Compositions and methods for fumonisin detoxification

Abstract

Compositions and methods for the complete detoxification of fumonisin and fumonisin degradation products are provided. Particularly, nucleotide sequences corresponding to the detoxification enzymes are provided. The sequences find use in preparing expression cassettes for the transformation of a broad variety of host cells and organisms.

Description

FIELD OF THE INVENTION

The invention relates to compositions and methods for detoxification or degradation of fumonisin or AP1. The method has broad application in agricultural biotechnology and crop agriculture and in the improvement of food grain quality.

BACKGROUND OF THE INVENTION

Fungal diseases are common problems in crop agriculture. Many strides have been made against plant diseases as exemplified by the use of hybrid plants, pesticides, and improved agricultural practices. However, as any grower or home gardener can attest, the problems of fungal plant disease continue to cause difficulties in plant cultivation. Thus, there is a continuing need for new methods and materials for solving the problems caused by fungal diseases of plants.

These problems can be met through a variety of approaches. For example, the infectious organisms can be controlled through the use of agents that are selectively biocidal for the pathogens. Another method is interference with the mechanism by which the pathogen invades the host crop plant. Yet another method, in the case of pathogens that cause crop losses, is interference with the mechanism by which the pathogen causes injury to the host crop plant. In the case of pathogens that produce toxins that are undesirable to mammals or other animals that feed on the crop plants, interference with toxin production, storage, or activity can be beneficial.

Since their discovery and structural elucidation in 1988 (Bezuidenhout et al. (1988) Journal Chem. Soc., Chem. Commun. 1988:743-745), fumonisins have been recognized as a potentially serious problem in maize-fed livestock. They are linked to several animal toxicoses including leukoencephalomalacia (Marasas et al. (1988) Onderstepoort J. Vet. Res. 55:197-204; Wilson et al. (1990) American Association of Veterinary Laboratory Diagnosticians: Abstracts 33 rd Annual Meeting, Denver, Colo., Madison, Wis., USA) and porcine pulmonary edema (Colvin et al. (1992) Mycopathologia 11 7:79-82). Fumonisins are also suspected carcinogens (Geary et al. (1971) Coord. Chem. Rev. 7:81; Gelderblom et al. (1991) Carcinogenesis 12:1247-1251; Gelderblom et al. (1992) Carcinogenesis 13:433-437). Fusarium isolates in section Liseola produce fumonisins in culture at levels from 2 to >4000 ppm (Leslie et al. (1992) Phytopathology 82:341-345). Isolates from maize (predominantly mating population A) are among the highest producers of fumonisin (Leslie et al., supra). Fumonisin levels detected in field-grown maize have fluctuated widely depending on location and growing season, but both preharvest and postharvest surveys of field maize have indicated that the potential for high levels of fumonisins exists (Murphy et al. (1993) J. Agr. Food Chem. 41:263-266). Surveys of food and feed products have also detected fumonisin (Holcomb et al. (1993) J. Agr. Food Chem. 41:764-767; Hopmans et al. (1993) J. Agr. Food Chem. 41:1655-1658); Sydenham et al. (1991) J. Agr. Food Chem. 39:2014-2018). The etiology of Fusarium ear mold is poorly understood, although physical damage to the ear and certain environmental conditions can contribute to its occurrence (Nelson et al. (1992) Mycopathologia 117:29-36). Fusarium can be isolated from most field grown maize, even when no visible mold is present. The relationship between seedling infection and stalk and ear diseases caused by Fusarium is not clear. Genetic resistance to visible kernel mold has been identified (Gendloff et al. (1986) Phytopathology 76:684-688; Holley et al. (1989) Plant Dis. 73:578-580), but the relationship to visible mold to fumonisin production has yet to be elucidated.

Fumonisins have been shown in in vitro mammalian cell studies to inhibit sphingolipid biosynthesis through inhibition of the enzyme sphingosine N-acetyl transferase, resulting in the accumulation of the precursor sphinganine (Norred et al. (1992) Mycopathologia 117:73-78; Wang et al. (1991) Biol. Chem. 266:14486; Yoo et al. (1992) Toxicol. Appl. Pharmacol. 114:9-15; Nelson et al. (1993) Annu. Rev. Phytpathol. 31:233-252). It is likely that inhibition of this pathway accounts for at least some of fumonisin's toxicity, and support for this comes from measures of sphinganine:sphingosine ratios in animals fed purified fumonisin (Wang et al. (1992) J. Nutr. 122:1706-1716). Fumonisins also affect plant cell growth (Abbas et al. (1992) Weed Technol. 6:548-552; Van Asch et al. (1992) Phytopathology 82:1330-1332; Vesonder et al. (1992) Arch. Environ. Contam. Toxicol. 23:464-467). Kuti et al. (1993) (Abstract, Annual Meeting American Phytopathological Society, Memphis, Tenn.: APS Press) reported on the ability of exogenously added fumonisins to accelerate disease development and increase sporulation of Fusarium moniliform and F. oxysporum on tomato.

Enzymes that degrade the fungal toxin fumonisin to the compound AP1 have been identified in U.S. Pat. No. 5,716,820 and pending U.S. patent application Ser. Nos. 08/888,949 and 08/888,950, both filed Jul. 7, 1997, and hereby incorporated by reference. Plants expressing a fumonisin esterase enzyme, infected by fumonisin producing fungus, and tested for fumonisin and AP1 were found to have low levels of fumonisin but high levels of AP1. AP1 is less toxic than fumonisin to plants and probably also animals, but contamination with AP1 is still a concern. The best result would be complete detoxification of fumonisin to a non-toxic form. Therefore enzymes capable of degrading AP1 are necessary for the further detoxification of fumonisin.

SUMMARY OF THE INVENTION

Compositions and methods for catabolism and detoxification of fumonisin and fumonisin-degradation products as well as fumonisin-related toxins are provided. In particular, proteins involved in catabolism and transmembrane transport of fumonisin and fumonisin catabolic products are provided. Nucleotide sequences corresponding to the proteins are also included. The compositions are useful in the detoxification and degradation of fumonisin. The nucleotide sequences can be used in expression cassettes for transformation of host cells of interest. The compositions and methods of the invention are steps in a catabolic pathway for fumonisin. Thus, organisms can be genetically modified to provide for the catabolism and detoxification of fumonisin and fumonisin-related toxins.

In particular, expression cassettes for expression of the enzymes in plants and other organisms are provided as well as transformed plants and other host cells.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 sets forth the proposed pathway for fumonisin degradation by Exophiala spinifera.

FIG. 2 schematically illustrates a plasmid vector comprising the gene for one of the fumonisin degradative enzymes of the invention operably linked to the ubiquitin promoter.

DETAILED DESCRIPTION OF THE INVENTION

The catabolic pathway for detoxification and degradation of fumonisin is provided. Particularly, enzymes involved in the degradation of fumonisin from Exophiala spinifera (American Type Culture Collection Deposit No. 74269) and nucleotide sequences encoding such enzymes are disclosed. Such enzymes and nucleotide sequences find use in the breakdown of fumonisin and fumonisin-related toxins as well as degradation products thereof. In this regard, enzymes can be synthesized and utilized or, alternatively, organisms can be transformed with the DNA sequences of the invention and used to detoxify fumonisin.

A proposed pathway for the degradation of fumonisin by Exophiala spinifera is provided in FIG. 1 . The present invention encompasses enzymes and nucleotide sequences encoding the enzymes involved in this degradation pathway for fumonisin. Compositions of the invention include a flavin monooxygenase, an aldehyde dehydrogenase, a permease, and a p-glycoprotein that are involved in the fumonisin degradation pathway. In particular, the present invention provides for isolated nucleic acid molecules comprising nucleotide sequences encoding the amino acid sequences shown in SEQ ID NOS:3, 5, 8, and 11, or the nucleotide sequences encoding the DNA sequences obtained from the overlapping clones deposited in a bacterial host with the American Type Culture Collection and assigned Accession Number PTA-299. By “DNA sequence obtained from the overlapping clones” is intended that the DNA sequence of the fumonisin degrading enzymes can be obtained by sequencing the individual clones which together comprise the entire fumonisin degrading enzymes. Further provided are polypeptides having an amino acid sequence encoded by a nucleic acid molecule described herein, for example those set forth in SEQ ID NOS:1, 2, 4, 6, 7, 9 and 10, the DNA sequences obtained from the overlapping clones deposited in a bacterial host with the American Type Culture Collection and assigned Accession Number PTA-299, and fragments and variants thereof.

Ten plasmids containing overlapping clones were deposited with the American Type Culture Collection, Manassas, Va., and assigned Accession Number PTA-299. The plasmids designated as F_perm3.5 and F_perm4.4 contain common sequences at the regions were they overlap to form the nucleotide sequence encoding a permease. The plasmids designated as F_p-glyco1L4, F_p-glyco5.13, and F_p-glyco6.43 contain common sequences at the regions were they overlap to form the nucleotide sequence encoding a p-glycoprotein. And the plasmids designated F_Alde1.1, F_Alde2.2, and F_Alde2.5 contain common sequences at the regions were they overlap to form the nucleotide sequence of an aldehyde dehydrogenase. One of skill in the art by sequencing the clones and aligning the overlap may obtain the entire sequence of the permease, the p-glycoprotein, and the aldehyde dehydrogenase.

These deposits will be maintained under the terms of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure. These deposits were made merely as a convenience for those of skill in the art and are not an admission that a deposit is required under 35 U.S.C. §112.

The invention encompasses isolated or substantially purified nucleic acid or protein compositions. An “isolated” or “purified” nucleic acid molecule or protein, or biologically active portion thereof, is substantially free of other cellular material, or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized. Preferably, an “isolated” nucleic acid is free of sequences (preferably protein encoding sequences) that naturally flank the nucleic acid (i.e., sequences located at the 5′ and 3′ ends of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived. For example, in various embodiments, the isolated nucleic acid molecule can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb, or 0.1 kb of nucleotide sequences that naturally flank the nucleic acid molecule in genomic DNA of the cell from which the nucleic acid is derived. A protein that is substantially free of cellular material includes preparations of protein having less than about 30%, 20%, 10%, 5%, (by dry weight) of contaminating protein. When the protein of the invention or biologically active portion thereof is recombinantly produced, preferably culture medium represents less than about 30%, 20%, 10%, or 5% (by dry weight) of chemical precursors or non-protein-of-interest chemicals.

Fragments and variants of the disclosed nucleotide sequences and proteins encoded thereby are also encompassed by the present invention. By “fragment” is intended a portion of the nucleotide sequence or a portion of the amino acid sequence and hence protein encoded thereby. Fragments of a nucleotide sequence may encode protein fragments that retain the biological activity of the native protein and hence degrade or catabolize fumonisn. Alternatively, fragments of a nucleotide sequence that are useful as hybridization probes generally do not encode fragment proteins retaining biological activity. Thus, fragments of a nucleotide sequence may range from at least about 20 nucleotides, about 50 nucleotides, about 100 nucleotides, and up to the full-length nucleotide sequence encoding the proteins of the invention.

A fragment of a fumonisin-degrading nucleotide sequence that encodes a biologically active portion of a fumonisin-degrading protein of the invention will encode at least 15, 25, 30, 50, 100, 150, 200, or 250, 300, 400, 500, 600, 700, 800, 900, 1,000, 1,100, 1,200 contiguous amino acids, or up to the total number of amino acids present in a full-length fumonisin-degrading protein of the invention (for example, 545, 487, 525, 1,263 amino acids for SEQ ID NOS:3, 5, 8 and 11, respectively). Fragments of a fumonisin-degrading nucleotide sequence that are useful as hybridization probes for PCR primers generally need not encode a biologically active portion of a fumonisin-degrading protein.

Thus, a fragment of a fumonisin-degrading nucleotide sequence may encode a biologically active portion of a fumonisin-degrading protein, or it may be a fragment that can be used as a hybridization probe or PCR primer using methods disclosed below. A biologically active portion of a fumonisin-degrading protein can be prepared by isolating a portion of one of the fumonisin-degrading nucleotide sequences of the invention, expressing the encoded portion of the fumonisin-degrading protein (e.g., by recombinant expression in vitro), and assessing the activity of the encoded portion of the fumonisin-degrading protein. Nucleic acid molecules that are fragments of a fumonisin-degrading nucleotide sequence comprise at least 16, 20, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 800, 900, 1,000, 1,100, 1,200, 1,300, or 1,400, 1500, 1, 600, 1,800, 2,000, 2,200, 2,400, 2,600, 2,800, 3,000, 3,200, 3,400, 3,600, 3,800, 3,900 nucleotides, or up to the number of nucleotides present in a full-length fumonisin-degrading nucleotide sequence disclosed herein (for example, 1,691, 1,638, 1,464, 1,764, 1,578, 3,999, 3,792 nucleotides for SEQ ID NOS:1, 2, 4, 6, 7, 9, and 10 respectively).

By “variants” is intended substantially similar sequences. For nucleotide sequences, conservative variants include those sequences that, because of the degeneracy of the genetic code, encode the amino acid sequence of one of the fumonisin-degrading polypeptides of the invention. Naturally occurring allelic variants such as these can be identified with the use of well-known molecular biology techniques, as, for example, with polymerase chain reaction (PCR) and hybridization techniques as outlined below. Variant nucleotide sequences also include synthetically derived nucleotide sequences, such as those generated, for example, by using site-directed mutagenesis but which still encode a fumonisin-degrading protein of the invention. Generally, nucleotide sequence variants of the invention will have at least 40%, 50%, 60%, 70%, generally, 80%, preferably 85%, 90%, up to 95%, 98% sequence identity to its respective native nucleotide sequence.

By “variant” protein is intended a protein derived from the native protein by deletion (so-called truncation) or addition of one or more amino acids to the N-terminal and/or C-terminal end of the native protein; deletion or addition of one or more amino acids at one or more sites in the native protein; or substitution of one or more amino acids at one or more sites in the native protein. Such variants may result from, for example, genetic polymorphism or from human manipulation.

The proteins of the invention may be altered in various ways including amino acid substitutions, deletions, truncations, and insertions. Methods for such manipulations are generally known in the art. For example, amino acid sequence variants of the fumonisin-degrading proteins can be prepared by mutations in the DNA. Methods for mutagenesis and nucleotide sequence alterations are well known in the art. See, for example, Kunkel (1985) Proc. Natl. Acad. Sci. USA 82:488-492; Kunkel et al. (1987) Methods in Enzymol. 154:367-382; U.S. Pat. No. 4,873,192; Walker and Gaastra, eds. (1983) Techniques in Molecular Biology (MacMillan Publishing Company, New York) and the references cited therein. Guidance as to appropriate amino acid substitutions that do not affect biological activity of the protein of interest may be found in the model of Dayhoff et al. (1978) Atlas of Protein Sequence and Structure (Natl. Biomed. Res. Found., Washington, D.C.), herein incorporated by reference. Conservative substitutions, such as exchanging one amino acid with another having similar properties, may be preferred.

Thus, the genes and nucleotide sequences of the invention include both the naturally occurring sequences as well as mutant forms. Likewise, the proteins of the invention encompass both naturally occurring proteins as well as variations and modified forms thereof. Such variants will continue to possess the desired ability to degrade or catabolize fumonisin. Obviously, the mutations that will be made in the DNA encoding the variant must not place the sequence out of reading frame and preferably will not create complementary regions that could produce secondary mRNA structure. See, EP Patent Application Publication No. 75,444.

The deletions, insertions, and substitutions of the protein sequences encompassed herein are not expected to produce radical changes in the characteristics of the protein. However, when it is difficult to predict the exact effect of the substitution, deletion, or insertion in advance of doing so, one skilled in the art will appreciate that the effect will be evaluated by routine screening assays. That is, the activity can be evaluated by a decrease or loss in the toxic activity of fumonisin or AP1.

Variant nucleotide sequences and proteins also encompass sequences and proteins derived from a mutagenic and recombinogenic procedure such as DNA shuffling. With such a procedure, one or more different fumonisin-degrading coding sequences can be manipulated to create a new fumonisin-catabolizing possessing the desired properties. In this manner, libraries of recombinant polynucleotides are generated from a population of related sequence polynucleotides comprising sequence regions that have substantial sequence identity and can be homologously recombined in vitro or in vivo. For example, using this approach, sequence motifs encoding a domain of interest may be shuffled between the fumonisin-degrading genes of the invention and other known fumonisin-catabolizing genes to obtain a new gene coding for a protein with an improved property of interest, such as an increased K m in the case of an enzyme. Strategies for such DNA shuffling are known in the art. See, for example, Stemmer (1994) Proc. Natl. Acad. Sci. USA 91:10747-10751; Stemmer (1994) Nature 370:389-391; Crameri et al. (1997) Nature Biotech. 15:436-438; Moore et al. (1997) J. Mol. Biol. 272:336-347; Zhang et al. (1997) Proc. Natl. Acad. Sci. USA 94:4504-4509; Crameri et al. (1998) Nature 391:288-291; and U.S. Pat. Nos. 5,605,793 and 5,837,458.

The carboxylesterase and amine oxidase have been previously described in U.S. Pat. No. 5,716,820 and pending U.S. patent application Ser. Nos. 08/888,949 and 08/888,950. Such disclosures are herein incorporated by reference. Thus, the sequences of the invention can be used in combination with those previously disclosed or disclosed in co-pending application Ser. Nos. 09/352,168 and 09/352,159, entitled “Amino Polyolamine Oxidase Polynucleotides and Related Polypeptides and Methods of Use”, herein incorporated by reference. The enzymes and nucleotide sequences of the present invention provide a means for continued catabolism of the fumonisin-degradation products obtained after degradation with at least the carboxylesterase and amine oxidase.

The practice of the present invention will employ, unless otherwise indicated, conventional techniques of botany, microbiology, tissue culture, molecular biology, chemistry, biochemistry, and recombinant DNA technology, which are within the skill of the art. Such techniques are explained fully in the literature. See, e.g., Langenheim and Thimann, (1982) Botany: Plant Biology and Its Relation to Human Affairs (John Wiley); Vasil, ed. (1984) Cell Culture and Somatic Cell Genetics of Plants, Vol. 1; Stanier et al. (1986) The Microbial World (5th ed., Prentice-Hall); Dhringra and Sinclair (1985) Basic Plant Pathology Methods (CRC Press); Maniatis et al. (1982) Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.); Glover, ed. (1985) DNA Cloning, Vols. I and II; Gait, ed. (1984) Oligonucleotide Synthesis; Hames and Higgins, eds. (1984) Nucleic Acid Hybridization; and the series Methods in Enzymology (Colowick and Kaplan, eds., Academic Press, Inc.).

In describing the present invention, the following terms will be employed, and are intended to be defined as indicated below.

By “microbe” is meant any microorganism (including both eukaryotic and prokaryotic microorganisms), such as fungi, yeast, bacteria, actinomycetes, algae, and protozoa, as well as other unicellular structures.

A “fumonisin-producing microbe” is any microbe capable of producing the mycotoxin fumonisin or analogues thereof. Such microbes are generally members of the fungal genus Fusarium, as well as recombinantly derived organisms that have been genetically altered to enable them to produce fumonisin or analogues thereof.

By “degrading or catabolizing fumonisin” is meant any modification to the fumonisin or AP1 molecule that causes a decrease or loss in its toxic activity. Such a change can comprise cleavage of any of the various bonds, oxidation, reduction, the addition or deletion of a chemical moiety, or any other change that affects the activity of the molecule. In a preferred embodiment, the modification includes hydrolysis of the ester linkage in the molecule as a first step and then oxidative deamination. Furthermore, chemically altered fumonisin can be isolated from cultures of microbes that produce an enzyme of this invention, such as by growing the organisms on media containing radioactively-labeled fumonisin, tracing the label, and isolating the degraded toxin for further study. The degraded fumonisin can be compared to the active compound for its phytotoxicity or mammalian toxicity in known sensitive species, such as porcines and equines. Such toxicity assays are known in the art. For example, in plants a whole leaf bioassay can be used in which solutions of the active and inactive compound are applied to the leaves of sensitive plants. The leaves may be treated in situ or, alternatively, excised leaves may be used. The relative toxicity of the compounds can be estimated by grading the ensuing damage to the plant tissues and by measuring the size of lesions formed within a given time period. Other known assays can be performed at the cellular level, employing standard tissue culture methodologies, e.g., using cell suspension cultures.

For purposes of the invention, the fumonisin or fumonisin degradation products will be degraded to at least about 50% to about 10% or less of the original toxicity, preferably about 30% to about 5% or less, more preferably about 20% to about 1% or less.

By “fumonisin esterase” is meant any enzyme capable of hydrolysis of the ester linkage in fumonisin. Two examples of such enzymes are ESP1 and BEST1 found in U.S. Pat. No. 5,716,820 and pending U.S. application Ser. Nos. 08/888,949 and 08/888,950, both filed Jul. 7, 1997.

By “structurally related mycotoxin” is meant any mycotoxin having a chemical structure related to a fumonisin such as fumonisin B1, for example AAL toxin, fumonisin B2, fumonisin B3, fumonisin B4, fumonisin C1, fumonisin A1 and A2, and their analogues, as well as other mycotoxins having similar chemical structures that would be expected to be detoxified by activity of the fumonisin degradative enzymes elaborated by Exophiala spinifera, American Type Culture Collection Accession No. 74269, Rhinocladiella atrovirens, American Type Culture Collection Accession No. 74270, or the bacterium of American Type Culture Collection Accession No. 55552.

By “amplified” is meant the construction of multiple copies of a nucleic acid sequence or multiple copies complementary to the nucleic acid sequence using at least one of the nucleic acid sequences as a template. Amplification systems include the polymerase chain reaction (PCR) system, ligase chain reaction (LCR) system, nucleic acid sequence based amplification (NASBA, Cangene, Mississauga, Ontario), Q-Beta Replicase systems, transcription-based amplification system (TAS), and strand displacement amplification (SDA). See, e.g., Persing et al., ed. (1993) Diagnostic Molecular Microbiology: Principles and Applications (American Society for Microbiology, Washington, D.C.). The product of amplification is termed an amplicon.

By “host cell” is meant a cell that contains a vector and supports the replication and/or expression of the expression vector. Host cells may be prokaryotic cells such as E. coli, or eukaryotic cells such as yeast, insect, amphibian, or mammalian cells. Preferably, host cells are monocotyledonous or dicotyledonous plant cells, including but not limited to maize, sorghum, sunflower, soybean, wheat, alfalfa, rice, cotton, and tomato. A particularly preferred monocotyledonous host cell is a maize host cell.

The term “hybridization complex” includes reference to a duplex nucleic acid structure formed by two single-stranded nucleic acid sequences selectively hybridized with each other.

As used herein, “operably linked” includes reference to a functional linkage between a promoter and a second sequence, wherein the promoter sequence initiates and mediates transcription of the DNA sequence corresponding to the second sequence. Generally, operably linked means that the nucleic acid sequences being linked are contiguous and, where necessary to join two protein coding regions, contiguous and in the same reading frame.

As used herein, “polynucleotide” includes reference to a deoxyribopolynucleotide, ribopolynucleotide, or analogues thereof that have the essential nature of a natural ribonucleotide in that they hybridize, under stringent hybridization conditions, to substantially the same nucleotide sequence as naturally occurring nucleotides and/or allow translation into the same amino acid(s) as the naturally occurring nucleotide(s). A polynucleotide can be full-length or a subsequence of a native or heterologous structural or regulatory gene. Unless otherwise indicated, the term includes reference to the specified sequence as well as the complementary sequence thereof. Thus, DNAs or RNAs with backbones modified for stability or for other reasons are “polynucleotides” as that term is intended herein. Moreover, DNAs or RNAs comprising unusual bases, such as inosine, or modified bases, such as tritylated bases, to name just two examples, are polynucleotides as the term is used herein. It will be appreciated that a great variety of modifications have been made to DNA and RNA that serve many useful purposes known to those of skill in the art. The term polynucleotide as it is employed herein embraces such chemically, enzymatically, or metabolically modified forms of polynucleotides, as well as the chemical forms of DNA and RNA characteristic of viruses and cells, including inter alia, simple and complex cells.

As used herein, “promoter” includes reference to a region of DNA upstream from the start of transcription and involved in recognition and binding of RNA polymerase and other proteins to initiate transcription. A “plant promoter” is a promoter capable of initiating transcription in plant cells. Exemplary plant promoters include, but are not limited to, those that are obtained from plants, plant viruses, and bacteria that comprise genes expressed in plant cells, such as Agrobacterium or Rhizobium. Examples are promoters that preferentially initiate transcription in certain tissues, such as leaves, roots, seeds, fibers, xylem vessels, tracheids, or sclerenchyma. Such promoters are referred to as “tissue preferred”. A “cell type” specific promoter primarily drives expression in certain cell types in one or more organs, for example, vascular cells in roots or leaves. An “inducible” promoter is a promoter that is under environmental control. Examples of environmental conditions that may effect transcription by inducible promoters include anaerobic conditions or the presence of light. Another type of promoter is a developmentally regulated promoter. For example, a promoter that drives expression during pollen development. Tissue-preferred, cell type specific, developmentally regulated, and inducible promoters constitute the class of “non-constitutive” promoters. A “constitutive” promoter is a promoter that is active under most environmental conditions. Constitutive promoters are known in the art and include, for example, 35S promoter (Meyer et al. (1997) J. Gen. Virol. 78:3147-3151); ubiquitin; as well as those disclosed in U.S. Pat. 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.

As used herein, “recombinant” includes reference to a cell or vector that has been modified by the introduction of a heterologous nucleic acid or that the cell is derived from a cell so modified. Thus, for example, recombinant cells express genes that are not found in identical form within the native (nonrecombinant) form of the cell or express native genes that are otherwise abnormally expressed, underexpressed, or not expressed at all as a result of deliberate human intervention. The term “recombinant” as used herein does not encompass the alteration of the cell or vector by naturally occurring events (e.g., spontaneous mutation, natural transformation/transduction/transposition) such as those occurring without deliberate human intervention.

As used herein, a “recombinant expression cassette” is a nucleic acid construct, generated recombinantly or synthetically, with a series of specified nucleic acid elements that permit transcription of a particular nucleic acid in a target cell. The recombinant expression cassette can be incorporated into a plasmid, chromosome, mitochondrial DNA, plastid DNA, virus, or nucleic acid fragment. Typically, the recombinant expression cassette portion of an expression vector includes, among other sequences, a nucleic acid to be transcribed, and a promoter.

The nucleotide sequences of the invention can be used to isolate corresponding sequences from other organisms. In this manner, methods such as PCR, hybridization, and the like can be used to identify such sequences based on their sequence homology to the sequences set forth herein. Sequences isolated based on their sequence identity to the entire fumonisin-degrading sequences set forth herein or to fragments thereof are encompassed by the present invention.

In a PCR approach, oligonucleotide primers can be designed for use in PCR reactions to amplify corresponding DNA sequences from cDNA or genomic DNA extracted from any organism of interest. Methods for designing PCR primers and PCR cloning are generally known in the art and are disclosed in Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Plainview, N.Y.). See also Innis et al., eds. (1990) PCR Protocols: A Guide to Methods and Applications (Academic Press, New York); Innis and Gelfand, eds. (1995) PCR Strategies (Academic Press, New York); and Innis and Gelfand, eds. (1999) PCR Methods Manual (Academic Press, New York). Known methods of PCR include, but are not limited to, methods using paired primers, nested primers, single specific primers, degenerate primers, gene-specific primers, vector-specific primers, partially-mismatched primers, and the like.

In hybridization techniques, all or part of a known nucleotide sequence is used as a probe that selectively hybridizes to other corresponding nucleotide sequences present in a population of cloned genomic DNA fragments or cDNA fragments (i.e., genomic or cDNA libraries) from a chosen organism. The hybridization probes may be genomic DNA fragments, cDNA fragments, RNA fragments, or other oligonucleotides, and may be labeled with a detectable group such as 32 P, or any other detectable marker. Thus, for example, probes for hybridization can be made by labeling synthetic oligonucleotides based on the fumonisin-degrading sequences of the invention. Methods for preparation of probes for hybridization and for construction of cDNA and genomic libraries are generally known in the art and are disclosed in Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Plainview, N.Y.).

For example, the entire fumonisin-degrading sequences disclosed herein, or one or more portions thereof, may be used as a probe capable of specifically hybridizing to corresponding fumonisin-degrading sequences and messenger RNAs. To achieve specific hybridization under a variety of conditions, such probes include sequences that are unique among fumonisin-degrading sequences and are preferably at least about 10 nucleotides in length, and most preferably at least about 20 nucleotides in length. Such probes may be used to amplify corresponding fumonisin-degrading sequences from a chosen organism by PCR. This technique may be used to isolate additional coding sequences from a desired organism or as a diagnostic assay to determine the presence of coding sequences in an organism. Hybridization techniques include hybridization screening of plated DNA libraries (either plaques or colonies; see, for example, Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Plainview, N.Y.).

Hybridization of such sequences may be carried out under stringent conditions. By “stringent conditions” or “stringent hybridization conditions” is intended conditions under which a probe will hybridize to its target sequence to a detectably greater degree than to other sequences (e.g., at least 2-fold over background). Stringent conditions are sequence-dependent and will be different in different circumstances. By controlling the stringency of the hybridization and/or washing conditions, target sequences that are 100% complementary to the probe can be identified (homologous probing). Alternatively, stringency conditions can be adjusted to allow some mismatching in sequences so that lower degrees of similarity are detected (heterologous probing). Generally, a probe is less than about 1000 nucleotides in length, preferably less than 500 nucleotides in length.

Typically, stringent conditions will be those in which the salt concentration is less than about 1.5 M Na ion, typically about 0.01 to 1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30° C. for short probes (e.g., 10 to 50 nucleotides) and at least about 60° C. for long probes (e.g., greater than 50 nucleotides). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide. Exemplary low stringency conditions include hybridization with a buffer solution of 30 to 35% formamide, 1 M NaCl, 1% SDS (sodium dodecyl sulphate) at 37° C., and a wash in 1× to 2× SSC (20×SSC=3.0 M NaCl/0.3 M trisodium citrate) at 50 to 55° C. Exemplary moderate stringency conditions include hybridization in 40 to 45% formamide, 1.0 M NaCl, 1% SDS at 37° C., and a wash in 0.5× to 1×SSC at 55 to 60° C. Exemplary high stringency conditions include hybridization in 50% formamide, 1 M NaCl, 1% SDS at 37° C., and a wash in 0.1×SSC at 60 to 65° C.

Specificity is typically the function of post-hybridization washes, the critical factors being the ionic strength and temperature of the final wash solution. For DNA-DNA hybrids, the T m can be approximated from the equation of Meinkoth and Wahl (1984) Anal. Biochem. 138:267-284: T m =81.5° C.+16.6 (log M)+0.41 (%GC)−0.61 (% form)−500/L; where M is the molarity of monovalent cations, %GC is the percentage of guanosine and cytosine nucleotides in the DNA, % form is the percentage of formamide in the hybridization solution, and L is the length of the hybrid in base pairs. The T m is the temperature (under defined ionic strength and pH) at which 50% of a complementary target sequence hybridizes to a perfectly matched probe. T m is reduced by about 1° C. for each 1% of mismatching; thus, T m , hybridization, and/or wash conditions can be adjusted to hybridize to sequences of the desired identity. For example, if sequences with ≧90% identity are sought, the T m can be decreased 10° C. Generally, stringent conditions are selected to be about 5° C. lower than the thermal melting point (T m ) for the specific sequence and its complement at a defined ionic strength and pH. However, severely stringent conditions can utilize a hybridization and/or wash at 1, 2, 3, or 4° C. lower than the thermal melting point (T m ); moderately stringent conditions can utilize a hybridization and/or wash at 6, 7, 8, 9, or 10° C. lower than the thermal melting point (T m ); low stringency conditions can utilize a hybridization and/or wash at 11, 12, 13, 14, 15, or 20° C. lower than the thermal melting point (T m ). Using the equation, hybridization and wash compositions, and desired T m , those of ordinary skill will understand that variations in the stringency of hybridization and/or wash solutions are inherently described. If the desired degree of mismatching results in a T m of less than 45° C. (aqueous solution) or 32° C. (formamide solution), it is preferred to increase the SSC concentration so that a higher temperature can be used. An extensive guide to the hybridization of nucleic acids is found in Tijssen (1993) Laboratory Techniques in Biochemistry and Molecular Biology—Hybridization with Nucleic Acid Probes, Part I, Chapter 2 (Elsevier, New York); and Ausubel et al., eds. (1995) Current Protocols in Molecular Biology, Chapter 2 (Greene Publishing and Wiley-Interscience, New York). See Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Plainview, N.Y.).

In general, sequences that encode for a fumonisin-degradative protein and hybridize to the fumonisin-degrading sequences disclosed herein will be at least 40% to 50% homologous, about 60% to 70% homologous, and even about 80%, 85%, 90%, 95% to 98% homologous or more with the disclosed sequences. That is, the sequence similarity of sequences may range, sharing at least about 40% to 50%, about 60% to 70%, and even about 80%, 85%, 90%, 95% to 98% sequence similarity.

The following terms are used to describe the sequence relationships between two or more nucleic acids or polynucleotides: (a) “reference sequence”, (b) “comparison window”, (c) “sequence identity”, (d) “percentage of sequence identity”, and (e) “substantial identity”.

(a) As used herein, “reference sequence” is a defined sequence used as a basis for sequence comparison. A reference sequence may be a subset or the entirety of a specified sequence; for example, as a segment of a full-length CDNA or gene sequence, or the complete CDNA or gene sequence.

(b) As used herein, “comparison window” makes reference to a contiguous and specified segment of a polynucleotide sequence, wherein the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. Generally, the comparison window is at least 20 contiguous nucleotides in length, and optionally can be 30, 40, 50, 100, or longer. Those of skill in the art understand that to avoid a high similarity to a reference sequence due to inclusion of gaps in the polynucleotide sequence a gap penalty is typically introduced and is subtracted from the number of matches.

Methods of alignment of sequences for comparison are well known in the art. Optimal alignment of sequences for comparison may be conducted by the local homology algorithm of Smith et al. (1981) Adv. Appl. Math. 2:482; by the homology alignment algorithm of Needleman et al. (1970) J. Mol. Biol. 48:443; by the search for similarity method of Pearson et al. (1988) Proc. Natl. Acad. Sci. 85:2444; by computerized implementations of these algorithms, including, but not limited to: CLUSTAL in the PC/Gene program by Intelligenetics, Mountain View, Calif.; GAP, BESTFIT, BLAST, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group (GCG), 575 Science Drive, Madison, Wis., USA; the CLUSTAL program is well described by Higgins et al. (1988) Gene 73:237-244 (1988); Higgins et al. (1989) CABIOS 5:151-153; Corpet et al. (1988) Nucleic Acids Res. 16:10881-90; Huang et al. (1992) Computer Applications in the Biosciences 8:155-65, and Person et al. (1994) Meth. Mol. Biol. 24:307-331; preferred computer alignment methods also include the BLASTP, BLASTN, and BLASTX algorithms (see Altschul et al. (1990) J. Mol. Biol. 215:403-410). Alignments are performed using the default parameters of the above mentioned programs. Alignment is also often performed by inspection and manual alignment.

(c) As used herein, “sequence identity” or “identity” in the context of two nucleic acid or polypeptide sequences makes reference to the residues in the two sequences that are the same when aligned for maximum correspondence over a specified comparison window. When percentage of sequence identity is used in reference to proteins it is recognized that residue positions which are not identical often differ by conservative amino acid substitutions, where amino acid residues are substituted for other amino acid residues with similar chemical properties (e.g., charge or hydrophobicity) and therefore do not change the functional properties of the molecule. When sequences differ in conservative substitutions, the percent sequence identity may be adjusted upwards to correct for the conservative nature of the substitution. Sequences that differ by such conservative substitutions are said to have “sequence similarity” or “similarity”. Means for making this adjustment are well known to those of skill in the art. Typically this involves scoring a conservative substitution as a partial rather than a fill mismatch, thereby increasing the percentage sequence identity. Thus, for example, where an identical amino acid is given a score of 1 and a non-conservative substitution is given a score of zero, a conservative substitution is given a score between zero and 1. The scoring of conservative substitutions is calculated, e.g., as implemented in the program PC/GENE (Intelligenetics, Mountain View, Calif.).

(d) As used herein, “percentage of sequence identity” means the value determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison, and multiplying the result by 100 to yield the percentage of sequence identity.

(e)(i) The term “substantial identity” of polynucleotide sequences means that a polynucleotide comprises a sequence that has at least 70% sequence identity, preferably at least 80%, more preferably at least 90%, and most preferably at least 95%, compared to a reference sequence using one of the alignment programs described using standard parameters. One of skill in the art will recognize that these values can be appropriately adjusted to determine corresponding identity of proteins encoded by two nucleotide sequences by taking into account codon degeneracy, amino acid similarity, reading frame positioning, and the like. Substantial identity of amino acid sequences for these purposes normally means sequence identity of at least 60%, more preferably at least 70%, 80%, 90%, and most preferably at least 95%.

Another indication that nucleotide sequences are substantially identical is if two molecules hybridize to each other under stringent conditions. Generally, stringent conditions are selected to be about 5° C. lower than the thermal melting point (T m ) for the specific sequence at a defined ionic strength and pH. However, stringent conditions encompass temperatures in the range of about 1° C. to about 20° C., depending upon the desired degree of stringency as otherwise qualified herein. Nucleic acids that do not hybridize to each other under stringent conditions are still substantially identical if the polypeptides they encode are substantially identical. This may occur, e.g., when a copy of a nucleic acid is created using the maximum codon degeneracy permitted by the genetic code. One indication that two nucleic acid sequences are substantially identical is when the polypeptide encoded by the first nucleic acid is immunologically cross reactive with the polypeptide encoded by the second nucleic acid.

(e)(ii) The term “substantial identity” in the context of a peptide indicates that a peptide comprises a sequence with at least 70% sequence identity to a reference sequence, preferably 80%, more preferably 85%, most preferably at least 90% or 95% sequence identity to the reference sequence over a specified comparison window. Preferably, optimal alignment is conducted using the homology alignment algorithm of Needleman et al. (1970) J. Mol. Biol. 48:443. An indication that two peptide sequences are substantially identical is that one peptide is immunologically reactive with antibodies raised against the second peptide. Thus, a peptide is substantially identical to a second peptide, for example, where the two peptides differ only by a conservative substitution. Peptides that are “substantially similar” share sequences as noted above except that residue positions that are not identical may differ by conservative amino acid changes.

As indicated, the enzymes and nucleotide sequences encoding such enzymes are involved in the degradation of fumonisin and fumonisin-like compounds. Such enzymes and nucleotide sequences can be utilized alone or in combination to engineer microbes or other organisms to metabolize fumonisin and resist its toxic effects.

Fumonisin is produced in the intercellular spaces (apoplast) of Fusarium-infected maize cells. Thus, the apoplast is the preferred location for esterase and deaminase, flavin amine oxidase and possibly other catabolic enzymes. It is possible that some fumonisin could diffuse or be transported into the maize cells before it is broken down by the apoplastic enzymes and may escape catabolism. Thus, it may be beneficial to express a fumonisin pump and reroute the fumonisin or degradation products in such cells. In this manner, any fumonisin entering the cell will be pumped out and reexposed to catabolic enzymes. Similar toxin pumps exist in other toxin-producing fungi that show resistance to toxins or antibiotics. Such a pump useful in the invention and disclosed herein is a P-glycoprotein homolog.

More complete catabolism of fumonisin in transgenic organisms may be provided by esterase and deaminase enzymes. Exophiala enzymes that can further oxidize fumonisin breakdown-products are not detected extracellularly. Such enzymes in all likelihood exist in the cytoplasm, where adequate cofactors such as NAD + or NADP are found. The fumonisin-induced metabolite transporter is predicted to provide transport of degradation products into cells where they can be further broken down by other enzymes. In this manner, a permease enzyme may be utilized in a heterologous system to transport either AP1 precursors or fumonisin degradation products into the cytoplasm.

The monooxygenase is expected to result in the oxidation of 2-OP to a compound that lacks a keto group, having instead a terminal aldehyde group, or possibly a carboxylate group. See, for example, Trudgill et al. (1984) in Microbial Degradation of Organic Compounds, ed. Gibson (Microbiology Series Vol. 13, Marcel Dekker, New York), Chapter 6; and Davey and Trudgill (1977) Eur. J. Biochem. 74:115.

This reaction is due to a type of enzymatic oxidation referred to as Baeyer-Villiger oxidation, in which monooxygen is inserted adjacent to a keto function, resulting in a lactone or ester linkage. The metabolism of trans-cyclohexane-1,2 diol by Acinetobacter provides a model for the activity of a Baeyer-Villiger monooxygenase on 2-OP. This diol is first oxidized to ortho hydroxy cyclohexanone and then a monooxygen is inserted between the quinone and hydroxy functions by the Baeyer-Villiger enzyme, cyclohexanone monooxygenase. This intermediate spontaneously rearranges to a linear aldehyde carboxylic acid. By analogy, for 2-OP it is predicted oxygen is inserted between carbons 2 and 3 followed by spontaneous cleavage to a C22 aldehyde and acetic acid. Further oxidation by an aldehyde dehydrogenase would convert this compound to a carboxylic acid; other catabolic products would also be possible given the high reactivity of the aldehyde group. Additional steps include the use of an aldehyde dehydrogenase to result in the oxidation of the aldehyde product of fumonisin to a hydroxy carboxylic acid.

It is recognized that the DNA sequences of the invention can be inserted into expression cassettes and used to transform a variety of organisms. Enzymes produced recombinantly may be tested for their ability to modify fumonisin or a fumonisin byproduct using labeled starting material and appropriate buffer and cofactor conditions. For example, to test aldehyde dehydrogenase activity, the aldehyde dehydrogenase produced in a recombinant manner would be incubated with cofactors, NAD+ or NADP, and 14 C-labeled 2-OP for various times and then an aliquot of the reaction mix spotted on TLC. Enzyme activity would be indicated by the appearance of a new radiolabeled spot at a different Rf on the TLC plate.

The sequences of the invention can be introduced into any host organism. The sequences to be introduced may be used in expression cassettes for expression in the host of interest where expression in the host is necessary for transcription.

Where expression cassettes are needed, such expression cassettes will comprise a transcriptional initiation region linked to the coding sequence or antisense sequence of the nucleotide of interest. Such an expression cassette is provided with a plurality of restriction sites for insertion of the sequence to be under the transcriptional regulation of the regulatory regions. The expression cassette may additionally contain selectable marker genes.

The marker gene confers a selectable phenotype on the transformed cells. Usually, the selectable marker gene will encode antibiotic resistance, with suitable genes including genes coding for resistance to the antibiotic spectinomycin (e.g., the aada gene), the streptomycin phosphotransferase (SPT) gene coding for streptomycin resistance, the neomycin phosphotransferase (NPTII) gene encoding kanamycin or geneticin resistance ; the hygromycin phosphotransferase (HPT) gene coding for hygromycin resistance, genes coding for resistance to herbicides which act to inhibit the action of acetolactate synthase (ALS), in particular the sulfonylurea-type herbicides (e.g., the acetolactate synthase (ALS) gene containing mutations leading to such resistance in particular the S4 and/or Hra mutations), genes coding for resistance to herbicides which act to inhibit action of glutamine synthase, such as phosphinothricin or basta (e.g., the bar gene), or other such genes known in the art. The bar gene encodes resistance to the herbicide basta, and the ALS gene encodes resistance to the herbicide chlorsulfuron.

The transcriptional initiation region, the promoter, may be native or analogous or foreign or heterologous to the host as well as to the coding sequence. Additionally, the promoter may be the natural sequence or alternatively a synthetic sequence. By foreign is intended that the transcriptional initiation region is not found in the native plant into which the transcriptional initiation region is introduced. As used herein a chimeric gene comprises a coding sequence operably linked to a transcription initiation region that is heterologous to the coding sequence.

The transcriptional cassette will include in the 5′-to-3′ direction of transcription, a transcriptional and translational initiation region, a DNA sequence of interest, and a transcriptional and translational termination region functional in the host. 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. For use in plants or plant cells, convenient termination regions are available from the Ti-plasmid of A. tumefaciens, 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 Acids Res. 15:9627-9639.

Nucleotide sequences of the invention are provided in expression cassettes for expression in the host cell of interest. The cassette will include 5′ and 3′ regulatory sequences operably linked to the sequence of interest. The cassette may additionally contain at least one additional sequence to be cotransformed into the organism. Alternatively, the additional sequence(s) can be provided on another expression cassette.

Where appropriate, the gene(s) may be optimized for increased expression in the transformed plant. That is, the genes can be synthesized using plant-preferred codons for improved expression. See, for example, Campbell and Gowri (1990) Plant Physiol. 92:1-11 for a discussion of host-preferred codon usage. Methods are available in the art for synthesizing plant-preferred genes. See, for example, U.S. Pat. Nos. 5,380,831, 5,436,391, and Murray et al. (1989) Nucleic Acids Res. 17:477-498, herein incorporated by reference.

Additional sequence modifications are known to enhance gene expression in a cellular host. These include elimination of sequences encoding spurious polyadenylation signals, exon-intron splice site signals, transposon-like repeats, and other such well-characterized sequences that may be deleterious to gene expression. The G-C content of the sequence may be adjusted to levels average for a given cellular host, as calculated by reference to known genes expressed in the host cell. When possible, the sequence is modified to avoid predicted hairpin secondary mRNA structures.

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 et al. (1989) PNAS USA 86:6126-6130); potyvirus 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 et al. (1991) Nature 353:90-94); untranslated leader from the coat protein mRNA of alfalfa mosaic virus (AMV RNA 4) (Jobling et al. (1987) Nature 325:622-625); tobacco mosaic virus leader (TMV) (Gallie et al. (1989) in Molecular Biology of RNA, ed. Cech (Liss, New York), pp. 237-256); and maize chlorotic mottle virus leader (MCMV) (Lommel et al. (1991) Virology 81:382-385). See also, Della-Cioppa et al. (1987) Plant Physiol. 84:965-968. Other methods known to enhance translation can also be utilized, for example, introns, and the like.

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. Toward 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, resubstitutions, e.g., transitions and transversions, may be involved.

In the same manner, a plant can be transformed with the nucleotide sequences of the invention to provide complete detoxification of fumonisin in the transformed plant and plant products. Such plants include, for example, species from the genera Cucurbita, Rosa, Vitis, Juglans, Fragaria, Lotus, Medicago, Onobrychis, Trifolium, Trigonella, Vigna, Citrus, Linum, Geranium, Manihot, Daucus, Arabidopsis, Brassica, Raphanus, Sinapis, Atropa, Capsicum, Datura, Hyoscyamus, Lycopersicon, Nicotiana, Solanum, Petunia, Digitalis, Majorana, Ciahorium, Helianthus, Lactuca, Bromus, Asparagus, Antirrhinum, Heterocallis, Nemesis, Pelargonium, Panieum, Pennisetum, Ranunculus, Senecio, Salpiglossis, Cucumis, Browaalia, Glycine, Pisum, Phaseolus, Lolium, Oryza, Zea, Avena, Hordeum, Secale, Triticum, Sorghum, Picea, Caco, and Populus.

As used herein, “transgenic plant” includes reference to a plant that comprises within its genome a heterologous polynucleotide. Generally, the heterologous polynucleotide is stably integrated within the genome such that the polynucleotide is passed on to successive generations. The heterologous polynucleotide may be integrated into the genome alone or as part of a recombinant expression cassette. “Transgenic” is used herein to include any cell, cell line, callus, tissue, plant part or plant, the genotype of which has been altered by the presence of heterologous nucleic acid including those transgenics initially so altered as well as those created by sexual crosses or asexual propagation from the initial transgenic. The term “transgenic” as used herein does not encompass the alteration of the genome (chromosomal or extra-chromosomal) by conventional plant breeding methods or by naturally occurring events such as random cross-fertilization, nonrecombinant viral infection, nonrecombinant bacterial transformation, nonrecombinant transposition, or spontaneous mutation.

Transformation protocols as well as protocols for introducing nucleotide sequences into plants may vary depending on the type of plant or plant cell, i.e., monocot or dicot, targeted for transformation. Suitable methods of introducing nucleotide sequences into plant cells and subsequent insertion into the plant genome 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 (Townsend et al., U.S. Pat. No. 5,563,055), direct gene transfer (Paszkowski et al. (1984) EMBO J. 3:2717-2722), and ballistic particle acceleration (see, for example, Sanford et al., U.S. Pat. No. 4,945,050; Tomes et al. (1995) “Direct DNA Transfer into Intact Plant Cells via Microprojectile Bombardment,” in Plant Cell, Tissue, and Organ Culture: Fundamental Methods, ed. Gamborg and Phillips (Springer-Verlag, Berlin); and McCabe et al. (1988) Biotechnology 6:923-926). Also see Weissinger et al. (1988) Ann. 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); Finer and McMullen (1991) In vitro Cell Dev. Biol. 27P: 175-182 (soybean); Singh et al. (1998) Theor. Appl. Genet. 96:319-324 (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); Tomes, U.S. Pat. No. 5,240,855; Buising et al., U.S. Pat. Nos. 5,322,783 and 5,324,646; Tomes et al. (1995) “Direct DNA Transfer into Intact Plant Cells via Microprojectile Bombardment,” in Plant Cell, Tissue, and Organ Culture: Fundamental Methods, ed. Gamborg (Springer-Verlag, Berlin) (maize); Klein et al. (1988) Plant Physiol. 91:440-444 (maize); Fromm et al. (1990) Biotechnology 8:833-839 (maize); Hooykaas-Van Slogteren et al. (1984) Nature ( London ) 311:763-764; Bowen et al., U.S. Pat. No. 5,736,369 (cereals); 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. Chapman et al. (Longman, New York), pp. 197-209 (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 modified plant 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.

The degradative enzymes can be fermented in a bacterial host and the resulting bacteria processed and used as a microbial spray. Any suitable microorganism can be used for this purpose. See, for example, Gaertner et al. (1993) in Advanced Engineered Pesticides, Kim (Ed.).

The genes of the invention can be introduced into microorganisms that multiply on plants (epiphytes) to deliver enzymes to potential target crops. Epiphytes can be gram-positive or gram-negative bacteria, for example.

The microorganisms that have been genetically altered to contain at least one degradative gene and protein may be used for protecting agricultural crops and products. In one aspect of the invention, whole, i.e., unlysed, cells of the transformed organism are treated with reagents that prolong the activity of the enzyme produced in the cell when the cell is applied to the environment of a target plant. A secretion signal sequence may be used in combination with the gene of interest such that the resulting enzyme is secreted outside the host cell for presentation to the target plant.

Plant signal sequences, including, but not limited to, signal-peptide encoding DNA/RNA sequences which target proteins to the extracellular matrix of the plant cell (Dratewka-Kos et al., (1989) J. Biol. Chem. 264:4896-4900), the Nicotiana plumbaginifolia extension gene (DeLoose, et al. (1991) Gene 99:95-100), signal peptides which target proteins to the vacuole like the sweet potato sporamin gene (Matsuka et al. (1991) PNAS 88:834) and the barley lectin gene (Wilkins et al. (1990) Plant Cell 2:301-313), signal peptides which cause proteins to be secreted such as that of PRIb (Lind et al. (1992) Plant Mol. Biol. 18:47-53), or the barley alpha amylase (BAA) (Rahmatullah et al. (1989) Plant Mol. Biol. 12:119) and hereby incorporated by reference, or from the present invention the signal peptide from the ESP1 or BEST1 gene, or signal peptides which target proteins to the plastids such as that of rapeseed enoyl-Acp reductase (Verwaert et al. (1994) Plant Mol. Biol. 26:189-202) are useful in the invention.

In this manner, at least one of the genes encoding a degradation enzyme of the invention may be introduced via a suitable vector into a microbial host, and said transformed host applied to the environment or plants or animals. Microorganism hosts that are known to occupy the “phytosphere” (phylloplane, phyllosphere, rhizosphere, and/or rhizoplane) of one or more crops of interest may be selected for transformation. These microorganisms are selected so as to be capable of successfully competing in the particular environment with the wild-type microorganisms, to provide for stable maintenance and expression of the gene expressing the polypeptide pesticide, and, desirably, to provide for improved protection of the enzymes of the invention from environmental degradation and inactivation.

Such microorganisms include bacteria, algae, and fungi. Of particular interest are microorganisms, such as bacteria, e.g., Pseudomonas, Erwinia, Serratia, Klebsiella, Xanthomonas, Streptomyces, Rhizobium, Rhodopseudomonas, Methylius, Agrobacterium, Acetobacter, Lactobacillus, Arthrobacter, Azotobacter, Leuconostoc, and Alcaligenes; fungi, particularly yeast, e.g., Saccharomyces, Pichia, Cryptococcus, Kluyveromyces, Sporobolomyces, Rhodotorula, and Aureobasidium. Of particular interest are such phytosphere bacterial species as Pseudomonas syringae, Pseudomonas fluorescens, Serratia marcescens, Acetobacter xylinum, Agrobacteria, Rhodopseudomonas spheroides, Xanthomonas campestris, Rhizobium melioti, Alcaligenes entrophus, Clavibacter xyli, and Azotobacter vinlandii; and phytosphere yeast species such as Rhodotorula rubra, R. glutinis, R. marina, R. aurantiaca, Cryptococcus albidus, C. diffluens, C. laurentii, Saccharomyces rosei, S. pretoriensis, S. cerevisiae, Sporobolomyces rosues, S. odorus, Kluyveromyces veronae, and Aureobasidium pullulans.

Illustrative prokaryotes, both Gram-negative and -positive, include Enterobacteriaceae, such as Escherichia, Erwinia, Shigella, Salmonella, and Proteus; Bacillaceae; Rhizobiaceae, such as Rhizobium; Spirillaceae, such as photobacterium, Zymomonas, Serratia, Aeromonas, Vibrio, Desulfovibrio, Spirillum; Lactobacillaceae; Pseudomonadaceae, such as Pseudomonas and Acetobacter; Azotobacteraceae; and Nitrobacteraceae. Among eukaryotes are fungi, such as Phycomycetes and Ascomycetes, which includes yeast, such as Saccharomyces and Schizosaccharomyces; and Basidiomycetes yeast, such as Rhodotorula, Aureobasidium, Sporobolomyces, and the like.

Characteristics of particular interest in selecting a host cell for purposes of production include ease of introducing the protein gene into the host, availability of expression systems, efficiency of expression, stability of the protein in the host, and the presence of auxiliary genetic capabilities. Other considerations include ease of formulation and handling, economics, storage stability, and the like.

A number of ways are available for introducing a gene expressing the degradation enzyme into the microorganism host under conditions that allow for stable maintenance and expression of the gene. For example, expression cassettes can be constructed that include the DNA constructs of interest operably linked with the transcriptional and translational regulatory signals for expression of the DNA constructs, and a DNA sequence homologous with a sequence in the host organism, whereby integration will occur, and/or a replication system that is functional in the host, whereby integration or stable maintenance will occur.

Transcriptional and translational regulatory signals include but are not limited to promoter, transcriptional initiation start site, operators, activators, enhancers, other regulatory elements, ribosomal binding sites, an initiation codon, termination signals, and the like. See, for example, U.S. Pat. Nos. 5,039,523 and 4,853,331; EPO 0480762A2; Sambrook et al. supra; Maniatis et al., eds. (1982) Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.); Davis et al., eds. (1980) Advanced Bacterial Genetics (Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.); and the references cited therein.

It is recognized that the construction of a catabolic pathway in a transformed organism is a complicated feat. Therefore, any means for assembling the enzymes of interest into an organism of interest is encompassed. For example, a single nucleotide sequence encoding all of the desired enzymes or multiples thereof may be transformed into the host organism. When microorganisms are to be applied to the environment or to a plant, several microorganisms, each transformed with one, two, three, or more nucleotide sequences of the invention, may be utilized. In this manner, all of the enzymes necessary to bring about detoxification of fumonisin and related products may be presented to the environment or to the plant by applying a mixture of transformed organisms or a single organism capable of expressing the entire pathway or at least expressing enough of the pathway to detoxify fumonisin.

In plants, nucleotide sequences for an enzyme may be transformed into a plant and crossed with plants expressing a different enzyme. In this manner, progeny can be obtained having the entire sequence or enough of the sequence to detoxify fumonisin. Alternatively, a plant can be transformed with nucleotides encoding several enzymes at the same time. In some tissue culture systems it is possible to transform callus with one nucleotide sequence, establish a stable culture line, and then transform the callus a second time with a second nucleotide sequence. The process may be repeated to introduce additional sequences.

To facilitate the expression of more than one enzyme in a cell, e.g. a plant cell, fusion proteins may be created. Generally, a spacer region is included between the proteins. The spacer region may comprise a cleavage site for cleavage by an endogenous or introduced protease.

The present invention also relates to a method of detoxifying a fumonisin or a structurally related mycotoxin with the enzymes from Exophiala spinifera (American Type Culture Collection Accession No. 74269), during the processing of grain for animal or human food consumption, during the processing of plant material for silage, or in food crops contaminated with a toxin-producing microbe, such as but not limited to, tomato. Since the atmospheric ammoniation of corn has proven to be an ineffective method of detoxification (see Haumann (1995) INFORM 6:248-257), such a methodology during processing is particularly critical where transgenic detoxification is not applicable.

In this embodiment, the fumonisin degradative enzymes found in Exophiala spinifera (American Type Culture Collection Accession No. 74269), are presented to grain, plant material for silage, or a contaminated food crop, or during the processing procedure, at the appropriate stages of the procedure and in amounts effective for detoxification of fumonisins and structurally related mycotoxins. Detoxification by this method can occur not only during the processing, but also any time prior to or during the feeding of the grain or plant material to an animal or incorporation of the grain or food crop into a human food product, or before or during ingestion of the food crop. The enzymes or microorganisms can be introduced during processing in appropriate manners, for example, as a wash or spray, or in dried or lyophilized form or powered form, depending upon the nature of the milling process and/or the stage of processing at which the enzymatic treatment is carried out. See generally, Hoseney, R.C. (1990) Principles of Cereal Science and Technology, American Assn. of Cereal Chemists, Inc. (especially Chapters 5, 6 and 7); Jones, J. M. (1992) Food Safety, Eagan Press, St. Paul, Minn. (especially Chapters 7 and 9); and Jelen, P. (1985) Introduction to Food Processing, Restan Publ. Co., Reston, Va. Processed grain or silage to be used for animal feed can be treated with an effective amount of the enzymes in the form of an inoculant or probiotic additive, for example, or in any form recognized by those skilled in the art for use in animal feed. The enzymes of the present invention are expected to be particularly useful in detoxification during processing and/or in animal feed prior to its use, since the enzymes display relatively broad ranges of pH activity. The esterase from Exophiala spinifera (American Type Culture Collection Accession No. 74269), showed a range of activity from about pH 3 to about pH 6, and the esterase from the bacterium of the American Type Culture Collection Accession No. 55552 showed a range of activity from about pH 6 to about pH 9 (U.S. Pat. No. 5,716,820, supra). The APAO enzyme from Exophiala spinifera (American Type Culture Collection Accession No. 74269) has a pH range of activity from pH 6 to pH 9.

The active ingredients of the present invention are normally applied in the form of compositions and can be applied to the crop area or plant to be treated, simultaneously or in succession, with other compounds. These compounds can be both fertilizers or micronutrient donors or other preparations that influence plant growth. They can also be selective herbicides, insecticides, fungicides, bactericides, nematicides, mollusicides, or mixtures of several of these preparations, if desired, together with further agriculturally acceptable carriers, surfactants, or application-promoting adjuvants customarily employed in the art of formulation. Suitable carriers and adjuvants can be solid or liquid and correspond to the substances ordinarily employed in formulation technology, e.g., natural or regenerated mineral substances, solvents, dispersants, wetting agents, tackifiers, binders, or fertilizers.

The enzymes can be introduced during processing in appropriate manners, for example as a wash or spray, or in dried or lyophilized form or powered form, depending upon the nature of the milling process and/or the stage of processing at which the enzymatic treatment is carried out. See generally, Hoseney (1990) Principles of Cereal Science and Technology (American Association of Cereal Chemists, Inc.), especially Chapters 5, 6, and 7; Jones (1992) Food Safety (Eagan Press, St. Paul, Minn.), especially Chapters 7 and 9; and Jelen (1985) Introduction to Food Processing (Restan Publishing Company, Reston, Va.). Processed grain or silage to be used for animal feed can be treated with an effective amount of the enzymes in the form of an inoculant or probiotic additive, for example, or in any form recognized by those skilled in the art for use in animal feed. The enzymes of the present invention are expected to be particularly useful in detoxification during processing and/or in animal feed prior to its use, since the enzymes display relatively broad ranges of pH activity. The enzymes from Exophiala spinifera, American Type Culture Collection Accession No. 74269, showed a range of activity for esterase from about pH 3 to about pH 7 (U.S. Pat. No. 5,716,820, supra). The APAO enzyme from Exophiala spinifera, American Type Culture Collection Accession No. 74269, has a pH range of activity from pH 6 to pH 9.

In another embodiment, ruminal microorganisms can be genetically engineered to contain and express at least one of the fumonisin degradation enzymes of the invention. The genetic engineering of microorganisms is now an art-recognized technique, and ruminal microorganisms so engineered can be added to feed in any art-recognized manner, for example as a probiotic or inoculant. In addition, microorganisms, plants, or other organisms or their cultured cells in vitro capable of functioning as bioreactors can be engineered so as to be capable of mass producing the degradative enzymes of Exophiala spinifera (American Type Culture Collection Accession No. 74269).

Another embodiment of the present invention is the use of the enzymes of the present invention as detection reagents for fumonisins and related compounds. The enzymes of the present invention can be used as detection reagents because of the high specificity of the esterase and deaminase enzymes, and the fact that hydrolysis followed by amine oxidation can be monitored by detection of hydrogen peroxide or ammonia using standard reagents (analogous to a glucose detection assay using glucose oxidase). Hydrogen peroxide is often measured by linking a hydrogen peroxide-dependent peroxidase reaction to a colored or otherwise detectable peroxidase product (e.g., Demmano et al. (1996) European Journal of Biochemistry 238(3):785-789). Ammonia can be measured using ion-specific electrodes: Fritsche et al. (1991) Analytica Chimica Acta 244(2):179-182; West et al. (1992) Analytical Chemistry 64(5):533-540, and all herein incorporated by reference) or by GC or other chromatographic method.

For example, recombinant or non-recombinant, active fumonisin esterase, APAO, and proteins of the invention are added in catalytic amounts to a sample tube containing an unknown amount of fumonisins (FB1, FB2, FB3, FB4, or partial or complete hydrolysis products of these). The tube is incubated under pH and temperature conditions sufficient to convert any fumonisin in the sample to AP1, the AP1 to 2-OP, ammonia, and hydrogen peroxide, and to further degradation products. Then suitable reagents are added for quantification of the hydrogen peroxide or ammonia that were generated stoichiometrically from fumonisins. By comparison with control tubes that received no esterase or APAO enzyme, the amount of fumonisin present can be calculated in direct molar proportion to the hydrogen peroxide or ammonia detected, relative to a standard curve.

This invention can be better understood by reference to the following nonlimiting examples. It will be appreciated by those skilled in the art that other embodiments of the invention may be practiced without departing from the spirit and the scope of the invention as herein disclosed and claimed.

Experimental

EXAMPLE 1

Fungal and Bacterial Isolates

Exophiala isolates from maize were isolated as described in U.S. Pat. No. 5,716,820 and pending U.S. application Ser. Nos. 08/888,949 and 08/888,950, both filed Jul. 7, 1997, and herein incorporated by reference.

Isolation Methods

Direct isolation of black yeasts from seed was accomplished by plating 100 microliters of seed wash fluid onto YPD or Sabouraud agar augmented with cycloheximide (500 mg/liter) and chloramphenicol (50 mg/liter). Plates were incubated at room temperature for 7-14 days, and individual pigmented colonies that arose were counted and cultured for analysis of fumonisin-degrading ability as described above.

Analysis of Fumonisins and Metabolism Products

Analytical thin-layer chromatography was carried out on 100% silanized C 18 silica plates (Sigma #T-7020; 10×10 cm; 0.1 mm thick) by a modification of the published method of Rottinghaus (Rottinghaus et al. (1992) J. Vet. Diagn. Invest. 4:326, and herein incorporated by reference).

To analyze fumonisin esterase activity, sample lanes were pre-wet with methanol to facilitate sample application. After application of from 0.1 to 2 μl of aqueous sample, the plates were air-dried and developed in MeOH:4% KCl (3:2) or MeOH:0.2 M KOH (3:2) and then sprayed successively with 0.1 M sodium borate (pH 9.5) and fluorescence (0.4 mg/ml in acetonitrile). Plates were air-dried and viewed under long-wave UV.

For analysis of APAO activity, an alternative method was used. Equal volumes of sample and 14 C-AP1 (1 mg/ml, pH 8) substrate were incubated at room temperature for six days. Analytical thin-layer chromatography was then carried out on C60 HPK silica gel plates (Whatman #4807-700; 10×10 cm; 0.2 mm thick). After application of from 0.1 to 2 μl of aqueous sample, the plates were air dried and developed in CHCl 3 :MeOH:CH 3 COOH:H 2 O (55:36:8:1). Plates were then air dried and exposed to PhosphorImager screen or autoradiographic film. A Storm PhosphorImager was used to scan the image produced on the screen.

Alkaline Hydrolysis of FB1 to AP1

FB1 or crude fumonisin C 8 material was suspended in water at 10-100 mg/ml and added to an equal volume of 4 N NaOH in a screw-cap tube. The tube was sealed and incubated at 60° C. for 1 hr. The hydrolysate was cooled to room temperature and mixed with an equal volume of ethyl acetate, centrifuged at 1000 RCF for 5 minute and the organic (upper) layer recovered. The pooled ethyl acetate layers from two successive extractions were dried under N 2 and resuspended in distilled H 2 O. The resulting material (the aminopentol of FB1 or “AP1”) was analyzed by TLC.

Enzyme Activity of Culture Filtrate and Mycelium

Exophiala spinifera isolate 2141.10 was grown on YPD agar for 1 week, and conidia were harvested, suspended in sterile water, and used at 105 conidia per ml to inoculate sterile Fries mineral salts medium containing 1 mg/ml purified FB1 (Sigma Chemical Co.). After 2 weeks incubation at 28° C. in the dark, cultures were filtered through 0.45 micron cellulose acetate filters and rinsed with Fries mineral salts. Fungal mycelium was suspended in 15 mL of 0.1% FB1, pH 5.2+1 mM EDTA+3 μg/mL Pepstatin A+1.5 μg/mL Leupeptin and disrupted in a Bead Beater™ using 0.1 mm beads and one minute pulses, with ice cooling. Hyphal pieces were collected by filtering through Spin X™ (0.22 μm), and both mycelial supernatant and original culture filtrates were assayed for fumonisin modification by methods outlined above.

Preparation of Crude Culture Filtrate

Agar cultures grown as above were used to inoculate YPD broth cultures (500 ml) in conical flasks at a final concentration of 105 conidia per ml culture. Cultures were incubated 5 days at 28° C. without agitation and mycelia harvested by filtration through 0.45 micron filters under vacuum. The filtrate was discarded, and the mycelial mat was washed and resuspended in sterile carbon-free, mineral salts medium (1 g/liter NH 3 NO 4 ; 1 g/liter NaH 2 PO 4 ; 0.5 g/liter MgCl 2 ; 0.1 g/liter NaCl; 0.13 g/liter CaCl 2 ; 0.02 g/liter FeSO 4 .7H 2 O, pH 4.5) containing 0.5 mg/ml alkaline hydrolyzed crude FB1. After 3-5 days at 28° C. in the dark with no agitation the cultures were filtered through low protein binding 0.45 micron filters to recover the culture filtrate. Phenylmethyl sulfonyl fluoride (PMSF) was added to a concentration of 2.5 mM and the culture filtrate was concentrated using an Amicon™ YM10 membrane in a stirred cell at room temperature and resuspended in 50 mM sodium acetate, pH 5.2 containing 10 mM CaCl 2 . The crude culture filtrate (approx. 200-fold concentrated) was stored at −20° C.

To obtain preparative amounts of enzyme-hydrolyzed fumonisin, 10 mg of FB1 (Sigma) was dissolved in 20 mL of 50 mM sodium acetate at pH 5.2+10 mM CaCl 2 , and 0.25 mL of 200× concentrated crude culture filtrate of 2141.10 was added. The solution was incubated at 37° C. for 14 hours, and then cooled to room temperature. The reaction mixture was brought to approximately pH 9.5 by addition of 0.4 mL of 4 N KOH, and the mixture was extracted twice with 10 mL ethyl acetate. The combined organic layers were dried under LN 2 and resuspended in dH 2 O. 2.5 milligrams of organic extracted material were analyzed by Fast Atom Bombardment (FAB) mass spectrometry. The resulting mass spectrum showed a major ion at M/z (+1)=406 mass units, indicating the major product of enzymatic hydrolysis was AP1, which has a calculated molecular weight of 405.

EXAMPLE 2

Preparation of AP1-induced and Non-induced Mycelium

Liquid cultures of Exophiala spinifera isolate 2141.10 were prepared from YPD agar plates (Yeast Extract 10 gm, Bacto-Peptone 20 gm, Dextrose 0.5 gm, Bacto-Agar 15 gm per liter of water). Aliquots (400-500 uL) of a water suspension of E. spinifera cells from YPD agar were spread uniformly onto 150×15 mm YPD agar plates with 4 mm sterile glass beads. The plates were incubated at room temperature for 6-7 days. The mycelia/conidia were transferred from the agar plates into Mineral Salts Medium (MSM) (Na 2 HPO 4 .7H 2 O 0.2 gm, NH 4 Cl 1.0 gm, CaCl 2 .2H 2 O 0.01 gm, FeSO 4 .7H 2 O 0.02 gm per liter of distilled water, pH 4.5) and centrifuged at 5000×g, 4° C., 20 minutes to pellet the cells. The cell pellet was rinsed once in 40 mL MSM and recentrifuged. The rinsed cell pellet was used to inoculate MSM at a 1:19 ratio of packed cells:MSM. The culture was supplemented with AP1 to a final concentration of 0.5-1.0 mg/ml and incubated at 28° C., 100 rpm, in the dark to induce catabolic enzymes. The supernatants were removed by filtration through 0.45 cellulose acetate. The remaining mycelial mat was washed with sterile MSM and then frozen in liquid nitrogen for storage.

EXAMPLE 3

Effect of FB1 and AP1 on Maize Coleoptiles

Maize coleoptiles from 4 day dark-grown germinated maize seeds were excised above the growing point and placed in 96-well microliter plates in the presence of 60 microliters of sterile distilled water containing FB1 or AP1 at approximately equimolar concentrations of 1.5, 0.5, 0.15, 0.05, 0.015, 0.005, 0.0015, or 0.0005 millimolar, along with water controls. After 2 days in the dark at 28° C. the coleoptiles were placed in the light and incubated another 3 days. Injury or lack thereof was evaluated as follows:

	0	.0005	.0015	.005	.015	.05	.15	.5	1.5	mM
FB1	−	−	−	−	+/−	+	+	+	+
AP1	−	−	−	−	−	−	−	−	+
+ = brown necrotic discoloration of coleoptile
− = no symptoms (same as water control)

The results (see table above) indicate there is at least a 30-fold difference in toxicity between FB1 and AP1 to maize coleoptiles of this genotype. This is in general agreement with other studies where the toxicity of the two compounds was compared for plant tissues. In Lemna tissues, AP1 was approximately 40-fold less toxic (Vesonder et al. (1992) Arch. Environ. Contam. Toxicol. 23:464-467 (1992)). Studies with both AAL toxin and FB1 in tomato also indicate the hydrolyzed version of the molecule is much less toxic (Gilchrist et al. (1992) Mycopathologia 117: 57-64). Lamprecht et al. also observed an approximate 100-fold reduction in toxicity to tomato by AP1 versus FB1 (Lamprecht et al. (1994) Phytopathology 84:383-391).

EXAMPLE 4

Effect of FB1 and AP1 on Maize Tissue Cultured Cells (Black Mexican Sweet, BMS)

FB1 or AP1 at various concentrations was added to suspensions of BMS cells growing in liquid culture medium in 96-well polystyrene plates. After 1 week the cell density in wells was observed under low power magnification and growth of toxin-treated wells was compared to control wells that received water. Growth of BMS cells was significantly inhibited at 0.4 micromolar FB1, but no inhibition was observed until 40 micromolar AP1. This represents an approximate 100-fold difference in toxicity to maize tissue-cultured cells. Similarly Van Asch et al. observed significant inhibition of maize callus grown on solid medium at 1.4 micromolar FB1 (Van Asch et al. (1992) Phytopathology 82:1330-1332). AP1 was not tested in that study, however.

EXAMPLE 5

The polynucleotides were identified using a proprietary transcript imaging method that compares transcript patterns in two samples and allows cloning of differentially expressed fragments. This technology was developed by CuraGen® (New Haven, Conn.) (see Published PCT Patent Application No. WO 97/15690, published May 1, 1997, and hereby incorporated by reference). Fluorescently-tagged, PCR amplified cDNA fragments representing expressed transcripts can be visualized as bands or peaks on a gel tracing, and the cDNA from differentially expressed (induced or suppressed) bands can be recovered from a duplicate gel, cloned, and sequenced. Known cDNAs can be identified without the need for cloning, by matching the predicted size and partially known sequence of specific bands on the tracing.

Two RNA samples were obtained from cultures of E. spinifera grown for a specified period in a mineral salts medium containing either AP1 (induced condition) or gamma-aminobutyric acid (ABA; non-induced condition) as a sole carbon source. In the induced condition, fumonisin esterase, amine oxidase, enzyme activities are detected, whereas in the non-induced condition these activities are not detected. The methods used for induction of and detection of enzyme activity are described earlier (see Example 2 and Example 5). RNA was extracted from induced mycelium by Tri-Reagent methods (Molecular Research Center Inc., Cincinnati, Ohio) only using frozen tissue samples ground with a mortar and pestle 2-fold and up to 79-fold and greater until slushy and adding an additional extraction after the phase separation by extracting the aqueous phase one time with phenol, and two times with a phenol:chloroform:isoamyl alcohol mixture. The RNAs were submitted for CuraGen® transcript imaging to detect cDNA fragments that are induced specifically in the presence AP1. In the resulting gel tracing several bands were found which showed induction of at least 10-fold in AP1-grown cells as compared to cells grown in ABA. One set of induced fragments can be matched to the fumonisin esterase cDNA. The cloned bands and possible functions are provided in Table 2. Highly induced bands and their likely function are provided in Tables 2 and 3.

TABLE 2
	Best BLAST
Clone ID	Hit	BLAST Hit Name, source, size	Prob	from-to	Function
Monooxygenase
M1a0-388	A28550	cyclohexanone monooxygenase, Acinetobacter	1.4e − 22	339-414	Baeyer-Villiger oxidation of
		(flavin monooxygenase or FMO)EC 1.14.13.22			2-OP1 (AP1-N1), utilizing
		Length = 543			molecular oxygen and
					reduced NADPH
					Or NADH
Aldehyde dehydrogenase (EC 1.)
k0n0-235	Y09876	Aldehyde dehydrogenase (Nicotiana tabacum);	1.1e − 07	152-191	Oxidation of aldehyde
passed		Length = 542			product of FMO to
					carboxylic acid
Permease
r0v0-239	S64084	Choline transport protein, yeast	9.3e − 0.5	337-397	Transport of 2-OP1 into the
		Length = 563			cytoplasm
r0w0-424	S51169	amino acid transporter AAP4-Arabidopsis	0.98	8-76	Transport of 2-OP1 into the
w0h0-268		thalina			cytoplasm
		len = 466
r0w0-205	P53744	KAPA/DAPA permease, yeast BIO5	2.1e − 07	446-488	Transport of 2-OP1 into the
p0t0-308		Length = 561			cytoplasm
(contig)
Transmembrane pump (P-glycoprotein homolog)
r0g1-420	S20548	Leptomycin resistance protein, pmd1,	1.8e − 37	1255-1359	Transmembrane pump that
		Schizosaccharomyces pombe.		or	removes FB1 from the
		Length = 1362		564-668	cytoplasm as a means of
					protection against its toxic
					activity
g0s0-142		Leptomycin resistance protein, pmd1,		527-588	Transmembrane pump that
		Schizosaccharomyces pombe.			removes FB1 from the
		Length = 1362			cytoplasm as a means of
					protection against its toxic
					activity
l0c0-129		Leptomycin resistance protein, pmd1,
		Schizosaccharomyces pombe.
		Length = 1362
r0s0-180		Leptomycin resistance protein, pmd1,		959-1009	Transmembrane pump that
		Schizosaccharomyces pombe.			removes FB1 from the
		Length = 1362			cytoplasm as a means of
					protection against its toxic
					activity
r0c0-193		Leptomycin resistance protein, pmd1,		885-945	Transmembrane pump that
		Schizosaccharomyces pombe.			removes FB1 from the
		Length = 1362			cytoplasm as a means of
					protection against its toxic
					activity
r0s0-330		Leptomycin resistance protein, pmd1,		1024-1110	Transmembrane pump that
		Schizosaccharomyces pombe.			removes FB1 from the
		Length = 1362			cytoplasm as a means of
					protection against its toxic
					activity
Loc0-129	S20548	Leptomycin resistance protein, pmd1,	.0082	949-988	Transmembrane pump that
		Schizosaccharomyces pombe.			removes FB1 from the
		Length = 1362			cytoplasm as a means of
					protection against its toxic
					activity
r0h1-262		Leptomycin resistance protein, pmd1,		1135-1218	Transmembrane pump that
		Schizosaccharomyces pombe.			removes FB1 from the
		Length = 1362			cytoplasm as a means of
					protection against its toxic
					activity
i0c0-116	e219956	ATP binding cassette multidrug transporter,		1026-1114	Transmembrane pump that
		Emericella nidulans Length = 1466			removes FB1 from the
					cytoplasm as a means of
					protection against its toxic
					activity

TABLE 3
Cloned	Homology,
Bands	Comments	Predicted function	Predicted Product
1. Transmembrane pump (P-glycoprotein homolog)
r0g1-420 g0s0-142 l0c0-129 r0s0-180 r0c0-193 r0s0-330 r0h1-262 i0c0-116	Homology to Leptomycin resis- tance protein, Pmd1, Schizosaccharo- myces pombe , Length = 1362, or other ABC trans- porter gene family member. { } All 9 bands show homology to mem- bers of the ABC transporter superfamily.	FB1 Pump: Trans- membrane pump that removes FB1 from the cytoplasm as a means of
# protection against its toxicity
2. Small Molecule Permease
r0v0-239 r0w0-205 p0t0-308.4 r0w0-424? w0h0-268?	Homology to choline transport protein, yeast Length = 563 { } Two bands (r0w0-205 and p0t0-308) contig with each other.	2-OP permease: Transport of 2- OP and/or AP1 into the cytoplasm
3. Flavin Monooxygenase (EC 1.14.13.22)
m1a0-388	Homology to cyclo- hexanone mono- oxygenase., Acinetobacter. Oxidation of ketone resulting in carbon-carbon bond breakage to form aldehyde. Utilizes NAD+ or NADP+	2-OP monooxy- genase: Intracellular oxidation of 2-OP1 to a hydroxy alde- hyde (HA-1) plus acetic acid
4. Aldehyde dehydrogenase
k0n0-235	Homology to alde- hyde dehydrogenase ( Nicotiana tabacum ); Length = 542	HA-1 deydrogense: Oxidation of aldehyde product of FMO to a hydroxy carboxylic acid (HCA-1)

Using sequence derived from each clone, a partial cDNA was obtained by 3′ and 5′ RACE-PCR (Chenchik et al. (1995) CLONTECHniquesX 1:5-8); Chenchik et al. (1996) in A Laboratory Guide to RNA: Isolation, Analysis, and Synthesis, ed. Krieg (Wiley-Liss, Inc.), pp. 273-321. A RACE cloning kit from Clontech was used to obtain the RACE amplicons. Briefly, poly A+ RNA is transcribed to make first strand cDNA using a “ock-docking” poly T, cDNA synthesis primer, the second strand is synthesized, and the Marathon cDNA adaptor is ligated to both ends of the ds cDNA. Diluted template is then used with the Marathon adapter primer and in separate reactions either a 5′ Gene Specific Primer (GSP) or a 3′GSP is used to produce the 3′ or 5′ RACE amplicon. After characterization of the RACE product(s) and sequencing, full-length cDNAs may be generated by 1) end-to-end PCR using distal 5′ and 3′ GSPs with the adapter-ligated ds cDNA as template, or 2) the cloned 5′ and 3′-RACE fragments may be digested with a restriction enzyme that cuts uniquely in the region of overlap, and the fragments isolated and ligated. Subsequently, the RACE-generated full-length cDNAs from 1) and 2) may be cloned into a suitable vector.

EXAMPLE 6

Pichia Expression of Degradative Enzymes

For cloning into Pichia pastoris expression vector, pPicZalphaA, oligonucleotide primers were designed that contain a 22 bp overlap of the 5′ end (sense strand) and 3′ end (antisense strand), respectively of the open reading frame of the degradative nucleotide of interest, including the stop codon. In addition, each oligo has a 5′ extension with digestible restriction sites that allows cloning of the amplified insert in-frame both into EcoRI/NotI digested pPicZalphaA. pPicZalphaA is an E. coli compatible Pichia expression vector containing a functional yeast alpha-factor secretion signal and peptide processing sites, allowing high efficiency, inducible secretion into the culture medium of Pichia. After the generation of the 5′ and 3′ RACE products, the resulting band was cloned into EcoRI/NotI digested pPicZalphaA plasmid.

Pichia can be transformed as described in Invitrogen Manual, Easy Select™ Pichia Expression Kit, Version B, #161219, with the enzyme polynucleotide of interest with either an intron (negative control, no expression) or without an intron (capable of making an active protein). The Pichia culture fluids and pellets are assayed for enzyme activity as described earlier. The six day culture fluids from the same cultures are used to spike with crude fungal enzyme for positive controls.

The sample 50 μl cell pellets are resuspended in 150 μl cold 50 mM Na-phosphate, pH8.0 and divided into two fresh 500 UL tubes. One tube is kept on ice with no treatment, the pellet suspension, and one tube is used for lysis. An equal volume of 0.1 mm zirconia-silica beads is added to each tube. The tubes are BeadBeat™ for 15 seconds then cooled on ice 5 minutes. This is repeated three times. The crude lysate is then transferred to another tube for assay or lysate suspension. The TLC assays are performed as follows:

1) pellet suspensions (“PELL”); 10 uL

2) lysate suspensions (“LYS”); 10 uL

3) media controls-mixed 5 uL media with 5 uL crude fungal enzyme (if available); 10 uL

4) positive control-used crude fungal enzyme undiluted; 10 uL

5) substrate control-used 50 mM Na-phosphate, pH8.0; 10 uL

cofactor (if required) is added to each reaction mixture

incubate 10 uL each sample+10 uL 14 C-substrate (fumonisin, metabolite, or other potential substrate) (1 mg/nL, pH8) at room temperature for 6 days

spot 1.0 uL onto C18 and C60 TLC plates

develop C18 plates in MeOH:4% KCl (3:2)

develop C60 plates in CHCl 3 :MeOH:CH 3 COOH:H 2 O (55:36:8:1)

air-dry plates

expose plates to PhosphorScreen 2-3 days

use Storm PhosphorImager (Molecular Dynamics) to develop images

EXAMPLE 7

Expression of Degradative Enzymes in E. coli

A vector for expressing the enzymes in E. coli is a prokaryotic glutathione S-transferase (GST) fusion vector for inducible, high-level intracellular expression of genes or gene fragments as fusions with Schistosoma japonicum GST. GST gene fusion vectors include the following features: a lac promoter for inducible, high-level expression; an internal lac Iq gene for use in any E. coli host; and the thrombin factor Xa or PreScission Protease recognition sites for cleaving the desired protein from the fusion product. The insert of interest is cloned into the 5′ EcoRI site and a 3′ NotI site allowing in-frame expression of the fusion peptide. The generation of such an insert is described in the previous example.

E. coli is transformed with the vector containing the coding sequence for the degradative enzyme as described in BRL catalogue, Life Technologies, Inc., catalogue; Hanahan (1983) J. Mol. Biol. 166:557; Jessee et al. (1984) J. Focus 6:4; King et al. (1986) Focus 8:1, and hereby incorporated by reference. The transformed E. coli is induced by addition of IPTG (isopropyl b-D-thiogalactopyranoside). Samples of soluble extract and Samples of insoluble inclusion bodies are tested for enzyme activity as described in Example 7.

EXAMPLE 8

Transformation and Regeneration of Transgenic Plants

Immature maize embryos from greenhouse donor plants are bombarded with a plasmid containing the fumonisin-degradation/transporter enzyme nucleotide sequences operably linked to a ubiquitin promoter (FIG. 2 ). This plasmid also contains the selectable marker gene PAT (Wohlleben et al. (1988) Gene 70:25-37) that confers resistance to the herbicide Bialaphos. The preferred construct for expression in maize is the nucleotide sequence of the degradative enzyme either fused to the barley alpha amylase signal sequence or organellar targeting sequence, or left intact for expression in the cytoplasm.Transformation is performed as follows. All media recipes are in the Appendix.

Preparation of Target Tissue

The ears are surface sterilized in 30% Chlorox bleach plus 0.5% Micro detergent for 20 minutes, and rinsed two times with sterile water. The immature embryos are excised and placed embryo axis side down (scutellum side up), 25 embryos per plate, on 560Y medium for 4 hours and then aligned within the 2.5-cm target zone in preparation for bombardment.

Preparation of DNA

A plasmid vector comprising the fumonisin-degradation/transporter enzyme operably linked to the ubiquitin promoter is made. This plasmid DNA also contains a PAT selectable marker. The plasmid is precipitated onto 1.1 μm (average diameter) tungsten pellets using a CaCl 2 precipitation procedure as follows:

100 μl prepared tungsten particles in water

10 μl (1 μg) DNA in TrisEDTA buffer (1 μg total)

100 μl 2.5 M CaCl 2

10 μl 0.1 M spermidine

Each reagent is added sequentially to the tungsten particle suspension, while maintained on the multitube vortexer. The final mixture is sonicated briefly and allowed to incubate under constant vortexing for 10 minutes. After the precipitation period, the tubes are centrifuged briefly, liquid removed, washed with 500 ml 100% ethanol, and centrifuged for 30 seconds. Again the liquid is removed, and 105 μl 100% ethanol is added to the final tungsten particle pellet. For particle gun bombardment, the tungsten/DNA particles are briefly sonicated and 10 μl spotted onto the center of each macrocarrier and allowed to dry about 2 minutes before bombardment.

Particle Gun Treatment

The sample plates are bombarded at level #4 in particle gun #HE34-1 or #HE34-2. All samples receive a single shot at 650 PSI, with a total of ten aliquots taken from each tube of prepared particles/DNA.

Subsequent Treatment

Following bombardment, the embryos are kept on 560Y medium for 2 days, then transferred to 560R selection medium containing 3 mg/liter Bialaphos, and subcultured every 2 weeks. After approximately 10 weeks of selection, selection-resistant callus clones are transferred to 288J medium to initiate plant regeneration. Following somatic embryo maturation (2-4 weeks), well-developed somatic embryos are transferred to medium for germination and transferred to the lighted culture room. Approximately 7-10 days later, developing plantlets are transferred to 272V hormone-free medium in tubes for 7-10 days until plantlets are well established. Plants are then transferred to inserts in flats (equivalent to 2.5″ pot) containing potting soil and grown for 1 week in a growth chamber, subsequently grown an additional 1-2 weeks in the greenhouse, then transferred to classic 600 pots (1.6 gallon) and grown to maturity. Plants are monitored for the expression of a fumonisin-degrading/transporter protein.

APPENDIX
272 V
	Ingredient	Amount	Unit
	D-I H 2 O	950.000	M1
	MS Salts (GIBCO 11117-074)	4.300	G
	Myo-Inositol	0.100	G
	MS Vitamins Stock Solution ##	5.000	M1
	Sucrose	40.000	G
	Bacto-Agar @	6.000	G
	Directions:
	@ = Add after bringing up to volume
	Dissolve ingredients in polished D-I H 2 O in sequence
	Adjust to pH 5.6
	Bring up to volume with polished D-I H 2 O after adjusting pH
	Sterilize and cool to 60° C.
	## = Dissolve 0.100 g of Nicotinic Acid; 0.020 g of Thiamine.HCL; 0.100 g of Pyridoxine.HCL; and 0.400 g of Glycine in 875.00 ml of polished D-I H 2 O in sequence. Bring up to volume with polished D-I H 2 O. Make in 400 ml portions. Thiamine.HCL & Pyridoxine.HCL are in Dark Desiccator. Store for one month, unless contamination or precipitation occurs, then make fresh stock.
	Total Volume (L) = 1.00

288 J
	Ingredient	Amount	Unit
	D-I H 2 O	950.000	M1
	MS Salts	4.300	g
	Myo-Inositol	0.100	g
	MS Vitamins Stock Solution ##	5.000	ml
	Zeatin .5 mg/ml	1.000	ml
	Sucrose	60.000	g
	Gelrite @	3.000	g
	Indoleacetic Acid 0.5 mg/ml #	2.000	ml
	0.1 mM Abscisic Acid	1.000	ml
	Bialaphos 1 mg/ml #	3.000	ml
	Directions:
	@ = Add after bringing up to volume
	Dissolve ingredients in polished D-I H 2 O in sequence
	Adjust to pH 5.6
	Bring up to volume with polished D-I H 2 O after adjusting pH
	Sterilize and cool to 60° C.
	Add 3.5 g/L of Gelrite for cell biology.
	## = Dissolve 0.100 g of Nicotinic Acid; 0.020 g of Thiamine.HCL; 0.100 g of Pyridoxine.HCL; and 0.400 g of Glycine in 875.00 ml of polished D-I H 2 O in sequence. Bring up to volume with polished D-I H 2 O. Make in 400 ml portions. Thiamine.HCL & Pyridoxine.HCL are in Dark Desiccator. Store for one month, unless contamination or precipitation occurs, then make fresh stock.
	Total Volume (L) = 1.00

560 R
Ingredient	Amount	Unit
D-I Water, Filtered	950.000	ml
CHU (N6) Basal Salts (SIGMA C-1416)	4.000	g
Eriksson's Vitamin Mix (1000X SIGMA-1511	1.000	ml
Thiamine.HCL 0.4 mg/ml	1.250	ml
Sucrose	30.000	g
2,4-D 0.5 mg/ml	4.000	ml
Gelrite @	3.000	g
Silver Nitrate 2 mg/ml #	0.425	ml
Bialaphos 1 mg/ml #	3.000	ml
Directions:
@ = Add after bringing up to volume
# = Add after sterilizing and cooling to temp.
Dissolve ingredients in D-I H 2 O in sequence
Adjust to pH 5.8 with KOH
Bring up to volume with D-I H 2 O
Sterilize and cool to room temp.
Total Volume (L) = 1.00

560 Y
Ingredient	Amount	Unit
D-I Water, Filtered	950.000	ml
CHU (N6) Basal Salts (SIGMA C-1416)	4.000	g
Eriksson's Vitamin Mix (1000X SIGMA-1511	1.000	ml
Thiamine.HCL 0.4 mg/ml	1.250	ml
Sucrose	120.000	g
2,4-D 0.5 mg/ml	2.000	ml
L-Proline	2.880	g
Gelrite @	2.000	g
Silver Nitrate 2 mg/ml #	4.250	ml
Directions:
@ = Add after bringing up to volume
# = Add after sterilizing and cooling to temp.
Dissolve ingredients in D-I H 2 O in sequence
Adjust to pH 5.8 with KOH
Bring up to volume with D-I H 2 O
Sterilize and cool to room temp.
**Autoclave less time because of increased sucrose**
Total Volume (L) = 1.00

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.

11 1 1691 DNA Exophiala spinifera misc_feature (0)...(0) flavin monooxygenase with intron 1atgtcggcca ccagcaactc cagaggcgat tgttccgtcg catgcgacgc catcatcgtt 60ggagccggcc tcagcggcat ctctgctgtg tacaaattgc gaaagctcag actcaacgcc 120aaaatcttcg agggagcccc cgattttggc ggcgtctggc actggaaccg ctaccctggc 180gctcgtgttg attcggagac gcccttctac caactgaaca ttcccgaagt atggaaagac 240tggacctggt cttgccgcta tcctgaccag aaagagttgc tgtcatatgt tcaccactgt 300gacaagatcc ggggcttgag aaaagacgtc tacttcggag ctgaggtggt tgatgcgcgg 360tatgccagag atctgggcac ctggactgtc aagacgtcgg ctggccatgt tgcgacggca 420aagtatctca ttctcgctac ggggttgctc cacaggaagc acactcccgc actccccggc 480ctcgccgatt tcaacgggaa ggtgattcat tcgagtgcct ggcacgaaga cttcgacgca 540gagggccaga gagtcgccgt catcggtgcc ggggccacaa gcatccagat tgttcaggag 600ttggccaaga aggctgacca ggtaaccatg tttatgcgaa ggccgagcta ttgtctgccc 660atgcggcaac gaacgatgga taggaacgaa cagacagcct ggaaggccta ctaccccacg 720ctgtttgaag cgagtcgaaa gtctcggatt ggattcccgg tccaggcacc gtcggttggc 780atctttgaag tcagccccga gcagcgggag gcctatttcg aagagttgtg ggagcgtggg 840gcctttaatt ttcttgcttg ccagtaccga gaagtcatgg ttgacaaaaa ggccaaccga 900ctggtctatg acttctgggc caaaaagact cgatctcgta tcgtcaatcc ggcaaagaga 960gatctcatgg ctcctctgga gccgccgtac tggttcggta ccaagcgctc cccactggag 1020agcgactact acgaaatgct ggacaagccg agcgtcgaaa ttgtgaatct agaacaatcg 1080cccattgtgg ctgttacaaa gacaggtgtg ctcttgagtg acggcagcaa gagggaatgc 1140gacacgatcg tgctggcgac gggtttcgac agtttcactg gctcgtgagt gtgctcgatc 1200atggctccga gtccggacgt ttggctgacc ttgaaagatt gacacatatg ggcttgaaaa 1260acaagcacgg agtggacctg aaggaggtgt ggaaagatgg catatctact tatatgggag 1320tcttctctca tggcttcccc aatgccttct tcgtcgccac ggctcaagcc ccgaccgtcc 1380tttccaacgg cccaacgatc atagaaaccc aagtcgactt gatcgccgat acaattgcaa 1440agttggaggc cgagcacgcc acgtccgttg aggcgacgaa atcagcacaa gaggcatggt 1500cgattatgat tgccaagatg aacgagcaca ctctgttccc cttgacggat tcgtggtgga 1560ctggaggcaa catccctggg aaagcaacac gtgctttaac cttcataggc gggattgctc 1620tctatgagca gatctgtcaa gagaaggtgg ccaattggga tgggtttgat gtgcttcatg 1680ctccctgcta a 1691 2 1638 DNA Exophiala spinifera misc_feature (0)...(0) flavin monooxygenase, fully spliced 2atgtcggcca ccagcaactc cagaggcgat tgttccgtcg catgcgacgc catcatcgtt 60ggagccggcc tcagcggcat ctctgctgtg tacaaattgc gaaagctcag actcaacgcc 120aaaatcttcg agggagcccc cgattttggc ggcgtctggc actggaaccg ctaccctggc 180gctcgtgttg attcggagac gcccttctac caactgaaca ttcccgaagt atggaaagac 240tggacctggt cttgccgcta tcctgaccag aaagagttgc tgtcatatgt tcaccactgt 300gacaagatcc ggggcttgag aaaagacgtc tacttcggag ctgaggtggt tgatgcgcgg 360tatgccagag atctgggcac ctggactgtc aagacgtcgg ctggccatgt tgcgacggca 420aagtatctca ttctcgctac ggggttgctc cacaggaagc acactcccgc actccccggc 480ctcgccgatt tcaacgggaa ggtgattcat tcgagtgcct ggcacgaaga cttcgacgca 540gagggccaga gagtcgccgt catcggtgcc ggggccacaa gcatccagat tgttcaggag 600ttggccaaga aggctgacca ggtaaccatg tttatgcgaa ggccgagcta ttgtctgccc 660atgcggcaac gaacgatgga taggaacgaa cagacagcct ggaaggccta ctaccccacg 720ctgtttgaag cgagtcgaaa gtctcggatt ggattcccgg tccaggcacc gtcggttggc 780atctttgaag tcagccccga gcagcgggag gcctatttcg aagagttgtg ggagcgtggg 840gcctttaatt ttcttgcttg ccagtaccga gaagtcatgg ttgacaaaaa ggccaaccga 900ctggtctatg acttctgggc caaaaagact cgatctcgta tcgtcaatcc ggcaaagaga 960gatctcatgg ctcctctgga gccgccgtac tggttcggta ccaagcgctc cccactggag 1020agcgactact acgaaatgct ggacaagccg agcgtcgaaa ttgtgaatct agaacaatcg 1080cccattgtgg ctgttacaaa gacaggtgtg ctcttgagtg acggcagcaa gagggaatgc 1140gacacgatcg tgctggcgac gggtttcgac agtttcactg gctcattgac acatatgggc 1200ttgaaaaaca agcacggagt ggacctgaag gaggtgtgga aagatggcat atctacttat 1260atgggagtct tctctcatgg cttccccaat gccttcttcg tcgccacggc tcaagccccg 1320accgtccttt ccaacggccc aacgatcata gaaacccaag tcgacttgat cgccgataca 1380attgcaaagt tggaggccga gcacgccacg tccgttgagg cgacgaaatc agcacaagag 1440gcatggtcga ttatgattgc caagatgaac gagcacactc tgttcccctt gacggattcg 1500tggtggactg gaggcaacat ccctgggaaa gcaacacgtg ctttaacctt cataggcggg 1560attgctctct atgagcagat ctgtcaagag aaggtggcca attgggatgg gtttgatgtg 1620cttcatgctc cctgctaa 1638 3 545 PRT Exophiala spinifera 3Met Ser Ala Thr Ser Asn Ser Arg Gly Asp Cys Ser Val Ala Cys Asp 1 5 10 15Ala Ile Ile Val Gly Ala Gly Leu Ser Gly Ile Ser Ala Val Tyr Lys 20 25 30Leu Arg Lys Leu Arg Leu Asn Ala Lys Ile Phe Glu Gly Ala Pro Asp 35 40 45Phe Gly Gly Val Trp His Trp Asn Arg Tyr Pro Gly Ala Arg Val Asp 50 55 60Ser Glu Thr Pro Phe Tyr Gln Leu Asn Ile Pro Glu Val Trp Lys Asp65 70 75 80Trp Thr Trp Ser Cys Arg Tyr Pro Asp Gln Lys Glu Leu Leu Ser Tyr 85 90 95Val His His Cys Asp Lys Ile Arg Gly Leu Arg Lys Asp Val Tyr Phe 100 105 110Gly Ala Glu Val Val Asp Ala Arg Tyr Ala Arg Asp Leu Gly Thr Trp 115 120 125Thr Val Lys Thr Ser Ala Gly His Val Ala Thr Ala Lys Tyr Leu Ile 130 135 140Leu Ala Thr Gly Leu Leu His Arg Lys His Thr Pro Ala Leu Pro Gly145 150 155 160Leu Ala Asp Phe Asn Gly Lys Val Ile His Ser Ser Ala Trp His Glu 165 170 175Asp Phe Asp Ala Glu Gly Gln Arg Val Ala Val Ile Gly Ala Gly Ala 180 185 190Thr Ser Ile Gln Ile Val Gln Glu Leu Ala Lys Lys Ala Asp Gln Val 195 200 205Thr Met Phe Met Arg Arg Pro Ser Tyr Cys Leu Pro Met Arg Gln Arg 210 215 220Thr Met Asp Arg Asn Glu Gln Thr Ala Trp Lys Ala Tyr Tyr Pro Thr225 230 235 240Leu Phe Glu Ala Ser Arg Lys Ser Arg Ile Gly Phe Pro Val Gln Ala 245 250 255Pro Ser Val Gly Ile Phe Glu Val Ser Pro Glu Gln Arg Glu Ala Tyr 260 265 270Phe Glu Glu Leu Trp Glu Arg Gly Ala Phe Asn Phe Leu Ala Cys Gln 275 280 285Tyr Arg Glu Val Met Val Asp Lys Lys Ala Asn Arg Leu Val Tyr Asp 290 295 300Phe Trp Ala Lys Lys Thr Arg Ser Arg Ile Val Asn Pro Ala Lys Arg305 310 315 320Asp Leu Met Ala Pro Leu Glu Pro Pro Tyr Trp Phe Gly Thr Lys Arg 325 330 335Ser Pro Leu Glu Ser Asp Tyr Tyr Glu Met Leu Asp Lys Pro Ser Val 340 345 350Glu Ile Val Asn Leu Glu Gln Ser Pro Ile Val Ala Val Thr Lys Thr 355 360 365Gly Val Leu Leu Ser Asp Gly Ser Lys Arg Glu Cys Asp Thr Ile Val 370 375 380Leu Ala Thr Gly Phe Asp Ser Phe Thr Gly Ser Leu Thr His Met Gly385 390 395 400Leu Lys Asn Lys His Gly Val Asp Leu Lys Glu Val Trp Lys Asp Gly 405 410 415Ile Ser Thr Tyr Met Gly Val Phe Ser His Gly Phe Pro Asn Ala Phe 420 425 430Phe Val Ala Thr Ala Gln Ala Pro Thr Val Leu Ser Asn Gly Pro Thr 435 440 445Ile Ile Glu Thr Gln Val Asp Leu Ile Ala Asp Thr Ile Ala Lys Leu 450 455 460Glu Ala Glu His Ala Thr Ser Val Glu Ala Thr Lys Ser Ala Gln Glu465 470 475 480Ala Trp Ser Ile Met Ile Ala Lys Met Asn Glu His Thr Leu Phe Pro 485 490 495Leu Thr Asp Ser Trp Trp Thr Gly Gly Asn Ile Pro Gly Lys Ala Thr 500 505 510Arg Ala Leu Thr Phe Ile Gly Gly Ile Ala Leu Tyr Glu Gln Ile Cys 515 520 525Gln Glu Lys Val Ala Asn Trp Asp Gly Phe Asp Val Leu His Ala Pro 530 535 540Cys545 4 1464 DNA Exophiala spinifera misc_feature (0)...(0) aldehyde dehydrogenase, fully spliced cDNA 4atggttcttt cgcctgacga atacaagagt gaactcttca tcaacaatga attcgtctcc 60tccaaggggt ccgagagatt aacgctcacg aacccgtggg acgaatccac cgttgccact 120gatgttcacg tggccaacgc ggccgatgtc gacagtgcag tagccgcttc ggtgcaggcg 180gtcaaaaagg gcccatggaa gaagttcaca ggtgcacaac gcgcggcgtg catgcttaag 240ttcgcggacc tcgccgagaa gaacgccgag aagctcgctc gtctggagtc gctgcccacc 300ggtagaccgg tgtcgatgat cactcatttc gacattccaa acatggtctc cgtgtttcgc 360tactatgcag gctgggccga caagatcgcc ggaaagacct ttcccgagga caacggcaag 420ccgaattggc gttacgagcc gatgggggtg tgtgctggta ttgccagctg gaacgcgact 480tttctttacg tcggctggaa gatagccccc gccctcgccg ccggctgctc cttcatcttc 540aaagcctcgg agaaatcccc gctgggcgtt ctgggcctcg ctcctctctt cgcagaagcc 600ggattccctc ctggagtcgt gcagttcctc actggagcac gagtgacggg tgaagcattg 660gcgtcgcaca tggacattgc gaagatcagc ttcacaagat ctgtcggcgg tggccgcgcc 720gtcaagcaag caacactcaa gtccaacatg aagcgcgtca ctctagaact gggggaaaag 780ccaaccatcg tcttcaacga agctcctctc gaacggcagt cgggggaatc ggcaaaggat 840ttctcaaaat tcgggcaaat ttgggtcccc ccctcctgtt tgctagtgca atggggaaat 900ttagcggaga aattccatgg agtccgtcat ggctcatttg gaggctgtca gagatggctt 960ggccagaacc cattggaacc caagaggacg catggtccct tcgtcgacaa gtcccagtac 1020gacagagtct tgggtaacat tgacgttggc aaggataccg cgcagctcct cactggcgtt 1080ggtagaaagg gcgacaaggg attcgcgatt gaaccgacga tatttgtcaa tcccaaacca 1140ggcagcaaaa tttggtttga ggagatcttt ggccccgtct tgtccattaa gacgttcaag 1200acggaagaag aggccattga gattgccaat gacacgactt atgggctagc ctcggtcatt 1260tataccaaat ctctcaacag gggtctccgt gtctcgtcgg cgctcgagac cggtggcgtc 1320tcgatcaact tcccctttat ccccgagaca caaactccgt ttggcggcat gaaacaatcg 1380ggctcaggca gagagctagg cgaagaaggg ctcaaggcgt acttggagcc caagaccatt 1440aatatccacg tcaacataga gtga 1464 5 487 PRT Exophiala spinifera 5Met Val Leu Ser Pro Asp Glu Tyr Lys Ser Glu Leu Phe Ile Asn Asn 1 5 10 15Glu Phe Val Ser Ser Lys Gly Ser Glu Arg Leu Thr Leu Thr Asn Pro 20 25 30Trp Asp Glu Ser Thr Val Ala Thr Asp Val His Val Ala Asn Ala Ala 35 40 45Asp Val Asp Ser Ala Val Ala Ala Ser Val Gln Ala Val Lys Lys Gly 50 55 60Pro Trp Lys Lys Phe Thr Gly Ala Gln Arg Ala Ala Cys Met Leu Lys65 70 75 80Phe Ala Asp Leu Ala Glu Lys Asn Ala Glu Lys Leu Ala Arg Leu Glu 85 90 95Ser Leu Pro Thr Gly Arg Pro Val Ser Met Ile Thr His Phe Asp Ile 100 105 110Pro Asn Met Val Ser Val Phe Arg Tyr Tyr Ala Gly Trp Ala Asp Lys 115 120 125Ile Ala Gly Lys Thr Phe Pro Glu Asp Asn Gly Lys Pro Asn Trp Arg 130 135 140Tyr Glu Pro Met Gly Val Cys Ala Gly Ile Ala Ser Trp Asn Ala Thr145 150 155 160Phe Leu Tyr Val Gly Trp Lys Ile Ala Pro Ala Leu Ala Ala Gly Cys 165 170 175Ser Phe Ile Phe Lys Ala Ser Glu Lys Ser Pro Leu Gly Val Leu Gly 180 185 190Leu Ala Pro Leu Phe Ala Glu Ala Gly Phe Pro Pro Gly Val Val Gln 195 200 205Phe Leu Thr Gly Ala Arg Val Thr Gly Glu Ala Leu Ala Ser His Met 210 215 220Asp Ile Ala Lys Ile Ser Phe Thr Arg Ser Val Gly Gly Gly Arg Ala225 230 235 240Val Lys Gln Ala Thr Leu Lys Ser Asn Met Lys Arg Val Thr Leu Glu 245 250 255Leu Gly Glu Lys Pro Thr Ile Val Phe Asn Glu Ala Pro Leu Glu Arg 260 265 270Gln Ser Gly Glu Ser Ala Lys Asp Phe Ser Lys Phe Gly Gln Ile Trp 275 280 285Val Pro Pro Ser Cys Leu Leu Val Gln Trp Gly Asn Leu Ala Glu Lys 290 295 300Phe His Gly Val Arg His Gly Ser Phe Gly Gly Cys Gln Arg Trp Leu305 310 315 320Gly Gln Asn Pro Leu Glu Pro Lys Arg Thr His Gly Pro Phe Val Asp 325 330 335Lys Ser Gln Tyr Asp Arg Val Leu Gly Asn Ile Asp Val Gly Lys Asp 340 345 350Thr Ala Gln Leu Leu Thr Gly Val Gly Arg Lys Gly Asp Lys Gly Phe 355 360 365Ala Ile Glu Pro Thr Ile Phe Val Asn Pro Lys Pro Gly Ser Lys Ile 370 375 380Trp Phe Glu Glu Ile Phe Gly Pro Val Leu Ser Ile Lys Thr Phe Lys385 390 395 400Thr Glu Glu Glu Ala Ile Glu Ile Ala Asn Asp Thr Thr Tyr Gly Leu 405 410 415Ala Ser Val Ile Tyr Thr Lys Ser Leu Asn Arg Gly Leu Arg Val Ser 420 425 430Ser Ala Leu Glu Thr Gly Gly Val Ser Ile Asn Phe Pro Phe Ile Pro 435 440 445Glu Thr Gln Thr Pro Phe Gly Gly Met Lys Gln Ser Gly Ser Gly Arg 450 455 460Glu Leu Gly Glu Glu Gly Leu Lys Ala Tyr Leu Glu Pro Lys Thr Ile465 470 475 480Asn Ile His Val Asn Ile Glu 485 6 1764 DNA Exophiala spinifera misc_feature (0)...(0) permease, partially spliced cDNA 6aactatggac tccagaccaa gtggatacgg cgagaaaggc gggacaaggc agacaacgaa 60gaacacagag acggcggcgg caggtggtgc gtccgagtcc ctgaacgttc ctctggagaa 120gaaacaattt ggcaccatca ccatcgtgtc cttggccttt gtgatttgca acagttgggc 180tggtatctca ggcagtctcc agctcgccct actagcgggg gggcccgtca ctctccttta 240cggcatccta atcagtactc tcgtctacat ctgcatcgct ttctcattag ccgaactgac 300cagcgtctac ccgactgccg gtggccaata tcattttgcg tcgatcctgg caccaaaatc 360aatcaatcgg agcatttcat acgtgtgcgg actcgtgtcg ttgctttcat ggatcgctat 420cggaagctca gtgaccatga tacctgctca acagatcccg gcgctgatag ccgcctatag 480tcacacatac tcccaggatt cgtggcatgt cttcctcatc tacgagggag tcgcgctggt 540ggtgctcttg ttcaacttgt ttgccctgaa aagaaaccct tgggttcatg aaatcggatt 600cggcctcacg atcgctctct tcgtgatctc ctttatcgcc attctagcgc ggtccaaccc 660caaggctcca aactcacagg tatggactgc ttggagcaac tatactggct ggtccgacgg 720cgtctgcttc atcctgggcc tttcgacatc ctgcttcatg ttcattggct tggacgcagc 780aatgcatctg gctgaagaat gcacagatgc tgctcgtacg gtacccaaag cagtggtcag 840tgcaatcata attggcttct gcaccgcctt tccatataca atcgcagttc tgtatggaat 900tacagatctc gactctattc taagttccgc cggctatatt ccattcgaga caatgacgca 960gtcccttcgg tcgctcagtt ttgcaacggt cctctcatgt ggcggtatcg tgatggcctt 1020cttcgccctc aacgctgtac aagagactgc gtctcgactc acctggagct ttgcccggga 1080caatgggctg gtattttcca ctcatctcga acgcattcat ccccgctggc aagttcctgt 1140ttggtctcta ttcgcgacct ggggaattct ggccacatgc ggatgtatat ttctaggttc 1200tagcacagct ttcaatgcct tggtcaattc cgccgttgta ctccagcaac tctccttcct 1260gatcccaatc gccctactcc tctaccaaaa gcgagatcca aagttcttgc cgagcactcg 1320tgcttttgtg ttaccgcgtg gaatcgggtt tctggtcaat gtgctagcgg tggtcttcac 1380gtccgtcacc actgtgtttt tcagcttccc actgaccgtg cctacggccg cgtcaaccat 1440gaattacaca agtgcgatta taggcgttgc acttgctctt ggtgtcttga actgggtcgt 1500gcatgccagg aagcattatc agggacccca cttggagctt gacggacggg tcgtcggagc 1560agaatttcaa gttgggccat gaattggacg aaatggagac gcgtgtgcaa tgtcaaaaat 1620tgctggggtg gtactgagag tctggattag ctgcaacgcg ggacaaccga gggtagaaca 1680ctctgcaatc gagcaggaca atatcaatta ggcaachasv caaaaaaaaa aaaaaaaaaa 1740aaaaaagcgg ccgctgaatt ctag 1764 7 1578 DNA Exophiala spinifera misc_feature (0)...(0) permease, fully spliced cDNA 7atggactcca gaccaagtgg atacggcgag aaaggcggga caaggcagac aacgaagaac 60acagagacgg cggcggcagg tggtgcgtcc gagtccctga acgttcctct ggagaagaaa 120caatttggca ccatcaccat cgtgtccttg gcctttgtga tttgcaacag ttgggctggt 180atctcaggca gtctccagct cgccctacta gcgggggggc ccgtcactct cctttacggc 240atcctaatca gtactctcgt ctacatctgc atcgctttct cattagccga actgaccagc 300gtctacccga ctgccggtgg ccaatatcat tttgcgtcga tcctggcacc aaaatcaatc 360aatcggagca tttcatacgt gtgcggactc gtgtcgttgc tttcatggat cgctatcgga 420agctcagtga ccatgatacc tgctcaacag atcccggcgc tgatagccgc ctatagtcac 480acatactccc aggattcgtg gcatgtcttc ctcatctacg agggagtcgc gctggtggtg 540ctcttgttca acttgtttgc cctgaaaaga aacccttggg ttcatgaaat cggattcggc 600ctcacgatcg ctctcttcgt gatctccttt atcgccattc tagcgcggtc caaccccaag 660gctccaaact cacaggtatg gactgcttgg agcaactata ctggctggtc cgacggcgtc 720tgcttcatcc tgggcctttc gacatcctgc ttcatgttca ttggcttgga cgcagcaatg 780catctggctg aagaatgcac agatgctgct cgtacggtac ccaaagcagt ggtcagtgca 840atcataattg gcttctgcac cgcctttcca tatacaatcg cagttctgta tggaattaca 900gatctcgact ctattctaag ttccgccggc tatattccat tcgagacaat gacgcagtcc 960cttcggtcgc tcagttttgc aacggtcctc tcatgtggcg gtatcgtgat ggccttcttc 1020gccctcaacg ctgtacaaga gactgcgtct cgactcacct ggagctttgc ccgggacaat 1080gggctggtat tttccactca tctcgaacgc attcatcccc gctggcaagt tcctgtttgg 1140tctctattcg cgacctgggg aattctggcc acatgcggat gtatatttct aggttctagc 1200acagctttca atgccttggt caattccgcc gttgtactcc agcaactctc cttcctgatc 1260ccaatcgccc tactcctcta ccaaaagcga gatccaaagt tcttgccgag cactcgtgct 1320tttgtgttac cgcgtggaat cgggtttctg gtcaatgtgc tagcggtggt cttcacgtcc 1380gtcaccactg tgtttttcag cttcccactg accgtgccta cggccgcgtc aaccatgaat 1440tacacaagtg cgattatagg cgttgcactt gctcttggtg tcttgaactg ggtcgtgcat 1500gccaggaagc attatcaggg accccacttg gagcttgacg gacgggtcgt cggagcagaa 1560tttcaagttg ggccatga 1578 8 525 PRT Exophiala spinifera 8Met Asp Ser Arg Pro Ser Gly Tyr Gly Glu Lys Gly Gly Thr Arg Gln 1 5 10 15Thr Thr Lys Asn Thr Glu Thr Ala Ala Ala Gly Gly Ala Ser Glu Ser 20 25 30Leu Asn Val Pro Leu Glu Lys Lys Gln Phe Gly Thr Ile Thr Ile Val 35 40 45Ser Leu Ala Phe Val Ile Cys Asn Ser Trp Ala Gly Ile Ser Gly Ser 50 55 60Leu Gln Leu Ala Leu Leu Ala Gly Gly Pro Val Thr Leu Leu Tyr Gly65 70 75 80Ile Leu Ile Ser Thr Leu Val Tyr Ile Cys Ile Ala Phe Ser Leu Ala 85 90 95Glu Leu Thr Ser Val Tyr Pro Thr Ala Gly Gly Gln Tyr His Phe Ala 100 105 110Ser Ile Leu Ala Pro Lys Ser Ile Asn Arg Ser Ile Ser Tyr Val Cys 115 120 125Gly Leu Val Ser Leu Leu Ser Trp Ile Ala Ile Gly Ser Ser Val Thr 130 135 140Met Ile Pro Ala Gln Gln Ile Pro Ala Leu Ile Ala Ala Tyr Ser His145 150 155 160Thr Tyr Ser Gln Asp Ser Trp His Val Phe Leu Ile Tyr Glu Gly Val 165 170 175Ala Leu Val Val Leu Leu Phe Asn Leu Phe Ala Leu Lys Arg Asn Pro 180 185 190Trp Val His Glu Ile Gly Phe Gly Leu Thr Ile Ala Leu Phe Val Ile 195 200 205Ser Phe Ile Ala Ile Leu Ala Arg Ser Asn Pro Lys Ala Pro Asn Ser 210 215 220Gln Val Trp Thr Ala Trp Ser Asn Tyr Thr Gly Trp Ser Asp Gly Val225 230 235 240Cys Phe Ile Leu Gly Leu Ser Thr Ser Cys Phe Met Phe Ile Gly Leu 245 250 255Asp Ala Ala Met His Leu Ala Glu Glu Cys Thr Asp Ala Ala Arg Thr 260 265 270Val Pro Lys Ala Val Val Ser Ala Ile Ile Ile Gly Phe Cys Thr Ala 275 280 285Phe Pro Tyr Thr Ile Ala Val Leu Tyr Gly Ile Thr Asp Leu Asp Ser 290 295 300Ile Leu Ser Ser Ala Gly Tyr Ile Pro Phe Glu Thr Met Thr Gln Ser305 310 315 320Leu Arg Ser Leu Ser Phe Ala Thr Val Leu Ser Cys Gly Gly Ile Val 325 330 335Met Ala Phe Phe Ala Leu Asn Ala Val Gln Glu Thr Ala Ser Arg Leu 340 345 350Thr Trp Ser Phe Ala Arg Asp Asn Gly Leu Val Phe Ser Thr His Leu 355 360 365Glu Arg Ile His Pro Arg Trp Gln Val Pro Val Trp Ser Leu Phe Ala 370 375 380Thr Trp Gly Ile Leu Ala Thr Cys Gly Cys Ile Phe Leu Gly Ser Ser385 390 395 400Thr Ala Phe Asn Ala Leu Val Asn Ser Ala Val Val Leu Gln Gln Leu 405 410 415Ser Phe Leu Ile Pro Ile Ala Leu Leu Leu Tyr Gln Lys Arg Asp Pro 420 425 430Lys Phe Leu Pro Ser Thr Arg Ala Phe Val Leu Pro Arg Gly Ile Gly 435 440 445Phe Leu Val Asn Val Leu Ala Val Val Phe Thr Ser Val Thr Thr Val 450 455 460Phe Phe Ser Phe Pro Leu Thr Val Pro Thr Ala Ala Ser Thr Met Asn465 470 475 480Tyr Thr Ser Ala Ile Ile Gly Val Ala Leu Ala Leu Gly Val Leu Asn 485 490 495Trp Val Val His Ala Arg Lys His Tyr Gln Gly Pro His Leu Glu Leu 500 505 510Asp Gly Arg Val Val Gly Ala Glu Phe Gln Val Gly Pro 515 520 525 9 3999 DNA Exophiala spinifera misc_feature (0)...(0) p-glycoprotein, with introns 9tatttsccat ctmckatgaa tggcagatga atcggagaaa cctcgaccaa accaagatgg 60cagtgagtcg tcctcacacc ctcccccaga aaaggaaacc gaaggcagta tttcagacta 120tctacgaatc ttcagatatg ccgacaaata cgactggact ctcaatgtca tcgcgctcat 180ctgcgccatc ggatccgggg cttcccttcc tctgatgtcg atcatcttcg gtagcttcac 240caacaagttc aacaattaca attcgggcga cgggagtcct gaagcgttca aggccgatgt 300ggatcatttc gtcctgtggt tcgtctacct ctttattggg aagtttgtcc tcacgtacgt 360ttccacggct gccattacca tttcagctat acgaaccact cgaactcttc gacgagtgtt 420ccttgaatgc accttgcggc aagaggtctg gcatttcgac aagcagagca atggagcaat 480cgccactcag gtcactacca atggcaaccg tatacaaaca ggtattgccg agaaattggt 540ctttaccgtg caggcacttt caatgttctt ttctgcattt gtggtcgctt tggcgtctca 600gtggaagcta gctttaatca ccatgtccgt catccctgcc attttcctgg tcaccggcat 660ctgcatagca attgatgccg ctcaggaggc caggatcacc aggatctact cacgcgccgc 720tgtcctcgca gaagaagtct tatcatccat ccggacagtc catgctttct acgcccagaa 780gaaaatggtc gaaaaatatg atgtcttttt gcagcaagca caccaagaag ggaagaagaa 840atcgccaaat tatggggtct tgttctcaac tgagtacttt tgcatttacg ctgctatcgc 900actgggcctt ttgggaaagg tttttcgcat gtatcagaat ggcgaggttg ccgacgttgg 960caaagtcttt actgttgcct ttccgtcacc tttagcagcc acgtccatct caatgcttgc 1020gccttcaggt tcagtcgttt accaacgccg catcttcggc ctccgaatta ttcagtatca 1080ttgacaaacc cacgcagctc gacccttctc gacccttttt ggaaagcagc cagagggctg 1140cttaggtcaa attgagatcc aaaacctggc atttgcctac ccctcccgac catctgccca 1200agtacttcga gatttcaact tgacaattcc agctggcaag acgacggccc tcgtcggtgc 1260atcaggtagc ggcaaaagca caatggtcgg cttacttgaa cggtggtatc tgcccagttc 1320ggggaggata ttacttgatg ggttggaact gggacaatac aatgtgaaat ggctgagaag 1380ccgcattcgc ctcgttcaac aggaacctgt gttgtttcgt ggcacaatct tccagaacat 1440tgccaacggt ttcatggatg agcaacgaga tctgcctcgc gaaaaacaaa tggagcttgt 1500gcaaaaagct tgcaaagcag caatgccgac gtgttcatta atgagcttcc gaacggttat 1560gagactgaag ttggcgagcg agccggagcc ttgagtggag gtcaacaagc cgaattgcaa 1620tcgcacgaag tatcatatcg gatcccaaga tcctgttact cgatgaagct accagcgccc 1680ttgacccgaa ggcggagaaa gtggtccagg aggccttgaa ccgagtgtcc aaagaccgca 1740ctactttggt cattgcccac aaactagcca ctgtcatacg actcactatt agggcgaatt 1800gggccctcta gatgcatgct cgagcggccg ccagtgtgac gaattgatgc agaattcggc 1860ttgtcattac gccgcactgg tgcgtgcaca ggacctcggg gctgacgaac aagaagaaca 1920tgagaagacc ctgcacgaaa aggcagcacg agaagctgct ggtgaacgac cggcacttga 1980gcgcactcac accactgcca catctcaagc tggagacctg gagaagcgga aggtgccggt 2040cgggactttg ggctactcgc tcctaaaatg catcctaatc atgttctacg aacaaaaaaa 2100tctctactgg tgcttcttgt tgtcaacaat agcggttctg atatgcgcgg ccacatttcc 2160aggacaagcc cttttgtttt cgagattgct cactgtcttc gagttgagtg gtcatgcggc 2220acaggaacgg gcagactttt atagtctgat gttctttgtc gtggctctag gaaatctagt 2280aggatatttc acgattggct ggacatgcaa cgttgtttca caagttgtca cccatcgcta 2340tcgagccgaa atgttccaac gagtactgga tcaagacatc gaattcttcg acatcccgga 2400gaatacttct ggtgctctca catcgcaact gtcagctcta cccacgcagt tgcaggagtt 2460gatatcaaca aattcttctc atttttatcg ttgtcgtaca acatcctctc gagcagtgct 2520ctagcactag cctatggatg gaaactgggc ctggtggttg tgtttggtgc acttccaccc 2580ctgcttttgg ctggctacct cagaattcgt cttgagacga agctagaagc cggaaactcg 2640gcaaactttg cagaaagtgc tgggcttgca agcgaagcag ttaccgcgat ccggaccgtc 2700tcatctttga ctctcgaagg scatgttctc caacagtact cggacatgtt gagcaaggtc 2760gtgctaagat catccaaagc tttggtttgg acgatgtttt ggttctcact gtcacagtcg 2820atcgagtttc tggctatggc cctgggaatt ttggtatggg aagtcgacta ctggcttcag 2880gtgaggtacg acacaactca attttatatc atcttcgtgg gcgttttgtt tgccggtcca 2940agcagcagcc cagaagccga attactccac gagtcttacc aaggctcggt cggctgcgaa 3000ctatatcctc tggctgcgga cattgaagcc gaccatccgc gaaacggagg agaacaagaa 3060aaaagggcca gtgggtggat gccctgtcga cctcgaggac attgaattca ggtatcgtca 3120acgtgattcg gctcgagttc tccgcggggt ttccatgaca atcgagccag gacaatttgt 3180agcttatgtg ggcgcttctg gctgtggcaa gtcaacgttg atcgctttgt tggaacgatt 3240ctacgacccg acctcgggcc gaatttcatt tgcacacgag aatattgcag aaatgtcgcc 3300gcgcttgtac cgcggccata tgtctttggt ccaacaggaa cccacayttt accaaggctc 3360cgttcgcgag aatgtgacgt tggccctcga agccgaatta tcagaagagc tttgtcaagg 3420acgccttccc gcaaggccaa tgctttggat tttgtcatct ctttaccaga aggctttgaa 3480acgccttgcg gctcaacgag ggatgcagtt ctccggcggg caacgacagc ggatcgccat 3540cgcaagagca ttgattcgaa atccaaagct gttgctactt gacgaagcga cgtcagccct 3600cgacacgcaa tcggaacgtc tggttcaagc tgccctcgat gaggcatcca cgagccgaac 3660gacaatagca gtggcgcacc gactttccac tattcggaat gttgatgtta tttttgtgtt 3720tgccaacggg agaatcgccg aaacgggcac tcacgcggaa ctacaacgac tgagaggaag 3780atattacgag atgtgtttgg cacaatcttt agaccaagca tgagcgttca cagagaagcg 3840gaaaagggcg gtgggatctt ttaggatagg tttagtggcg tgttacttac tacaggcgtt 3900tggattcagg tacgacaact tgtacaataa gtagcataga gcatgtaatg aaagggtact 3960cgtcccggaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaa 3999 10 3792 DNA Exophiala spinifera misc_feature (0)...(0) p-glycoprotein, fully spliced cDNA 10atggcagatg aatcggagaa acctcgacca aaccaagatg gcagtgagtc gtcctcacac 60cctcccccag aaaaggaaac cgaaggcagt atttcagact atctacgaat cttcagatat 120gccgacaaat acgactggac tctcaatgtc atcgcgctca tctgcgccat cggatccggg 180gcttcccttc ctctgatgtc gatcatcttc ggtagcttca ccaacaagtt caacaattac 240aattcgggcg acgggagtcc tgaagcgttc aaggccgatg tggatcattt cgtcctgtgg 300ttcgtctacc tctttattgg gaagtttgtc ctcacgtacg tttccacggc tgccattacc 360atttcagcta tacgaaccac tcgaactctt cgacgagtgt tccttgaatg caccttgcgg 420caagaggtct ggcatttcga caagcagagc aatggagcaa tcgccactca rgtcactacc 480aatggcaacc gtatacaaac aggtattgcc gagaaattgg tctttaccgt gcaggcactt 540tcaatgttct tttctgcatt tgtggtcgct ttggcgtctc agtggaagct agctttaatc 600accatgtccg tcatccctgc cattttcctg gtcaccggca tctgcatagc aattgatgcc 660gctcaggagg ccaggatcac caggatctac tcacgcgccg ctgtcctcgc agaagaagtc 720ttatcatcca tccggacagt ccatgctttc tacgcccaga agaaaatggt cgaaaaatat 780gatgtctttt tgcagcaagc acaccaagaa gggaagaaga aatcgccaaa taatggsgtc 840ttgttctcaa ctgagtactt ttgcatttac gctgctatcg cactggcctt ttggaaaggt 900tttcgcatgt atcagaatgg cgaggttgcc gacgttggca aagtctttac tgttgtcctt 960tccgtcacct tagcagccac gtccatctca atgcttgcgc cttcaggttc agtcgtttac 1020caacgccgca tcttcggctc cgaattattc agtatcattg acaaacccac gcagctcgac 1080cctctcgacc cttctggaaa gcagccagag ggctgcctag gtcaaattga gatccaaaac 1140ctggcatttg cctacccctc ccgaccatct gcccaagtac ttcgagattt caacttgaca 1200attccagctg gcaagacgac ggccctcgtc ggtgcatcag gtagcggcaa aagcacaatg 1260gtcggcttac ttgaacggtg gtatctgccc agttcgggga ggatattact tgatgggttg 1320gaactgggac aatacaatgt gaaatggctg agaagccgca ttcgcctcgt tcaacaggaa 1380cctgtgttgt ttcgtggcac aatcttccag aacattgcca acggtttcat ggatgagcaa 1440cgagatctgc ctcgcgaaaa acaaatggag cttgtgcaaa aagcttgcaa agccagcaat 1500ggcgacgtgt tcattaatga gcttccgaac ggttatgaga ctgaagttgg cgagcgagcc 1560ggagccttga gtggaggtca acgacaacga attgcaatcg cacgaagtat catatcggat 1620cccaagatcc tgttactcga tgaagctacc agcgcccttg acccgaaggc ggagaaagtg 1680gtccaggagg ccttgaaccg agtgtccaaa gaccgcacta ctttggtcat tgcccacaaa 1740ctagccactg tcaaaagtgc tggcaacatc gcagtcattt cccaggggaa aatcgtcgag 1800caaggcacac accacgaatt gatcgaattc ggctgtcatt acgccgcact ggtgcgtgca 1860caggacctcg gggctgacga acaacaagaa catgagaaga ccctgcacga aaaggcagca 1920cgagaagctg ctggtgaacg accggcactt gagcgcactc acaccactgc cacatctcaa 1980gctggagacc tggagaagcg gaaggtgccg gtcgggactt tgggctactc gctcctaaaa 2040tgcatcctaa tcatgttcta cgaacaaaaa aatctctact ggtgcttctt gttgtcaaca 2100ataacggttc tgatatgcgc ggccacattt ccaggacaag cccttttgtt ttcgagattg 2160ctcactgtct tcgagttgag tggtcatgcg gcacaggaac gggcagactt ttatattctg 2220atgttctttg tcgtggctct aggaaatcta gtaggatatt tcacgattgg ctggacatgc 2280aacgttattt cacaagttgt cacccatcgc tatcaagccg caatgttcca acgagtactg 2340gatcaagaca tcgaactcct cgacatcccg gagcaaattt ctggtgctct cacatcgcaa 2400ctgtcagctc tacccacgca gttgcaagag ttgatatcag caaattttct catttatatc 2460gttgtcggtc aacatcgtct cgagcagtgc tctaccacta gcctatggat ggaaactggg 2520cctggtggtt gtgtttggtg cacttccacc cctgcttttg gctggctacc tcagaattcg 2580tctagagacg aagctagaag ccggaaactc ggcaaacttt gcagaaagtg ctgggcttgc 2640aagcgaagca gttaccgcga tccggaccgt ctcatctttg actctcgaag gccatgttct 2700ccaacagtac tcggacatgt tgagcaaggt cttgctaaga tcatccaaag cttttggttt 2760ggacgatgtt ttggttttca cttgtcacag tcgatggagt ttttggctat tgccctggga 2820ttttgtattg cagtcgataa ttggcttcag gtgagtacga cacaactcaa ttttatatca 2880tcttcgtggg cgttttgttt gccggtccaa gcagcagccc agtatttggc ttactccacg 2940agttttacca aggctcggtc ggctgcgaac tatatcctct ggctgcggac attgaagccg 3000accatccgcg aaacggagga gaacaagaaa aaaggcccag tgggtggatg ccctgtcgac 3060ctcgaggaca ttgaattcag gtatcgtcaa cgtgattcgg ctcgagttct ccgcggggtt 3120tccatgacaa tcgagccagg acaatttgta gcttatgtgg gcgcttctgg ctgtggcaag 3180tcaacgttga tcgctttgtc ggaacgattc tacgacccga cctcgggccg aatttcattt 3240gcacacgaga atattgcaga aatgtcgccg cgcttgtacc gcggccatat gtctttggtc 3300caacaggaac ccacacttta ccaaggctcc gttcgcgaga atgtgacgtt ggccctcgaa 3360gccgaattat cagaagagct ttgtcaagga cgccttcccg caaggccaat gctttggatt 3420ttgtcatctc tttaccagaa ggctttgaaa cgccttgcgg ctcaacgagg gatgcagttc 3480tccggcgggc aacgacagcg gatcgccatc gcaagagcat tgattcgaaa tccaaagctg 3540ttgctacttg acgaagcgac gtcagccctc gacacgcaat cggaacgtct ggttcaagct 3600gccctcgatg aggcatccac gagccgaacg acaatagcag tggcgcaccg actttccact 3660attcggaatg ttgatgttat ttttgtgttt gccaacggga gaatcgccga aacgggcact 3720cacgcggaac tacaacgact gagaggaaga tattacgaga tgtgtttggc acaatcttta 3780gaccaagcat ga 3792 11 1263 PRT Exophiala spinifera VARIANT 157 Xaa = Any Amino Acid 11Met Ala Asp Glu Ser Glu Lys Pro Arg Pro Asn Gln Asp Gly Ser Glu 1 5 10 15Ser Ser Ser His Pro Pro Pro Glu Lys Glu Thr Glu Gly Ser Ile Ser 20 25 30Asp Tyr Leu Arg Ile Phe Arg Tyr Ala Asp Lys Tyr Asp Trp Thr Leu 35 40 45Asn Val Ile Ala Leu Ile Cys Ala Ile Gly Ser Gly Ala Ser Leu Pro 50 55 60Leu Met Ser Ile Ile Phe Gly Ser Phe Thr Asn Lys Phe Asn Asn Tyr65 70 75 80Asn Ser Gly Asp Gly Ser Pro Glu Ala Phe Lys Ala Asp Val Asp His 85 90 95Phe Val Leu Trp Phe Val Tyr Leu Phe Ile Gly Lys Phe Val Leu Thr 100 105 110Tyr Val Ser Thr Ala Ala Ile Thr Ile Ser Ala Ile Arg Thr Thr Arg 115 120 125Thr Leu Arg Arg Val Phe Leu Glu Cys Thr Leu Arg Gln Glu Val Trp 130 135 140His Phe Asp Lys Gln Ser Asn Gly Ala Ile Ala Thr Xaa Val Thr Thr145 150 155 160Asn Gly Asn Arg Ile Gln Thr Gly Ile Ala Glu Lys Leu Val Phe Thr 165 170 175Val Gln Ala Leu Ser Met Phe Phe Ser Ala Phe Val Val Ala Leu Ala 180 185 190Ser Gln Trp Lys Leu Ala Leu Ile Thr Met Ser Val Ile Pro Ala Ile 195 200 205Phe Leu Val Thr Gly Ile Cys Ile Ala Ile Asp Ala Ala Gln Glu Ala 210 215 220Arg Ile Thr Arg Ile Tyr Ser Arg Ala Ala Val Leu Ala Glu Glu Val225 230 235 240Leu Ser Ser Ile Arg Thr Val His Ala Phe Tyr Ala Gln Lys Lys Met 245 250 255Val Glu Lys Tyr Asp Val Phe Leu Gln Gln Ala His Gln Glu Gly Lys 260 265 270Lys Lys Ser Pro Asn Asn Gly Val Leu Phe Ser Thr Glu Tyr Phe Cys 275 280 285Ile Tyr Ala Ala Ile Ala Leu Ala Phe Trp Lys Gly Phe Arg Met Tyr 290 295 300Gln Asn Gly Glu Val Ala Asp Val Gly Lys Val Phe Thr Val Val Leu305 310 315 320Ser Val Thr Leu Ala Ala Thr Ser Ile Ser Met Leu Ala Pro Ser Gly 325 330 335Ser Val Val Tyr Gln Arg Arg Ile Phe Gly Ser Glu Leu Phe Ser Ile 340 345 350Ile Asp Lys Pro Thr Gln Leu Asp Pro Leu Asp Pro Ser Gly Lys Gln 355 360 365Pro Glu Gly Cys Leu Gly Gln Ile Glu Ile Gln Asn Leu Ala Phe Ala 370 375 380Tyr Pro Ser Arg Pro Ser Ala Gln Val Leu Arg Asp Phe Asn Leu Thr385 390 395 400Ile Pro Ala Gly Lys Thr Thr Ala Leu Val Gly Ala Ser Gly Ser Gly 405 410 415Lys Ser Thr Met Val Gly Leu Leu Glu Arg Trp Tyr Leu Pro Ser Ser 420 425 430Gly Arg Ile Leu Leu Asp Gly Leu Glu Leu Gly Gln Tyr Asn Val Lys 435 440 445Trp Leu Arg Ser Arg Ile Arg Leu Val Gln Gln Glu Pro Val Leu Phe 450 455 460Arg Gly Thr Ile Phe Gln Asn Ile Ala Asn Gly Phe Met Asp Glu Gln465 470 475 480Arg Asp Leu Pro Arg Glu Lys Gln Met Glu Leu Val Gln Lys Ala Cys 485 490 495Lys Ala Ser Asn Gly Asp Val Phe Ile Asn Glu Leu Pro Asn Gly Tyr 500 505 510Glu Thr Glu Val Gly Glu Arg Ala Gly Ala Leu Ser Gly Gly Gln Arg 515 520 525Gln Arg Ile Ala Ile Ala Arg Ser Ile Ile Ser Asp Pro Lys Ile Leu 530 535 540Leu Leu Asp Glu Ala Thr Ser Ala Leu Asp Pro Lys Ala Glu Lys Val545 550 555 560Val Gln Glu Ala Leu Asn Arg Val Ser Lys Asp Arg Thr Thr Leu Val 565 570 575Ile Ala His Lys Leu Ala Thr Val Lys Ser Ala Gly Asn Ile Ala Val 580 585 590Ile Ser Gln Gly Lys Ile Val Glu Gln Gly Thr His His Glu Leu Ile 595 600 605Glu Phe Gly Cys His Tyr Ala Ala Leu Val Arg Ala Gln Asp Leu Gly 610 615 620Ala Asp Glu Gln Gln Glu His Glu Lys Thr Leu His Glu Lys Ala Ala625 630 635 640Arg Glu Ala Ala Gly Glu Arg Pro Ala Leu Glu Arg Thr His Thr Thr 645 650 655Ala Thr Ser Gln Ala Gly Asp Leu Glu Lys Arg Lys Val Pro Val Gly 660 665 670Thr Leu Gly Tyr Ser Leu Leu Lys Cys Ile Leu Ile Met Phe Tyr Glu 675 680 685Gln Lys Asn Leu Tyr Trp Cys Phe Leu Leu Ser Thr Ile Thr Val Leu 690 695 700Ile Cys Ala Ala Thr Phe Pro Gly Gln Ala Leu Leu Phe Ser Arg Leu705 710 715 720Leu Thr Val Phe Glu Leu Ser Gly His Ala Ala Gln Glu Arg Ala Asp 725 730 735Phe Tyr Ile Leu Met Phe Phe Val Val Ala Leu Gly Asn Leu Val Gly 740 745 750Tyr Phe Thr Ile Gly Trp Thr Cys Asn Val Ile Ser Gln Val Val Thr 755 760 765His Arg Tyr Gln Ala Ala Met Phe Gln Arg Val Leu Asp Gln Asp Ile 770 775 780Glu Leu Leu Asp Ile Pro Glu Gln Ile Ser Gly Ala Leu Thr Ser Gln785 790 795 800Leu Ser Ala Leu Pro Thr Gln Leu Gln Glu Leu Ile Ser Ala Asn Phe 805 810 815Leu Ile Tyr Ile Val Val Gly Gln His Arg Leu Glu Gln Cys Ser Thr 820 825 830Thr Ser Leu Trp Met Glu Thr Gly Pro Gly Gly Cys Val Trp Cys Thr 835 840 845Ser Thr Pro Ala Phe Gly Trp Leu Pro Gln Asn Ser Ser Arg Asp Glu 850 855 860Ala Arg Ser Arg Lys Leu Gly Lys Leu Cys Arg Lys Cys Trp Ala Cys865 870 875 880Lys Arg Ser Ser Tyr Arg Asp Pro Asp Arg Leu Ile Phe Asp Ser Arg 885 890 895Arg Pro Cys Ser Pro Thr Val Leu Gly His Val Glu Gln Gly Leu Ala 900 905 910Lys Ile Ile Gln Ser Phe Trp Phe Gly Arg Cys Phe Gly Phe His Leu 915 920 925Ser Gln Ser Met Glu Phe Leu Ala Ile Ala Leu Gly Phe Cys Ile Ala 930 935 940Val Asp Asn Trp Leu Gln Val Ser Thr Thr Gln Leu Asn Phe Ile Ser945 950 955 960Ser Ser Trp Ala Phe Cys Leu Pro Val Gln Ala Ala Ala Gln Tyr Leu 965 970 975Ala Tyr Ser Thr Ser Phe Thr Lys Ala Arg Ser Ala Ala Asn Tyr Ile 980 985 990Leu Trp Leu Arg Thr Leu Lys Pro Thr Ile Arg Glu Thr Glu Glu Asn 995 1000 1005Lys Lys Lys Gly Pro Val Gly Gly Cys Pro Val Asp Leu Glu Asp Ile 1010 1015 1020Glu Phe Arg Tyr Arg Gln Arg Asp Ser Ala Arg Val Leu Arg Gly Val1025 1030 1035 1040Ser Met Thr Ile Glu Pro Gly Gln Phe Val Ala Tyr Val Gly Ala Ser 1045 1050 1055Gly Cys Gly Lys Ser Thr Leu Ile Ala Leu Ser Glu Arg Phe Tyr Asp 1060 1065 1070Pro Thr Ser Gly Arg Ile Ser Phe Ala His Glu Asn Ile Ala Glu Met 1075 1080 1085Ser Pro Arg Leu Tyr Arg Gly His Met Ser Leu Val Gln Gln Glu Pro 1090 1095 1100Thr Leu Tyr Gln Gly Ser Val Arg Glu Asn Val Thr Leu Ala Leu Glu1105 1110 1115 1120Ala Glu Leu Ser Glu Glu Leu Cys Gln Gly Arg Leu Pro Ala Arg Pro 1125 1130 1135Met Leu Trp Ile Leu Ser Ser Leu Tyr Gln Lys Ala Leu Lys Arg Leu 1140 1145 1150Ala Ala Gln Arg Gly Met Gln Phe Ser Gly Gly Gln Arg Gln Arg Ile 1155 1160 1165Ala Ile Ala Arg Ala Leu Ile Arg Asn Pro Lys Leu Leu Leu Leu Asp 1170 1175 1180Glu Ala Thr Ser Ala Leu Asp Thr Gln Ser Glu Arg Leu Val Gln Ala1185 1190 1195 1200Ala Leu Asp Glu Ala Ser Thr Ser Arg Thr Thr Ile Ala Val Ala His 1205 1210 1215Arg Leu Ser Thr Ile Arg Asn Val Asp Val Ile Phe Val Phe Ala Asn 1220 1225 1230Gly Arg Ile Ala Glu Thr Gly Thr His Ala Glu Leu Gln Arg Leu Arg 1235 1240 1245Gly Arg Tyr Tyr Glu Met Cys Leu Ala Gln Ser Leu Asp Gln Ala 1250 1255 1260

Claims

1. A microorganism having stably incorporated at least one expression cassette comprising a promoter active in said microorganism operably linked to a nucleotide sequence, wherein:a) said microorganism is a bacterium or a fungus; b) said microorganism is heterologous to said nucleotide sequence; and c) said nucleotide sequence is selected from the group consisting of the nucleotide sequence set forth in SEQ ID NO: 6 or 7.

2. The microorganism of claim 1, wherein said expression cassette is stably incorporated into the genome of said microorganism.

3. A microorganism having stably incorporated at least one expression cassette comprising a promoter active in said microorganism operably linked to a nucleotide sequence, wherein said microorganism is heterologous to said nucleotide sequence and wherein:a) said nucleotide sequence has at least 90% identity to the nucleotide sequence set forth in SEQ ID NO: 6 or 7; and, b) expression of said sequence reduces the pathogenicity of a fungus producing fumonisin.

4. The microorganism of claim 3, wherein said nucleotide sequence encodes the amino acid sequence set forth in SEQ ID NO: 8.

5. A microorganism having stably incorporated at least one expression cassette comprising a nucleotide sequence, wherein:a) said microorganism is a bacterium or a fungus; b) said nucleotide sequence is operably linked to a promoter that is active in said microorganism; c) said promoter is heterologous to the nucleotide sequence; and, d) said sequence is selected from the group consisting of the nucleotide sequence set forth in SEQ ID NO: 6 or 7.

6. The microorganism of claim 5, wherein said expression cassette is stably integrated into the genome of said microorganism.

7. A microorganism having stably incorporated at least one expression cassette comprising a nucleotide sequence, wherein:a) said microorganism is a bacterium or a fungus; b) said nucleotide sequence is operably linked to a promoter that is active in said microorganism; c) said promoter is heterologous to said nucleotide sequence; d) said nucleotide sequence encodes the amino acid sequence set forth in SEQ ID NO: 8; and, e) expression of said sequence reduces the pathogenicity of a fungus producing fumonisin.

8. The microorganism of claim 7, wherein said expression cassette is stably integrated into the genome of the microorganism.

9. A microorganism having stably incorporated at least one expression cassette comprising a nucleotide sequence, wherein:a) said microorganism is a bacterium or a fungus; b) said nucleotide sequence is operably linked to a promoter that is active in said microorganism; b) said promoter is heterologous to the nucleotide sequence; c) said nucleotide sequence has at least 90% identity to the nucleotide sequence set forth in SEQ ID NO: 6 or 7; and, d) expression of said sequence reduces the pathogenicity of a fungus producing fumonisin.

10. An expression cassette comprising a nucleotide sequence having at least 90% identity to the nucleotide sequence of SEQ ID NO:6, wherein said nucleotide sequence encodes a protein that retains permease activity and wherein said nucleotide sequence is operably linked to a promoter that drives expression in a microorganism, wherein said microorganism is a bacterium or fungus.

11. The expression cassette of claim 10, wherein said nucleotide sequence is set forth in SEQ ID NO: 6.

12. An expression cassette comprising a nucleotide sequence having at least 90% identity to the nucleotide sequence set forth in SEQ ID NO: 7, wherein said nucleotide sequence retains permease activity and wherein said nucleotide sequence is operably linked to a promoter that drives expression in a microorganism, wherein said microorganism is a bacterium or a fungus.

13. The expression cassette of claim 12, wherein said sequence comprises the nucleotide sequence set forth in SEQ ID NO: 7.

14. A microorganism which is a bacterium or a fungus, said microorganism having stably incorporated at least one DNA construct comprising a promoter active in said microorganism operably linked to a nucleotide sequence having at least 90% identity to the nucleotide sequence set forth in SEQ ID NO:7, wherein said nucleotide sequence retains permease activity and wherein said promoter is heterologous to said nucleotide sequence.

15. The microorganism of claim 14, wherein said sequence comprises the nucleotide sequence set forth in SEQ ID NO:7.

16. A microorganism which is a bacterium or a fungus, said microorganism having stably incorporated at least one nucleotide sequence having at least 90% identity to the nucleotide sequence set forth in SEQ ID NO:7, wherein:a) said nucleotide sequence is operably linked to a promoter active in said microorganism; b) said nucleotide sequence is heterologous to the microorganism; and c) said sequence retains permease activity.

17. The microorganism of claim 16, wherein said sequence comprises the nucleotide sequence set forth in SEQ ID NO: 7.

18. A method of producing a polypeptide useful in degrading fumonisin, said method comprising:a) culturing the microorganism of claim 1; and, b) purifying the polypeptide encoded by said nucleotide sequence.

19. A method of producing a polypeptide useful in degrading fumonisin, said method comprising:a) culturing the microorganism of claim 5; and, b) purifying the polypeptide encoded by said nucleotide sequence.

20. The microorganism of claim 3, wherein said nucleotide sequence has at least 95% identity to the nucleotide sequence set forth in SEQ ID NO:6 or 7.

21. The microorganism of claim 9, wherein said nucleotide sequence has at least 95% identity to the nucleotide sequence set forth in SEQ ID NO:6 or 7.