Method of Controlling Insects

This invention relates to a method of controlling insects, including lepidopterans and boll weevils, by use of an enzyme which may be applied directly to the plant or produced thereon by microorganisms or by genetically modifying the cotton plant to produce the enzyme, and to genes, microorganisms, and plants useful in that method.

The use of natural products, including proteins, is a well known method of controlling many insect pests. For example, endotoxins of Bacillus thuringiensis (B.t.) are used to control both lepidopteran and coleopteran insect pests. Genes producing these endotoxins have been introduced into and expressed by various plants, including cotton, tobacco, and tomato. There are, however, several economically important insect pests that are not susceptible to B.t. endotoxins. One such important pest is the cotton boll weevil. There is also a need for additional proteins which control insects for which B.t. provides control in order to manage any development of resistance in the population.

It is therefore an object of the present invention to provide proteins capable of controlling insects, such as boll weevil and lepidopterans, and genes useful in producing such proteins. It is a further object of the present invention to provide genetic constructs for and methods of inserting such genetic material into microorganisms and plant cells. It is another object of the present invention to provide transformed micro-organisms and plants containing such genetic material.

It has been discovered that proteins that catalyze the oxidation of 3-hydroxysteroids, for example, cholesterol, will control lepidopteran insects and boll weevils. They are lethal to boll weevil larvae and will interrupt the reproductive cycle of adults. They cause mortality and stunting of larvae of lepidopteran insects. The enzymes may be applied directly to plants or introduced in other ways such as through the application of plant-colonizing microorganisms or by the plants themselves, which have been transformed to produce the enzymes.

3-Hydroxysteroid oxidases (E.C.1.1.3.6) are naturally produced by microorganisms such as Streptomyces sp., Pseudomonas sp., Mycobacterium sp., Schizophyllum commune, Nocardia sp., and Rhodococcus sp. [Smith et al., 1976, and Long et al., 1990.]. Preparations of enzymes from several different sources are available from Sigma Chemical Company, St. Louis, Mo.

New Streptomyces genes that control the expression of 3-hydroxysteroid oxidase have been isolated and sequenced. These new genes or genes from other known producers of 3-hydroxysteroid oxidase may be inserted into a transformation vector cassette which is used to transform plant-colonizing microorganisms which when applied to plants express the genes producing a 3-hydroxysteroid oxidase, thereby providing control of lepidopterans and boll weevil. Alternatively, genes which function in plants and encode the subject enzymes may be inserted into transformation vector cassettes which may be incorporated into the genome of the plant, which then protects itself from attack by expressing the gene and producing a 3-hydroxysteroid oxidase. Additionally, the plant may also be transformed to co-express B.t. genes which express proteins for the control of other insects. Examples of plants transformed to express B.t. genes are disclosed in European Patent Publication No. 0 385 962, which corresponds to U.S. Ser. No. 476,661, filed Feb. 12, 1990 [Fischhoff et al.], which is incorporated herein by reference.

In accomplishing the foregoing, there is provided, in accordance with one aspect of the present invention, a method of controlling insect infestation of plants comprising providing a 3-hydroxysteroid oxidase for ingestion by the insect.

In accordance with another aspect of the present invention, there is provided a recombinant, double-stranded DNA molecule comprising in operative sequence:

a) a promoter which functions in plant cells to cause the production of an RNA sequence; and

b) a structural coding sequence that encodes 3-hydroxysteroid oxidase;

c) a 3' non-translated region which functions in plant cells to cause the addition of polyadenylate nucleotides to the 3' end of the RNA sequence,

wherein said promoter is heterologous with respect to the structural coding sequence and wherein said promoter is operatively linked with said structural coding sequence, which is in turn operably linked with said non-translated region.

In accordance with another aspect of the present invention, there is provided a method of producing genetically transformed plants which express an effective amount of a 3-hydroxysteroid oxidase, comprising the steps of:

a) inserting into the genome of a plant cell a recombinant, double-stranded DNA molecule comprising

(i) a promoter which functions in plant cells to cause the production of an RNA sequence;

(ii) a structural coding sequence that encodes for 3-hydroxysteroid oxidase; and

(iii) a 3' non-translated region which functions in said plant cells to cause the addition of polyadenylate nucleotides to the 3' end of the RNA sequence,

wherein said promoter is heterologous with respect to the structural coding sequence and wherein said promoter is operatively linked with said structural coding sequence, which is in turn operably linked with said non-translated region;

b) obtaining transformed plant cells; and

c) regenerating from the transformed plant cells genetically transformed plants which express an insecticidally effective amount of sterol oxidase.

There is also provided, in accordance with another aspect of the present invention, bacterial and transformed plant cells that contain DNA comprised of the above-mentioned elements (i), (ii), and (iii).

As used herein, the term "controlling insect infestation" means reducing the number of insects which cause reduced yield, either through mortality, retardation of larval development (stunting), or reduced reproductive efficiency.

As used herein, the term "structural coding sequence" means a DNA sequence which encodes for a polypeptide, which may be made by a cell following Transcription of the DNA to mRNA, followed by translation to the desired polypeptide.

3-Hydroxysteroid oxidases catalyze the oxidation of the 3-hydroxy group of 3-hydroxysteroids to produce ketosteroids and hydrogen peroxide. They are capable of catalyzing the oxidation of various 3-hydroxysteroids, such as, for example, cholesterol. Most of the previously known 3-hydroxysteroid oxidases are called "cholesterol oxidases" (enzymatically catalogued as E.C. #1.1.3.6) but cholesterol is only one of the 3-hydroxysteroid substrates, not the only one. The use of all 3-hydroxysteroid oxidases and the genes encoding such proteins, for the purpose of controlling insects, are within the scope of the present invention.

3-Hydroxysteroid oxidases are commercially available for use as reagents for serum cholesterol assays. For example, Sigma Chemical Company, St. Louis, Mo., offers three such 3-hydroxysteroid oxidases (denominated as cholesterol oxidases), one from a Streptomyces sp., one from a Pseudomonas fluorescens, and one from a Brevibacterium. Two other sources of 3-hydroxysteroid oxidase, two streptomycetes denominated A19241 and A19249, each of which produce a 3-hydroxysteroid oxidase, have been isolated. The organisms were collected in Madagascar. When these organisms were cultured according to usual methods the culture filtrates were found to affect insect larvae as described below.

A seed culture of A19249 was started in 55 mL sterile Tryptone-Yeast Extract broth, pH 6.8, in a 250 mL shake flask. The seed was agitated at 250 rpm on a rotary shaker for 3 days at 30.degree. C. A New Brunswick Biofio II Bioreactor with a 2 L working volume was filled with "medium 202" (MgSO.sub.4 .multidot.7H.sub.2 O 2 g/L, KH.sub.2 PO.sub.4 0.5 g/L, NaCl 0.5 g/L, CaCO.sub.3 1 g/L, ZnSO.sub.4 .multidot.H.sub.2 O (1 mg/mL stock) 5 mL/L, 100 mM FeEDTA 0.5 mL/L, Soluble Starch 5 g/L, Dextrose 2.5 g/L, Malt Extract 2.5 g/L, Soytone 5 g/L). The pH was adjusted to 6.5 with 2.5N NaOH or 1N HCl, and 1 mL/L of P2000, an antifoam agent was added. The bioreactor was sealed and autoclaved for 25 min at. 250 .degree. C. The seed, at 3 days growth, was used to inoculate the fermentor at 2% or 40 mL. The fermentation took place at 30.degree. C. with an airflow of 1 L/min and agitation running at 500 rpm. The fermentation was harvested after 40 h.

Each of these enzymes has demonstrated control of insects as shown below. The P. fluorescens 3-hydroxysteroid oxidase is immunologically distinct from the Streptomyces enzymes, but it also controls insects.

Other organisms producing 3-hydroxysteroid oxidases of the present invention may be identified by assaying culture filtrates or individual proteins for 3-hydroxysteroid oxidase activity using a spectrophotometric assay, described below, which measures hydrogen peroxide production in the presence of a 3-hydroxysteroid, for example, cholesterol [Gallo, 1981].

Assays for activity against boll weevil larvae are carried out by incorporating the test sample into a agar liquid diet similar to that for southern corn rootworm [Martone et al., 1985]. The test sample is substituted for the 20% water component. Neonate larvae are allowed to feed on the diet and mortality and growth stunting are evaluated.

The results of the assays of the 3-hydroxysteroid oxidases identified above are given in Table 1. Protein concentrations were determined spectrophotometrically with BCA protein reagent [Smith et al., 1985].

Lepidopteran larvae were tested on artificial diet treated with the indicated amount of the A19249 3-hydroxysteroid oxidase (cholesterol oxidase) for six days. The results are shown in Table 2.

An extended test was performed with tobacco budworm larvae to test the effect of the stunting noted in the six-day test. Tobacco budworm eggs were added to artificial diet (as described above) containing either buffer or 100 ppm A19249 3-hydroxysteroid oxidase (cholesterol oxidase). After seven days, some mortality as compared to the controls was noted. Surviving larvae were moved to fresh diet (control or treated, as appropriate). Percent mortality (corrected for control mortality) is reported for the 7 day and 10 day periods in Table 2A. The corrected number of larvae was 23.

The diet incorporation study described above was performed to determine relative sensitivities of neonate and older (2nd instar) boll weevil larvae to the Sigma Streptomyces 3-hydroxysteroid oxidase (cholesterol oxidase). The mortality results shown in Table 3 reflect an eight-fold difference in susceptibility at six days exposure. This difference disappears after two weeks of exposure.

3-Hydroxysteroid oxidases, in addition to lethal effects on larvae, will also affect the reproductive cycle of adult boll weevils, as demonstrated by the following experiment.

Pre-oviposition: Approximately 220 adult boll weevils, collected within 2 clays of emergence, were divided into two groups. One was fed standard diet and the other was fed standard diet containing 48 ppm 3-hydroxysteroid oxidase front Sigma (Strepto-myces). The adults were allowed to feed and mate for four days at which time mortality was determined. The results are reported in Table 4.

Oviposition study: These two groups of adults were then divided into two subgroups and individually placed on artificial, enzyme-containing or control bolls. Artificial bolls were constructed of standard diet, with or without 48 ppm 3-hydroxysteroid oxidase, and encased in paraffin containing 1% cottonseed oil. After three days at 27.degree. C., the adults were removed and ten bolls from each of the four groups were removed and examined for eggs. The remaining bolls were incubated for an additional 7 days at 27.degree. C. to allow development of larvae. The bolls were then dissected and the eggs and larvae, dead and surviving, were counted. The results are reported in Tables 5 and 6.

Group 1=Control Adults placed on control bolls

Group 2=Control Adults placed on treated bolls

Group 3=Enzyme-fed adults placed on control bolls

Group 4=Enzyme-fed adults placed on treated bolls

The above results confirm the effects of 3-hydroxysteroid oxidase on boll weevil larvae when apparently normal larvae are challenged with the enzyme in their diet. Data in Table 5 indicate that adults fed 3-hydroxysteroid oxidase do not oviposit normally, even when presented with control bolls. It is also apparent that normal adults will readily oviposit in bolls containing the enzyme (Table 5). Table 6 data suggest a reduction of egg viability when adults are fed 3-hydroxysteroid oxidase during the pre-oviposition period. Although no direct mortality in adults was observed (Table 4) during the observation period, there is evidence of profound 3-hydroxysteroid oxidase effects on the adults' ability to develop and/or oviposit viable eggs.

The following studies show that 3-hydroxysteroid oxidase has a direct effect on the insect itself and that the activity demonstrated in the experiments described above cannot be attributed to the enzymes effect on the insect's diet, for example by sterol depletion. Lepidopteran larvae and boll weevils are most susceptible to the enzyme. It is believed that this specificity is due to the effect of 3-hydroxysteroid oxidase on the midgut of the insect as explained in more detail below. It has been observed that the boll weevil midgut has a proteinase composition which is more like lepidopterans than that of coleopteran (Purcell, et al., 1992), which probably explains why boll weevils and lepidopterans are the most sensitive to the enzyme. Other insects with similar midgut physiologies may also be controlled by 3-hydroxysteroid oxidase. In addition, 3-hydroxysteroid oxidases other than those tested and reported herein may control a different spectrum of insects with different midgut physiologies.

The Southern corn rootworm diet used in the assay described above was the control. Two treatment diets were made by mixing 30 g of one of two types of cottonseed flour into 170 mL of a 1.6% agar solution at 50.degree. C., containing 0.13% propionic acid, 0.014% phosphoric acid, and 30 mg each of streptomycin sulfate and chlor-tetracycline. Before mixing, 10% KOH was used to adjust the pH to 6.2. One test diet utilized raw cottonseed flour (Sigma) as the nutrient source; the other utilized Pharmamedia.TM. (Traders Protein), a flour made up of cottonseed embryos. The diets were incubated in a water bath at 40.degree. C. Dilutions of the Sigma Streptomyces 3-hydroxysteroid oxidase (cholesterol oxidase) were incorporated into the diets as described above. Boll weevil larvae were allowed to feed and mortality rates were determined after six days. The results shown in Table 7 demonstrate that the enzyme is lethal to boll weevil larvae in the presence of cotton plant components.

In addition, tobacco budworm larvae are 68% stunted when exposed to 3-hydroxysteroid oxidase (100 ppm) in cottonseed diet (made with Pharmamedia.TM. flour).

In order to test 3-hydroxysteroid oxidase against boll weevil larvae in a host tissue diet environment, a study was conducted in which cotton leaf tissue was the only nutritional component of an agar-based diet. Two cotton leaves (each approx. 5 inches wide) with stems were homogenized at 50.degree. C. into 170 mL of a 1.6% agar solution containing 0.13% propionic acid, 0.014% phosphoric acid, and 30 mg each of streptomycin sulfate and chlortetracycline. Before addition of the leaves, 10% KOH was used to adjust the pH of the agar solution to 6.2. The leaf "diet" was allowed to cool to 40.degree. C. Dilutions of cholesterol oxidase and a water control were incorporated into the leaf "diet", poured into insect diet trays and allowed to cool. Boll weevil eggs were added to the diet wells. The assay was evaluated six clays later. The results shown in Table 8 demonstrate that the enzyme maintains its insecticidal activity in the presence of cotton leaf tissue. This illustrates that the enzyme is insecticidal in the presence of intact cotton tissue and cells. Since the sterols in these leaf homogenates would presumably not all be accessible to the exogenously added 3-hydroxysteroid oxidase, this suggests that the enzyme is not depleting the diet of all the necessary sterols and that the mode of action of 3-hydroxysteroid oxidase is not dependent on sterol depletion of the nutrient source. These results demonstrate that successful control of boll weevil should be attained when the 3-hydroxysteroid oxidase gene is expressed in cotton tissue.

Two experiments were performed using cotton callus as the assay feeding substrate. In both cases the arena was a 96-well insect diet tray; each well contained 0.5 mL of gelled 2% agar, with 0.13% propionic acid and 0.0 14% phosphoric acid, covered with a 1/2 inch filter paper disc. For each replicate, fifty to one hundred milligrams of cotton callus (Coker 312) was soaked in either water (control) or a 400 ppm cholesterol oxidase solution and placed in a diet tray well. A second instar boll weevil larva was added to each well, and the tray was covered with a sheet of mylar and sealed with a lacking iron. Assay duration was six days. The results shown in Table 9 demonstrate that the enzyme is active in cotton callus bioassays. This illustrates that the enzyme is insecticidal in the presence of intact cotton tissue and the bioactivity is seen when the enzyme is ingested with whole cotton cells and tissue. Since the sterols in this callus would presumably not all be accessible to the exogenously added 3-hydroxysteroid oxidase, this further demonstrates that the mode of action of 3-hydroxysteroid oxidase is not dependent on sterol depletion of the nutrient source. These results demonstrate that successful control of boll weevil should be attained when the 3-hydroxysteroid oxidase gene is expressed in cotton tissue.

The table below summarizes the activity of 3-hydroxysteroid oxidase on boll weevil neonate larvae when presented in various diets. If the activity depended upon depletion of sterols from the diet then one would expect that the amount of enzyme required for boll weevil mortality would depend greatly upon the diet that is utilized in the assay since the amount and accessibility of the constituent sterols would vary widely in these assay mediums. The data shown in Table 10 demonstrate that LC.sub.50 values obtained are very consistent from one type of assay to the next. This further demonstrates that the mode of action of 3-hydroxysteroid oxidase is not dependent on sterol depletion of the nutrient source but that the enzyme is directly active upon the boll weevil. These results demonstrate that successful control of boll weevil should be attained when the 3-hydroxysteroid oxidase gene is expressed in cotton tissue.

Three other coleopteran species, three other insects, and one mite species were evaluated for susceptibility to Sigma Streptomyces 3-hydroxysteroid oxidase (Table 11). Bioassays were evaluated after 4 to 7 days to measure acute effects of the enzyme on the insects' growth and survival. No significant acute effects (mortality or stunting of larval growth) were observed in these short term assays. The long term effects of exposure to cholesterol oxidase were not determined for these insects. The bioactivity against tobacco budworm demonstrates that longer exposure to the enzyme resulted in greater mortality (Table 2a, above). Thus deleterious effects on larval growth and development of the insects listed in Table 11 and other insects may result from chronic ingestion of 3-hydroxysteroid oxidase. ("lv"- larvae)

While not being bound by this theory, it is believed that the 3-hydroxysteroid oxidase enzyme kills or stunts boll weevil larvae and stunts the growth of lepidopteran larvae by some action in the gut after ingestion. There are no lethal or stunting effects from feeding boll weevil or lepidopteran larvae a diet sample that was incubated for one week with a 3-hydroxysteroid oxidase and then boiled prior to using it in the above assay. This further demonstrates that the mode of action of 3-hydroxysteroid oxidase is not dependent on sterol depletion of the nutrient source but that the enzyme is directly active upon the insect.

Nor do cholestenone or hydrogen peroxide, the products of enzymatic action on cholesterol, exhibit any lethal effects against boll weevil when incorporated at up to 200 .mu.M in the standard diet described above. The addition of catalase (E.C. #1.11.1.6) to 3-hydroxysteroid oxidase in the bioassay does not block the lethal effects of 3-hydroxysteroid oxidase on boll weevil, providing further evidence that in vitro generation of hydrogen peroxide is not the mode of action.

The enzymatic action in the gut is believed to be oxidation of the 3-hydroxysteroid(s) of the cell membranes in the lining of the gut. The effects of ingested 3-hydroxysteroid oxidase on the midguts of boll weevil larvae have been studied. The midguts of boll weevil larvae (neonate and second instar at initiation of assay) feeding on diets containing sublethal doses of 3-hydroxysteroid oxidase were dissected out. Representative midguts were immediately placed in fixative and analyzed microscopically for morphological changes. Disruption of the epithelial cell layer was observed in the guts of larvae ingesting low doses, and complete lysis of the cells was observed from the higher doses. There was a good correlation of the morphological changes with the observed mortality over the 3-hydroxysteroid oxidase concentration range in the diet. Parallel midguts were dissected and homogenized and found to contain active 3-hydroxysteroid oxidase in enzymatic assays. This study demonstrates that the mode of action of 3-hydroxysteroid oxidase on insect larvae involves lysis of the epithelial cell layer, possibly by oxidation of its membrane cholesterol or other 3-hydroxysteroid.

The active proteins from the Madagascar Streptomyces microorganisms were isolated, purified, partially sequenced, and identified as 3-hydroxysteroid oxidases.

Each culture filtrate was purified by first sizing on YM10 membranes (Amicon) to a [>10 kDa] fraction, followed by multiple chromatography runs on an FPLC Mono Q HR10/10 (Pharmacia LKB, Piscataway, N.J.) column. For chromatography on the Mono Q column, the samples were loaded on the column in 25 mM Hepes pH 7.5 and eluted with a gradient to 1.0M KCl in 25 mM Hepes pH 7.5. Fractions were collected and aliquots of each were filtered through 0.2.mu. Acrodisc syringe tip filters. Each was tested in the boll weevil assay described above. Aliquots of insecticidally active fractions were electrophoresed on SDS-PAGE [Laemmli, 1970] using a Daiichi Double Gel Device and 10-20% mini-gel. Proteins were visualized by silver staining using Daiichi silver stain reagent kit. The active enzymes of the present invention, isolated from the novel microorganisms, were found to be a 52.5 kDa protein.

An SDS-PAGE gel of the protein produced by Streptomyces A19241, isolated as above, was blotted onto PVDF paper (Immobilon, Millipore Corp.) using the protocol of Matsudaira [Matsudaira, 1987]. The N-terminus was sequenced using automated Edman degradation chemistry. A gas phase sequencer (Applied Biosystems, Inc.) was used for the degradation using the standard sequencer cycle. The respective PTH-aa derivatives were identified by reverse phase HPLC analysis in an on-line fashion employing a PTH analyzer (Applied Biosystems, Inc.) fitted with a Brownlee 2.1 mm i.d. PTH-C18 column. For internal sequences, digestions were carried out on purified 3-hydroxysteroid oxidase from A19249 using trypsin (TPCK-treated, from Worthington Biochemicals Corp., Freehold, N.J.). Fragments were then purified by reverse phase HPLC and sequenced in an N-terminal fashion.

The resulting partial sequences were compared to known proteins and a strong (71%) homology was found with the reported fourteen amino acid sequence at the N-terminus of a 3-hydroxysteroid oxidase isolated from a Streptomyces species [Ishizaki et al., 1989]. The reported enzyme has an M.sub.r of 54.9 kDa which agrees well with the M.sub.r of 52.5 kDa of the isolated enzyme.

Six internal fragments of the purified enzyme from A19249, also having homology to six regions of the reported enzyme, were sequenced. The fragments had 95, 76, 64, 58, 89, and 100 percent sequence identities.

The amino acid composition of the 3-hydroxysteroid oxidase produced by A19249 was determined and compared with the composition of the reported Streptomyces enzyme. The samples were subjected to acid hydrolysis (6N HCl, vapor phase hydrolysis using a Water's Picotag workstation, 24 hr, 110.degree. C.). All analyses were performed after post-column derivation of the hydrolysates using ninhydrin [Moore et al., 1963]. A Beckman Model 6300 Auto analyzer was employed for the actual determinations. The S delta n/N statistic is used to compare two compositions in order to make a prediction about their relatedness. The formula for the statistic is:

where A is one con,position, B is the other composition, i is each amino acid, n is the number of each amino acid, N is the toted number of amino acids in the protein. If S delta n/N is <0.42, then there is a greater than 95% chance that the proteins are related. The smaller the value, the more closely the determined compositions match.

The S delta n/N statistic for the A19249 protein compared to the reported enzyme is 0.36, indicating that the two ace highly related.

The identity of the enzyme was confirmed by testing its ability to oxidize a 3-hydroxysteroid, specifically cholesterol. The enzyme is added to a reagent mixture comprising horseradish peroxidase (20 U/mL), phenol (14 mM), 4-amino antipyrine (0.82 mM), Triton.RTM. X-100 (0.05%) and phosphate buffer (100 mM, pH 7). The sterol in isopropanol is then added and the absorbance at 500 nm monitored. One unit of activity is defined as the amount of enzyme required to oxidize 1 .mu.mole of sterol per minute at 20.degree. C.

The activity levels of the enzymes are reported in Table 12 for 3-hydroxysteroids representative of various classes of 3-hydroxysteroids. The enzyme sources are as follows:

1.mu.A19249

2=A19241

3=Sigma Streptomyces

4=Sigma Pseudomonas

The Sigma Streptomyces enzyme is immunologically related to the 3-hydroxysteroid oxidases produced by the isolates of the present invention, numbers A19241 and 19249, as demonstrated by Western blotting [Burnette et al., 1981] using polyclonal antisera generated against the Sigma Streptomyces enzyme. The antisera recognized both enzymes produced by the isolates. The 3-hydroxysteroid oxidase from P. fluorescens was not recognized by the antisera. This demonstrates that immunologically distinct 3-hydroxysteroid oxidases are lethal to boll weevils.

The 3-hydroxysteroid oxidase gene was isolated from one of the Streptomyces microorganisms isolated in Madagascar and its sequence determined.

As discussed above, peptide sequences of purified 3-hydroxysteroid oxidase from A19249 were obtained for four regions of the protein. These peptide sequences were compared to a database of known protein sequences, and this comparison revealed that the A19249 protein showed a high degree of homology to a known 3-hydroxysteroid oxidase from Streptomyces [Ishizaki]. Comparing the A19249 peptide sequences to this known protein sequence, these peptides were assigned to their likely positions in the A19249 protein sequence. The sequence derived from the intact 3-hydroxysteroid oxidase from A19249 corresponded to a region near the N-terminus of the secreted form of the enzyme from the published sequence. From this it was concluded that the A19249 N-terminal peptide sequence was also, likely to correspond to a "mature" secreted form of the protein lacking its putative secretory signal sequence. This was later confirmed by the DNA sequence analysis of the A19249 gene (see below). Three peptides were used to construct hybridization probes for isolation of the A19249 3-hydroxysteroid oxidase gene. Peptide N2 (SEQ ID NO:1 ) corres-ponded to N-terminal amino acids 29-43 of the known mature protein sequence (sequence without the putative signal peptide); peptide C1 (SEQ ID NO:2) to amino acids 434-449 of the mature protein sequence; and peptide C2 (SEQ ID NO:3) to amino acids 464-475 of the mature protein sequence.

Based on these peptide sequences, three long nondegenerate oligonucleotides, corresponding to 3-hydroxysteroid oxidase peptide sequences from A19249 were designed using Streptomyces preferred codons. The oligonucleotides N2 (SEQ ID NO:4), C1 (SEQ ID NO:5), and C2 (SEQ ID NO:6) correspond to the peptides N2, C1, and C2 described above.

Probes N2 (SEQ ID NO:4), C1 (SEQ ID NO:5), and C2 (SEQ ID NO:6) were all used as hybridization probes on Southern blots of A19249 genomic DNA. All three probes hybridized to the same 2.2 kb band in BamHI digested DNA, but N2 (SEQ ID NO:4) hybridized to a different fragment than C1 (SEQ ID NO:5) and C2 (SEQ ID NO:6) did in SalI and BglII digests. This indicated that SalI and BglII cut within the coding sequence of the 3-hydroxysteroid oxidase gene from A 19249, which was confirmed by DNA sequence analysis.

The 3-hydroxysteroid oxidase gene from A19249 was isolated using the three synthetic oligonucleotides as hybridization probes on a library of DNA fragments of A19249 DNA in a lambda phage vector. A library was made in lambda EMBL3 using partial-digest Mbo1 DNA fragments of A19249. These probes were used to screen approximately 72,000 lambda phage plaques from the primary library. Primary plaque screening was performed using N2 (SEQ ID NO:4) plus C2 (SEQ ID NO:6). A total of 12 recombinant plaques that hybridized to the N and C-terminal probes were picked and purified by a second round of hybridization screeching with probes N2 (SEQ ID NO:4) and C2 (SEQ ID NO:6). Southern blot analysis revealed that, in five of six lambda clones analyzed, a 2.2 kb BamHI fragment hybridized to both the N and C-terminal probes. This result confirmed the earlier Southern hybridization analysis that indicated a 2.2 kb BamHI fragment contained the 3-hydroxysteroid oxidase gene. This 2.2 kb DNA fragment was cloned into plasmid vector pUC18 [Yanisch-Perron et al., 1985] in both orientations for further analysis. Restriction mapping showed that there were internal SalI and BglII sites as predicted by the Southern hybridization analysis. These sites are also conserved compared to the published 3-hydroxysteroid oxidase gene sequence. The BamHI fragment was further subcloned into four fragments for direct DNA sequencing.

A total of 1865 nucleotides of DNA sequence from the 2.2 kb BamHI fragment were determined by direct DNA sequence analysis of subclones of this fragment using the dideoxy chain termination method. This sequence is identified as SEQ ID NO:7. This DNA sequence contains noncoding flanking regions at both the 3' and 5' ends. Analysis of this DNA sequence revealed a single long open reading frame that encodes a secretory signal peptide and the mature 3-hydroxysteroid oxidase protein of 43 and 504 amino acids, respectively. It is 84.37% identical to the published 3-hydroxysteroid oxidase nucleotide sequence. The derived amino acid sequence is 81.685% identical to the published 3-hydroxysteroid oxidase sequence. It is identified as SEQ ID NO:8. Examination of the A19249 DNA sequence and comparison to the N-terminal amino acid sequence of intact 3-hydroxysteroid oxidase from A19249 revealed that the A19249 gene encoded a protein that includes a signal peptide sequence, which is apparently cleaved during secretion of the protein from the cells. Thus the N-terminus of the mature protein from A19249 begins with Ser-Gly-Gly-Thr-Phe, identified as SEQ ID NO:12.

A 3-hydroxysteroid oxidase gene can be isolated from novel organisms or may be obtained from known sources, such as the Rhodococcus sp. described by Long et al., in WO 90 05,788. This gene may then be used to transform bacterial cells or plant cells to enable the production of 3-hydroxysteroid oxidase and carry out methods of this invention. Examples of how this may be done with the gene of A19249 are given below.

In order to incorporate the A19249 gene into vectors appropriate for expression of the 3-hydroxysteroid oxidase in heterologous bacterial or plant hosts, it was necessary to introduce appropriate restriction sites near the ends of the gene. The goals of this mutagenesis were to create cassettes that included the protein coding sequence with minimal noncoding flanking sequences and to incorporate useful restriction sites to mobilize these cassettes. Cassettes were designed that would allow mobilization of the intact coding sequence including the signal peptide or just the mature coding sequence. To incorporate these cassettes into appropriate bacterial or plant expression vectors, an NcoI restriction site was engineered at the N-terminus of the intact protein sequence or at the N-terminus of the mature protein sequence. A BamHI site was engineered just after the termination codon of the intact coding sequence. Three mutagenesis primers were designed to create these cassettes, as shown below. Mutagenesis with primer Chossn (SEQ ID NO:9) substituted three amino acids (MAT) for valine and asparagine at the N-terminus of the signal peptide of the intact protein and Chomnr (SEQ ID NO:10) added two amino acids (MA) at the N-terminus of the mature protein. This was necessary to allow incorporation of the NcoI restriction site. Mutagenesis with primer Cho3br (SEQ ID NO:11) incorporated a BamHI site at the 3' end of the coding sequence. Primers Chomnr and Cho3br were used to direct formation of the antisense strand of DNA.

The resulting modified sequences were identified as SEQ ID NO:13 encoding for the intact protein and SEQ ID NO:14 for the mature protein.

The NcoI-BamHI fragments containing either the intact protein coding sequence or the mature protein coding sequence were inserted into a vector designed for protein expression in E. coli, vector pKK233-2 (Pharmacia LKB, Piscataway, N.J.). pKK233-2 contains the IPTG-inducible trc promoter. The vector containing the intact (full length) protein coding sequence as modified (SEQ ID NO:13) is designated pMON20909. The vector containing the mature protein coding sequence as modified (SEQ ID NO:14) is designated pMON20907. E. coli XL1 Blue cells (Statagene, San Diego, Calif.) modified with pMON20909 expressed 3-hydroxysteroid oxidase at higher levels of enzymatic activity than cells modified with pMON20907. The protein was extracted and purified from 4 liters of IPTG-induced E. coli containing pMON 20909. The soluble fraction from sonicated bacterial lysate was concentrated and dialyzed, and then partially purified by Mono Q chromatography to yield 11 units of 3-hydroxysteroid oxidase activity. Western blot analysis indicates that the signal sequence of the intact protein is cleaved in E. coli, but the exact site of cleavage was not determined. Analysis of the recovered protein showed a five-fold reduction in enzymatic activity relative to the A19249 protein, but the loss has not been explained by DNA sequencing which found no alterations that would explain loss of enzymatic activity in plant protoplasts or E. coli.

The recovered protein was used in artificial diet overlay assays to determine the effects on boll weevil viability. The dose response curve for activity against boll weevil, based upon enzymatic activity units, was very similar to that originally observed with the Streptomyces and A19249 enzymes. The results are shown in Table 13. In addition, the recovered protein was tested against tobacco budworm and resulted in 88% stunting at a dose of 100 .mu.g/ml.

To control boll weevil, it may be desirable to express 3-hydroxysteroid oxidase in plant colonizing bacteria, and then apply this bacteria to the plant. As the boll weevil feeds on the plant, it ingests a toxic dose of 3-hydroxysteroid oxidase produced by the plant colonizers. Plant colonizers can be either those that inhabit the plant surface, such as Pseudomonas or Agrobacterium species, or endophytes that inhabit the plant vasculature such as Clavibactcr species. For surface colonizers, the 3-hydroxysteroid oxidase gene may be inserted into a broad host range vector capable of replicating in these Gram-negative hosts. Examples of these such vectors are pKT231 of the IncQ incompatibility group [Bagdasarian et al., 1981] or pVK100 of the IncP group [Knauf, 1982]. For endophytes the 3-hydroxysteroid oxidase gene can be inserted into the chromosome by homologous recombination or by incorporation of the gene onto an appropriate transposon capable of chromosomal insertion in these endophytic bacteria.

The expression of a plant gene which exists in double-stranded DNA form involves transcription of messenger RNA (mRNA) from one strand of the DNA by RNA polymerase enzyme, and the subsequent processing of the mRNA primary transcript inside the nucleus. This processing involves a 3' non-translated region which adds polyadenylate nucleotides to the 3' end of the RNA. Transcription of DNA into mRNA is regulated by a region of DNA usually referred to as the "promoter." The promoter region contains a sequence of bases that signals RNA polymerase to associate with the DNA and to initiate the transcription of mRNA using one of the DNA strands as a template to make a corresponding strand of RNA.

A number of promoters which are active in plant cells have been described in the literature. Such promoters may be obtained from plants or plant viruses and include, but are not limited to, the nopaline synthase (NOS) and octopine synthase (OCS) promoters (which are carried on tumor-inducing plasmids of Agrobacterium tumefaciens), the cauliflower mosaic virus (CaMV) 19S and 35S promoters, the light-inducible promoter from the small subunit of ribulose 1,5-bis-phosphate carboxylase (ssRUBISCO, a very abundant plant polypeptide), and the Figwort Mosaic Virus (FMV) 35S promoter. All of these promoters have been used to create various types of DNA constructs which have been expressed in plants (see e.g., PCT publication WO 84/02913).

The particular promoter selected should be capable of causing sufficient expression of the enzyme coding sequence to result in the production of an effective amount of 3-hydroxysteroid oxidase. A preferred promoter is a constitutive promoter such as FMV35S. It has been observed to provide more uniform expression of heterologous genes in the flowering portions of plants. Use of suck, a promoter with the 3-hydroxysteroid oxidase gene may provide greater protection of cotton bolls and squares from boll weevil damage, than other promoters.

The promoters used in the DNA constructs (i.e. chimeric plant genes) of the present invention may be modified, if desired, to affect their control characteristics. For example, the CaMV35S promoter may be ligated to the portion of the ssRUBISCO gene that represses the expression of ssRUBISCO in the absence of light, to create a promoter which is active in leaves but not in roots. The resulting chimeric promoter may be used as described herein. For purposes of this description, the phrase "CaMV35S" promoter thus includes variations of CaMV35S promoter, e.g., promoters derived by means of ligation with operator regions, random or controlled mutagenesis, etc. Furthermore, the promoters may be altered to contain multiple "enhancer sequences" to assist in elevating gene expression. Examples of such enhancer sequences have been reported by Kay et al. (1987).

The RNA produced by a DNA construct of the present invention also contains a 5' non-translated leader sequence. This sequence can be derived from the promoter selected to express the gene, and can be specifically modified so as to increase translation of the mRNA. The 5' non-translated regions can also be obtained from viral RNA's, from suitable eukaryotic genes, or from a synthetic gene sequence. The present invention is not limited to constructs wherein the non-translated region is derived from the 5' non-translated sequence that accompanies the promoter sequence. As shown below, a plant gene leader sequence which is useful in the present invention is the petunia heat shock protein 70 (Hsp7C)) leader. [Winter et al.]

As noted above, the 3' non-translated region of the chimeric plant genes of the present invention contains a polyadenylation signal which functions in plants to cause the addition of adenylate nucleotides to the 3' end of the RNA. Examples of preferred 3' regions are (1) the 3' transcribed, non-translated regions containing the polyadenylate signal of Agrobacterium tumor-inducing (Ti) plasmid genes, such as the nopaline synthase (NOS) gene and (2) plant genes like the soybean 7s storage protein genes and the pea ssRUBISCO E9 gene. [Fischhoff et al.]

A chimeric plant gene containing a structural coding sequence of the present invention can be inserted into the genome of a cotton plant by any suitable method. Suitable plant transformation vectors include those derived from a Ti plasmid of Agrobacterium tumefaciens, as well as those disclosed, e.g., by Herrera-Estrella (1983), Bevan (1983), Klee (1985) and EPO publication 0 120 516 (Schilperoort et al.). In addition to plant transformation vectors derived from the Ti or root-inducing (Ri) plasmids of Agrobacterium, alternative methods can be used to insert the DNA constructs of this invention into plant cells. Such methods may involve, for example, the use of liposomes, electroporation, chemicals that increase free DNA uptake, free DNA delivery via microprojectile bombardment, and transformation using viruses or pollen.

A particularly useful Ti plasmid cassette vector for transformation of dicotyledonous plants is pMON11782. The expression cassette pMON11782 consists of the FMV35S promoter, the petunia Hsp70 5' untranslated leader, and the 3' end including polyadenylation signals from the pea ssRUBISCO E9 gene. Between the leader and the 3' polyadenylation signals is a multilinker containing multiple restriction sites, including a BamHI site for the insertion of genes. pMON 11782 also contains a HindIII site before the promoter sequence.

The remainder of pMON11782 contains a segment of pBR322 (New England Biolabs, Beverly, Mass.) which provides an origin of replication in E. coli; the oriV region from the broad host range plasmid RK1 which allows replication in Agrobacterium strain ABI; the streptomycin/spectinomycin resistance gene from Tn7; and a chimeric NPTII gene, containing the CaMV35S promoter and the nopaline synthase (NOS) 3' end, which provides kanamycin resistance in transformed plant cells.

Both 3-hydroxysteroid oxidase gene cassettes, that is the gene encoding intact protein with the signal sequence and that encoding only the mature protein, each modified at the N-terminus as described above, were mobilized as NcoI-BamHI fragments and inserted into a transient expression vector that had been cut with NcoI and BamHI. A transient expression vector is a simple plasmid containing a plant promoter with a 5' nontranslated leader, a 3' nontranslated polyadenylation sequence, and between them a multi linker having multiple restriction, sites for insertion of a protein coding sequence. The constructed vectors placed the 3-hydroxysteroid exidase gene under the control of the FMV35S promoter with the petunia HSP70 leader sequence discussed above. At the 3' end terminator region is the non-translated polyadenylation signal terminator region of the nopaline synthase gene. A plasmid containing the intact protein coding sequence (SEQ ID NO:13) was identified and named pMON 20910. A plasmid containing the modified mature protein coding sequence (SEQ ID NO:14) was identified and named pMON20908.

pMON20910 and pMON20908 are vectors for expression of 3-hydroxysteroid oxidase genes in plant cells, but these vectors lack appropriate sequences for use in Agrobacterium-mediated plant transformation. However, these vectors can be used for either transient expression of 3-hydroxysteroid oxidase in plant cells, or they can be used to generate stably transformed cotton plants via free DNA delivery such as biolistic bombardment of cotton meristems.

For transient expression analysis, plasmid DNA samples from pMON20908 and pMON20910 vectors were purified and introduced into tobacco via electroporation. Freeze-thaw extraction followed by a nine-fold concentration of soluble fractions on Centricon-10 filter concentrators allowed unambiguous detection of 3-hydroxysteroid oxidase activity in all cell lysates, immunologically by Western blot assay and enzymatically. The activity of the lysate from cells containing pMON20908, that is the coding sequence for the modified mature protein, was approximately ten-fold lower than that recovered from cells containing pMON20910. Western blot analysis indicated that the signal sequence is cleaved in protoplasts, although not necessarily with the fidelity necessary to generate a processed protein identical in form and activity to that naturally secreted by Streptomyces A19249.

pMON20910 containing the intact coding sequence was used to construct a vector for stable transformation of cotton plants with Agrobacterium. It was cut with restriction enzymes HindlII and BamHI. Such digestion creates HindIII-BamHI DNA fragments that contain the FMV35S promoter, the petunia Hsp70 leader, and the intact (full length) 3-hydroxysteroid oxidase coding sequence. These HindIII-BamHI fragments are inserted into plasmid pMON11782, discussed above, which has been previously digested with HindIII-BamHI. pMON20912 was identified as containing the oxidase coding sequence. pMON20912 is thus composed of the FMV35S promoter, the petunia Hsp70 leader, the intact, 3-hydroxysteroid oxidase coding sequence, and the 3' polyadenylation signal from the pea ssRUBISCO E9 gene.

This vector was introduced into disarmed Agrobacterium host ABI and used to transform cotton explants in tissue culture. Selection for kanamycin resistance led to several lines of cotton callus, which have been found to produce 3-hydroxysteroid oxidase as demonstrated by enzymatic activity and Western blot assay. After plant regeneration, whole cotton plants containing the 3-hydroxysteroid oxidase coding sequences will be recovered.

Vectors containing the intact or mature 3-hydroxysteroid oxidase cassette express the active enzyme in the cytoplasm of the plant cell. There has been no evidence of secretion outside the transformed cells. Some bacterial secretory signal sequences have been shown to function in plant cells. It may be desirable to direct most or all of the 3-hydroxysteroid oxidase protein into the plant secretory pathway. To achieve this, it may be advantageous to use a signal sequence derived from a plant gene rather than a bacterial signal. An example of such a signal is that from the tobacco PR1b gene, described by Cornelissen et al. pMON10824, disclosed in EP Publ. 0 385 962, is a plant transformation vector designed for the expression of the lepidopteran active B.t. kurstaki protein. In pMON10824, the B.t.k. coding sequence is fused to the PR1b signal sequence plus 10 amino acids of the mature PR1b coding sequence. This B.t.k. fusion gene is driven by the CaMV35S promoter containing a duplicated enhancer. To create a vector in which the PR1 b signal is fused to the 3-hydroxysteroid oxidase gene, pMON10824 is first cut with BglII, and the large vector fragment is isolated. In a separate reaction, pMON10824 is cut with BglII and NcoI and the small BglII-NcoI fragment that contains the PR1b signal is isolated. In a ligation reaction, the BglII-cut pMON10824 is mixed with the small BglII-NcoI pMON10824 fragment plus the 3-hydroxysteroid oxidase NcoIoBamHI cassette. This reaction constructs a plasmid in which the 3-hydroxysteroid oxidase coding sequence (either mature protein or intact protein cassette) is fused to the secretory signal from the PR1b gene and driven by the CaMV35S promoter. A similar plasmid may be constructed wherein the 3-hydroxysteroid oxidase gene is under the control of the FMV35S promoter. Such plasmids are mobilized into a disarmed Agrobact-erium host and used to transform cotton plants. Thus, these plants produce a 3-hydroxysteroid oxidase that is secreted into the extracellular space.

In some cases, proteins that enter the plant secretory pathway are targeted to different cellular compartments such as the vacuole. It may be desirable to direct the 3-hydroxysteroid oxidase to the vacuole of plant cells. In this case, the vectors described above in which the PR1b signal is used are further modified to include vacuolar targeting sequences derived from known plant vacuolar enzyme genes.

It may also be advantageous to direct the localization of the 3-hydroxysteroid oxidase protein to another cellular compartment, the chloroplast. Proteins can be directed to the chloroplast by including at their N-termini a chloroplast transit peptide (CTP). One CTP that has worked to localize heterologous proteins to the chloroplast is that derived from the RUBISCO small subunit gene of Arabidopsis, denoted ats1A. A variant of this transit peptide that encodes the transit peptide, 23 amino acids of mature RUBISCO sequence, plus a reiteration of the transit peptide cleavage site has been constructed for the successful chloroplast localization of the B.t.k. protein. Vectors containing the rabidopsisd ats1A transit peptide fused to the B.t.k. gene may be used as the base for constructing vectors for the chloroplast localization of the 3-hydroxysteroid oxidase protein. For example, pMON10821, constructed of an enhanced CaMV35S promoter, the ats1A chloroplast transit peptide and a truncated B.t.k. enzyme gene, disclosed in EP Publ. 0 385 962, is cut with BglII and the large BglII vector fragment is isolated. In a separate reaction, pMON 10821 is cut with BglII and NcoI and the small BglII-NcoI fragment that contains the PR1b signal is isolated. In a ligation reaction, the BglII-cut pMON 10821 is mixed with the small BglII-NcoI pMON10821 fragment plus the 3-hydroxysteroid oxidase NcoI-BamHI cassette. This reaction constructs a plasmid in which the 3-hydroxy-steroid oxidase coding sequence (either mature protein or intact protein cassette) is fused to the Arabidopsis transit peptide with 23 amino acids of mature RUBISCO, and driven by the CaMV35S promoter. Alternatively, a similar plasmid may be constructed to replace the promoter with the FMV35S promoter. Such plasmids are mobilized into disarmed Agrobacterium hosts and used to transform cotton plants. Thus, these plants produce a 3-hydroxysteroid oxidase that is localized to the chloroplast.

A 3-hydroxysteroid oxidase gene may be stably incorporated into the genome of monocots using the vectors and methods described in co-pending U.S. patent application Ser. No. 07/855,857, filed Mar. 19, 1992 (Brown et al.), which is hereby incorporated by reference. The gene can be inserted in an appropriate vector, for example pMON19653 and 19643, described by Brown et al. The resulting construct contains a cassette of the CaMV E35S promoter, the Hsp70 intron, the CP4 glyphosate selection marker, and the NOS terminator; a cassette of the CaMV E35S promoter, the Hsp70 intron, the GOX glyphosate selection marker, and the NOS terminator; and a single NotI site for insertion of a gene expression cassette containing a 3-hydroxysteroid oxidase gene, such as SEQ ID NO:13 or SEQ ID NO:14.

This vector is inserted by bombardment of embryogenic tissue culture cells using a biolistic particle gun as described by Brown et al. Transformed cells are selected for glyphosate resistance and whole plants are regenerated. Insect-resistant plants may be confirmed to be expressing the gene by Western blot analysis, esterase activity assay, or insect resistance assay.

Targeting of the protein to certain cellular compartments is also possible in monocots using the signal sequences described above.

All publications and patents mentioned in this specification are herein incorporated by reference as if each individual publication or patent was specifically and individually stated to be incorporated by reference.

From the foregoing, it will be seen that this invention is one well adapted to attain all the ends and objects hereinabove set forth together with advantages which are obvious and which are inherent to the invention.

It will be understood that certain features and subcombinations are of utility and may be employed without reference to other features and subcombinations. This is contemplated by and is within the scope of the claims.

Since many possible embodiments may be made of the invention without departing from the scope thereof, it is to be understood that all matter herein set forth or shown in the accompanying drawings is to be interpreted as illustrative and not in a limiting sense.

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