WO1998040504A1 - Methods for altering benzoxazinone levels in plants - Google Patents

Abstract

Transformed plants, plant cells, and seeds having altered hydroxamic acid levels are provided. The plants, plant cells, and seeds are transformed with at least one hydroxamic acid biosynthesis gene. Methods find use in altering the levels of particular benzoxazinones in plants as a plant defense mechanism for insect and disease resistance and for increasing herbicide tolerance in the plant. At the same time, the transformed plants can provide a source for tryptophan.

Description

METHODS FOR ALTERING BENZOXAZINONE LEVELS IN PLANTS

FIELD OF THE INVENTION

The invention relates to the genetic manipulation of plants, particularly to increasing insect and disease resistance in plants.

BACKGROUND OF THE INVENTION Hydroxamic acids (Hx) are known to function in the chemical defense of many cereal crops against a diverse group of pests including insects, fungi and bacteria. Efforts to exploit hydroxamic acids as natural pesticides have met with limited success despite their widespread occurrence in the gramineae. Besides being associated with resistance to pests, hydroxamic acids have been associated with triggering the reproduction of grass-feeding mammals and with allelopathic effects of cereals. The presence of hydroxamic acids has also been related to the detoxification of herbicides and pesticides, and to the mineral nutrition of the plant.

Hydroxamic acid concentrations are high in certain maize seedlings and provide substantial resistance against the first brood European corn borer, Ostrinia nubilalis, but may not be effective against the second brood European cor borer, which feeds after hydroxamic acid levels have dropped to lower levels. Attempts at selecting maize lines which maintain high hydroxamic acid concentrations throughout development have been unsuccessful.

A series of 1 ,4-benzoxazin-3-ones were discovered in rye plants in relation to resistance of the plants to fungal infection. The compounds were later found in maize and wheat. Relationships were established between levels of the compounds in the plants and the degree of resistance of the plants to insects, fungi and bacteria. Since this first discovery, the range of known activities of these compounds has substantially widened.

1 ,4-benzoxazin-3-ones are naturally present in the plants as 2-/3- O-D-glucosides, which may be isolated if care is taken to inactivate the enzymes in the tissues before extraction. A review of hydroxamic acids including structure and biological activity can be found in Niemeyer, H.N. (1988) Phytochemistry 27:3349-3358.

Biochemical studies have demonstrated that synthesis of hydroxamic acids, particularly benzoxazinones synthesis, has some intermediates in common with the tryptophan biosynthetic pathway . Labelled tryptophan precursors such as anthranilic acid, ribose and indole have been shown to be incorporated into DIMBOA (2,4-dihydroxy-7-methoxy-l,4- benzoxazin-3-one) as representative of the graminae-specific secondary metabolites termed benzoxazinones. However, labelled tryptophan was not incorporated into DIMBOA indicating indole as the formal branch point of the two pathways.

Plant pests result in losses to farmers which run into multi- millions of dollars per year. The rising cost of pesticides, the increasing resistance of insects to pesticides, and their undesirable effects on the environment stresses the need to exploit host plant resistance to pests and diseases. Thus, a mechanism is needed to protect plants against pest attack.

SUMMARY OF THE INVENTION Transformed plants, plant cells, and seeds having altered hydroxamic acid levels are provided. Additionally, plants, plant cells, and seeds of the invention may have altered levels of tryptophan. The plants, plant cells, and seeds are transformed with at least one hydroxamic acid biosynthesis gene. The compositions and methods of the invention find use in altering the levels of particular benzoxazinones in plants as a plant defense mechanism for insect and disease resistance and for increasing herbicide tolerance in the plant. At the same time, the transformed plants can provide a source for tryptophan.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1 sets forth the structure and chromosomal location of the Bx genes. (A) Schematic representation of the Bx gene cluster on chromosome 4. Genetic distance is indicated in centi Morgan. (B) Exon/intron structure of Bx2 to Bx5. Exons are represented by boxes. Translation start and stop codons and poly(A) addition sites are shown. The insertion of a Mu element in the bx3: :Mu allele is designated by an arrow. The complete sequences of the genes have been deposited in the EMBL data bank (Accession numbers Bx2: Y11368, Bx3: Y11404; Bx4: X81828; Bx5: Y11403). (C) The insertion sites of Mu in bx3: :Mu are shown. The characteristic 9-bp host sequence duplication associated with Mu insertion is underlined with an arrow. In bx3: :Mu the insertion occurred at position 827, M. Frey et al. (1995) Mol. Gen. Genet. 246: 100 (the new gene designation is: Bx2 = CYP71C4, Bx3 = CYP71C2, Bx4 = CYP71C1, Bx5 = CYP71C3), of the published DNA sequences.

Fig. 2 sets forth the detection of metabolites of the DIMBOA pathway by HPLC. Metabolites are indicated at the position of chromatographic peaks. S represents the solvent peak. (A) and (B): Feeding of standard mutant seedling shoots with ImM indole (A) or ImM tryptophan (B). 1 g seedling material was extracted, B.A. Bailey et al. (1991) Plant Physiol. 95:792, and analyzed on a Merck LiChroCART RP-18 HPLC column (4 x 125 mm). Elution was for 5 min under isocratic conditions with solvent A (H₂O/HOAc, 9: 1) followed by a linear gradient from 100% solvent A to 100% B (MeOH/H₂O/HOAc, 70:27:3) over 7 min. (C-F). Analysis of maize P450 enzymes expressed in yeast microsomes. Reaction mixtures of 0.2 ml contained 50mM K-Pl pH 7.5, 0.8 mM NADPH, 0.1 mM to 0.5 mM of the respective substrates, and 1 mg microsomal protein. Incubation was for 30 min at 25 °C. HPLC analysis was as described above. (C) Bx2 microsomes incubated with indole. (D) Bx3 microsomes incubated with indolin-2-one, (E) Bx4 microsomes incubated with 3-hydroxy-indolin-2-one, (F) Bx5 microsomes incubated with HBO A.

Fig. 3 sets forth the pathway for DIMBOA biosynthesis and tryptophan biosynthesis and their relationship.

DETAILED DESCRIPTION OF THE INVENTION In accordance with the subject invention, methods for increasing the plant defense mechanism for insect and disease resistance are provided. The method involves transforming a plant with at least one hydroxamic acid biosynthesis gene. This has the effect of altering hydroxamic acid biosynthesis, particularly increasing the production of downstream products. Additional genes can then be utilized to shunt the metabolic activity to the production of particular benzoxazinones. Of particular interest are the benzoxazinones which occur in hydroxamic acid and lactam forms. Such compounds are a part of the biosynthesis pathway of DIMBOA. See Fig. 3 for the pathway of DIMBOA biosynthesis and its relationship with the tryptophan synthesis pathway.

The hydroxamic acid forms of benzoxazinones possess an N- hydroxyl group and are generally found in high concentrations. Lactam members lack the N-hydroxyl group and occur in lower concentrations. The hydroxamic acid 2,4-dihyroxy-l,4-benzoxazin-3-one (DIBOA) has as its lactam counterpart 2-hydroxy-l,4-benzoxazin-3-one (HBOA). The hydroxamic acids of the invention include the defense chemicals, the benzoxazinones, and more particularly the intermediates in the DIMBOA biosynthesis pathway, as well as DIMBOA. Such intermediate compounds include HMBOA, DIBOA, HBOA, 3-hydroxy-indolin-2-one, indolin-2-one, indole, and the like. Of particular interest in the invention are the genes which encode the DIMBOA biosynthesis enzymes, referred to herein as biosynthesis genes. Such genes can be used to produce transformed plants with increased or altered benzoxazinones.

These biosynthesis genes of the invention include the CYP71C (Bx) genes included in DIMBOA biosynthesis. These genes are described in Frey et al. (1995) Mol. Gen. Genet. 246: 100-109, and are designed as the CYPzm genes, CYPzm 1-4.

The biosynthesis genes of the invention also encompass other genes encoding cytochrome P450 enzymes and P450 reductases. Cytochrome P450 enzymes are membrane-bound, heme-containing enzymes implicated in a variety of biosynthetic reactions. See, for example, Donaldson and Luster (1991) Plant Physiol 96:669-674; West, CA (1980) m Davis DD (ed), The Biochemistry of Plants. Vol. 2, Academic Press, New York, pp. 317-364; and Butt and Lamb (1981) In: Conn E.E. (ed), The Biochemistry of Plants. Vol. 7, Academic Press, New York, pp. 627-665. Gene sequences for several P450 enzymes are available. See, Bozak et al. (1990) Proc. Natl. Acad. Sci USA 87:3904-3908; Vetter et al. (1992) Plant Physiol 100:998-1007; Mitzutani et al. (1993) Biochem. Biophys. Res. Commun. 190:875-880; Teutsch et al. (1993) Proc. Natl. Acad. Sci. USA 90:4102-4106; Fahrendorf and Dixon 1993) Arch Biochem. Biophys. 305:509-515; Holten et al. (1993) Nature 366:276-279; Toguri et al. (1993) Biochem. Biophys. Acta 1216: 165-169; Toguri et al. (1993) Plant Mol. Biol. 23:933-946; Umemoto et al. (1993) FEBS Lett 12957: 169-173; Mangold et al. (1994) Plant Sci. 96: 129-136; Larson and Bussard (1996) Plant Physiol. 80:483-486; Bailey and Larson (1991) Plant Physiol 95:792-796; Okeefe et al. (1994) Plant Physiol 105:473-482; Pierrel et al. (1994) Eur. J. Biochem. 224:835-844; Urban et al. (1994) J. Biochem. 222:843-850; Shiota et al. (1994) Plant Physiol. 106: 17-23; herein incorporated by reference.

Also included as biosynthesis genes are other alkalaid biosynthetic genes. See, Kutchan, T.M. 196) Gene 179:73-81, and the references cited therein. A variety of sources are available for the biosynthesis genes of the invention and, for the most part, a gene from any source can be utilized. In fact, because of the similarity of the P-450 systems in bacteria, yeast, plants, and mammals, genes from any of these sources can be utilized. It is recognized that because of co-suppression, the native plant gene or one having high homology due to the plant gene may not be preferred when increased expression of a compound is intended.

Sequences of the Bx2, Bx3, Bx4, and Bx5 genes have been published. See, Frey et al. (1995) Mol. Gen. Genet. 246: 100-109, herein incorporated by reference. In general, see also Paril et al. (1971) J. Biol. Chem. 246:6953-6955 (mammalian); Lee et al. (1974) Biochem. Biophys. Acta. 344:205-240 (mammalian); Yu et al. (1974) J. Biol. Chem. 249:94-101 (bacterial); West CA. (1980) Hvdroxylases. Monooxygenases and Cytochrome. pp. 450, In D.D. Davies (ed.) The Biochemistry of Plants, Academic Press, New York, pp. 317-364 (plant).

Transformation of a plant with an early DIMBOA biosynthesis gene leads to significant increases in the production of DIMBOA in the transformed plant. Thus, the transformed plant has a heightened defense mechanism for insect and disease resistance.

By increased plant defense mechanism for insect and disease resistance is intended that the plant is protected against attacks from pests. That is, the plants have an enhanced resistance to typical pests as compared to untransformed plants. Enhanced resistance to insects is measured by growth- inhibitory activity, including larval mortality, smaller individuals, slower development, poorer matings, and fewer offspring. Resistance to pathogens is measured by a decrease or absence of disease symptoms, as well as infection ratings. The compositions and methods of the invention also find use in altering allelopathic effects of plants. DIBOA and BOA have been shown to be involved in the allelopathic effects of rye. See, Fuerst and Putman (1983) J. Chem. Ecol. 9:937; and Barnes and Putman (1987) J. Chem. Ecol. 13:889. DIBOA has been shown to be the most active compound against monocots while BOA was most active against dicots. Barnes and Putnam (1987) J. Chem. Ecol. 13:889.

The compositions and methods also find use in the detoxification of herbicides and pesticides. Detoxification of the 2-chloro-s-triazine derived herbicides occur by hydroxylation, dealkylation, or glutathione conjugation. Altering the hydroxamic acid levels in the plant can increase tolerance to these and other herbicides. Plants can be transformed with biosynthesis genes and tested for tolerance to the herbicide of interest. Hurter, J. (1966) Experimentia 22:741; Shimabukuro, R.H. (1967) Plant Physiol. 42: 1269; Shimabukuro et al. (1970) Plant Physiol. 46: 103; Malan et al. (1986) S. Afr. J. Plant Soil 3: 115; Malan et al. (1984) S. Afr. J. Plant Soil 1 : 103; Hamilton and Moreland (1962) Science 135:373; Nakano et al. (1973) J. Org. Chem. 38:4396; Anderson, R.N. (1964) Weeds 12:60; and Palmer and Grogan (1965) Weeds 13:219. As indicated, for the production of increased DIMBOA, transformation of the plant with at least one biosynthesis gene which encodes an enzyme which catalyzes an early step in the pathway (an early biosynthesis gene) is sufficient. Such enzymes include, but are not limited to, Bx2, Bx3, Bx4, Bx5, the CYPzm genes, etc.

The DIMBOA biosynthesis pathway can also be directed for the production of a particular compound or compounds. In this manner, a plant is transformed with an early DIMBOA biosynthesis gene, such as Bx2 or Bx3. Transformation with such an early gene (referred to also as the primary gene) increases the metabolic activity for the production of downstream compounds. Once the biosynthetic activity has been increased, the pathway can be diverted for the production of specific compounds. The diversion involves the action of at least one second gene of interest (the secondary gene). The secondary gene can encode an enzyme to force the production of a particular compound or alternatively can encode an antisense RNA to stop the pathway for the accumulation of a particular compound. For example, for the production of HBOA, antisense Bx5 is utilized as the secondary gene.

In this manner, the pathway can be modified for high production of particular compounds of interest. Such plants can be tested for increased resistance to particular pathogens.

The DIMBOA biosynthesis genes can also be used in combination with any of the genes from the tryptophan biosynthetic pathway. Because DIMBOA synthesis has some intermediates in common with the tryptophan biosynthesis pathway, early genes in the tryptophan pathway can be utilized to prime the pathway for the production of DIMBOA. For example, the anthranilate synthase trp4 can be utilized to increase the biosynthesis of tryptophan. However, before indole is converted into tryptophan, the indole can be diverted to DIMBOA biosynthesis by transforming the plant with Bx2. Likewise, other genes in the tryptophan pathway can be utilized including phosphoribosylanthranilate synthase (trp 1), phosphoribosylanthranilate isomerase; indole-3-glycerolphosphate synthase; tryptophan synthase (trp 3); and the like. See, Radwomski et al. (1995) Plant Cell 7:921. Additionally, a sequence encoding the antisense for tryptophan synthase 3 can be utilized to divert the pathway from tryptophan production to DIMBOA production. Because tryptophan biosynthesis is associated with the DIMBOA biosynthesis, plant cells which overproduce tryptophan can be utilized in the transformation experiments. Such tryptophan overproducing mutants of cereal crops are known in the art. See, for example, U.S. Patent No. 4,581,847, herein incorporated by reference. Such plants are useful as a source for cells in transformation experiments.

It is also likely that a cytochrome P-450 reductase will improve the activity of the compounds of the invention. Accordingly, in addition to the biosynthesis genes, the plant can be transformed with a gene encoding a P-450 reductase. Such genes have been cloned from yeast and maize and are available in the art. See, for example, U.S. Patent No. 5,114,852; Murakami et al. (1990) J. Biochem 108:859-865, Tokyo: Japanese Biochemical Society. Any means for producing a plant comprising both the primary and secondary genes, or the biosynthesis gene and any of the other genes described herein, are encompassed by the present invention. For example, the secondary gene of interest can be used to transform a plant at the same time as the primary gene (cotransformation). The secondary gene can be introduced into a plant which has already been transformed by the primary gene.

Alternatively, transformed plants, one expressing the primary gene and one expressing the secondary gene can be crossed to bring the genes together in the same plant.

By combining the genes with tissue-specific promoters, the hydroxamic acid levels can be altered in particular tissues of the plant. While any promoter or promoter element capable of driving expression of a coding sequence can be utilized, of particular interest are root promoters (Bevan et al. (1993) in Gene Conservation and Exploitation. Proceedings of The 20th Stadler Genetics Symposium. Gustafson et al. (eds.), Plenum Press, New York pp. 109-129; Brears et al. (1991) Plant J. 1 :235-244; Lorenz et al. (1993) Plant J. 4:545-554; U.S. Patent Nos. 5,459,252; 5,608,149; 5,599,670); ; pith (U.S. Patent Nos. 5,466,785; 5,451 ,514; 5,391,725); or other tissue specific and constitutive promoters (See, for example, U.S. Patent Nos. 5,608, 149; 5,608,144; 5,604,121; 5,569,597; 5,466,785; 5,399,680; 5,268,463; 5,608, 142).

The primary, secondary, or other genes encoding the enzymes of interest can be used in expression cassettes for expression in the transformed plant tissues. To alter the hydroxamic acid levels in a plant of interest, the plant is transformed with at least one expression cassette comprising a transcriptional initiation region linked to the gene of interest. Such an expression cassette is provided with a plurality of restriction sites for insertion of the gene of interest to be under the transcriptional regulation of the regulatory regions.

The transcriptional initiation region, the promoter, may be native or analogous or foreign or heterologous to the host. By foreign is intended that the transcriptional initiation region is not found in the wild-type host into which the transcriptional initiation region is introduced. As used herein a chimeric gene comprises a coding sequence operably linked to transcription initiation region which is heterologous to the coding sequence.

The transcriptional cassette will include the in 5 '-3' direction of transcription, a transcriptional and translational initiation region, a DNA sequence of interest, and a transcriptional and translational termination region functional in plants. The termination region may be native with the transcriptional initiation region, may be native with the DNA sequence of interest, or may be derived from another source. Convenient termination regions are available from the Ti-plasmid of A. tunefaciens, such as the octopine synthase and nopaline synthase termination regions. See also,

Guerineau et al. , (1991) Mol. Gen. Genet. 262: 141-144; Proudfoot (1991) Cell 64:671-674; Sanfacon et al. (1991) Genes Dev. 5: 141-149; Mogen et al. (1990) Plant Cell 2: 1261-1272; Munroe et al. (1990) Gene 91: 151-158; Ballas et al. (1989) Nucleic Acids Res. 17:7891-7903; Joshi et al. (1987) Nucleic Acid Res. 15:9627-9639.

The nucleotide sequences encoding the proteins or polypeptides of the invention are useful in the genetic manipulation of plants. In this manner, the genes of the invention are provided in expression cassettes for expression in the plant of interest. The cassette will include 5' and 3' regulatory sequences operably linked to the gene of interest. The cassette may additionally contain at least one additional gene to be cotransformed into the organism. Alternatively, the gene(s) of interest can be provided on another expression cassette. Where appropriate, the gene(s) may be optimized for increased expression in the transformed plant. Where mammalian, yeast, or bacterial P450 enzymes are used in the invention, they can be synthesized using plant preferred codons for improved expression. Methods are available in the art for synthesizing plant preferred genes. See, for example, U.S. Patent Nos. 5,380,831, 5,436, 391, and Murray et al. (1989) Nucleic Acids Res. 17:477- 498, herein incorporated by reference.

The expression cassettes may additionally contain 5' leader sequences in the expression cassette construct. Such leader sequences can act to enhance translation. Translation leaders are known in the art and include: picornavirus leaders, for example, EMCV leader (Encephalomyocarditis 5' noncoding region) (Elroy-Stein, O. , Fuerst, T.R. , and Moss, B. (1989) PN S USA 56:6126-6130); poty virus leaders, for example, TEV leader (Tobacco Etch Virus) (Allison et al. (1986); MDMV leader (Maize Dwarf Mosaic Virus); Virology 154:9-20), and human immunoglobulin heavy-chain binding protein (BiP), (Macejak, D.G. , and P. Sarnow (1991) Nature 353:90-94; untranslated leader from the coat protein mRNA of alfalfa mosaic virus (AMV RNA 4), (Jobling, S.A. , and Gehrke, L. , (1987) Nature 325:622-625; tobacco mosaic virus leader (TMV), (Gallie, D.R. et al. (1989) Molecular Biology of RNA, pages 237-256; and maize chlorotic mottle virus leader (MCMV) (Lommel, S.A. et al. (1991) Virology 81 :382-385). See also, Della-Cioppa et al. (1987) Plant Physiology 84:965-968. Other methods known to enhance translation can also be utilized, for example, introns, and the like.

It may be beneficial to target the gene products of the invention to a cellular organelle. Leader sequences and methods are available in the art for targeting to organelles, such as chloroplasts (Lancelin et al. (1994) EEβS Letters 343:261-266; Tsutsumi et al. (1994) Gene 141 :215-220; Kubo et al. (1993) Platn and Cell Physiol 34: 1259-1266; Tang et al. (1994) Plant Physiol 104: 1081-1082; U.S. Patent Nos. 5,45,818; 5,545,817; 5,608, 149); and mitochondria (Arai et al. (1996) Biochem. Biophys. Res. Com. 227:433-439; Hsu et al. (1996) Biochemistry 35:9797-9806; Galanis et al. (1991) FEBS Lett. 282:425-430; Tamura et al. (1996) Biochem Biophys Res. Com. 222:659-663; Balzan et al. (1995) Proc. Natl. Acad. Sci. USA 92:4219-4223; U.S. Patent No. 5,530,191).

In preparing the expression cassette, the various DNA fragments may be manipulated, so as to provide for the DNA sequences in the proper orientation and, as appropriate, in the proper reading frame. Towards this end, adapters or linkers may be employed to join the DNA fragments or other manipulations may be involved to provide for convenient restriction sites, removal of superfluous DNA, removal of restriction sites, or the like. For this purpose, in vitro mutagenesis, primer repair, restriction, annealing, resection, ligation, PCR, or the like may be employed, where insertions, deletions or substitutions, e.g. transitions and trans versions, may be involved.

The compositions and methods of the invention can be used to transform any plant. It is recognized that DIMBOA biosynthesis genes are present in cereals. Thus, the DIMBOA biosynthesis of such plants can be modified by transformation of the plant with a single or several genes. For those plants, including dicots, which do not natively synthesize DIMBOA, a pathway for the synthesis of a particular compound or compounds can be constructed. Such pathways provide new approaches to insect and disease resistance in these plants. As discussed, the compositions and methods of the present invention can be used to transform any plant. In this manner, genetically modified plants, plant cells, plant tissue, seed, and the like can be obtained. Transformation protocols may vary depending on the type of plant or plant cell, i.e. monocot or dicot, targeted for transformation. Suitable methods of transforming plant cells include microinjection (Crossway et al. (1986) Biotechniques 4:320-334), electroporation (Riggs et al. (1986) Proc. Natl. Acad. Sci. USA 83:5602-5606, Agrobacterium mediated transformation (Hinchee et al. (1988) Biotechnology 6:915-921), direct gene transfer (Paszkowski et al. (1984) EMBO J. 3:2717-2722), and ballistic particle acceleration (see, for example, Sanford et al. , U.S. Patent 4,945,050; and McCabe et al. (1988) Biotechnology 6:923-926). Also see, Weissinger et al. (1988) Annual Rev. Genet. 22:421-477; Sanford et al. (1987) Particulate Science and Technology 5:27-37 (onion); Christou et al. (1988) Plant Physiol. 87:671-674(soybean); McCabe et al. (1988) Bio/Technology 6:923-926 (soybean); Datta et al. (1990) Biotechnology 8:736-740(rice); Klein et al. (1988) Proc. Natl. Acad. Sci. USA 85:4305-4309(maize); Klein et al. (1988) Biotechnology 6:559-563 (maize); Klein et al. (1988) Plant Physiol.

91 :440-444(maize); Fromm et al. (1990) Biotechnology 8:833-839; and Tomes et al. "Direct DNA transfer into intact plant cells via microprojectile bombardment" In: Gamborg and Phillips (Eds.) Plant Cell, Tissue and Organ Culture: Fundamental Methods, Springer- Verlag, Berlin (1995) (maize); Hooydaas-Van Slogteren & Hooykaas (1984) Nature (London) 311 :763-764;

Bytebier et al. (1987) Proc. Natl. Acad. Sci. USA 84:5345-5349 (Liliaceae); De Wet et al. (1985) In The Experimental Manipulation of Ovule Tissues ed. G.P. Chapman et al. , pp. 197-209. Longman, NY (pollen); Kaeppler et al. (1990) Plant Cell Reports 9:415-418; and Kaeppler et al. (1992) Theor. Appl. Genet. 84:560-566 (whisker-mediated transformation); D'Halluin et al. (1992) Plant Cell 4: 1495-1505 (electroporation); Li et al. (1993) Plant Cell Reports 12:250- 255 and Christou and Ford (1995) Annals of Botany 75:407-413 (rice); Osjoda et al. (1996) Nature Biotechnology 14:745-750 (maize via Agrobacterium tumefaciens); all of which are herein incorporated by reference. The cells which have been transformed may be grown into plants in accordance with conventional ways. See, for example, McCormick et al. (1986) Plant Cell Reports 5:81-84. These plants may then be grown, and either pollinated with the same transformed strain or different strains, and the resulting hybrid having the desired phenotypic characteristic identified. Two or more generations may be grown to ensure that the subject phenotypic characteristic is stably maintained and inherited and then seeds harvested to ensure the desired phenotype or other property has been achieved. As noted earlier, the nucleotide sequences of the invention can be utilized to protect plants from insect and disease pests. For purposes of the present invention, pests include but are not limited to insects, pathogens including fungi, bacteria, nematodes, viruses or viroids, and the like. Insect pests include insects selected from the orders Coleoptera, Diptera,

Hymenoptera, Lepidoptera, Mallophaga, Homoptera, Hemiptera, Orthroptera, Thysanoptera, Dermaptera, Isoptera, Anoplura, Siphonaptera, Trichoptera, etc. , particularly Coleoptera and Lepidoptera. Insect pests of the invention for the major crops include: Maize: Ostrinia nubilalis, European corn borer; Agrotis ipsilon, black cutworm; Helicoverpa zea, corn earworm; Spodoptera frugiperda, fall armyworm; Diatraea grandiosella, southwestern corn borer; Elasmopalpus lignosellus, lesser cornstalk borer; Diatraea saccharalis, surgarcane borer; Diabrotica virgifera, western corn rootworm; Diabrotica longicornis barberi, northern corn rootworm; Diabrotica undecimpunctata howardi, southern corn rootworm; Melanotus spp. , wireworms; Cyclocephala borealis, northern masked chafer (white grub); Cyclocephala immaculata, southern masked chafer (white grub); Popillia japonica, Japanese beetle; Chaetocnema pulicaria, corn flea beetle; Sphenophorus maidis, maize billbug; Rhopalosiphum maidis, corn leaf aphid; Anuraphis maidiradicis , corn root aphid; Blissus leucopterus leucopterus, chinch bug; Melanoplus femurrubrum, redlegged grasshopper; Melanoplus sanguinipes, migratory grasshopper; Hylemya platura, seedcorn maggot; Agromyza parvicornis, corn bloth leafminer; Anaphothrips obscrurus, grass thrips; Solenopsis milesta, thief ant; Tetranychus urticae, twospotted spider mite; Sorghum: Chilo partellus, sorghum borer; Spodoptera frugiperda, fall armyworm; Helicoverpa zea, corn earworm; Elasmopalpus lignosellus, leser cornstalk borer; Feltia subterranea, granulate cutworm; Phyllophaga crinita, white grub; Eleodes, Conoderus, and Aeolus spp. , wireworms; Oulema melanopus, cereal leaf beetle; Chaetocnema pulicaria, corn flea beetle; Sphenophorus maidis, maize billbug; Rhopalosiphum maidis; corn leaf aphid; Sip ha flava, yellow sugarcane aphid; Blissus leucopterus leucopterus, chinch bug; Contarinia sorghicola, sorghum midge; Tetranychus cinnabarinus , carmine spider mite; Tetranychus urticae, twospotted spider mite; Wheat: Pseudaletia unipunctata, army worm; Spodoptera frugiperda, fall armyworm; Elasmopalpus lignosellus, lesser cornstalk borer; Agrotis orthogonia, plae western cutworm; Elasmopalpus lignosellus, lesser cornstalk borer; Oulema melanopus, cereal leaf beetle; Hypera punctata, clover leaf weevil; Diabrotica undecimpunctata howardi, southern corn rootworm; Russian wheat aphid; Schizaphis graminum, greenbug; Macrosiphum avenae, English grain aphid; Melanoplus femurrubrum, redlegged grasshopper; Melanoplus differentiali , differential grasshopper; Melanoplus sanguinipes, migratory grasshopper; Mayetiola destructor, Hessian fly; Sitodiplosis mosellana, wheat midge; Meromyza americana, wheat stem maggot; Hylemya coarctata, wheat bulb fly; Frankliniella fusca, tobacco thrips; Cephus cinctus, wheat stem sawfly; Aceria tulipae, wheat curl mite; Sunflower: Suleima helianthana, sunflower bud moth; Homoeosoma electellum, sunflower moth; zygogramma exclamationis , sunflower beetle; Bothy rus gibbosus, carrot beetle; Neolasioptera murtfeldtiana, sunflower seed midge; Cotton: Heliothis virescens, cotton boll worm; Helicoverpa zea, cotton bollworm; Spodoptera exigua, beet armyworm; Pectinophora gossypiella, pink bollworm; Anthonomus grandis, bool weevil; Aphis gossypii, cotton aphid; Pseudatomoscelis seriatus, cotton fleahopper; Trialeurodes abutilonea, bandedwinged whitefly; Lygus lineolaris, tarnished plant bug; Melanoplus femurrubrum, redlegged grasshopper; Melanoplus differentialis, differential grasshopper; Thrips tabaci, onion thrips; Franklinkiella fusca, tobacco thrips; Tetranychus cinnabarinus , carmine spider mite; Tetranychus urticae, twospotted spider mite; Rice: Diatraea saccharalis, sugarcane borer; Spodoptera frugiperda, fall armyworm; Helicoverpa zea, corn earworm; Colaspis brunnea, grape colaspis;

Lissorhoptrus oryzophilus, rice water weevil; Sitophilus oryzae, rice weevil; Nephotettix nigropictus, rice leafhoper; Blissus leucopterus leucopterus, chinch bug; Acrosternum hilare, green stink bug; Soybean: Pseudoplusia includens, soybean looper; Anticarsia gemmatalis, velvetbean caterpillar; Plathypena scabra, green cloverworm; Ostrinia nubilalis, European corn borer; Agrotis ipsilon, black cutworm; Spodoptera exigua, beet armyworm; Heliothis virescens, cotton boll worm; Helicoverpa zea, cotton bollworm; Epilachna varivestis, Mexican bean beetle; Myzus persicae, green peach aphid; Empoasca fabae, potato leafhopper; Acrosternum hilare, green stink bug; Melanoplus femurrubrum, redlegged grasshopper; Melanoplus differentialis, differential grasshopper; Hylemya platura, seedcorn maggot; Sericothrips variabilis, soybean thrips; Thrips tabaci, onion thrips; Tetranychus turkestani, strawberry spider mite; Tetranychus urticae, twospotted spider mite; Barley: Ostrinia nubilalis, European corn borer; Agrotis ipsilon, black cutworm; Schizaphis graminum, greenbug; Blissus leucopterus leucopterus, chinch bug; Acrosternum hilare, green stink bug; Euschistus servus, brown stink bug; Jylemya platura, seedcorn maggot; Mayetiola destructor, Hessian fly; Petrobia latens, brown wheat mite; Oil Seed Rape: Vrevicoryne brassicae, cabbage aphid.

Generally, Viruses include tobacco or cucumber mosaic virus, ringspot virus, necrosis virus, maize dwarf mosaic virus, etc. Specific pathogens for the major crops include: Soybeans: Phytophthora megasperma fsp. glycinea, Macrophomina phaseolina, Rhizoctonia solani, Sclerotinia sclerotiorum, Fusarium oxysporum, Diaporthe phaseolorum var. sojae (Phomopsis sojae), Diaporthe phaseolorum var. caulivora, Sclerotium rolfsii, Cercospora kikuchii, Cercospora sojina, Peronospora manshurica, Colletotrichum dematium (Colletotichum truncatum), Corynespora cassiicola, Septoria glycines, Phyllosticta sojicola, Alternaria alternata, Pseudomonas syringae p. v. glycinea, Xanthomonas campestris p. v. phaseoli, Microsphaera diffusa, Fusarium semitectum, Phialophora gregata, Soybean mosaic virus, Glomerella glycines, Tobacco Ring spot virus, Tobacco Streak virus, Phakopsora pachyrhizi, Pythium aphanidermatum, Pythium ultimum, Pythium debaryanum, Tomato spotted wilt virus, Heterodera glycines Fusarium solani; Canola: Albugo Candida, Alternaria brassicae, Leptosphaeria maculans, Rhizoctonia solani, Sclerotinia sclerotiorum, Mycosphaerella brassiccola, Pythium ultimum, Peronospora parasitica, Fusarium roseum, Alternaria alternata; Alfalfa: Clavibater michiganese subsp. insidiosum, Pythium ultimum, Pythium irregulare, Pythium splendens, Pythium debaryanum, Pythium aphanidermatum, Phytophthora megasperma, Peronospora trifoliorum, Phoma medicaginis var. medicaginis, Cercospora medicaginis, Pseudopeziza medicaginis, Leptotrochila medicaginis, Fusarium oxysporum, Rhizoctonia solani, Uromyces striatus, Colletotrichum trifolii race 1 and race 2, Leptosphaerulina briosiana, Stemphylium botryosum, Stagonospora meliloti, Sclerotinia trifoliorum, Alfalfa Mosaic Virus, Verticillium albo-atrum, Xanthomonas campestris p. v. alfalfae, Aphanomyces euteiches, Stemphylium herbarum, Stemphylium alfalfae; Wheat: Pseudomonas syringae p. v. atrofaciens, Urocystis agropyri, Xanthomonas campestris p. v. translucens, Pseudomonas syringae p. v. syringae, Alternaria alternata, Cladosporium herbarum, Fusarium graminearum, Fusarium avenaceum, Fusarium culmorum, Ustilago tritici, Ascochyta tritici, Cephalosporium gramineum, Collotetrichum graminicola, Erysiphe graminis f.sp. tritici, Puccinia graminis f.sp. tritici, Puccinia recondita f.sp. tritici, Puccinia striiformis , Pyrenophora tritici- repentis, Septoria nodorum, Septoria tritici, Septoria avenae, Pseudocercosporella herpotrichoides , Rhizoctonia solani, Rhizoctonia cerealis, Gaeumannomyces graminis var. tritici, Pythium aphanidermatum, Pythium arrhenomanes , Pythium ultimum, Bipolaris sorokiniana, Barley Yellow Dwarf Virus, Brome Mosaic Virus, Soil Borne Wheat Mosaic Virus, Wheat Streak Mosaic Virus, Wheat Spindle Streak Virus, American Wheat Striate Virus, Claviceps purpurea, Tilletia tritici, Tilletia laevis, Ustilago tritici, Tilletia indica, Rhizoctonia solani, Pythium arrhenomannes, Pythium gramicola, Pythium aphanidermatum, High Plains Virus, European wheat striate virus; Sunflower: Plasmophora halstedii, Sclerotinia sclerotiorum, Aster Yellows, Septoria helianthi, Phomopsis helianthi, Alternaria helianthi, Alternaria zinniae, Botrytis cinerea, Phoma macdonaldii, Macrophomina phaseolina, Erysiphe cichoracearum, Rhizopus oryzae, Rhizopus arrhizus, Rhizopus stolonifer,

Puccinia helianthi, Verticillium dahliae, Erwinia carotovorum pv. carotovora, Cephalosporium acremonium, Phytophthora cryptogea, Albugo tragopogonis; Corn: Fusarium moniliforme var. subglutinans, Erwinia stewartii, Fusarium moniliforme, Gibberella zeae (Fusarium graminearum) , Stenocarpella maydi (Diplodia maydis), Pythium irregulare, Pythium debaryanum, Pythium graminicola, Pythium splendens, Pythium ultimum, Pythium aphanidermatum, Aspergillus flavus, Bipolaris maydis O, T (Cochliobolus heterostrophus) , Helminthosporium carbonum I, II & III (Cochliobolus carbonum), Exsewhilum turcicum I, II & III, Helminthosporium pedicellatum, Physoderma maydis, Phyllosticta maydis, Kabatiella zeae, Colletotrichum graminicola, Cercospora zeae-maydis, Cercospora sorghi, Ustilago maydis, Puccinia sorghi, Puccinia polysora, Macrophomina phaseolina, Penicillium oxalicum, Nigrospora oryzae, Cladosporium herbarum, Curvularia lunata, Curvularia inaequalis, Curvularia pallescens, Clavibacter michiganense subsp. nebraskense, Trichoderma viride, Maize Dwarf Mosaic Virus A & B, Wheat Streak Mosaic Virus, Maize Chlorotic Dwarf Virus, Claviceps sorghi, Pseudonomas avenae, Erwinia chrysanthemi pv. zea, Erwinia corotovora, Cornstunt spiroplasma, Diplodia macrospora, Sclerophthora macrospora, Peronosclerospora sorghi, Peronosclerospora philippinensis , Peronosclerospora maydis, Peronosclerospora sacchari, Spacelotheca reiliana, Physopella zeae, Cephalosporium maydis, Caphalosporium acremonium, Maize Chlorotic Mottle Virus, High Plains Virus, Maize Mosaic Virus, Maize Rayado Fino Virus,

Maize Streak Virus, Maize Stripe Virus, Maize Rough Dwarf Virus; Sorghum: Exsewhilum turcicum, Colletotrichum graminicola (Glomerella graminicola) , Cercospora sorghi, Gloeocercospora sorghi, Ascochyta sorghina, Pseudomonas syringae p. v. syringae, Xanthomonas campestris p. v. holcicola, Pseudomonas andropogonis, Puccinia purpurea, Macrophomina phaseolina, Perconia circinata, Fusarium moniliforme, Alternaria alternate, Bipolaris sorghicola, Helminthosporium sorghicola, Curvularia lunata, Phoma insidiosa, Pseudomonas avenae (Pseudomonas alboprecipitans) , Ramulispora sorghi, Ramulispora sorghicola, Phyllachara sacchari, Sporisorium reilianum (Sphacelotheca reiliana), Sphacelotheca cruenta, Sporisorium sorghi, Sugarcane mosaic H, Maize Dwarf Mosaic Virus A & B, Claviceps sorghi, Rhizoctonia solani, Acremonium strictum, Sclerophthona macrospora, Peronosclerospora sorghi, Peronosclerospora philippinensis, Sclerospora graminicola, Fusarium graminearum, Fusarium oxysporum, Pythium arrhenomane , Pythium graminicola, etc.

Plant tissue cultures and recombinant plant cells containing the proteins and nucleotide sequences, or the purified protein, of the invention may also be used in an assay to screen chemicals for potential as herbicidal compounds. Such an assay is useful as a general screen to identify chemicals which are herbicide candidates.

EXPERIMENTAL Synthesis and deposition of secondary metabolites is one major strategy of defense evolved by plants. A substantial number of these metabolites are dedicated to this function, as for example the cyclic hydroxamic acids which are a class of secondary metabolites found almost exclusively in Gramineae. DIMBOA (2,4-dihydroxy-7-methoxy-l ,4-benzoxazin-3-one) and its precursor DIBOA (2,4-dihydroxy-4-benzoxaxin-3-one) are present in maize. DIMBOA has been shown to be important in the resistance of maize to first brood European com borer (Ostrinia nubilalis), northern corn leaf blight (Helminithosporium turcicum), maize plant louse (Thophalosiphum maydis) and stalk rot (Diplodia maydis), as well as to the herbicide atrzine, H.M. Niemeyer (1988) Phytochemistry 27:3349. DIBOA is the main hydroxamic acid in rye, whereas DIMBOA is the predominant form in wheat and maize H.M. Niemeyer (1988) Phytochemistry 27:3349. Biochemical studies have demonstrated that DIMBOA synthesis has some intermediates in common with the tryptophan biosynthetic pathway. Labeled tryptophan precursors such as anthranilic acid, ribose and indole were shown to be incorporated into

DIMBOA, whereas labeled tryptophan was not incorporated into DIMBOA, S.R. Disai et al. (1996) Chem. Commun. 1321 , implicating indole as the formal branchpoint of the two pathways. The accumulation of benzoxazinone derivatives in maize is absent in a line homozygous for the bxl mutation, R.H. Hamilton (1964) Weeds 12:27; R. M. Couture et al. (1971) Phisiol. PI. Path. 1 :515. Homozygous bxl plants grow normally although they are extremely susceptible to the above mentioned pests.

(The maize inbred line C31A was the source for all wildtype cDNA and genomic clones. Cloning was performed as described) (M. Frey et al. (1995) Mol. Gen. Genet. 246: 100 (the new gene designation is: Bx2 = CYP71C4, Bx3 = CYP71C2, Bx4 = CYP71C1, Bx5 = CYP71C3). Bxl was mapped with the Recombinant inbred system, B. Burr et al. (1991) Trends Genet 7:55, and proved to be located on the short arm of chromosome 4 and, within the limits of the method, at exactly the same map position as Bx2 (15, Fig. 1). The Bxl gene has been mapped to the same region by classical genetic means, K. D. Simcox et al. (1985) Crop. Sci.

25:827, DNA sequence comparison showed that the Bxl gene and the Bx2 gene are close neighbors in the genome.

The cDNA sequences of four maize cytochrome P450 genes have been previously reported, M. Frey et al. (1995) Mol. Gen. Genet 246: 100 (the new gene designation is: Bx2 = CYP71C4, Bx3 = CYP71C2, Bx4 =

CYP71C1, Bx5 = CYP71C3). On the basis of amino acid homology, these genes have been grouped into the CYP71C subfamily of plant cytochrome P450 genes. These genes are strongly expressed in young maize seedlings, share an overall amino acid identity of 45 to 60%, and are clustered on the short arm of chromosome 4 (Fig. 1). The developmental expression pattern of the genes in the young maize plants, occurring predominantly in tissues that are exposed to the environment, M. Frey et al. (1995) Mol. Gen. Genet 246: 100 (the new gene designation is: Bx2 = CYP71C4, Bx3 = CYP71C2, Bx4 = CYP71C1, Bx5 = CYP71C3), matches well with the defense related DIMBOA accumulation. This observation and the finding that all oxygen atoms of DIMBOA are incorporated from molecular oxygen, E. Glawischnig et al. , Phytochemistry (in press), led to the speculation that these cytochrome P450 enzymes might be involved in this pathway. The genes encoding these enzymes are therefore designated in the following Bx2, Bx3, Bx4 and Bx5, M. Frey et al. (1995) Mol. Gen. Genet 246: 100 (the new gene designation is: Bx2 = CYP71C4, Bx3 = CYP71C2, Bx4 = CYP71C1, Bx5 = CYP71C3).

Genomic clones of Bx2-Bx5 were isolated from a λ library and sequenced (the maize inbred line C31A was the source for all wildtype cDNA and genomic clones. Cloning was performed as described (Frey et al. (1995) Mol. Gen. Genet 246: 100 (the new gene designation is: Bx2 = CYP71C4, Bx3 = CYP71C2, Bx4 = CYP71C1, Bx5 = CYP71C3)). Comparison of the genomic DNA sequences to the cDNA sequences yielded the exon/introl structure of the genes (Fig. 1). The position of one intron is identical for all genes, indicating a common evolutionary origin. The position of the second intron, present only in Bx3 and Bx5 is also conserved. The structural similarities and the chromosomal clustering of Bx2-Bx5 suggest that they might have been generated by gene duplication events.

In order to test a possible function of the four P450 enzymes in the DIMBOA pathway, we used the yeast expression system established by G. Truan et al. (1993) Gene 125:49. The cDNAs of Bx2-Bx5 were inserted into the pYeDP60 expression vector P. Urban et al. (1994) Eur. J. Biochem. 222:843 (full size P450 cDNAs were inserted in the expression vector, beginning with the AUG translation start codon. This was accomplished by PCR amplification of the relevant cDNA sequences. These constructs were used to transform the WAT11 yeast strain. In WAT11, a galactose inducible Arabidopsis thaliana microsomal NADPH-P450 reductase (ATR1) replaces the yeast reductase D. Pompon et al. (1996) Methods Enzymol. 272:51.

Microsomes were isolated from the transgenic yeast strains and tested for enzymatic activity G. Truan et al. (1993) Gene 125:49; D. Pompon et al. (1996) Methods Enzymol. 272:51. In summary, indole was converted to DIBOA by the stepwise action of the four cytochrome P450 enzymes (Fig. 2C- F). When [3-¹³C] indole was incubated with yeast microsomes containing Bx2 protein, [3-¹³C]indolin-2-one was produced in the reaction assay. A sufficient amount of [3-¹³C]indolin-2-one was produced by this enzyme catalyzed reaction in order to test for subsequent enzymatic conversions. Incubation of [3- ¹³C]indolin-2-one with microsomes containing Bx3 resulted in [3-¹ C]hydroxy- indolin-2-one production. For further analysis, unlabelled 3-hydroxy-indolin-2- one was obtained by reduction of isatin (commercially available isatin (indole- 2,3-dione) was reduced to 3-hydroxy-indolin-2-one in a yeast culture and HPLC purified (Glawischnig et al. unpublished). The structure of 3-hydroxy-indolin- 2-one was confirmed by H NMR (Tab. 1)). The conversion of 3-hydroxy- indolin-2-one to 2-hydroxy-l,4-benzoxazin-3-one (HBOA) was catalyzed by microsomes containing Bx4. The reaction mechanism for this unusual ring expansion is as yet unknown. Finally, HBOA was converted to DIBOA by microsomes containing Bx5. This reaction was previously described for maize microsomes B. A. Bailey et al. (1991) Plant Physiol. 95:792. The identity of the reaction products was confirmed by cochromatography with the authentic substances and by either UV spectra. The reaction products indolin-2-one and 3-hydroxy-indol-2-one were further identified by their Η-NMR-spectra (Tab. 1). The identity of HBOA and DIBOA was corroborated by GC/MS analysis, M. D. Woodward et al. (1979) Plant Physiol 63:9 (it was determined that TMS₂-HOBA m/e 309 (100%), 294 (29%), 266 (28%), 220 (12%), 208 (14%), 193 (17%), 192 (35%), 191 (15 %), 147 (95 %); TMS₂-DIBOA m/e 325 (31 %), 310 (84%), 297 (17%), 208 (43 %), 192 (54%), 191 (30%), 179 (36%), 164 (72%), 151 (24%), 150 (23 %), 147 (100%), 136 (73%)).

Although the four cytochrome P450 enzymes are homologous proteins, they are substrate specific. Only one substrate was converted by each respective P450 enzyme to a specific product. No detectable conversions occurred in other enzyme/substrate combinations (data not shown). Enzymatic reactions, identical to the ones with the different yeast microsomal preparations, could be performed with maize microsomes, indicating that these reactions occur natively in maize. These findings suggest a similar in vivo reaction sequence from indole to HBOA as outlined in Fig. 3. According to this scheme, benzoxazinone would not be a natural intermediate for DIMBOA synthesis, as proposed earlier on the basis of feeding experiments P. Kumar et al. (1994) Phytochemistry 36:893. Whether alternative routes for DIMBOA synthesis exist remains to be determined.

The four cytochrome P450 genes represent a sufficient set of genes for the conversion of indole-3-glycerol phosphate to the secondary metabolite DIBOA (Fig. 3). Indole-3-glycerol phosphate appears to be the real branchpoint from the tryptophan pathway. DIMBOA is the 7-methoxy derivative of DIBOA. The conversion of DIBOA to DIMBOA most likely requires two further enzymatic reactions. Since the O atom at C-7 is incorporated from molecular O₂, E. Glawischnig et al. , Phytochemistry (in press), hyroxylation by another cytochrome P450 enzyme followed by a methyltransferase reaction would be expected. These enzymes which are probably present only in some Gramineae, H. M. Niemeyer (1988) Phytochemistry 27:3349, remain to be isolated.

Independent evidence for the involvement of the cytochrome P450 genes in DIMBOA biosynthesis is provided by a mutant Bx3 allele isolated by a reverse genetic approach to screen for Mu insertions in the P450 genes (the Pioneer Hi-Bred collection of 24,000 FI maize plants mutagenized by means of a Robertson's Mutator element was screened for w-containing alleles of the Bxl gene by a reverse genetics-based technology [R.J. Bensen et al. (1995) Plant Cell 7] . PCR amplifications were done as described in M. Mean et al. (1996) Science 274: 1537. A Bx3 mutant allele, designated Bx3: :Mu, was identified. Sequencing of the PCR amplified Mu-flanking genomic DNA fragments showed that Bx3: :Mu has a Mu transposon inserted in the second exon of the gene (Fig. 1). In maize seedlings homozygous for the recessive mutant allele, no DIMBOA could be detected by staining roots with Fe²⁺, K.D. Simcox et al. (1985) Crop Sci. 25:827, or by HPLC analysis. In contrast, DIMBOA was detected in seedlings that were either heterozygous or homozygous wild type. Consegregation of the recessive mutant phenotype was established by genomic blotting analysis of 27 F2 individuals (four homozygous recessives). These results demonstrate that an intact Bx3 gene is required for DIMBOA biosynthesis.

We estimate that the DIMBOA concentration in maize seedlings is approximately 0.1 % of the fresh weight. This value exceeds the total tryptophan content of the seedling by a factor of about 10-20, E. R. Radwanski et al. (1995) Plant Cell 7:921. Hence most of the metabolites of tryptophan pathway would be channeled into the secondary metabolic DIMBOA pathway. The synthesis of several other secondary metabolites in plants, such as the indole glucosinates, anthranilate-derived alkaloids and tryptamine derivatives E. R. Radwanski et al. 91995) Plant Cell 7:921; T. M. Kutchan (1995) Plant Cell 7: 1059, depends on the tryptophan pathway. Indole-3 -glycerol phosphate was also proposed as a branchpoint from the tryptophan pathway for the synthesis of the indolic phytoalexin camalexin (3-thiazol-2'yl-indole) in Arabidopsis thaliana. For this secondary metabolic pathway, a coordinate regulation of gene expression of the tryptophan pathway and camalexin synthesis was established, J. Zhao et al. (1996) Plant Cell 8:2235. With the isolation of five genes specific for DIMBOA biosynthesis, the regulation of gene expression of "primary" and "secondary" metabolic genes can now be studied in maize in a developmental and tissue specific manner.

Evolution of maize has located the five DIMBOA-specific genes within one cluster on chromosome 4. It is the first time that the clustering of genes for one pathway has been observed in maize. This is reminiscent of gene clusters of secondary metabolic genes in certain fungi, e.g. the _/3-lactam antibiotic biosynthetic genes, D. J. Smith et al. (1990) EMBO J. 9:1 Al, and the aflatoxin pathway genes, J. Yu et al. (1995) Appl. Environ. Microbiol 61 :2365. Whether gene clustering has any meaning for expression of these genes is not yet known. Future analysis will show if this cluster is conserved in the genomes of other grasses.

Table 1 Η-NMR Analysis of Indole Derived Enzymatic Products in Maize

Indolin-2-one 3-Hvdroxy-indolin-2-one DIMBOA pos. δ ΗH pos. δ ΗH pos. δ ΗH

4 7.215(d) 7.5 4 7.42(d) 7.5 5 7.258(d) 8.8 6 7.176(tq) 7.8,0.9 6 7.264(t) 7.7 6 6.668(dd) 8.8, 2.6

5 6.973(tq) 7.5,0.8 5 7.072(t) 7.5 8 6.625(d) 2.6 7 6.863(d) 7.8 7 6.842(d) 7.7 2 5.674(s)

3 3.488(s) 3 3 5.049(s) O-CH, 3.751

The ¹³C label from [3-¹³C]indole has been incorporated into position 3 of indolin-2-one and 3-hydroxy-indolin-2-one, and into position 2 of DIMBOA (determined by ¹³C-decoupled 'H-NMR spectroscopy). The signal assignments are based on two-dimensional NMR analysis (GRASP-HMQC,GRASP-HMBC, GRASP-DQF-COSY, data not shown).

Abbreviations: pos. = position, δ = chemical shift in ppm, referenced to solvent signals, Signal multiplicities in brackets (s = singlet, d = duplet, t = triplet, q = quartet); ^JHH = coupling constant in Hz. All publications and patent applications mentioned in the specification are indicative of the level of those skilled in the art to which this invention pertains. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be obvious that certain changes and modifications may be practiced within the scope of the appended claims.

Claims

THAT WHICH IS CLAIMED:

1. A method for altering the levels of benzoxazinones in a plant, said method comprising transforming said plant with a gene comprising in the 5 '-3' direction of transcription and operably linked, a transcriptional initiation region which is expressed in a plant cell, a DNA coding sequence of at least one primary biosynthesis gene, and a transcriptional termination region, wherein said primary gene is selected from the group consisting of DIMBOA biosynthesis genes, tryptophan biosynthesis genes, genes encoding P450 reductases and genes encoding cytochrome P450 enzymes.

2. The method of claim 1, wherein said gene is a chimeric gene.

3. The method of claim 1 , wherein said transcriptional initiation region is heterologous to the DNA coding sequence.

4. The method of claim 3, wherein said transcriptional initiation region is selected from the group consisting of pith, root, constitutive, and leaf transcription initiation region.

5. The method of claim 1 , wherein said plant is a monocotyledonous plant.

6. The method of claim 1, wherein said plant is a dicotyledonous plant.

7. The method of claim 1, further comprising transforming said plant with a secondary gene comprising in the 5 '-3' direction of transcription and operably linked, a transcriptional initiation region which is expressed in a plant cell, a DNA coding sequence of at least one biosynthesis gene, and a transcriptional termination region, wherein said secondary gene is a DIMBOA biosynthesis gene.

8. The method of claim 7, wherein said secondary gene is a chimeric gene.

9. The method of claim 7, wherein said transcriptional initiation region of the secondary gene is heterologous to the DNA coding sequence.

10. The method of claim 9, wherein said transcriptional initiation region is selected from the group consisting of pith, root, constitutive, and leaf transcription initiation region.

11. The method of claim 7, wherein said plant is a monocotyledonous plant.

12. The method of claim 7, wherein said plant is a dicotyledonous plant.

13. The method of claim 1, wherein said benzoxaninone is selected from HBOA, DIBOA, HMBOA, and DIMBOA.

14. The method of claim 1 , wherein said DIMBOA biosynthesis genes are selected from the group consisting of Bx2, Bx3, Bx4, and Bx5.

15. The method of claim 1, wherein said DNA coding sequence is an antisense sequence for at least one DIMBOA biosynthesis gene.

16. The method of claim 7, wherein said DNA coding sequence of the secondary gene is an antisense sequence for at least one DIMBOA biosynthesis gene.

17. The method of claim 1, wherein said plant overproduces tryptophan.

18. The method of claim 1, wherein said DNA coding sequence is a tryptophan biosynthesis gene which increases the expression of tryptophan.

19. The method of claim 18, wherein said gene is anthranilate synthase.

20. The method of claim 1 wherein said gene is a mammalian, bacterial, or plant gene.

21. The method of claim 1, wherein said plant has increased insect and disease resistance.

22. The method of claim 7, wherein said plant has increased insect and disease resistance.

23. The method of claim 1 , wherein said plant has increased tolerance to a herbicide.

24. The method of claim 7, wherein said plant has increased tolerance to a herbicide.

25. A transformed plant having altered levels of at least one benzoxazinone as compared to levels in an untransformed plant, said plant having been transformed with at least one gene wherein said gene comprises a primary biosynthesis gene operably linked to a promoter region, wherein said primary gene is selected from the group consisting of DIMBOA biosynthesis genes, tryptophan biosynthesis genes, genes encoding P450 reductases and genes encoding cytochrome P450 enzymes.

26. The method of claim 25, wherein said gene is a chimeric gene.

27. The method of claim 25, wherein said promoter is heterologous to the DNA coding sequence.

28. The method of claim 27, wherein said transcriptional initiation region is selected from the group consisting of pith, root, constitutive, and leaf transcription initiation region.

29. The method of claim 25, wherein said plant is a monocotyledonous plant.

30. The method of claim 25, wherein said plant is a dicotyledonous plant.

31. The plant of claim 25, further comprising transforming said plant with a secondary gene, said secondary gene comprising in the 5 '-3' direction of transcription and operably linked, a promoter which is expressed in a plant cell, a DNA coding sequence of at least one biosynthesis gene, and a transcriptional termination region, wherein said secondary gene is a DIMBOA biosynthesis gene.

32. The method of claim 31, wherein said secondary gene is a chimeric gene.

33. The method of claim 31, wherein said promoter of said secondary gene is heterologous to the DNA coding sequence.

34. The method of claim 33, wherein said promoter is selected from the group consisting of pith, root, constitutive, and leaf promoters.

35. The method of claim 31 , wherein said plant is a monocotyledonous plant.

36. The method of claim 31, wherein said plant is a dicotyledonous plant.

37. The plant of claim 25, wherein said benzoxaninone is selected from HBOA, DIBOA, HMBOA, and DIMBOA.

38. The plant of claim 31, wherein said benzoxaninone is selected from HBOA, DIBOA, HMBOA, and DIMBOA.

39. The plant of claim 37, wherein said DIMBOA biosynthesis genes are selected from the group consisting of Bx2, Bx3, Bx4, and Bx5.

40. The plant of claim 38, wherein said DIMBOA biosynthesis genes are selected from the group consisting of Bx2, Bx3, Bx4, and Bx5.

41. The plant of claim 25, wherein said DNA coding sequence is an antisense sequence for at least one DIMBOA biosynthesis gene.

42. The plant of claim 31 , wherein said DNA coding sequence is an antisense sequence for at least one DIMBOA biosynthesis gene.

43. The plant of claim 25, wherein said plant overproduces tryptophan.

44. The plant of claim 25, wherein said DNA coding sequence is a tryptophan biosynthesis gene which increases the expression of tryptophan.

45. The plant of claim 44, wherein said gene is anthranilate synthase.

46. The plant of claim 25 wherein said gene is a mammalian, bacterial, or plant gene.

47. The plant of claim 25, wherein said plant has increased insect and disease resistance.

48. The plant of claim 31 , wherein said plant has increased insect and disease resistance.

49. The plant of claim 25, wherein said plant has increased tolerance to a herbicide.

50. The plant of claim 31, wherein said plant has increased tolerance to a herbicide.