh5 function or HpF5 gene activity may be a peptide or non-peptide in nature. Non-peptide "small molecules" are often preferred for many agricultural or horticultural purposes due to their perceived stability.
There are several steps commonly taken in the design of a mimetic-type antagonist of HpCh5 from a compound. First, the particular parts of the compound that are critical and/or important in determining the target property are determined. In the case of a peptide, this can be done by systematically varying the amino acid residues in the peptide, e.g. by substituting each residue in turn. Alanine scans of peptides are commonly used to refine such peptide motifs. These parts or residues constituting the active region of the compound are known as its "agrichemicaphore".
Once the agrichemicaphore has been found, its structure is modeled according to its physical properties, e.g. stereochemistry, bonding, size and/or charge, using data from a range of sources, e.g. spectroscopic techniques, x-ray diffraction data and NMR. Computational analysis, similarity mapping (which models the charge and/or volume of a agrichemicaphore, rather than the bonding between atoms) and other techniques can be used in this modeling process.
In a variant of this approach, the three-dimensional structure of HpCh5 and a compound binding it. This can be especially useful where HpCh5 or its antagonist change conformation on binding, allowing the model to take account of this in the design of the mimetic. Modeling can be used to generate inhibitors which interact with the linear sequence or a three-dimensional configuration.
A template molecule is then selected onto which chemical groups which mimic the pharmacophore can be grafted. The template molecule and the chemical groups grafted onto it can conveniently be selected so that the agrichemicaphore is easy to synthesize and is likely to be agriculturally or horticulturally acceptable. Alternatively, where the agrichemicaphore is peptide-based, further stability can be achieved by cyclizing the peptide, increasing its rigidity. The agrichemicaphore or agrichemicaphores found by this approach can then be screened to see whether they have HpCh5 antagonistic property, or to what extent they exhibit it. Further optimization or modification can then be carried out to arrive at one or more final agents for testing.
Yet another aspect of the present invention provides a method for detecting an agent capable of binding or otherwise associating with a HpCh5 binding site or functional equivalent thereof said method involving the use of in-silico 3-D modeling to identify compounds that bind to HpCh5 and specifically, are not interfered with by Arginine 192.
The goal of rational HpCh5 antagonist design is to produce structural analogs of HpCh5 or of small molecules with which HpCh5 interacts (e.g. an antagonist or inhibitor) in order to fashion agents which are, for example, more inhibitory of HpCh5. See, e.g. Hodgson (Bio/Technology 9: 19-21, 1991). In one approach, one first determines the three-dimensional structure of HpCh5 by x-ray crystallography, by computer modeling or, most typically, by a combination of approaches. Useful information regarding the structure of a polypeptide may also be gained by modeling based on the structure of chymotrypsins. An example of rational drug design is the development of HIV protease inhibitors (Erickson et al., Science 249: 527-533, 1990). In addition, target molecules may be analyzed by an alanine scan (Wells, Methods Enzymol. 202: 2699-2705,1991). In this technique, an amino acid residue is replaced by Ala and its effect on the peptide's activity is determined. Each of the amino acid residues of the peptide is analyzed in this manner to determine the important regions of the peptide.In addition, compounds including antagonists may be directed to particular locations or regions or domains or HpCh.5.
It is also possible to isolate a HpCh5 -specific antibody and then to solve its crystal structure. In principle, this approach yields an agricore upon which subsequent agent design can be based. It is possible to bypass protein crystallography altogether by generating anti-idiotypic antibodies (anti-ids) to a functional antibody. As a mirror image of a mirror image, the binding site of the anti-ids is expected to be an analog of the original receptor. The anti-id could then be used to identify and isolate peptides from banks of chemically or biologically produced banks of peptides. Selected peptides would then act as the agrichemicaphore.
Chymotrypsin clone HpF2B is expressed in E. coli fused to a six histidine (6.H) tag at the C-terminus and is purified to homogeneity on Talon metal affinity resin (Figure 20) for injection into a rabbit for production of polyclonal antibodies. N-terminal sequencing of the purified product confirmed the expression of the chymotrypsin HpCh2B. After the fourth boost with antigen, the serum is collected and tested on protein blots of bacterially expressed protein and unfractionated gut extracts. The antibody detected the full-length recombinant chymotrypsin at a dilution of 1 in 2500 as well as several break-down products. Unfractionated gut extract and a sample of protein bound to the Cl affinity column were also stained with the anti-HpCh2B antibody which detected the mature native form of the enzyme.
Purified 6H,HpCh2B is used to test the detection limit of anti-HpCh2B antibody by comparison of immunoblots to silver stained SDS-PAGE gels. The antibody detected 20 ng of bacterially expressed chymotrypsinogen and also recognized the mature form of the native chymotrypsin isolated from gut of H. punctigera.
The cDNA (HpF5) encoding the NaPI-insensitive chymotrypsin (HpCh5) is expressed in E. coli in a similar manner except the six-histidine tag is fused to the N-terminus of the expressed protein. The polyclonal antiserum that is raised against the bacterially expressed chymotrypsin HpCh2B did not cross react with bacterially expressed NaPI- insensitive chymotrypsin (HpCh5) on protein blots (Figure 21). Likewise the antiserum raised against HpCh5 did not bind to HpCh2B. This indicates that these antisera can be used to specifically distinguish between and monitor levels of the NaPI -insensitive and sensitive chymotrypsins in unfractionated gut extracts
Accordingly, still another aspect of the present invention is directed to antibodies to HpCh5 and HpCh2B including catalytic antibodies.
In another aspect of the present invention, a method is provided for the isolation of and separation of individual isoforms of chymotrypsin, said method consisting of:
(i) affinity chromatography of insect gut extracts initially with benzamidine-sepharose to bind trypsins;
(ii) further affinity chromatography of the unbound proteins using immobilized N. alata serine proteinase inhibitor Cl to bind all NaPI inhibitable chymotypsins; and
(iii) affinity chromatography of the eluate from (ii) with immobilized PotI and PotII or chymostatin to bind the remainder. The putative NaPI-insensitive chymotrypsins are then eluted with 8 M urea.
The present invention extends to a genetic approach to down-regulating expression of an HpF5 or its homologs or variants. Such an approach uses nucleic acid molecules or molecules having a genetic component (e.g. RNAi) to induce pre- or post-transcriptional gene silencing.
The terms "nucleic acids", "nucleotide" and "polynucleotide" include RNA, cDNA, genomic DNA, synthetic forms and mixed polymers, both sense and antisense strands, and may be chemically or biochemically modified or may contain non-natural or derivatized nucleotide bases, as will be readily appreciated by those skilled in the art. Such modifications include, for example, labels, methylation, substitution of one or more of the naturally occurring nucleotides with an analog (such as the morpholine ring), internucleotide modifications such as uncharged linkages (e.g. methyl phosphonates, phosphotriesters, phosphoamidates, carbamates, etc.), charged linkages (e.g. phosphorothioates, phosphorodithioates, etc.), pendent moieties (e.g. polypeptides), intercalators (e.g. acridine, psoralen, etc.), chelators, alkylators and modified linkages (e.g. α-anomeric nucleic acids, etc.). Also included are synthetic molecules that mimic polynucleotides in their ability to bind to a designated sequence via hydrogen binding and other chemical interactions. Such molecules are known in the art and include, for example, those in which peptide linkages substitute for phosphate linkages in the backbone of the molecule.
Antisense polynucleotide sequences, for example, are useful in silencing transcripts of HpF5. Furthermore, polynucleotide vectors containing all or a portion of HpF5 gene locus may be placed under the control of a promoter in either the sense or antisense orientation and introduced into a cell. Expression of such a sense or antisense construct within a cell interferes with target transcription and/or translation. Furthermore, co-suppression (i.e. using sense-suppresion) and mechanisms to induce RNAi or siRNA may also be employed. Alternatively, antisense or sense molecules may be directly administered. In this latter embodiment, the antisense or sense molecules may be formulated in a composition and then administered by any number of means to target cells.
A variation on antisense and sense molecules involves the use of morpholinos, which are oligonucleotides composed of morpholine nucleotide derivatives and phosphorodiamidate linkages (for example, Summerton and Weller, Antisense and Nucleic Acid Drug Development 7: 187-195, 1997). Such compounds are injected into embryos and the effect of interference with mRNA is observed.
In one embodiment, the present invention employs compounds such as oligonucleotides and similar species for use in modulating the function or effect of nucleic acid molecules
encoding HpCh5, i.e. the oligonucleotides induce transcriptional or post-transcriptional gene silencing. This is accomplished by providing oligonucleotides which specifically hybridize with one or more nucleic acid molecules encoding the inhibitor. The oligonucleotides may be provided directly to a cell or generated within the cell. As used herein, the terms "target nucleic acid" and "nucleic acid molecule encoding HpCh5" have been used for convenience to encompass DNA encoding the inhibitor, RNA (including pre-mRNA and mRNA or portions thereof) transcribed from such DNA, and also cDNA derived from such RNA. The hybridization of a compound of the subject invention with its target nucleic acid is generally referred to as "antisense". Consequently, the preferred mechanism believed to be included in the practice of some preferred embodiments of the invention is referred to herein as "antisense inhibition." Such antisense inhibition is typically based upon hydrogen bonding-based hybridization of oligonucleotide strands or segments such that at least one strand or segment is cleaved, degraded, or otherwise rendered inoperable. In this regard, it is presently preferred to target specific nucleic acid molecules and their functions for such antisense inhibition.
The functions of DNA to be interfered with can include replication and transcription. Replication and transcription, for example, can be from an endogenous cellular template, a vector, a plasmid construct or otherwise. The functions of RNA to be interfered with can include functions such as translocation of the RNA to a site of protein translation, translocation of the RNA to sites within the cell which are distant from the site of RNA synthesis, translation of protein from the RNA, splicing of the RNA to yield one or more RNA species, and catalytic activity or complex formation involving the RNA which may be engaged in or facilitated by the RNA.
In the context of this invention, "hybridization" means the pairing of complementary strands of oligomeric compounds. In the present invention, the preferred mechanism of pairing involves hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary nucleoside or nucleotide bases (nucleobases) of the strands of oligomeric compounds. For example, adenine and thymine
are complementary nucleobases which pair through the formation of hydrogen bonds. Hybridization can occur under varying circumstances.
An antisense compound is specifically hybridizable when binding of the compound to the target nucleic acid interferes with the normal function of the target nucleic acid to cause a loss of activity, and there is a sufficient degree of complementarity to avoid non-specific binding of the antisense compound to non-target nucleic acid sequences under conditions in which specific binding is desired.
"Complementary" as used herein, refers to the capacity for precise pairing between two nucleobases of an oligomeric compound. For example, if a nucleobase at a certain position of an oligonucleotide (an oligomeric compound), is capable of hydrogen bonding with a nucleobase at a certain position of a target nucleic acid, said target nucleic acid being a DNA, RNA, or oligonucleotide molecule, then the position of hydrogen bonding between the oligonucleotide and the target nucleic acid is considered to be a complementary position. The oligonucleotide and the further DNA, RNA, or oligonucleotide molecule are complementary to each other when a sufficient number of complementary positions in each molecule are occupied by nucleobases which can hydrogen bond with each other. Thus, "specifically hybridizable" and "complementary" are terms which are used to indicate a sufficient degree of precise pairing or complementarity over a sufficient number of nucleobases such that stable and specific binding occurs between the oligonucleotide and a target nucleic acid.
According to the present invention, compounds include antisense oligomeric compounds, antisense oligonucleotides, ribozymes, external guide sequence (EGS) oligonucleotides, alternate splicers, primers, probes, and other oligomeric compounds which hybridize to at least a portion of the target nucleic acid. As such, these compounds may be introduced in the form of single-stranded, double-stranded, circular or hairpin oligomeric compounds and may contain structural elements such as internal or terminal bulges or loops. Once introduced to a system, the compounds of the invention may elicit the action of one or more enzymes or structural proteins to effect modification of the target nucleic acid. One
non-limiting example of such an enzyme is RNAse H, a cellular endonuclease which cleaves the RNA strand of an RNA:DNA duplex. It is known in the art that single-stranded antisense compounds which are "DNA-like" elicit RNAse H. Activation of RNase H, therefore, results in cleavage of the RNA target, thereby greatly enhancing the efficiency of oligonucleotide-mediated inhibition of gene expression. Similar roles have been postulated for other ribonucleases such as those in the RNase III and ribonuclease L family of enzymes.
While the preferred form of antisense compound is a single-stranded antisense oligonucleotide, in many species the introduction of double-stranded structures, such as double-stranded RNA (dsRNA) molecules, has been shown to induce potent and specific antisense-mediated reduction of the function of a gene or its associated gene products.
In the context of the subject invention, the term "oligomeric compound" refers to a polymer or oligomer comprising a plurality of monomeric units. In the context of this invention, the term "oligonucleotide" refers to an oligomer or polymer of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) or mimetics, chimeras, analogs and homologs thereof. This term includes oligonucleotides composed of naturally occurring nucleobases, sugars and covalent internucleoside (backbone) linkages as well as oligonucleotides having non-naturally occurring portions which function similarly. Such modified or substituted oligonucleotides are often preferred over native forms because of desirable properties such as, for example, enhanced cellular uptake, enhanced affinity for a target nucleic acid and increased stability in the presence of nucleases.
While oligonucleotides are a preferred form of the compounds of this invention, the present invention comprehends other families of compounds as well, including but not limited to oligonucleotide analogs and mimetics such as those herein described.
The open reading frame (ORF) or "coding region" which is known in the art to refer to the region between the translation initiation codon and the translation termination codon, is a region which may be effectively targeted. Within the context of the present invention, one
region is the intragenic region encompassing the translation initiation or termination codon of the open Beading frame (ORF) of a gene.
Other target regions include the 5' untranslated region (5'UTR), known in the art to refer to the portion of an mRNA in the 5' direction from the translation initiation codon, and thus including nucleotides between the 5' cap site and the translation initiation codon of an mRNA (or corresponding nucleotides on the gene), and the 3' untranslated region (3'UTR), known in the art to refer to the portion of an mRNA in the 3' direction from the translation termination codon, and thus including nucleotides between the translation termination codon and 3' end of an mRNA (or corresponding nucleotides on the gene). The 5' cap site of an mRNA comprises an N7-methylated guanosine residue joined to the 5'-most residue of the mRNA via a 5'-5' triphosphate linkage. The 5' cap region of an mRNA is considered to include the 5' cap structure itself as well as the first 50 nucleotides adjacent to the cap site. It is also preferred to target the 5' cap region.
Although some eukaryotic mRNA transcripts are directly translated, many contain one or more regions, known as "introns", which are excised from a transcript before it is translated. The remaining (and, therefore, translated) regions are known as "exons" and are spliced together to form a continuous mRNA sequence. Targeting splice sites, i.e. intron-exon junctions or exon-intron junctions, may also be particularly useful in situations where aberrant splicing is implicated in disease, or where an overproduction of a particular splice product is implicated in disease. Aberrant fusion junctions due to rearrangements or deletions ase also preferred target sites. mRNA transcripts produced via the process of splicing of two (or more) mRNAs from different gene sources are known as "fusion transcripts". It is also known that introns can be effectively targeted using antisense compounds targeted to, for example, DNA or pre-mRNA.
As is known in the art, a nucleoside is a base-sugar combination. The base portion of the nucleoside is normally a heterocyclic base. The two most common classes of such heterocyclic bases are the purines and the pyrimidines. Nucleotides are nucleosides that further include a phosphate group covalently linked to the sugar portion of the nucleoside.
For those nucleosides that include a pentofuranosyl sugar, the phosphate group can be linked to either the 2', 3' or 5' hydroxyl moiety of the sugar. In forming oligonucleotides, the phosphate groups covalently link adjacent nucleosides to one another to form a linear polymeric compound. In turn, the respective ends of this linear polymeric compound can be further joined to form a circular compound, however, linear compounds are generally preferred. In addition, linear compounds may have internal nucleobase complementarity and may, therefore, fold in a manner as to produce a fully or partially double-stranded compound. Within oligonucleotides, the phosphate groups are commonly referred to as forming the internucleoside backbone of the oligonucleotide. The normal linkage or backbone of RNA and DNA is a 3' to 5' phosphodiester linkage.
For topical delivery of antisense compounds, these oligonucleotides may contain modified backbones or non-natural internucleoside linkages. As defined in this specification, oligonucleotides having modified backbones include those that retain a phosphorus atom in the backbone and those that do not have a phosphorus atom in the backbone. For the purposes of this specification, and as sometimes referenced in the art, modified oligonucleotides that do not have a phosphorus atom in their internucleoside backbone can also be considered to be oligonucleosides.
Preferred modified oligonucleotide backbones containing a phosphorus atom therein include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3'-alkylene phosphonates, 5-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3'-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, selenophosphates and boranophosphates having normal 3'-5' linkages, 2'-5' linked analogs of these, and those having inverted polarity wherein one or more intemucleotide linkages is a 3' to 3', 5' to 5' or 2' to 2' linkage. Preferred oligonucleotides having inverted polarity comprise a single 3' to 3' linkage at the 3'-most internucleotide linkage i.e. a single inverted nucleoside residue which may be abasic (the
nucleobase is missing or has a hydroxyl group in place thereof). Various salts, mixed salts and free acid forms are also included.
In an alternative embodiment, genetic constructs including DNA "vaccines" are used to generate antisense or sense molecules in plant cells. Furthermore, many of the preferred features described above are appropriate for sense nucleic acid molecules.
A further aspect of the present invention relates to a method for control of insect populations, said method comprising administering to insects an effective amount of an agent for a time and under conditions sufficient to inhibit the expression of HpF5 or sufficient to inhibit the activity of HpCh5, wherein said modulation results in reduction of the biological fitness of said insects.
In one preferred embodiment of the present invention, the agent is one that can bind to the primary substrate binding pocket of HpCh5, and not be interfered with by the arginine residue found at position 192.
Reference to a "reduction of biological fitness" should be understood to be changes in the insect including, but not limited to, changes in body mass and/or viability. Preferably, these changes are understood as reductions in both body mass and/or viability. A reduction in biological fitness, therefore, includes a reduction in their growth and development.
In yet another aspect the present invention provides a method for detecting an agent capable of modulating the function of HpCh5 or functional equivalent or derivative thereof, said method comprising administering to an insect containing said HpCh5 or functional equivalent or derivative thereof with a putative agent and detecting an altered activity phenotype associated with modulation of function of HpCh5 or its functional equivalent or derivative.
Reference to "administration" of the modulator to HpCh5 refers to delivery of the modulating agent in any convenient means. In the agricultural setting this is likely to
include, but not be limited to:-
(i) the delivery of the agent as the active ingredient in a spray or powder formulation; or
(ii) the production of the agent either as a protein or the product of a metabolic pathway in a plant.
In a preferred embodiment of the invention, the administration of the agent is via the introduction of a nucleic acid encoding said agent into a plant for subsequent expression and production of the agent. Another preferred embodiment is the formulation of a spray or powder with said agent as the active ingredient.
"Introduction" of the agent is to be understood to cover all means known to those in the art of making genetic changes in a plant. These include, but are not limited to plant transformation methods such as particle bombardment, Agrobacterium-mediated transformation, electroporation and viral transfection; plant-breeding techniques and mutagenesis of native plant genes.
Accordingly, in the context of the present invention, nucleic acid molecules encoding an antisense or sense form of HpF5 or encoding an inhibitor of HpCh5 activity or HpF5 expression is operably linked to a promoter, generally in a vector or other suitable medium for introduction to a plant genome. Alternatively, an existing PI may be cloned and modified to render it active against HpCh.5.
The present invention further provides a genetically modified plant comprising cells which are capable of producing an antagonist of HpCh5 or HpF5 gene expression. In one particularly useful example, cotton or other crop plants are engineered to produce PotI or a combination of PotI and NaPI. Reference herein to "PotI" and "NaPI" includes reference to derivatives, variants and homologs including modifications to one or more domains in PotI or NaPI.
Reference herein to a "promoter" is to be taken in its broadest context and includes the transcriptional regulatory sequences of a classical genomic gene, including the TATA box which is required for accurate transcription initiation, with or without a CCAAT box sequence and additional regulatory elements (i.e. upstream activating sequences, enhancers and silencers) which alter gene expression in response to developmental and/or external stimuli, or in a tissue-specific manner. A promoter is usually, but not necessarily, positioned upstream or 5', or a structural gene region, the expression of which it regulates. Furthermore, the regulatory elements comprising a promoter are usually positioned within 2 kb of the start site of transcription of the gene.
In the present context, the term "promoter" is also used to describe a synthetic or fusion molecule, or derivative which confers, activates or enhances expression of a nucleic acid molecule in a cell.
Preferred promoters may contain additional copies of one or more specific regulatory elements, to further enhance expression of the sense molecule and/or to alter the spatial expression and/or temporal expression of said sense molecule. For example, regulatory elements which confer copper inducibility may be placed adjacent to a heterologous promoter sequence driving expression of a sense molecule, thereby conferring copper inducibility on the expression of said molecules.
Placing a nucleic acid molecule under the regulatory control of a promoter sequence means positioning the said molecule such that expression is controlled by the promoter sequence. Promoters are generally positioned 5' (upstream) to the genes that they control. In the construction of heterologous promoter/structural gene combinations, it is generally preferred to position the promoter at a distance from the gene transcription start site that is approximately the same as the distance between that promoter and the gene it controls in its natural setting, i.e. the gene from which the promoter is derived. As is known in the art, some variation in this distance can be accommodated without loss of promoter function. Similarly, the preferred positioning of a regulatory sequence element with respect to a
heterologous gene to be placed under its control is defined by the positioning of the element in its natural setting, i.e. the genes from which it is derived. Again, as is known in the art, some variation in this distance can also occur.
The promoter may regulate the expression of HpF5 or its variant or homolog constitutively, or differentially with respect to cell, the tissue or organ in which expression occurs or, with respect to the developmental stage at which expression occurs, or in response to external stimuli such as physiological stresses, or pathogens, or metal ions, amongst oiiers.
Preferably, the promoter is capable of regulating expression of a nucleic acid molecule in a plant cell, tissue or organ, at least during the period of time over which the target gene is expressed therein and more preferably also immediately preceding the commencement of detectable expression of the HpF5gene in said cell, tissue or organ.
Accordingly, strong constitutive promoters are particularly useful for the purposes of the present invention or promoters which may be induced by virus infection or the commencement of HpF5 gene expression.
Plant-operable promoters are particularly preferred for use in the construct of the present invention. Examples of suitable promoters include pCaMV 35S (Fang et al, Plant Cell 1: 141-150, 1989), PGEL1 (Hajdukiewicz et al, Plant Mol. Biol. 25: 989-994, 1994), class III chitinase (Samac and Shah, Plant Cell 3: 1063-1072, 1991),pin2 (Keil et al., EMBO J. 8: 1323-1330, 1989), PEP carboxylase (Pathirana et al., Plant J. 12: 293-304, 1997; MAP kinase (Schoenbeck et al., Molec. Plant-Microbe Interact, 1999), MSV (Legavre et al, In: Vth International Congress of Plant Molecular Biology, Singapore, 1997), pltp (Hsu et al, Plant Sci. 143: 63-70,1999), pmpi (Cordero et al., In: General Meeting of the International Program on Rice Biotechnology of the Rockefeller Foundation, Malacca, Malaysia, 1997) or glutamin synthase (Pujade-Renaud et al., Plant Physiol Biochem. 35: 85-93, 1997).
In the present context, the terms "in operable connection with" or "operably under the control" or similar shall be taken to indicate that expression of the nucleic acid molecule is under the control of the promoter sequence with which it is spatially connected; in a cell, tissue, organ or whole plant.
The construct preferably contains additional regulatory elements for efficient transcription, for example, a transcription termination sequence.
The term "terminator" refers to a DNA sequence at the end of a transcriptional unit which signals termination of transcription. Terminators are 3'-non-translated DNA sequences generally containing a polyadenylation signal, which facilitates the addition of polyadenylate sequences to the 3'-end of a primary transcript. Terminators active in plant cells are known and described in the literature. They may be isolated from bacteria, fungi, viruses, animals and/or plants or synthesized de nova.
As with promoter sequences, the terminator may be any terminator sequence which is operable in the cells, tissues or organs in which it is intended to be used.
Examples of terminators particularly suitable for use in the synthetic genes of the present invention include the SV40 polyadenylation signal, the HSV TK polyadenylation signal, the CYCl terminator, ADH terminator, SPA terminator, nopaline synthase (NOS) gene terminator of Agrobacterium tumefaciens, the terminator of the cauliflower mosaic virus (CaMV) 35S gene, the zein gene terminator from Zea mays, the Rubisco small subunit gene (SSU) gene terminator sequences, subclover stunt virus (SCSV) gene sequence terminators, any rho-independent E. coli terminator, or the lacZ alpha terminator, amongst others.
In a particularly preferred embodiment, the terminator is the SV40 polyadenylation signal or the HSV TK polyadenylation signal which are operable in animal cells, tissues and organs, octopine synthase (OCS) or nopaline synthase (NOS) terminator active in plant cells, tissue or organs, or the lacZ alpha terminator which is active in prokaryotic cells.
Those skilled in the art will be aware of additional terminator sequences which may be suitable for use in performing the invention. Such sequences may readily be used without any undue experimentation.
Means for introducing (i.e. transfecting or transforming) cells with the constructs are well-known to those skilled in the art.
The constructs described supra are capable of being modified further, for example, by the inclusion of marker nucleotide sequences encoding a detectable marker enzyme or a functional analogue or derivative thereof, to facilitate detection of the synthetic gene in a cell, tissue or organ in which it is expressed. According to this embodiment, the marker nucleotide sequences will be present in a translatable format and be expressed.
Those skilled in the art will be aware of how to produce the constructs described herein and of the requirements for obtaining the expression thereof, when so desired, in a specific cell or cell-type under the conditions desired. In particular, it will be known to those skilled in the art that the genetic manipulations required to perform the present invention may require the propagation of a genetic construct described herein or a derivative thereof in a prokaryotic cell such as an E. coli cell or a plant cell or an animal cell.
The constructs of the present invention may be introduced to a suitable cell, tissue or organ without modification as linear DNA, optionally contained within a suitable carrier, such as a cell, virus particle or liposome, amongst others. To produce a genetic construct, a nucleic acid (e.g. HpF5) is inserted into a suitable vector or episome molecule, such as a bacteriophage vector, viral vector or a plasmid, cosmid or artificial chromosome vector which is capable of being maintained and/or replicated and/or expressed in the host cell, tissue or organ into which it is subsequently introduced.
Accordingly, a further aspect of the invention provides a genetic construct which at least comprises a genetic element as herein described and one or more origins of replication and/or selectable marker gene sequences.
Usually, an origin of replication or a selectable marker gene suitable for use in bacteria is physically-separated from those genetic sequences contained in the genetic construct which are intended to be expressed or transferred to a plant cell, or integrated into the genome of a plant cell.
As used herein, the term "selectable marker gene" includes any gene which confers a phenotype an a cell on which it is expressed to facilitate the identification and/or selection of cells which are transfected or transformed with a genetic construct of the invention or a derivative thereof.
Suitable selectable marker genes contemplated herein include the ampicillin-resistance gene (Ampr), tetracycline-resistance gene (Tcr), bacterial kanamycin-resistance gene (Kanr), the zeocin resistance gene (Zeocin is a drug of the bleomycin family which is trade mark of InVitrogen Corporation), the AURI-C gene which confers resistance to the antibiotic aureobasidin A, phosphinothricin-resistance gene, neomycin phosphotransferase gen (nptII), hygromycin-resistance gene, ß-glucuronidase (GUS) gene, chloramphenicol acetyltransferase (CAT) gene, green fluorescent protein-encoding gene or the luciferase gene, amongst others.
Preferably, ihe selectable marker gene is the nptII gene or Kanr gene or green fluorescent protein (GFP)-encoding gene.
Those skilled in the art will be aware of other selectable marker genes useful in the performance of the present invention and the subject invention is not limited by the nature of the selectable marker gene.
The present invention extends to all genetic constructs essentially as described herein, which include further genetic sequences intended for the maintenance and/or replication of said genetic construct in prokaryotes or eukaryotes and/or the integration of said genetic construct or a part thereof into the genome of a eukaryotic cell or organism.
Standard methods described supra may be used to introduce the constructs into the cell, tissue or organ, for example, liposome-mediated transfection or transformation, transformation of cells with attenuated virus particles or bacterial cells, cell mating, transformation or transfection procedures known to those skilled in the art.
Additional means for introducing recombinant DNA into plant tissue or cells include, but are not lirniited to, transformation using CaCl2 and variations thereof, direct DNA uptake into protoplasts, PEG-mediated uptake to protoplasts, microparticle bombardment, electroporation, microinjection of DNA, microparticle bombardment of tissue explant or cells, vacuum-infiltration of tissue with nucleic acid, or in the case of plants, T-DNA-mediated transfer from Agrobacterium to the plant tissue.
For microparticle bombardment of cells, a microparticle is propelled into a cell to produce a transformed cell. Any suitable ballistic cell transformation methodology and apparatus can be used in performing the present invention. Exemplary apparatus and procedures are disclosed by Stomp et al. (U.S. Patent No. 5,122,466) and Sanford and Wolf (U.S. Patent No. 4,945,050). When using ballistic transformation procedures, the genetic construct may incorporate a plasmid capable of replicating in the cell to be transformed.
Examples of microparticles suitable for use in such systems include 1 to 5 jam gold spheres. The DNA construct may be deposited on the microparticle by any suitable technique, such as by precipitation.
In a further embodiment of the present invention, the genetic constructs described herein are adapted for integration into the genome of a cell in which it is expressed. Those skilled in the art will be aware that, in order to achieve integration of a genetic sequence or genetic construct into the genome of a host cell, certain additional genetic sequences may be required. In the case of plants, left and right border sequences from the T-DNA of the Agrobacteriun tumefaciens Ti plasmid will generally be required.
The present invention further extends to an isolated cell, tissue or organ comprising the constructs or parts thereof. The present invention extends further to regenerated tissues, organs and whole organisms derived from said cells, tissues and organs and to propagules and progeny thereof as well as seeds and other reproductive material.
For example, plants may be regenerated from transformed plant cells or tissues or organs on hormone-containing media and the regenerated plants may take a variety of forms, such as chimeras of transformed cells and non-transformed cells; clonal transformants (e.g. all cells transformed to contain the expression cassette); grafts of transformed and untransformed tissue (e.g. a transformed root stock grafted to an untransformed scion in citrus species). Transformed plants may be propagated by a variety of means, such as by clonal propagation or classical breeding techniques. For example, first generation (or Tl) transformed plants may be selfed to give homozygous second generation (or T2) transformed plants, and the T2 plants further propagated through classical breeding techniques.
Plants contemplated herein include cotton, sweet corn, tomato, tobacco, piniento, potato, sunflower, citrus, plums, sorghum, leeks, soybean, alfalfa, beans, pidgeon peas, chick peas, artichokes, curcurbits, lettuce, Dianthus (an ornamental plant), geraniums, cape gooseberry, maize, flax and linseed, lupins, broad beans, garden peas, peanuts, canola, snapdragons, cherry, sunflower, pot marigolds, Helichrysum (an ornamental plant), wheat, barley, oats, triticale, carrots, onions, orchids, roses and petunias
Another aspect of the present invention relates to the insensitive chymotrypsin as a selectable marker for the transformation of insects. At present, methods for the germ-line transformation of insects involves injection of insect embryos with a genetic construct comprising a transposable element, the gene of interest and a selectable marker. Somatic transformation of insects can also be achieved using viral vectors that include the gene of interest and a selectable marker (Peloquin et al., J. Cot. Sci. 5: 114-120, 2001). Presently, most insect transformation is done using white-eye mutants of the insect, to allow the detection of the commonly used selectable markers. Typically, selectable markers are genes that complement the white-eye mutation or Enhanced Green Fluorescent Protein (EGFP). In Drosophila, the white eye mutation is caused by mutant alleles such as the ώ1118 allele. These individuals can be returned to normal (red) eye pigmentation via the introduction of an allele conferring normal eye pigmentation such as white (Lidholm et al., Genetics 134: 859-868, 1993) or miniwhite (Lozovskaya et al., Genetics 142: 173-177, 1996). The reversion of ώ1118 mutants to normal eye pigmentation acts as the marker for introduction of the vector. In a similar way, EGFP has been used to indicate the presence of a vectof. Again, insects with non-pigmented eyes are used, and EGFP expression is detected in the eyes of these insects (Hediger et al, Insect Mol. Biol. 10: 113-119, 2001).
The selectable markers commonly used in the art require dissection of the insects to examine the eyes for either pigmentation or EGFP fluorescence, which is time consuming and requires destructive sampling of the insects. The present invention provides a means for the selection of transformed individuals without the need for insect dissection or inspection of individual insects. This would allow the recovery of live transformants and provides a non-laborious means of screening large numbers of putative transformants at one time. In addition, the present invention provides a means for the selection of transformants that does not rely on the availability of white-eye mutants.
The present invention contemplates the use of HpF5 or a derivative, homolog or analog thereof encoding a NaPI-insensitive chymotrypsin, as a selectable marker in an insect transformation vector. This vector, comprising HpF5, would have utility for the selection of transformants for any insect that is susceptible to the Cl serine proteinase inhibitor of N. alata. The contemplated insects may be naturally resistant to Cl, or may be NaPI-susceptible mutants or genetically modified NaPI susceptible strains of naturally NaPI-resistant insects. In a particularly preferred embodiment of the invention the insect host of the said vector would be Lepidopteran.
HpF5 or a derivative, homolog or analog thereof encoding an NaPI-insensitive chymotrypsin could be incorporated into any insect transformation vector using common molecular biology techniques known to those in the art. Upon transformation the insect would transcribe HpF5 and produce HpCh5, the NaPI-insensitive chymotrypsin. Transformed individuals could then be selected by incorporation of the Cl proteinase inhibitor of N. alata into the diet of the insects. In this case individuals that did not carry the insensitive chymotrypsin encoded by HpF5 in the vector would die, and those that did carry the vector encoding HpF5 would be insensitive to Cl.
Accordingly the present invention provides insect transformation vectors including baculovirus vectors comprising HpF5 or a derivative, homolog or analog thereof, as a selectable marker. The vector may be used for any purpose in the insect. Non-limiting examples include: gene cloning, gene expression and gene knockouts. Specific examples of insect transformation vectors into which HpF5 could be incorporated as a selectable marker include, but are not limited to: those that utilize the piggyBac mobilizable element (Hediger el al., 2001, supra); P-element based vectors (Cripps et al., J. Cell Biol. 126: 689-699, 1994); hobo element based vectors (Lozovskaya et al, 1996, supra); mariner element based vectors (Lidholm et al., 1993, supra); and viral vectors such as pTE/3'2J (Peloquin et al., 2001, supra),
The presents invention is further described by the following non-limiting Examples.
Effect of ingestion of the N. alata proteinase inhibitors on growth and development of Helicoverpa species
The effect of the N. alata PIs is examined on the digestive enzymes and the growth and development of Helicoverpa punctigera and Helicoverpa armigera larvae (Heath el al, J. Insect Physiol. 43: 833-842, 1997). The PIs suppressed total gut protease activity by 73% in an in vitro assay using 14C-casein as substrate. When incorporated into an artificial diet the PIs retarded the growth and development of both H. punctigera (Heath et al, 1997, supra) (Figure 2A and 2B) and H. armigera. Similar results are obtained when larvae from both species were fed on transgenic tobacco (N. tabacum) expressing the N. alata PIs at levels of 0,2% -0.5% soluble protein (Heath et al., 1997, supra).
Ingestion of NaPIs (Cl, C2 and T1-T4) changes the relative activity of the trypsins and chymotrypsins in the gut and faeces of H. punctigera larvae. Trypsin activity in the gut and the faeces is substantially lowered or abolished after exposure to NaPIs, whereas chymotrypsin activity is often unaffected or enhanced (Figures 2C and 2D). The lack of trypsin activity in the faeces of the NaPI fed larvae was not due to decreased production of trypsin. Indeed protein blots were used to demonstrate that these insects had overproduced trypsin in tesponse to the PIs, but it had been totally inactivated (Figure 2E).
Interestingly, the chymotrypsin produced after ingestion of NaPIs was not inhibitable by the NaPIs, whereas some of the chymotrypsin produced by control insects (not exposed to NaPIs) was inhibitable (Figure 2F). In control insects the degree of inhibition varied between individuals, and ranged from about 75% to no inhibition at all. This suggested that Helicoverpa larvae produce different classes of chymotrypsins; some that are inhibitable by the NaPIs (PI-sensitive) and some that are not inhibitable (Pi-insensitive). Thus, it appears ingestion of NaPIs inhibits the trypsin activity in the gut of Helicoverpa larvae, but larvae may not be severely impacted if they produce NaPI-insensitive chymotrypsins. In a subsequent experiment, the gut contents were removed from H. punctigera larvae that had been raised on a haricot bean diet without added proteinase inhibitors. The gut chymotrypsin activity was inhibited about 80% by the NaPI inhibitors (Cl, C2, T1-T4) and 70% by C1 alone (Figure 3). The Bowman Birk and trypsin inhibitors from Soybean and the lima bean trypsin inhibitor also failed to abolish all the chymotrysin activity, whereas activity was totally inhibited by the potato PotI inhibitor and chymostatin. Using this information a series of affinity columns were prepared for identification and purification of the NaPI-insensitive and -sensitive chymotrypsins from the gut of H, punctigera larvae.
Bioassays with Helicoverpa larvae on artificial diets
Haricot bean diet
Helicoverpa punctigera larvae were raised on artificial diets based on Haricot beans (Teakle et al, Journal of Invertebrate Pathology 46: 166-173, 1985). One litre of diet was composed of 58.5 g Haricot beans, 14 g agar, 700 ml water, 35 g Tortula yeast, 50 g wheatgerm, 3.5 g ascorbic acid, 1.1 g sorbic acid, 2.2 g p-hydroxybenzoic acid methyl ester, 0.2 g ampicillin, 0.2 g streptomycin, 16 mg prochloraz. The beans were soaked overnight in water, drained and homogenized to a fine paste. Wheatgerm, yeast and 300 ml of water were added. The agar was dissolved in 400 mL of boiling water and added to the mixture. The mixture was cooled to 50°C before the addition of the remaining ingredients. The blended diet was poured into trays and after setting was used immediately or stored at -20°C for no longer than two weeks. The test diet was supplemented with the NaPI (0.26% (w/v)) and the control diet had an equivalent amount of casein. Twenty newly emerged neonates were added to each diet and mortality was recorded every two days. Weight gain was recorded at the sixth day and then every second day thereafter. The larvae were reared in 1.5 ml eppendorf microfuge tubes (one larva/tube) until day eight when they were transferred to individual plastic containers with lids (SOLO [trademark] plastic portion cups, 28 mL). Larvae were fed small amounts of diet (40 mg) initially that was replaced as required to provide a continuous supply. The larvae were kept in a temperature controlled room at 25±l°C, 16:8(L:D).
Cotton leaf diet
Cotton leaf artificial diet was prepared from fresh young leaves from cotton plants (cultivar
Coker 315) which were grown in an insect-free and insecticide-free temperature controlled
cabinet at 26°C (±2°C) with a light regime of 16:8 (L:D). Following picking, the leaves were immediately frozen in liquid nitrogen and freeze dried. After drying, the leaves were ground to a fine powder in a mortar and pestle. The cotton leaf artificial diet was prepared in the same manner as haricot bean artificial diet using a recipe modified from potato leaf artificial diet (Gatehouse et al., J. Insect Physiol. 45 (6), 545-558, 1999). One hundred grams of cotton leaf artificial diet contained 3 g of cotton leaf powder, 0.08 mL linseed oil, 2 g yeast, 0.016 mL wheatgerm oil, 2.4 g wheat germ, 0.028 g ampicillin, 3.2 g ascorbic acid, 0.028 g streptomycin, 0.08 g sorbic acid, 3.2 g agar, 0.16 g paraben (mould inhibitor) plus NaPI or casein to required % ((w/v)).
Preparation of individual gut and frass extracts
All work was performed at 4°C. Each gut was dissected from the larva and placed in a 1.5 mL microfuge tube containing 500 µL of ice-cold 10 mM Tris-HCl, pH 8. Gut and contents were homogenized using a micropestle (Eppendorf). Insoluble material was removed by centrifugation at 13,000 g for 4 min and the supernatant was stored at -80°C. Frass extracts were prepared in the same manner using 200 mg frass/mL buffer. Total protein concentration was determined using the Bradford method (Bradford, Anal Biochem 72: 248-254, 1976) with reagents from Bio-Rad and BSA as a standard.
Trypsin and chymotrypsin activity
Gut proteinase activity was determined at pH 10 in 50 mM 3-(cyclohexylamino)-l-propanesulfonic acid (CAPS buffer) using the chromogenic substrate N-benzoyl-DL-arginine-p-nitroanilide (BApNA) for trypsin and N-succinyl-L-alanine-alanine-proline-phenylalanine-p-nitroanilide (SA2PFpNA) for chymotrypsin activity. Substrates were freshly prepared as 1 mM solutions in 10% ((w/v)) N,N-dimethylformamide (DMF), 50 mM CAPS buffer, pH 10. The assays were performed in duplicate or triplicate following the method of Heath et al, Eur.J.Biochem.230 (1): 250-257. Blanks, without enzyme, were used to account for any spontaneous breakdown of substrates. The release of p-nitroanilide was recorded .at 405 nm after 30 min at 30°C on a SpectraMax 250 microtitre plate reader (Molecular Devices).
Inhibition of trypsin and chymotrypsin activity by NaPI
Trypsin and chymotrypsin inhibition assays were conducted using the standard trypsin and chymotrypsin assays described above except samples were pre-incubated with T1 or Cl inhibitor (80nM) for 30 min at 30°C prior to the addition of substrate to initiate the reaction. NaPI monomers Tl and Cl were HPLC purified as described by Heath et al, 1999, supra.
Chymotrypsin assays in the presence and absence of proteinase inhibitors
Preparation of gut extracts
Fourth-instar larvae were killed using ethyl-acetate prior the removal of individual gut which were then homogenized in a mortar and pestle in an equal amount [(w/v)] of 50 mM Tris-HCl, pH 8.0 containing 100 µM benzamidine. Insoluble material was removed by centrifugatipn at 20,000 rpm for 15 min at 4°C and total protein concentration determined using the Bradford method (Bradford, Anal Biochem 72: 248-254, 1976) with reagents from BioRad and BSA as a standard.
Unfractionated gut extract containing approximately 1µg of buffer soluble protein was added to 10µL CAPS buffer (0.5 M, 3-[cyclohexylamino]-l-propane-sulfonic acid, pH 10) and made to a final volume of 100 µL in individual wells of a 96 well microtiter plate. Proteinase inhibitors were added over a range of concentrations and incubated for 15 min at 25°C. Bovine chymotrypsin (100 ng) was used as a positive control, both for activity and inhibition. The chromogenic artificial substrate SAAPFpNA (N-Succinyl-L-alanyl-L-alanyl-L-prolyl-L-phenylalanine 4-nitroanilide) was then added to a final concentration of 1 mM and hydrolysis of the substrate was measured at 405 run using the SpectraMax 250 microtiter plate reader (Molecular Devices).
Lima bean trypsin inhibitor (LBTI), soybean trypsin inhibitor (SBTI), soybean Bowman-Birk inhibitor (SBBI) and chymostatin were all purchased from Sigma-Aldrich Pty.Ltd. Potato Inhibitor I was purchased from Calbiochem-Novabiochem or purified from potato
tubers (Example 6). A crude mixture of Potato inhibitor I and II was also obtained from potatoes. N. alata proteinase inhibitors (NaPIs) were purified as described by Atkinson et al, 1993, supra and Heath et al, 1995, supra. The chymotrypsin inhibitor Cl was purified from bacterial expression cultures. The purity of the inhibitors was assessed by SDS-PAGE and silver staining.
EXAMPLE 2 Isolation of NAPI sensitive and insensitive chymotrypsins from H. punctigera gut
Most published work on Helicoverpa chymotrypsins has focused on cDNA clones or the measurement of enzyme activity in unfractionated gut extracts. There are few reports on purification of chymotrypsins from Helicoverpa or other lepidopteran species. Johnston and coworkers (1995, supra) described partial purification of chymotrypsins from H.armigera that employed ion exchange techniques. Peterson and coworkers (Insect Biochem. Mol. Biol. 25: 765-774, 1995) purified a chymotrysin from the midgut of the lepidopteran Manduca sexta by affinity chromatography on tryptophan methyl ester and Valiatis et al., Insect Biochemistry and Molecular Biology 29: 405-415, 1999 used immobilized potato proteinase inhibitor I (PotI) to isolate a chymotrypsin from the Western Spruce budworm. No one has described a procedure that separates individual chymotrypsin isozymes from one another and there is no description of the isolation of two chymotrypsins from a single species where one isozyme is inhibitable by a certain proteinase inhibitor while another is not inhibitable.
Preparation of pure enzymes and N-terminal sequencing
The midgut was dissected from 80 fourth instar larvae and buffer soluble extracts were prepared. The gut extract was depleted of trypsins by repeated passage through a benzamidine-Sepharose affinity column. The unbound protein was collected and applied to an affinity column composed of the immobilized chymotrypsin inhibitor Cl (Figure 1) that had been produced using a bacterial expression system. This column was expected to specifically bind NaPI-sensitive chymotrysins. Proteins that did not bind to this column were applied to a third affinity column composed of either immobilized Potato Type I
(PotI) and Type II inhibitors (PotII) or chymostatin. This column was designed to capture the chymotrypsins that did not bind to the Cl column, that is, the NaPI-insensitive chymotrypsins. Proteins that bound to the affinity columns were eluted with 8 M urea and were subjected to electrophoresis through an SDS-polyacrylamide gel before transfer to a PVDF membrane for N-terminal sequencing (Figures 4 and 5).
About 30 amino acids of N-terminal sequence were obtained that confirmed that the proteins were indeed chymotrypsins, and that the sensitive and insensitive chymotrypsins were products of different genes (Figures 4 and 5).
Preparation of NaPI-insensitive chymotrypsin depleted of trypsins and NaPI sensitive chymotrypsins for biochemical analysis
The midgut was dissected from 100 fourth instar larvae and buffer soluble extracts were prepared. The gut extract was depleted of NaPI-sensitive proteases by passage through an affinity column composed of immobilized NaPI protein (Cl, C2, T1-T4). All trypsins and NaPI-sensitive chymotrypsins bound to the column and the NaPI-insensitive chymotrypsin was unbound. This preparation of unbound material was used to study the pH optimum, substrate preference and effect of a range of proteinase inhibitors on the activity of the NaPI-insensitive proteinase. The effect of pH on activity of the insensitive chymotrypsins is illustrated in Figure 6. The enzyme is inactive below pH6 and is most active at pH10-12 consistent with its role in the alkaline midgut of larvae. The best substrate for enzyme assays was determined using seven different commercial substrates. The best substrate was N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide (sAAPF-pNA) followed by N-succinyl-Ala-Ala-Pro-Leu-p-nitroanilide (sAAPL-pNA) and N-methoxysuccinyl-Ala-Ala-Pro-Met-p-nitroanilide (mAAPM-pNA). N-succinyl-Ala-Ala-Ala-p-nitroanilide (sAAA-pNA), benzoyl-tyr- p-NA, Ac-Pro-Leu-Ser-p-NA and Ac-Asn-Gly-IIe-Pro-p-NA were not substrates.
Several proteinase inhibitors were tested for their ability to inhibit the activity of the insensitive chymotrypsins. The buffer used for all assays was 500 mM CAPS, 75 mM NaCl, 2.5 mM MgCl2 at pH 10. The inhibitors were preincubated with 100 ng of bovine
chymotrypsin or the amount of the gut chymotrypsin required to produce the same absorbance as 100 ng of bovine chymotrypsin (note that the Helicoverpa enzyme has been depleted of trypsins and sensitive chymotrypsins, but still contains other gut proteins) for 30 min at 30°C before the addition of substrate. The incubation was continued for a further 30 min at Boom temperature before absorbance was measured at 405 nm. PotI was the best of the proteinaceous inhibitors and NaPI did not inhibit (Table 3).
Benzamidine-agarose (1 mL; was purchased from ICN Biomedicals) and contained 35 umoles beazamidine per ml of gel.
Production of the Cl-affinity column
DNA encoding the Cl domain of NaPI (Figure 1, Atkinson et al., 1993, supra) was amplified from the pNa-PI-2-cDNA (Atkinson et al., 1993, supra) using oligonucleotide primers that incorporated BamHl (5' GACCAGCCGGATCCGATCGGATAT GCACCAAC) [SEQ ID N0:7] and HindIII (3' GGAGCCAAGCCAAGCTTTGAACGCG GGCAAACTC) [SEQ ID NO:8] sites for cloning into a pQE expression vector (Qiagen). The PCR product was sub-cloned into the pCR (registered trademark) 2.1-TOPO vector (Invitrogen) then excised with BamHl and HindIII and ligated into the pQE-30 vector. The expression vector incorporated a hexahistidine tag at the N-terminus of the expressed protein for metal affinity purification. The Cl/pQE-30 construct was transformed into the chemically competent E. coli strain Ml5 (Qiagen) prepared according to the method of Inoue et al, Gene 96: 23-28, 1990. Bacterial expression cultures were grown and induced according to the procedures outlined in the QiaExpress manual (Qiagen). Expression of the 6H.C1 recombinant protein was achieved by induction with 1 mM of IPTG. Samples (1 ml) were removed from the culture at hourly intervals, collected by centrifugation and resuspended in 1X SDS loading buffer (100µL) for SDS-PAGE analysis.
Recombinant Cl inhibitor was purified under denaturing conditions using Talon Metal
Affinity Resin (2 mL) (BD Biosciences Clontech) according to the manufacturer's protocol. Cl was eluted from the affinity matrix and examined using SDS-PAGE to confirm purity. Subsequent rounds of expression were conducted under the same conditions and Cl from a total of 2L of culture was purified using the Talon resin. After washing to iemove unbound proteins the C1 inhibitor remained bound to the Talon resin which was then used as the affinity column to purify chymotrypsins from gut preparations.
Potato Inhibitor 1 and potato Inhibitor II affinity column
Cyanogen bromide-activated Sepharose 4B (1 g) was swollen and washed according to the manufacturer's protocol. A mixture of potato 1 and II Inhibitors (10 mg) was dissolved in coupling buffer [0.1 M NaHCO3, 0.5 M NaCl, pH 8.3], combined with the gel suspension and incubated over night at 4°C in an end-over-end mixer. The gel was rinsed several times in blocking buffer [0.1 M Tris-HCl, pH 8.0] to remove excess ligand. Following the washes the conjugated Sepharose was mixed with fresh blocking buffer and incubated overnight at 4°C. The gel slurry was transferred to a column (Amersham Biosciences) and washed alternately (x3) with five column volumes of coupling buffer then five column volumes of rinse buffer [0.1 M NaOAc, 0.5 M NaCl, pH 4.0]. Finally the matrix was extensively washed with coupling buffer and stored in 20% (v/v) ethanol at 4°C.
Chymostatin affinity column
Chymostatin (Sigma-Aldrich) was immobilized on EAH Sepharose-4B (1.5 ml; Sigma-Aldrich) according to the manufacturer's instructions. Chymostatin (10 mg) was dissolved in 500 µL of glacial acetic acid then 1 mL of distilled water was added and the pH adjusted to 4.5 with dilute NaOH. The gel suspension and chymostatin solution were combined before EDC [N-ethyl-N'-(3-dimethylaminopropyl) carbodiimide] was slowly added to a final concentration of 0.1 M. The gel suspension was mixed overnight at 4°C before the gel was washed with Milli-Q filtered water to remove excess ligand and unreacted carbodiimide and the gel matrix was stored at 4°C in 20% [(v/v)] ethanol.
Purification of NaPI- sensitive and insensitive chymotrypsins
Eighty gut from fifth instar larvae that had been stored at -80°C were homogenized using a mortar and pestle in 10 ml of gut extraction buffer (20 mM CAPS pH 10, 350 mM NaCl). The gut extract was centrifuged at 15,000 g for 15 min at 4°C and the supernatant was filtered through a syringe filter (0.45 µM; Millipore) then briefly stored on ice before application to the benzamidine affinity column.
The filtered gut extract was passed through the benzamidine column five times to remove the trypsins. The unbound fraction (10 mL) was passed through the Cl affinity column (x3) before the column was washed with 20mL of extraction buffer and bound proteins were eluted with 8 M urea, pH 8.0 (5 mL). Proteins that did not bind to the Cl column were applied to the Potato Inhibitor I and II affinity column prior to washing with 10 column volumes of buffer [20 mM CAPS pH 10, 0.5 M NaCl] and elution of bound proteins with 8 M urea, pH 8.0 (5 mL).
Analysis of affinity purified gut proteins: SDS-PAGE
Samples of gut proteins (5-10 µg) were concentrated using TCA precipitation and resuspended in 0.2 M NaOH (10 µL). SDS-PAGE sample buffer (50 mM Tris-HCl, pH 6.8, 2% (w/v) SDS, 10% (v/v) glycerol, 5% (v/v) p-mercaptoethanol, 0.1% (w/v)] bromophenol blue) was added and samples were heated to 100°C for 5min prior to separation on 12.5% (w/v) reducing polyacrylamide gels using the MiniProtean II Electrophoresis apparatus (Bio-Rad) at 200 Volts. Broad range or peptide molecular size markers (Bio-Rad) were used to estimate relative molecular masses. Following electrophoresis the proteins were visualized by staining with Coomassie Brilliant Blue R-250 (0.1% (w/v) in 40% (v/v) methanol, 10% (v/v) acetic acid) for 60 min followed by destaining in 40% (v/v) methanol, 10% (v/v) acetic acid or were transferred to either nitrocellulose (0.22 µM pore size; Micron Separations Inc.) or Sequi-Blot Polyvinylidene Fluoride membrane (PVDF; Bio-Rad).
Immunoblotting and N-terminal sequencing
After electrophoresis gels Avere equilibrated in transfer buffer (192 mM glycine. 48 mM Tris-base, 20% (v/v) methanol) for 10 min prior to the transfer of proteins to a nitrocellulose membrane (0.22 µM pore size; Micron Separations Inc.) using the Mini Trans-Blot apparatus (BioRad) at 100 V for 60 min. Membranes were briefly washed in TBS (20 mM Tris-HCL 150 mM NaCl, pH 7.5) then stained with amido black (1:50 dilution of 0.1% (w/v) amido black, 40% (v/v) methanol, 10% (v/v) acetic acid) to confirm transfer of the proteins and to visualize the molecular size markers. Blots were then blocked by incubation with 3% (w/v) skim milk powder (Dutchjug) in TBST [0.1% (w/v) Tween-20 in TBS] for 1 h at RT, followed by a 1 h incubation with the α-chymotrypsin antibody (HpCH2B; 1:5000 dilution in 3% (w/v) skim milk powder in TBST). The nitrocellulose blots were then rinsed three times (5 min) in TBST and incubated for 1 hour at RT with the secondary antibody (anti-rabbit IgG conjugated to horseradish peroxidase diluted 1:5000 in TBST; Amersham Biosciences). Membranes were washed three times (5 min) with TBST and immunoreactive proteins were visualized with Enhanced Chemiluminescence (ECL) reagents and Hyperfilm ECL X-ray film (Amersham Biosciences) according to the manufacturer's instructions.
To obtain N-terminal sequence, samples were transferred to Sequi-Blot PVDF membrane (BioRad) equilibrated in electroblotting buffer (10 mM CAPS, 10% (v/v) methanol, 0.01% (w/v) SDS, pH 11) using the Mini Trans-Blot cell (BioRad) at 100 V for 45 minutes. Following transfer, the membrane was briefly rinsed in Milli-Q water, stained with Coomassie Brilliant Blue (0.1% Coomassie Blue R-250, 1% (v/v) acetic acid, 40% (v/v) methanol) and then destained (50% (v/v) methanol). Finally the membrane was rinsed with water and dried before excision of the appropriate proteins for N- terminal sequencing.
EXAMPLE 3 Cloning cDNAs encoding gut chymotrypsins from H. punctigera
cDNAs encoding the H. punctigera chymotrypsins were obtained using two approaches. Both approaches employed PCR amplification of cDNA produced from midgut mRNA extracted from H. punctigera larvae at the late fourth and early fifth instar stage of development.
Isolation of chymotrypsin clones using oligonudeotides complimentary to highly conserved regions in H.armigera chymotrypsins
Bown and colleagues (1997, supra) have described several cDNA clones encoding H.armigera chymotrypsins. Figure 7 shows the predicted proteins and the regions complementary to the oligonudeotides (Figure 8) chosen for PCR amplification of chymotrypgin cDNAs from H. punctigera. The PCR products were cloned and sequenced, and five distinct chymotrypsin sequences were obtained (FlApcr, FIBpcr, F2Bpcr, F3pcr and F4pcr, Figure 9), These PCR products were used to screen a cDNA library prepared from midgut mRNA isolated from late fourth instar and early fifth instar larvae. Seven distinct cDNA clones were isolated encoding chymotrypsins that were divided into four families based on sequence identity (Figure 10, Table 2). These chymotrypsins share high sequence identity with the H. armigera chymotrysins described by Bown et al., 1997, supra and with the small number of chymotrypsin sequences reported for H. virescem and H. zea (Table 6). The chymotrypsins encoded by the full length clones presented in Figure 10 characteristically encode zymogens of approximately 292-295 amino acids, including putative antino-terminal signal peptides of 16-17 residues predicted by the signal peptide prediction program PSORTII. The presence of the residues IVGG (positions 62-65) at the N-terminus of several active chymotrypsins results in the prediction of activation peptides ranging from 35-44 residues in length on the zymogens predicted from the cDNA clones. Furthermore these activation peptides consistently have the dipeptide arginine-isoleucine at their C-termini suggesting a role for trypsin in activating the chymotrypsins. The sequence identities for the mature domains of each translated protein are presented in Table 2. Family 4 was most divergent with less than 20% identity with the other families, while families 2 and 3 shared most similarity with scores of about 85%. The N-terminal sequence obtained from the NaPI inhibitable chymotrypsin (Figure 4) matched the N-terminal sequence predicted from the cDNA clones encoding chymotrypsins from family 2. The N-terminal sequence of the NaPI -insensitive chymotrypsins was not represented in the 4 families of chymotrypsins represented by the cDNA clones.
TABLE 5Protein sequence identity (%) betweeen members of the H. punctigera chymotrypsin gene family (mature chymotrypsin domain only)
H. punctigera chymotrypsins are closely related to chymotrypsins from other Helicoverpa species
Protein sequences most similar to H. punctigera were obtained using the BLAST search engine. Genbank accession numbers are listed. The mature activated proteins were compared and percentage of protein sequence identity determined.
The N-terminal region of the insensitive chymotrypsins had two unique stretches of sequence, designated Fl and F2 that were used to design oligonucleotides for PCR amplification of DNA encoding one of the insensitive chymotrypsins (Figure 11) A 641 bp fragment of DNA was obtained with the Fl oligonucleotide that encompassed most of the protein-coding region. This PCR product was used to screen for a full length clone in the H. punctigera midgut library. Approximately 0.2% of the 50,000 plaques screened hybridized strongly to the PCR product. Ten plaques were selected and and a full length clone was identified and sequenced (Figure 12). The 921 bp clone had an open reading frame of 828 bp and residues 41-76 of the deduced protein were an identical match to the N-terminal; sequence obtained from the purified protein (Figure 12). The clone lacked theScreening the cDNA library
The primary screen was performed using ten (15 cm) Petri dishes per probe, with about 50,000 phage;per plate. Preparation of the plates for subsequent plaque lifts and treatment of the membranes prior to hybridization was performed according to instructions provided with the Lambda Zap cDNA synthesis kit (Stratagene). The membranes (Hybond-N; Amersham Biosciences) were prehybridized in 50 ml of 2 x PIPES (0.8 M Nad, 16 mM Piperazine-l,4-bis(2-ethanesulphonic acid) pH 6.5) 50% (v/v) formamide, 0.5% (w/v) SDS and 100 µg/mL Herring sperm DNA (Boehringer Manningham) at 42°C for 3 hours. Following prehyridization, the incubation solution was replaced with 50 ml of 50% (v/v) formamide, 2 x PIPES buffer, 0.5% (w/v) SDS and 100 αg/mL denatured herring sperm DNA containing the labeled probe and left overnight at 42°C. The hybridization membranes were washed three times in 2 X SSPE/0.1% (w/v) SDS at room temperature and twice in 0.2 X SSPE/0.1% (w/v) SDS at room temperature, before they were blotted on 3 mm Whitman paper and exposed to X-ray film (Kodak XAR-5) for 48 hours at -70°C with intensifying screens.
Due to crowding and hence overlapping between positive clones and non-specific plaques a secondary screen was conducted to isolate individual clones. Two 8.5 cm plates each with 50 plaques were used for each probe. The secondary screen was conducted as described for the primary screen.
At least 50 positive plaques of varying intensities were selected from each screen probed with an individual RT-PCR product. Each plaque was transferred to a 1.5 mL microfuge tube containing 1 mL of SM buffer (0.1 M NaCl, 8 mM MgS04.7H20, 50 mM Tris-HCL pH 7.5, 0.01% (w/v) gelatin) and chloroform (20 uL) and stored at 4°C. Initially 10 plaques for each probe were excised and converted into pBluescriptll SK(-) phagemids using the ExAssist/SOLR system (Stratagene). The excised phagemids were transformed into the chemically competent E. coli strain XL 1-Blue cells and plasmid DNA was prepared and sequenced.
RT-PCR amplification of chymotrypsin genes
Chymotrygin genes from Helicoverpa armigera were aligned using ClustalW and regions of high conservation were identified for design of chymotrypsin specific primers. Primers were individually designed to the H. armigera clone (CAA72951) due to the high level of divergence to the other chymotrypsin sequences. Forward primers were used in combination with RVG4 to amplify the gene fragments using reverse transcriptase polymerase chain reaction (RT-PCR; Superscript Preamplification System, Gibco BRL) with the protocols supplied. The PCR products were gel purified (Qiagen gel extraction kit), sub-cloned into TOPO PCR2.1 TA cloning vector (Invitrogen) and transformed into chemically competent E. coli XL 1-Blue cells (Stratagene) prepared according to the method of (Inoue et al., 1990, supra). Plasmid DNA was prepared using the QIAprep (registered trademark) Spin Miniprep Kit (Qiagen).
Production and screening of a H. punctigera gut cDNA library: Library preparation
PolyA+ mRNA was isolated from the RNA (1.0 mg) using conventional protocols. A biotinylated oligonucleotide (dT) primer that binds to the polyA tail tags the transcripts that are subsequently captured on streptavidin paramagnetic particles (PolyATract (registered trademark), mRNA Isolation Systems, Promega). Purified mRNA (5 µg) was used to construct a cDNA library with the Lambda ZAP-cDNA synthesis kit and the Zap-cDNA GigapackIII Gold packaging extract (Stratagene) following the manufacturer's instructions. The amplified library titre was 2.8 x 1010 pfu/mL.
RT-PCR products (50 ng) were individually labelled with [α-32P] dCTP (Amersham Life Sciences) using the MEGAPRIME (trademark) DNA labeling system labeling (Amersham Life Sciences). Unincorporated radiolabeled nucleotides were removed using the Micro Bio-spin P-30 chromatography columns (BioRad) according to the manufacturer's protocol. The double stranded labelled probes were denatured by boiling (5 min), cooled on ice, and then added to the hybridization solution (as below).
5'signal peptide sequence, but comparison to the other chymotrypsin clones indicated the activation peptide was full length. The active enzyme is predicted to be 236aa in length, with a mass of 24.2 kDa. Protein sequence homology was determined by comparing the mature domains of each of the H. punctigera cDNAs. Family 2 and family 3 were most similar to HpCh5 (Rechla), with about 72 and 70% identity respectively. Family 1 members shared 53-57% identity while Family 4 had about 20% identity with HpCh5. Comprehensive Blast searches at the NCBI facility of both translated and protein databases produced no significant matches. The insensitive chymotrypsin belongs to a new family of Helicoverpa chymotrypsins designated Family 5 (Figure 13). A blast search performed on a Helicoverpa armigera midgut EST database matched a clone with over 97% sequence identity and 100% sequence similarity Figure 14.
Bovine chynaotrypsins A and B and the NaPI-sensitive chymotrypsin (HpCh2A) were compared to the insensitive chymotrypsin (HpCh5) to identify regions of variability that may be involved in low affinity binding to Cl (Figure 15).
Ten substitutions identified in the alignment did not appear to fall into functionally significant regions, whereas the eleventh substitution was associated with one of the P-strands that forms a wall of the primary substrate-binding pocket. The location of this substitution and conversion to an arginine is highly unusual for the SI domain that is predominantly lined with non-polar residues that define chymotrypsin specificity.
Preparation of RNA
Fifty late fourth and early fifth instar larvae were cold anaesthetized before the gut was removed and snap frozen in liquid nitrogen. The 50 gut were ground to a fine powder in the presence of liquid N2 using a mortar and pestle and were stored at -70°C. The gut tissue (100 mg) was added to 1 mL of TRIZOL (trademark) reagent (Life Technologies) and total RNA was purified and quantified according to the manufacturer's protocol.
DNA sequencing and analysis
The cDNA clones were grouped on the basis of restriction fragment patterns obtained using combinations of the endonucleases BamHl, XhoII, KpnI, Sad, SacII, and SalI (Promega). RT-PCR products and cDNA inserts were sequenced in both directions using Ml3 universal primers at either Micromon sequencing facility at Monash University (Melbourne) or SUPAMAC at the Royal Prince Alfred Hospital in Sydney. The sequence data was edited using the BioEdit v220.127.116.11 software written by Tom Hall, North Carolina State University freely available at the web address: www.mbio.ncsu.edu/BioEdit/ bioedit.html. Sequence homologies were assessed using the BLASTN search facility at National Centre for Biotechnology Information (NCBI) and further multiple sequence alignments were performed using ClustalW version 1.4. at the Network Protein Sequence Analysis facility (NPSA; http://npsa-pbil.ibcp.fr/cgi-bin/align_clustalw.pl) (Combet et al, TIBS. 25: 147-150,2000).
The web based program 'PSORT II' available at the Human Genome Centre at the University of Tokyo (http://psort.nibb.ac.jp/form2.html), was used to predict signal peptide cleavage points. UTRscan was used to detect functional elements in the 3' untranslated regions Of the cDNA clones [Pesole, Trends Genet, 15: 378, 1999]. (http://bigliost.area.ba.cnr.it/BIG/UTRScan/).
Isolation of a partial cDNA clone encoding a NaPI' insensitive chymotrypsin
A sample of RNA previously purified for the production of the cDNA library was thawed and used as template for the following RT-PCR reaction. Two 5' degenerate primers (5) were designed to unique regions of the N-terminal amino acid sequence and used in combination with the 3' primer RVG4. First strand cDNA synthesis was achieved using the SuperScriptII Preamplification system from Stratagene and was followed by PCR amplification of the target cDNA using the Perkin Elmer thermocycler [25 cycles for 1 min at 94°C, 1 min at 48°C and 1 min at 72°C then 7 mins at 72°C], PCR products were separated on 1% (w/v) agarose gel (SEAKEM (registered trademark); BioWhittaker Molecular Applications) and a band of approximately 650 bp was excised and purified using the Concert purification system (Gibco). The partial cDNA was cloned into the TOPO PCR2.l-TA vector (Invitrogen) and transformed into E. coli strain XL-BL1 (Stratagene).
Isolation of the insensitive chymotrypsin cDNA clone
The cDNA library prepared from fourth instar larval gut was screened using a partial fragment cloned according to the techniques described above
Homology modeling of the H. punctigera chymotrypsins
The deduced amino acid sequences from the cDNA clones HpF2B (sensitive) and HpF5 (insensitive) were modeled on the structures of the Bos taunts (bovine) and fire ant chymotrypsins, obtained from the Research Collaboratory for Structural Bioinformatics (RCSB) Protein Data Bank site (http://www.rcsb.org/pdb/). The Helicoverpa chymotrypsins are predicted to adopt similar structures to those reported for all the chymotrypsin structures available in the PDB databank. The modeled structure consists of the classic serine protease fold consisting of two, six-stranded anti-parallel p barrels with the catalytic triad located between the two domains. Two surface loops, 60 and 142 are considerably larger in the H. punctigera chymotrypsins (Figures 15 and 16). Due to the limitations of modelling, a small amount of ambiguity was present in several surface loops, some of which are cleaved in mammalian chymotrypsins (loop 142), but remain intact within insect chymotrypsins. The only reported crystal structure of an insect chymotrypsin is from the fire ant, Soenopsis invicta (Botos et al, Journal of Molecular Biology 298: 895-901, 2000) and this was used to help refine the orientation of the surface loops on the model of the Helicoverpa chymotrypsin.
Cl was modeled in complex with sensitive and insensitive chymotrypsins to investigate whether substitution of glutamine (or asparagine) 192 (Greer nomenclature, Figure 15) with an arginine would affect the binding capacity of the Helicoverpa chymotrypsins. The structure of the chymotrypsin inhibitor (Cl) was previously determined by 1H NMR (Nielson et al., 1994, supra). No structures of Cl complexes have been solved and therefore the related proteinase inhibitor PCI-1 from Solanum tuberosum in complex with Proteinase B from Streptomyces griseus (Greenblatt et al., 1989, supra) provided a basis for structural modeling. Figure 17 illustrates the binding region surrounding Gln 192 in the Cl-HpF2B chymotrypsin complex. The predicted model of HpF2B and Cl shows that glutamine 192 is not in conflict with any regions on the inhibitor molecule. Comparison to the cognate arginine residue in the insensitive chymotrypsin however suggests there is limited space to accommodate this much larger residue (Figure 18). The modeling viewing program 'Spdbv', predicted Arg 192 to be in direct conflict with tlireonine 5 of Cl. PotI has been identified as a strong inhibitor of the insensitive chymotrypsin from H. punctigera and was, therefore, modelled in complex with the insensitive chymotrypsin (Figure 19). Unlike Cl, PotI easily accommodates arginine 192 upon binding to the insensitive chymotrypsin.
In summary, the NaPI-insensitive chymotrypsin from Helicoverpa species has an arginine in place of an asparagine or glutamine at position 192 that extends into the S1 binding pocket and appears to interfere with Cl binding. Furthermore, it is clear that this arginine residue does not interfere with PotI binding, consistent with the observation that PotI is a much more efficient inhibitor of insect chymotrypsins than the NaPI inhibitors.
Production and characterization of polyclonal antisera to the NaPI inhibitable and NaPI- insensitive chymotrypsins from H. punctigera
Chymotrypsin clone HpF2B was expressed in E. coli fused to a six histidine (6.H) tag at the C-terminus and was purified to homogeneity on Talon metal affinity resin (Figure 20) for injection into a rabbit for production of polyclonal antibodies. N-terminal sequencing of the purified product confirmed the expression of the NaPI inhibitable chymotrypsin (HpCh2B). After the fourth boost with antigen, the serum was collected and tested on protein blots of bacterially expressed protein and unfractionated gut extracts. The antibody detected the full-length recombinant chymotrypsin at a dilution of 1 in 2500 as well as several break-down products. Unfractionated gut extract and a sample of protein bound to
the Cl affinity column were also stained with the anti-HpCh2B antibody which detected the mature native form of the enzyme.
Purified 6HHpCh2B was used to test the detection limit of anti-HpCh2B antibody by comparison of immunoblots to silver stained SDS-PAGE gels. The antibody detected 20 ng of bacterially expressed chymotrypsinogen and also recognized the mature form of the native chymotrypsin isolated from gut of H, punctigera.
The cDNA (HpF5) encoding the NaPI-insensitive chymotrypsin (HpCh5) was expressed in E. coli in a similar manner except the six.histidine tag was fused to the N-terminus of the expressed protein. The polyclonal antiserum that was raised against the bacterially expressed NaPI inhibitable chymotrypsin (HpCh2B) did not cross-react with bacterially expressed NaPI-insensitive chymotrypsin (HpCh5) on protein blots (Figure 21). Likewise the antiserum raised against HpCh5 did not bind to HpCh2B. This indicates that these antisera can be used to specifically distinguish between and monitor levels of the NaPI-insensitive and sensitive chymotrypsins in unfractionated gut extracts.
Preparation of antigen for immunization
The cDNA clone 'HpF2B'which encodes an NaPI sensitive chymotrypsin (Figure 11) was amplified by polymerase chain reaction (PCR) using two oligonucleotides (Table 7) that incorporated NcoI and BglII restriction sites at the 5' and 3' ends of the cDNA respectively.
Degenerate primers designed to tiw unique regions in the N-terminus of the insensitive chymotrypsin protein. Primer positions are shown in the ammo acid
sequence by matching typeface
TABLE 8 PCM amplification primers for bacterial expression of chymotrypsin HpF2B
The amplified region consisted of the pro-peptide and mature domain of the chymotrypsinogen, but lacked the putative secretion signal. Digests using Ncol and Bglll enzymes (Promega) were performed on the PCR amplified product and the pQE-60 expression vector (Qiagen). The pQE-60 vector provides a His-tag at the C-terminus of the expressed protein. Each restriction digest was purified using WIZARD (registered trademark) DNA clean up system (Promega) and the vector and chymotrypsin insert were subsequently ligated using standard molecular biology techniques (Sambrook and Russell, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 3rd edition 20)01) The ligation mix was heated at 65 °C for 10 min before being transformed into the E. coli strain XLl-Blue. Plasmid DNA was prepared for sequencing and subsequent transformation into the E. coli cell line Ml5 (Qiagen) using the Q1APREP (registered trademark) Spin Miniprep Kit (Qiagen).
Expression and purification of the recombinant chymotrypsinogen was performed under denaturing conditions according to the methods detailed in the QiaExpress manual (Qiagen). The purity of the expressed chymotrypsinogen was assessed by SDS-PAGE and the identity of the recombinant protein confirmed by N-terminal sequencing. Preparation of the chymotrypsin for injection consisted of removal of the urea by dialysis against 50 mM Tris-HCl pH 8.0. During this process most of the protein aggregated. The aggregated protein was collected by centrifugation and resuspended in 1 mL of 50 mM Tris-HCl, pH 8.0 for injection. The protein concentration was approximated by the comparison of a 10 µL sub-sample to a series of bovine trypsin standards (Sigma) using SDS-PAGE and Coomassie staining.
The cDNA clone 'HpF5' which encodes the NaPI-insensitive chymotrypsin (Figure 12) was PCR amplified essentially as described for clone HpF2B above except the forward primer (FwpMalRECH ) incorporated a Histidine tag at the N-terminus of the expressed protein and the reverse primer (RvRECH) contained a stop codon and thus prevented incorporation of a Histidine tag at the C-terminus from the pQE-60 expression vector.
Biassays mill PotI inhibitor and Helicoverpa larvae
Large quantities of the PotI inhibitor were purified from potato tubers to evaluate the combined effect of NaPI and PotI on the growth of H.armigera larvae. Bioassays confirmed that addition of PotIsignificantly enhances the activity of the NaPI inhibitors. Caterpillars fed NaPI and PotI in combination (0.26 and 0.34% (w/v), respectively) were 34% the size of control larvae at the fifth instar stage of development whereas caterpillars feeding on NaPIs alone were about 84% the size of the controls. Based on these results we decided to clone genes encoding the PotI inhibitors for transfer into cotton plants. Two PotI genes (PotIA and PotIB) were isolated using mRNA isolated from potato tubers and wounded potato leaves. These cDNA clones were used to construct vectors for bacterial expression of the PotI proteins. The bacterially produced PotI proteins totally inhibited the NaPI-insensitive chymotrypsin isolated from H. punctigera larvae. Both PotI genes were incorporated into constructs for cotton transformation. Purified PotI inhibitor from potato tubers was used to generate specific antibodies that were used to monitor levels of Poti produced by transgenic plants,
Purification of PotI inhibitor from potato tubers
Potato tubers (2 Kg, Solanun tuberosum var Russet Burbank) were diced and soaked overnight at 4°C in two litres of 50 mM H2SO4. The tissue was homogenized in a blender and insoluble material was removed by filtration through two layers of Miracloth followed by centrifugation (13,000 rpm, 15 min, 4°C). The supernatant was adjusted to pH 7.8 with 10 M NaOH, heated for 30 min in boiling water and cooled before precipitated material was separated by centrifugation (13,000 rpm, 12 min, 4°C). Soluble proteins were precipitated with ammonium sulphate (80% saturation), collected by centrifugation and redissolved in gel filtration buffer (150 mM KC1, l0mM Tris-HCl, pH 8) before they were applied to a Sephadex G75 column (85 x 2.54cm). Elution fractions (50mL) containing PotI were identified using SDS-polyacrylamide gel electrophoresis and inhibition assays with bovine chymotrypsin. PotI containing fractions were pooled, dialysed and freeze dried. The protein in these pooled fractions was examined by reverse phase -HPLC on a system Gold HPLC (Beckman, Fullerton, CA) coupled to a 166 detector (Beckman). The analytical RP-HPLC was conducted on a Brownlee Aquapore RP300 C8 column (4.6 x 100mm; Parkin-Elmer). The protein was eluted with a linear gradient of 0-100% (v/v) buffer B (60%[v/v] acetonitrile in 0.089% [v/v] trifluoroacetic acid) at a flow rate of 1 mL min-1 over 40min (Figure 22). The identity of the PotI proteins was confirmed by N-terminal sequencing and mass spectrometry and at least two PotI isoforms were identified.
Growth of H. armigera larvae on artificial diet containing NaPI and PotI
PotI protein isolated from potato tubers and NaPI peptides isolated from N. alata stigmas, were incorporated into a cotton leaf based artificial diet and fed to H.armigera neonates (Figure 23). PotI had an additive affect on larval growth. Larvae fed NaPI only were inhibited in growth by 16% compared to control larvae while larvae fed both NaPI and PotI were inhibited in growth by 75% compared to control larvae (Figure 23).
Isolation of cDNAs encoding Potato protein ase inhibitor 1 (PotI)
Potato proteinase inhibitor I cDNA was synthesized using reverse transcriptase-PCR and total RNA from potato tubers or wounded potato leaves. First strand cDNA was prepared using Thermoscript RT-PCR with Oligo (dT)2o primers (Life Technologies). PotI cDNA sequences were subsequently amplified using gene specific primers 5' CGG-GAT-CCA-TGG-AGT-CAA-AGT-TTG-C-3' [SEQ ID NO: 14] (sense) and 5'-GCG-TCG-ACG-CTT-AAG-CCA-CCC-TAG-G-3' [SEQ ID NO: 15] (antisense) that were designed to anneal to the 5' and 3' ends of the open reading frame of the published PotI genomic sequence Ml7108 (Cleveland et al, Plant Mol. Biol. 8: 199-207, 1987) and included restriction sites Bam HI and SalI respectively.
Two PotI homologs were isolated. StPotIA was derived from wounded leaf RNA and StPotIB was derived from tuber RNA. StPotIA and StPotIB cDNA share 92.6% nucleic acid sequence identity and share 86% amino acid sequence identity (Figure 25).
The predicted amino acid sequence and comparison to other members of the Potato Inhibitor I family are presented in Figure 24. StPot1B is very similar to published Pot1 sequences from potato tuber and shares a methionine at the P1 reactive site. In contrast, StPotIA contains an alanine residue at P1and also has an additional four amino acids at positions 41 to 44. Additional amino acids in this position have not been reported for other potato Poll isolates although they have been found in a wound induced Poti from tomato.
Expression of StPotIA and StPotIB in E. coll
DNA encoding PotI without the endoplasmic reticulum signal sequence (amino acids 37-111 in StPotIA and 37-107 in StPotIB) was amplified by PCR. Primers used were 5'-CGG-GAT-CCA-AGG-AAT-CGG-AAT-CTG-3' [SEQ ID NO: 16] (StPotIA sense), 5'-CGG-GAT-CCA.AGG-AAT-TTG-AAT-GC-3' [SEQ ID NO: 17] (StPotIB sense) and 5'-CGA-GCT-CTT-AAG-CCA-CCC-TAG-G-3' [SEQ ID NO: 18] (StPotlA/B antisense). PCR products were initially cloned into the pGEM T-Easy vector (Promega) before they were excised with BamHI and Sad and ligated into the bacterial expression vector pQE30 (Qiagen) which provides a 6x His-tag at the N-terminus of the expressed protein.
The His-tagged PotI proteins were expressed in E. Coli (BR21 DE3 Codon Plus strain (Stratagene) for StPotIA and Ml 5 strain (Qiagen) for StPotIB). Cells were induced with 1 mM IPTG, harvested by centrifugation and lysed in 8 M urea, 0.1 M NaH2PO4, 0.01 M Tris-HCl, pH 8.0. Cell debris was removed by centrifugation at 10,000 g for 5 min and the His-tagged PotI was purified from the supernatant by metal-affinity chromatography on Talon resin (Clontech). Bound protein was eluted from the resin with 8 M urea, 0.1 M NaH2P04, 0.01 M Tris-Cl, pH 4.0 and elution was monitored by SDS-PAGE. The StPotIA and StPotIB proteins were purified further by RP-HPLC (Figure 25) their identity was confirmed by N-terminal sequencing and mass spectrometry.
Inhibition of the NaPI-insensitive chymotrypsins from H. punctigera by StPotIA and StPotIB
The inhibitory activity of StPotIA and StPotIB against the NaPI-insensitive chymotrypsins from Hpunetigera was determined by preincubating the NaPI-insensitive protease (10 µL) with varying amounts of StPotIA and StPotIB (0-600 nM) in 133 mM CAPS buffer, pH 10.0 at 30°C in 96 well microtitre plates. After the 30 min preincubation, the incubation was started by the addition of substrate (SA2PFpNA, SA2PLpNA or SA2PMpNA) to a final concentratiop of 1 mM in a final volume of 100 µL. Absorbance was measured at 405 nm after 30 or 60 min.
StPotIA, StPotIB and the mix of PotI isoforms isolated from potato tuber inhibited the NaPI-insensitive chymotrypsin in the Helicoverpa punctigera gut extract (Figure 26). At least 75% of SA2PFpNA or SA2PLpNA hydrolysis by the NaPI-insensitive chymotrypsin was inhibited by the addition of 300 nM of StPotIA, StPotIB or a mix of PotI isoforms isolated from tuber. Inhibition of SA2PMpNA hydrolysis was lower (40%).
Production oftransgenic cotton expressing NaPI and StPotIA
Two gene constructs (pHEX2 and pHEX6) were prepared for transformation of cotton (Gossypium hirsutun). pHEX2 consists of a 35S promoter driving the NaPI gene with a 35S terminator, inserted into the binary vector pBIN 19 (Bevan, Nucl. Acids Research, 12: 8711-8721,1984). pHEX6 consists of a 35S promoter driving the StPotIA gene with a 35S terminator inserted into the binary vector pBIN 19.
Transgenic cotton was produced using the method of Umbek et al., Biotechnology, 5: 263-266, 1987) with modifications. Hypocotyl sections of cotton Cv Coker 315 were co-cultivated with Agrobacterium tumefaciens strain LBA 4404 containing the required binary vector. Callus was induced on media consisting of MS salts, B5 vitamins, 3% glucose, 0.9 g/L MgCl2 (hexahydrate), 1.9 g/L potassium nitrate, 2 g/L Gelrite, 0.1 mg/L Kinetin, 0.1 mg/L 2,4-D, 500 mg/L carbenicillin, 35 mg/L Kanamycin. Embryogenic callus was induced by growing the callus on the same media but without hormones. Embryos were excised and incubated on media in petri dishes (Stewart and Hsu, Planta 137: 113-117, 1977). Germinated embryos that had produced roots and true leaves were transferred to containers for further development and then transferred to soil and grown in a growth cabinet at 27°C.
Production of transgenic cotton expressing NaPI and StPotIA
To produce plants expressing both genes, pollen from a transgenic line expressing NaPI was used to pollinate a flower from a plant expressing StPotIA and the seed collected. One progeny plant (plant 3) was identified as expressing both genes by immunoblot analysis.
Leaves from plant 3 were used in a bioassay with H. armigera (Figure 27). While expression of either NaPI or StPotIA in the leaves only resulted in a small inhibition of larval growth compared to the control, expression of both proteins had a synergistic effect on larval growth.
EXAMPLE 7 Baculovirus expression of HpCh5
Addition of the signal peptide sequence to HpCh5
While the HpF5 clone encoded the entire chymotrypsinogen sequence of HpCh5 with the activation domain it did not encode the signal peptide required for correct targeting of the protein to the endoplasmic reticulum (ER). For baculovirus expression, an ER signal sequence was added to the HpF5cDNA using two overlapping oligonucleotides corresponding to the 19 amino acids of the ER signal peptide from HpCh2A (Figure 10). The ER signal sequence was added to the preactivation and mature domains of HpCh5 in a two-step PCR (Table 9).
PCR amplification primers for addition of an ER signal sequence to HpCh5
The first PCR used primers FwBacRECHl and RvRECH with the HpF5 cDNA template to add the first half of the ER signal sequence. The second PCR used FwBacRECH2 together with RvRECH and the product of the first PCR reaction as template. At each step, the amplification products were purified after electrophoresis on 0.7% (w/v) agarose gels before they were used for subsequent PCRs.
Cloning of HpF5/ER intopFastBac vector
The HpF5 cDNA with the ER signal sequence (HpF5/ER) was subcloned into the pCR (registered trademark)-2.1 TOPO vector (Invitrogen) and was sequenced at the Micromon, DNA sequencing facility, Monash University, Victoria, Australia. Recombinants with the correct sequence were digested with £coRI and gel purified before they were digested with BamHl and HindIII and ligated into the pFastBac vector (Invitrogen) and transformed into E. coll XL 1 Blue cells.
Transposition of pFastBac/HpF5/ER construct into E. coliDH10Bac cells
E. coll XL1 Blue cells were screened for the presence of the HpF5/ER cDNA in the pFastBac vector (pFastBac/HpF5/ER) by PCR and restriction digest. Minipreps were performed on positive transformants. E. coli DHlOBac competent cells containing bacmid DNA and the helper plasmid required for transposition of HpF5/ER to the bacmid DNA were thawed on ice. Approximately 1 ng of pFastBac/HpF5/ER recombinant plasmid was added to the cells (150 µL) and after gently mixing the mixture was transferred to a pre-chilled GENE PULSER (registered trademark)/E. COLI (trademark) pulser cuvette. The cuvette was placed in the electroporation apparatus (BioRad) and a pulse of 1.7 Amps was applied. LB (1 mL) was then added and the sample was transferred to a 10 mL capped tube and allowed to recover on a shaking incubator (190 rpm) at 37°C for 4 hours. A sample was withdrawn and serially diluted (10-1., 10-2, and 10-3) using LB medium before 100 µL of each dilution was spread evenly onto LB agar plates containing 50 µg/mL kanamycin, 7 µg/mL gentiamicin, 10 µg/mL tetracycline, 100 µg/mL 5-bromo-4-chloro-3-indolyl-p-D-galactopyraaoside (X-Gal) and 40µg/mL IPTG prior to incubation for 48 hours at 37°C. A pFastBac vector with no insert was treated the same way and used as a control.
Isolation of recombinant bacmid DNA
Recombination between the pFastBac/HpF5/ER vector and the bacmid DNA was detected by the presence of white colonies as apposed to blue colonies, indicating a disruption in the lacZa. gene. To confirm the recombination was stable, the colonies were restreaked on selective media and incubated for 24 hours. White colonies were used to inoculate 10 mL LB media containing 50 µg/mL kanamycin, 7 µg/mL gentiamicin and 10 µg/mL tetracycline for isolation of the recombinant bacmid DNA. The same procedure was performed with a blue colony (not recombinant) selected as a control. Cells were grown overnight at 37°C with shaking at 190 rpm. Cultures were centrifuged at 14,000 x g for 5 min. The supernatant was discarded and the pellet was resuspend in 0.3 ml of Solution I (15 mM Tris-HCl, pH 8.0, 10 mM EDTA, 100 µg/mL RNase A]. Solution II (0.2 N NaOH, 1% (w/v) SDS) was then added and gently mix and incubated at room temperature for 5 min. Potassium acetate (0.3 mL, 3 M, pH 5.5) was slowly added and the samples were placed on ice for 10 min before, they were centrifuged for 10 min at 14,000 x g to remove the thick white precipitate. The supernatant was then transferred to a 1.5 mL microfuge tube containing 0.8 ml absolute isopropanol and was mixed by inversion. After 10 min on ice samples were centrifuged for 15 min at 14,000 x g at room temperature. The supernatant was removed and pellet was washed twice with 0.5 mL of 70% (v/v) ethanol. The sample was then centrifuged for 5 min at 14,000 x g at room temperature before the 70% (v/v) ethanol was removed and the DNA pellet was air dried. The DNA was then dissolved in 60 µL TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) and stored at -20°C until needed. The bacmid was examined by electrophoresis on a 0.5% (w/v) agarose gel
prepared in TAE (40 mM Tris-acetate, 1 mM EDTA, pH 8) buffer at 23 V for 12 hours with λDNA/HindIII Fragment markers (MB1 Fragments). The bacmid was also screened using PCR with Ml 3 Forward and reverse primers.
Transfection of HIGH FIVE (trademark) cells with recombinant bacmid DNA
Approximately 9 x 105 HIGH FIVE (trademark) insect cells were placed into a 35-mm well in a 6-well plate (NUNCLON (trademark) A Surface) in 2 ml of EX-CELL (trademark) 405 media with 50 units/mL penicillin and 50 µl/mL streptomycin (JRH Biosciences). Cells were left at 27°C for 1 hour to allow them to attach to the plate. Two solutions were prepared for each transfection each adding a different amount of bacmid miniprep (5., 10 or 20 µL); Solution A: mini-prep of bacmid DNA into 100 µL EX-CELL (trademark) 405 media without antibiotics, and Solution B: 6 uL of CELLFECTIN (registered trademark) Reagent (Gibco BRL) into 100 uL EX-CELL (trademark) 405 media without antibiotics. Solutions A and B were combined and incubated for 1 hour at room temperature followed by 0.8 mL of selection free media added. After the cells had attached to the plate, they were washed once with 2 mL media without antibiotics. The media was removed from cells and was replaced with the DNA containing solution. Cells were incubated for 5 hours in a 27°C incubator before the transfection mixture was aspirated and replaced with 2 mL of media containing antibiotics. Cells were incubated further at 27°C for up to 72 hours.
After 24, 43 and 72 hours, 2 mL of the supernatant was transferred to a 10 mL sterile capped tube and centrifuged for 5 min at 500 x g. The virus-containing supernatant was transferred to a fresh tube and stored at 4°C, protected from light until required.
Infection of insect cells with recombinant baculovirus particles
Two 10 mL cultures were set up for each transfection. Each culture was infected with 100 µl of harvested virus (~2 x 107 pfu/mL). Infected cells were harvested at 24, 48, and 72 hours. These samples were later analyzed for protein expression by immunoblot analysis with the anti-HpCh5 antibody.
EXAMPLE 8 Baculovirus expression of HPCH5
Primer design for ER signal sequence
The original HpF5 cDNA clone did not encode an ER signal sequence. While the ER signal sequence was not required for bacterial expression it was essential for baculovirus expression. An ER signal sequence was thus constructed using the DNA sequence from chymotrypsin family 2A which is most closely related to the chymotrypsin family 5.
The sequence was added using PCR reactions with two primers encoding part of the ER signal sequence. The FwBacRECHl primer had a silent mutation to remove the BamHl restriction digest site and encoded half the ER signal sequence. The FwBacRECH2 primer encoded the remainder of the ER signal sequence and introduced a BamHl restriction site (Figure 28).
Amplification and ligation of HpCh5 into thepFastBac vector
PCR using primers the FwBacRECHl and RvRECH and HpFScDNA as the template yielded a product of ~900 bp. A second PCR using this PCR product together with FwBacRECH2 and RvRECH primers was performed to yield a product of ~930 bp that encoded the HpCh5 protein with an ER signal sequence. The amplified product was subcloned into pCR (registered trademark)-2.1 TOPO vector (Invitrogen) before transfection into TOP 10 competent cells. Colonies were screened for the presence of insert using Ml 3 forward and reverse primers. Restriction digests of the isolated plasmids with EcoRl then subsequently with BamHl and Hindlll yielded a product of the expected size of -930 bp.
The -930 bp fragment was subsequently ligated into the pFastBac vector to create pFastBac/MpF5/ER. The presence of the HpF5 cDNA insert in the pFastBac vector was confirmed by restriction digests with BamHl and Hindlll and PCR using FwBacRECH2 and RVRECH primers (Table 9). Plasmids containing the insert were sent to Micromon for DNA sequencing.
Analysis of Eacmid DNA
The transfection of the recombinant pFastBac vector into DHlOBac cells resulted in the transposition of the HpF5 insert into the bacmid DNA. Recombinant bacmid DNA was isolated from the transfected cells and was separated on a 0.5% (w/v) agarose gel.
The bacmid was checked for the HpF5 cDNA insert by PCR analysis with M13 forward and reverse primers. The expected ~3300 bp fragment was further analysed using the primers to HpF5/ER cDNA and Ml3 to ensure the insert was in the correct orientation. The positive bacmid DNA was subsequently used for the transfection of insect cells.
Virus formation and production of HpCh5 in the baculovirus expression system
Three different concentrations of bacmid DNA were used to transfect the insect cells for production of baculovirus. After transfection, the cells were incubated at 27°C for 72 hours before the culture medium containing the virus was collected. The medium was tested by immunoblot analysis with the α-HpCh5 antibodies for the production of HpCh5 protein, which indicated virus was being produced (Figure 29A). Many controls were used to monitor the transfection. These included pFastBac vector alone, transfection of DNA from a blue colony, incubation of cells with CELLFECTIN (registered tradmark) alone and non-transfected cells (Figure 29A). The medium containing virus was used in a subsequent experiment to infect more insect cells on a larger scale and a time course was performed over a 72 hour period to monitor the regulation of HpCh5 expression (Figure 29B).
Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any two or more of said steps or features.
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1. A synthetic or recombinant nucleic acid molecule encoding an amino acid sequence defining potato inhibitor 1A (PotIA), which PotIA comprises the amino acid sequence set forth in SEQ ID NO:81 and inhibits the proteolytic activity of the protein having the amino acid sequence set forth in SEQ ID NO:2.
2. PotIA encoded by the nucleic acid molecule as claimed in claim 1.
3. A vector comprising the nucleic acid molecule as claimed in claim 1.
|Indian Patent Application Number||5343/DELNP/2005|
|PG Journal Number||46/2010|
|Date of Filing||22-Nov-2005|
|Name of Patentee||HEXIMA LIMITED.|
|Applicant Address||LEVEL 17, 200 QUEEN STREET, MELBOURNE, VICTORIA 3000, AUSTRALIA.|
|PCT International Classification Number||C12N 9/76|
|PCT International Application Number||PCT/AU2004/000524|
|PCT International Filing date||2004-04-23|