CA2350328A1 - Gene encoding oxalate decarboxylase from aspergillus phoenices - Google Patents

Abstract

A novel nucleic acid sequence encoding Aspergillus phoenices oxalate decarboxylase (APOXD) has been determined, as well as the encoded amino acid sequence. The gene and its encoded protein are useful in degrading oxalate, in diagnostic assays of oxalate, and as a selectable marker.

Description

Applicant Ref. 0561-PCT

- I -GENE ENCODING OXALATE DECARBOXYLASE FROM
ASPERGILLUS PHOENICES
Field of the Invention This invention relates to a novel nucleic acid sequence encoding oxalate decarboylyase isolated from Aspergillus phoenices and to use of the nucleic acid sequence to produce its encoded protein.
Background of the Invention Oxalic acid (oxalate) is a diffusable toxin associated with various plant diseases, particularly those caused by fungi. Some leafy green vegetables, including spinach and rhubarb, produce oxalate as a nutritional stress factor. When plants containing oxalate are consumed in large amounts, they can be toxic to humans.
Oxalate is used by pathogens to gain access into and subsequently throughout an infected plant. See for example, Mehta and Datta, The Jourhal of Biological Chemistry, 266:23548-23553, 1991; and published PCT Application W092/14824.
Field crops such as sunflower, bean, canola, alfalfa, soybean, flax, safflower, peanut, clover, as well as numerous vegetable crops, flowers, and trees are susceptible to oxalate-secreting pathogens. For example, fungal species including Sclerotinia and Sclerotium use oxalic acid to provide an opportunistic route of entry into plants, causing serious damage to crops such as sunflower.
Because of the role of oxalate in plant disease and toxicity, compounds that inhibit oxalate mediated disease, and particularly genes encoding such inhibitory degrading molecules, are greatly needed.
Enzymes that utilize oxalate as a substrate have been identified. These include oxalate oxidase and oxalate decarboxylase. Oxalate oxidase catalyzes the conversion of oxalate to COZ and H202. A gene encoding barley oxalate oxidase has been cloned from a barley root cDNA library and sequenced (See PCT publication No.
W092/14824). A gene encoding wheat oxalate oxidase activity (Germin) has been isolated and sequenced, (PCT publication No. WO 94/13790) and the gene has been introduced into a canola variety. Canola plants harboring the gene appeared to show Applicant Ref. 0561-PCT

some resistance to Sclerotinia sclerotiorum, in vitro (Dumas, et al., 1994, Abstracts: 4th Int'l. Congress of Plant Molecular Biology, #1906).
- Oxalate decarboxylase converts oxalate to C02 and formic acid. A gene encoding oxalate decarboxylase has been isolated from Collybia velutipes (now termed Flammulina velutipes) and the cDNA clone has been sequenced (W094/12622, published 9 June 1994). Oxalate decarboxylase activities have also been described in Aspergillus niger and Aspergillus phoenices (Emiliani et al., 1964, ARCH. Biochem.
Biophys.
105:488-493), however the amino acid sequence and nucleic acid sequence encoding these enzyme activities have not been isolated or characterized.
Enzymatic assays for clinical analysis of urinary oxalate provide significant advantages in sensitivity and qualification Obzansky, et al., 1983, Clinical Chem. 29:1815-1819. For many reasons, including reactivity with interfering analytes and the high cost of available oxalate oxidase used in this diagnostic assay, alternative enzymes are needed. (Lathika et al., 1995, Analytical Letters 28: 425-442).
In this application, we disclose the isolation, cloning, and sequencing of a unique gene encoding an oxalate decarboxylase enzyme from Aspergillus phoenices. The gene is useful in producing highly purified Aspergillus phoenices oxalate decarboxylase enzyme, in producing transgenic plant cells and plants expressing the enzyme in viva, and in diagnostic assays of oxalate.
Summary of the Invention The present invention provides a nucleic acid sequence encoding oxalate decarboxylase isolated from Aspergillus phoenices (APOXD). The gene sequence [Seq ID No: l ], the recombinant protein produced therefrom [Seq ID No:2], and vectors, transformed cells, and plants containing the gene sequence are provided as individual embodiments of the invention, as well as methods using the gene or its encoded protein.
The nucleic acid is useful for producing oxalate decarboxylase for commercial applications, including degradation of oxalic acid, protection against oxalic acid toxicity, and diagnostic assays to quantify oxalate.
The nucleic acid of the invention is also useful as a selectable marker.
Growth of plant cells in the presence of oxalic acid favors survival of plant cells transformed with the coding sequence of the gene.

Applicant Ref. 0561-PCT

The present invention also includes compositions and methods for degrading oxalic acid, in providing protection against oxalic acid toxicity, and in - combating and providing protection against plant pathogens that utilize oxalate to gain access to plant tissue or otherwise in the course of the pathogenesis of the disease.
Oxalate decarboxylase from Aspergillis phoenices (APOXD) of the present invention is combined with an appropriate carrier for delivery to the soil or plants.
Alternatively, plant cells are transformed with the nucleic acid sequence of the invention for expression of APOXD in vivo.
Brief Description of the Drawings Figure 1 is a diagram showing a first primer strategy for amplification of a portion of the nucleic acid sequence encoding APOXD.
Figure 2 is a diagram showing the primer position and design of nested, gene-specific primers (arrows above diagram) for 3' RACE and the single gene specific primer (arrow beneath diagram) used for 5' RACE.
Figure 3 is a diagram showing the construction of plasmid pPHP9723 containing the l.4kb nucleic acid sequence encoding APOXD including leader and pre-sequence.
Figure 4 is a diagram of the plasmid pPHP9723.
Figure 5 is a diagram showing the plasmid pPHP9762 containing the nucleic acid sequence encoding APOXD with the fungal leader and pre-sequence replaced by the plant signal sequence of the wheat oxalate oxidase gene, Germin.
Detailed Descriution of the Invention The purified oxalate decarboxylase of the present invention has many commercial uses, including inhibiting oxalate toxicity of plants and preventing pathogenic disease in plants where oxalic acid plays a critical role. It has been suggested that degradation of oxalic acid is a preventative measure, e.g., to prevent invasion of a pathogen into a plant, or during pathogenesis, when oxalic acid concentrations rise (Dumas, et al., 1994, Supra). The gene of the invention is also useful as a selectable marker of transformed cells, for diagnostic assay of oxalate, and for production of the enzyme in plants.

Applicant Ref. 0561-PCT

Nucleic Acid Seguence Encoding APOXD
A nucleic acid sequence encoding APOXD [Seq. ID No: 1 ] has now been determined by methods described more fully in the Examples below. Briefly, DNA
encoding APOXD was obtained by amplification of genomic A. phoenices DNA using a RACE strategy as described in Innis et. al., eds., 1990, PCR Protocols. A
Guide to Methods and Applications, Academic Press, San Diego, CA, pages 28-38. See also pages 39-45, "Degenerate primers". The nucleic acid sequence and its deduced amino acid sequence (Seq. ID No:2] are shown below in Table 1. The predicted signal peptide [Seq.
ID No: 3] and pre-protein (Seq. ID No: 4] are shown along with the potential cleavage site between them as determined by computer analysis using PC gene software (IntelliGenetics, Inc., Mountain View, CA). The mature protein [Seq. ID No: 5]
is also indicated. This 1.4 kb sequence encodes a 458 amino acid enzyme subunit with a calculated molecular weight of 51,994 daltons. Southern hybridization indicates that the enzyme is encoded by a single gene in the Aspergillis phoenices genome. The plasmid pPHP9685 containing the nucleic acid sequence encoding APOXD as an insert was deposited with the A.T.C.C. on March 18, 1997, having Accession No. 97959.

SEQUENCE OF FULL LENGTH APOXD DNA
(Signal Peptide W

(Met Gln Leu Thr Leu Pro Pro Arg Gln Leu Leu Leu Ser Phe Ala Thr Val Ala Ala Leu Leu Asp Pro Ser His Gly (Pre-protein ~

IGly Pro Val Pro Asn Glu Ala Tyr Gln Gln Leu Leu Gln Ile Pro Ala (Mature Protein fi TCA TCC CCA TCC ATT TTC TTC (CAA GAC AAG CCA TTC ACC CCC GAT CAT 197 Ser Ser Pro Ser Ile Phe Phe IGln Asp Lys Pro Phe Thr Pro Asp His Applicant Ref. 0561-PCT

NruI

Arg Asp Pro Tyr Asp His Lys Val Asp Ala Ile Gly Glu Gly His Glu Pro Leu Pro Trp Arg Met Gly Asp Gly Ala Thr Ile Met Gly Pro Arg Asn Lys Asp Arg Glu Arg Gln Asn Pro Asp Met Leu Arg Pro Pro Ser Thr Asp His Gly Asn Met Pro Asn Met Arg Trp Ser Phe Ala Asp Ser His Ile Arg Ile Glu Glu Gly Gly Trp Thr Arg Gln Thr Thr Val Arg Glu Leu Pro Thr Ser Lys Glu Leu Ala Gly Val Asn Met Arg Leu Asp Glu Gly Val Ile Arg Glu Leu His Trp His Arg Glu Ala Glu Trp Ala Tyr Val Leu Ala Gly Arg Val Arg Val Thr Gly Leu Asp Leu Glu Gly Gly Ser Phe Ile Asp Asp Leu Glu Glu Gly Asp Leu Trp Tyr Phe Pro Ser Gly His Pro His Ser Leu Gln Gly Leu Ser Pro Asn Gly Thr Glu Phe Leu Leu Ile Phe Asp Asp Gly Asn Phe Ser Glu Glu Ser Thr Phe Applicant Ref. 0561-PCT

Leu Leu Thr Asp Trp Ile Ala His Thr Pro Lys Ser Val Leu Ala Gly Asn Phe Arg Met Arg Pro Gln Thr Phe Lys Asn Ile Pro Pro Ser Glu Lys Tyr Ile Phe Gln Gly Ser Val Pro Asp Ser Ile Pro Lys Glu Leu Pro Arg Asn Phe Lys Ala Ser Lys Gln Arg Phe Thr His Lys Met Leu Ala Gln Lys Pro GIu His Thr Ser Gly Gly Glu Val Arg Ile Thr Asp Ser Ser Asn Phe Pro Ile Ser Lys Thr Val Ala Ala Ala His Leu Thr Ile Asn Pro GIy Ala Ile Arg Glu Met His Trp His Pro Asn Ala Asp Glu Trp Ser Tyr Phe Lys Arg Gly Arg Ala Arg Val Thr Ile Phe Ala Ala Glu Gly Asn Ala Arg Thr Phe Asp Tyr Val Ala Gly Asp val Gly Ile Val Pro Arg Asn Met Gly His Phe Ile Glu Asn Leu Ser Asp Asp Glu Glu Val Glu Val Leu Glu Ile Phe Arg Ala Asp Arg Phe Arg Asp ,. Applicant Ref. 0561-PCT

- '7 -Phe Ser Leu Phe Gln Trp Met Gly Glu Thr Pro Gln Arg Met Val Ala Glu His Val Phe Lys Asp Asp Pro Asp Ala Ala Arg Glu Phe Leu Lys Ser Val Glu Ser Gly Glu Lys Asp Pro Ile Arg Ser Pro Ser Glu (Stop 9~;i.: ~~~i ~ a _,'~;~,.
l~ ~ .. ~ .. ~~. I
,~, x u, _ , .- ~,.. , 2: .'~ a s '~h. . . I , ._ ~, r, w I ~~:.m II -s ' r ~~. .
~ ~.' 1:. , ' , ' . ~fl. S
'.~T',~1 ~ ~.E~,G ~~I ~ . , ~7 CJ
~ r r. .
~... ~a. ~~: ~ ~~ ~, ~ ~,..d' ~;~
e~. N , S. l , ' w Y.
!~ ..7" 4.~,.mr:;~ ~i . .. ~$.''~h~t~~~IF~ 1 ,16 ,c S~ t , I?;; ,., ii ..7w..~9.d.".. W~IIe'a.
:~1. 'k, ~''i4 1 :F.x._;'., "x tV'~7d~Aw~a~..'"Y~~I~ _w'y _.~..-r.Ji~
;'.~wh~, n .-1.4 kb gene 1-1437 1 Encoded 24-1397 1-458 2 Protein Signal Peptide 24-101 1-26 3 Pre-protein 102-1397 27-458 4 Mature Protein 71-1394 50-457 5 Redundancy in the genetic code permits variation in the gene sequences shown in Table 1. In particular, one skilled in the art will recognize specific codon preferences by a specific host species and can adapt the disclosed sequence as preferred for the desired host. For example, rare codons having a frequency of less than about 20%
in known sequence of the desired host are preferably replaced with higher frequency codons. Codon preferences for a specific organism may be calculated, for example, codon usage tables available on the INTERNET at the following address:
http://www.dna.affrc.go.jp/~nakamura/codon.html. One specific program available for Arabidopsis is found at: http://genome-www.stanford.edu/Arabidopsis/codon usage.html.

Applicant Ref. 0561-PCT

Additional sequence modifications are known to enhance protein 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 which 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. Where possible, the sequence is modified to avoid predicted hairpin secondary mRNA structures.
Other useful modifications include the addition of a translational initiation consensus sequence at the start of the open reading frame, as described in Kozak, 1989, Mol Cell Biol. 9:5073 5080.
In addition, the native APOXD gene or a modified version of the APOXD
gene might be further optimized for expression by omitting the predicted signal and pre-sequence, replacing the signal sequence with another signal sequence, or replacing the signal and pre-sequence with another signal sequence. Any one of the possible APOXD
gene variations may work best when combined with a specific promoter and/or termination sequence.
The term "selectively hybridizes" includes a reference to hybridization, under stringent hybridization conditions, of a nucleic acid sequence to a specified nucleic acid target sequence to a detestably greater degree (e.g., at least 2-fold over background) than its hybridization to non-target nucleic acid sequences and to the substantial exclusion of non-target nucleic acids. Selectively hybridizing sequences typically have about at least 80% sequence identity, preferably 90% sequence identity, and most preferably 100%
sequence identity (i.e., complementary) with each other.
The terms "stringent conditions" or "stringent hybridization conditions"
include reference to conditions under which a probe will hybridize to its target sequence, to a detestably greater degree than 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 can be identified which are 100% complementary to the probe (homologous probing). Alternatively, stringency conditions can be adjusted to allow some mismatching in sequences so that lower degrees of similarity are detected Applicant Ref. 0561-PCT

(heterologous probing). Generally, a probe is less than about 1000 nucleotides in length, optionally 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 NaCI, 1 % SDS (sodium dodecyl sulphate) at 37°C, and a wash in 1X to 2X SSC (20X 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 M NaCl, 1% SDS at 37°C, and a wash in O.SX to 1X
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 O.1X 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 Tm can be approximated from the equation of Meinkoth and Wahl, Aaal. Biochem., 138:267-284 (1984): Tm = 81.5°C + 16.6 (log M) +
0.41 (%CG) -0.61 (% form) - 500/L; where M is the molarity of monovalent cations, %CG 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 Tm is the temperature (under defined ionic strength and pH) at which SO%
of a complementary target sequence hybridizes to a perfectly matched probe. Tm is reduced by about I°C for each 1% of mismatching; thus, Tm, 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 Tm can be decreased 10°C.
Generally, stringent conditions are selected to be about 5°C lower than the thermal melting point (Tm) 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 l, 2, 3, or 4° lower than the thermal melting point (Tm); moderately stringent conditions can utilize a hybridization and/or wash at 6, 7, 8, 9, or 10°C lower than the thermal melting point (Tm); low stringency conditions can utilize a hybridization and/or wash at 11, 12, Applicant Ref. 0561-PCT

- 1~ -13, 14, 15, or 20°C lower than the thermal melting point (Tn,). Using the equation, hybridization and wash compositions, and desired Tm, 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 Tm 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, Laboratory Techniques in Biochemistry and Molecular Biology-Hybridization with Nucleic Acid Probes, Part I, Chapter "Overview of principles of hybridization and the strategy of nucleic acid probe assays", Elsevier, New York (1993); and Current Protocols in Molecular Biology, Chapter 2, Ausubel, et al., Eds., Greene Publishing and Wiley-Interscience, New York (1995).
As used herein, "vector" includes reference to a nucleic acid used in transfection of a host cell and into which can be inserted a polynucleotide.
Vectors are often replicons. Expression vectors permit transcription of a nucleic acid inserted therein.
The following terms are used to describe the sequence relationships between two or more nucleic acids or polynucleotides: (a) "reference sequence", (b) "comparison windows", (c) "sequence identity", and (d) "percentage of sequence 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" means includes reference to a contiguous and specified segment of a polynucleotide sequence, wherein the polynucleotide sequence may be compared to a reference sequence and wherein the portion of 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.

Applicant Ref. 0561-PCT

- m -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 and Waterman. Adv. Appl. Math. 2: 482 (1981); by the homology alignment algorithm of Needleman and Wunsch, J. Mol. Biol 48: 443 (1970);
by the search for similarity method of Pearson and Lipman, PYOC. Natl. Acad.
Sci. 85:
2444 (1988); by computerized implementations of these algorithms, including, but not limited to: CLUSTAL in the PCIGene program by Intelligenetics, Mountain View, California, GAP, BESTFIT, BLAST, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group (GCG), 575 Science Dr., Madison, Wisconsin, USA; the CLUSTAL program is well described by Higgins and Sharp, Gene 73: 237-244 (I988); Higgins and Sharp, CABIOS 5: 151-153 (1989); Corpet, et al., Nucleic Acids Research 16: 10881-90 (1988); Huang, et al., Computer Applications in the Biosciences 8: 155-65 (1992), and Pearson, et al., Methods in Molecular Biology 24: 307-331 (1994). The BLAST family of programs which can be used for database similarity searches includes: BLASTN for nucleotide query sequences against nucleotide database sequences; BLASTX for nucleotide query sequences against protein database sequences;
BLASTP for protein query sequences against protein database sequences; TBLASTN
for protein query sequences against nucleotide database sequences; and TBLASTX for nucleotide query sequences against nucleotide database sequences. See, Current Protocols in Molecular Biology, Chapter 19, Ausubel, et al., Eds., Greene Publishing and Wiley-Interscience, New York (1995).
GAP uses the algorithm of Needleman and Wunsch (J Mol Biol 48: 443-453 (1970)) to find the alignment of two complete sequences that maximizes the number of matches and minimizes the number of gaps. GAP considers all possible alignments and gap positions and creates the alignment with the largest number of matched bases and the fewest gaps. Default gap creation penalty values and gap extension penalty values in Version 10 of the Wisconsin Genetics Software Package are 8 and 2, respectively, for protein sequences. For nucleotide sequences the default gap creation penalty is 50 while the default gap extension penalty is 3. The gap creation and gap extension penalties can be expressed as an integer selected form the group of integers consisting of form 0 to 100.
Thus, for example, the gap creation and gap extension penalties can be 0, l, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, or greater.

Applicant Ref. 0561-PCT

GAP presents one member of the family of best alignments. There may be many members of this family, but no other member has a better quality. GAP
displays four figures of merit for alignments: Quality, Ratio, Identity, and Similarity. The Quality is the metric maximized in order to align the sequences. Ratio is the quality divided by the number of bases in the shorter segment. Percent Identity is the percent of the symbols that actually match. Percent Similarity is the percent of the symbols that are similar.
Symbols that are across from gaps are ignored. A similarity is scored when the scoring matrix value for a pair of symbols is greater than or equal to 0.50, the similarity threshold.
The scoring matrix used in Version 10 of the Wisconsin Genetics Software Package is BLOSUM62 (see Henikoff and Henikoff, Proc Natl Acad Sci USA 89:10915).
Unless otherwise stated, sequence identity/similarity values provided herein refer to the value obtained using GAP version 10 of Wisconsin Genetic Software Package using default parameters.
APOXD Protein The recombinant APOXD protein produced from the disclosed nucleic acid sequence provides a substantially pure protein useful to degrade oxalate, particularly in applications where highly purified enzymes are required. The recombinant protein may be used in enzymatic assays of oxalate or added to compositions containing oxalate to induce oxalate degradation.
When used externally, the enzyme can be placed in a liquid dispersion or solution, or may be mixed with a carrier solid for application as a dust or powder. The particular method of application and carrier used will be determined by the particular plant and pathogen target. Such methods are known, and are described, for example, in U.S. Patent No. 5,488,035 to Rao:
Gene Delivery The nucleic acid sequence encoding APOXD may be delivered to plant cells for transient transfections or for incorporation into the plant's genome by methods know in the art. Preferably, the gene is used to stably transform plant cells for expression of the protein in vivo.
To accomplish such delivery, the gene containing the coding sequence for APOXD may be attached to regulatory elements needed for the expression of the gene in Applicant Ref. 0561-PCT

a particular host cell or system. These regulatory elements include, for example, promoters, terminators, and other elements that permit desired expression of the enzyme in a particular plant host, in a particular tissue or organ of a host such as vascular tissue, root, leaf, or flower, or in response to a particular signal.
Promoters A promoter is a DNA sequence that directs the transcription of a structural gene, e.g., that portion of the DNA sequence that is transcribed into messenger RNA
(mRNA) and then translated into a sequence of amino acids characteristic of a specific polypeptide. Typically, a promoter is located in the 5' region of a gene, proximal to the transcriptional start site. A promoter may be inducible, increasing the rate of transcription in response to an inducing agent. In contrast, a promoter may be constitutive, whereby the rate of transcription is not regulated by an inducing agent. A promoter may be regulated in a tissue-specific or tissue-preferred manner, such that it is only active in transcribing the operably linked coding region in a specific tissue type or types, such as plant leaves, roots, or meristem.
Inducible Promoters An inducible promoter useful in the present invention is operably linked to a nucleotide sequence encoding APOXD. Optionally, the inducible promoter is operably linked to a nucleotide sequence encoding a signal sequence which is operably linked to a nucleotide sequence encoding APOXD. With an inducible promoter, the rate of transcription increases in response to an inducing agent.
Any inducible promoter can be used in the present invention to direct transcription of APOXD, including those described in Ward, et al., 1993, Plant Molecular Biol. 22: 361:-366. Exemplary inducible promoters include that from the ACEl system which responds to copper (Mett et al., 1993, PNAS 90: 4567-4571); In2 gene promoter from maize which responds to benzenesulfonamide herbicide safeners (Hershey et al., 1991, Plant Mol. Biol. 17:679-690; and the Tet repressor from TnlO (Hersey, et al., 1991, Mol. Gen. Genetics 227:229-237; Gatz, et al., 1994, Mol.Gen. Genetics 243:32-38).
A particularly preferred inducible promoter is one that responds to an inducing agent to which plants do not normally respond. One example of such a promoter is the steroid hormone gene promoter. Transcription of the steroid hormone gene Applicant Ref. 0561-PCT

promoter is induced by glucocorticosteroid hormone. (Schena et al., 1991, PNAS
U.S.A.
88:10421) - In the present invention, an expression vector comprises an inducible promoter operably linked to a nucleotide sequence encoding APOXD. The expression vector is introduced into plant cells and presumptively transformed cells are exposed to an inducer of the inducible promoter. The cells are screened for the presence of APOXD
proteins by immunoassay methods or by analysis of the enzyme's activity.
Pathogen-Inducible Promoters A pathogen-inducible promoter of the present invention is an inducible promoter that responds specifically to the inducing agent, oxalic acid, or to plant pathogens such as oxalic acid-producing pathogens including Sclerotinia sclerotiorum.
Genes that produce transcripts in response to Sclerotinia and oxalic acid have been described in Mouley et al., 1992, Plant Science 85:51-59. One member of the prpl-1 gene family contains a promoter that is activated in potato during early stages of late blight infection and is described in Martini et al., 1993, Mol.Gen.Genet.
236:179-186.
Tissue-specific or Tissue-Preferred Promoters A tissue specific promoter of the invention is operably linked to a nucleotide sequence encoding APOXD. Optionally, the tissue-specific promoter is operably linked to a nucleotide sequence encoding a signal sequence which is operably linked to a nucleotide sequence encoding APOXD. Plants transformed with a gene encoding APOXD operably linked to a tissue specific promoter produce APOXD
protein exclusively, or preferentially, in a specific tissue.
Any tissue-specific or tissue-preferred promoter can be utilized in the instant invention. Examples of such promoters include a root-preferred promoter such as that from the phaseolin gene as described in Murai et al., 1983, Science 222:476-482 and in Sengupta-Gopalan et al., 1985, PNAS USA 82:3320-3324; a leaf specific and light-induced promoter such as that from cab or rubisco as described in Simpson et al., 1985, EMBO J. 4(11):2723-2729, and in Timko et al., 1985, Nature 318:579-582; an anther-specific promoter such as that from LAT52 as described in Twell et al., 1989, Mol. Gen.
Genet. 217:240-245; a pollen-specific promoter such as that from Zml3 as described in Guerrero et al., 1990, Mol.Gen. Genet. 224:161-168; and a microspore-preferred Applicant Ref. 0561-PCT

promoter such as that from apg as described in Twell et al., 1993, Sex. Plant Reprod.
6:217-224.
. Other tissue-specific promoters useful in the present invention include a phloem-preferred promoter such as that associated with the Arabidopsis sucrose synthase gene as described in Martin et al., 1993, The Plant Journal 4(2):367-377; a floral-specific promoter such as that of the Arabidopsis HSP 18.2 gene described in Tsukaya et al., 1993, Mol. Gen. Genet. 237:26-32 and of the Arabidopsis HMG2 gene as described in Enjuto et al., 1995, Plant Cell 7:517-527.
An expression vector of the present invention comprises a tissue-specific or tissue-preferred promoter operably linked to a nucleotide sequence encoding APOXD.
The expression vector is introduced into plant cells. The cells are screened for the presence of APOXD protein by immunological methods or by analysis of enzyme activity.
Constitutive Promoters A constitutive promoter of the invention is operably linked to a nucleotide sequence encoding APOXD. Optionally, the constitutive promoter is operably linked to a nucleotide sequence encoding a signal sequence which is operably linked to a nucleotide sequence encoding APOXD.
Many different constitutive promoters can be utilized in the instant invention to express APOXD. Examples include promoters from plant viruses such as the 35S promoter from cauliflower mosaic virus (CaMV), as described in Odell et al., 1985, Nature 313:810-812, and promoters from genes such as rice actin (McElroy et al., 1990, Plant Cell 2:163-171); ubiquitin (Christensen et al., 1989, Plant Mol. Biol.
12:619-632;
and Christensen et al., 1992, Plant Mol. Biol 18:675-689); pEMU (Last et al., 1991, Theor. Appl. Genet. 81:581-588); MAS (Velten et al., 1984, EMBO J. 3:2723-2730); and maize H3 histone (Lepetit et al., 1992, Mol.Gen.Genet. 231:276-285; and Atanassvoa et al., 1992, Plant Journal 2(3):291-300).
The ALS promoter, a Xba/NcoI fragment 5' to the Brassica napus ALS3 structural gene, or a nucleotide sequence having substantial sequence similarity to the XbaI/NcoI fragment, represents a particularly useful constitutive promoter, and is described in published PCT Application number WO 96/30530.
In the present invention, an expression vector comprises a constitutive promoter operably linked to a nucleotide sequence encoding APOXD. The expression r Applicant Ref. 0561-PCT

vector is introduced into plant cells and presumptively transformed cells are screened for the presence of APOXD proteins by immunoassay methods or by analysis of the enzyme's activity.
Additional regulatory elements that may be connected to the APOXD
nucleic acid sequence for expression in plant cells include terminators, polyadenylation sequences, and nucleic acid sequences encoding signal peptides that permit localization within a plant cell or secretion of the protein from the cell. Such regulatory elements and methods for adding or exchanging these elements with the regulatory elements of the APOXD gene are known, and include, but are not limited to, 3'termination and/or polyadenylation regions such as those of the Agrobacterium tumefaciens nopaline synthase (nos) gene (Bevan et al., 1983, Nucl. Acids Res. 11(2):369-385); the potato proteinase inhibitor II (PINII) gene (Keil. et al., 1986, Nucl. Acids Res.
14:5641-5650; and An et al., 1989, Plant Cell 1:115-122); and the CaMV 19S gene (Molten et al., 1990, Plant Cell 2:1261-1272).
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., J. Biol. Chem. 264:4896-4900, 1989) and the Nicotiana plumbaginifolia extensin gene (DeLoose, et al., Gene 99:95-100, 1991), or signal peptides which target proteins to the vacuole like the sweet potato sporamin gene (Matsuoka, et al., PNAS 88:834, 1991) and the barley lectin gene (Wilkins, et al., Plant Cell, 2:301-313, 1990), or signals which cause proteins to be secreted such as that of PRIb (Lund, et al., Plant Mol. Biol. 18:47-53, 1992), or those which target proteins to the plastids such as that of rapeseed enoyl-Acp reductase (Verwoert, et al., Plant Mol. Biol.
26:189-202, 1994) are useful in the invention.
Gene Transformation Methods Numerous methods for introducing foreign genes into plants are known and can be used to insert the APOXD gene into a plant host, including biological and physical plant transformation protocols. See, for example, Miki et al., 1993, "Procedure for Introducing Foreign DNA into Plants" in: Methods in Plant Molecular Biology and Biotechnology, Glick and Thompson, eds., CRC Press, Inc., Boca Raton, pages 67-88.
The methods chosen vary with the host plant, and include chemical transfection methods such as calcium phosphate, microorganism-mediated gene transfer such as Applicant Ref. 0561-PCT

- i~ -Agrobacterium (Horsch, et al., Science 227:1229-31, 1985), electroporation, micro-injection, and biolistic bombardment.
Expression cassettes and vectors and in vitro culture methods for plant cell or tissue transformation and regeneration of plants are known and available.
See, for example, Gruber, et al., 1993, "Vectors for Plant Transformation" In: Methods in Plant Molecular Biology and Biotechnology, Glick and Thompson, eds., CRC Press, Inc., Boca Raton, pages 89-119.
A~robacte~ium-mediated Transformation The most widely utilized method for introducing an expression vector into plants is based on the natural transformation system of Agrobacterium. A.
tumefaciens and A. rhizogenes are plant pathogenic soil bacteria which genetically transform plant cells. The Ti and Ri plasmids of A. tumefaciens and A. rhizogenes, respectfully, carry genes responsible for genetic transformation of plants. See, for example, Kado, 1991, Crit. Rev.Plant Sci. 10(1):1-32. Descriptions of the Agrobacterium vector systems and methods for Agrobacterium-mediated gene transfer are provided in Gruber et al., supra;
Miki, et al., supra; and Moloney, et al., 1989, Plant Cell Reports 8:238.
Direct Gene Transfer Despite the fact that the host range for Agrobacterium-mediated transformation is broad, some major cereal crop species and gymnosperms have generally be recalcitrant to this mode of gene transfer, even though some success has recently been achieved in rice (Hiei et al., 1994, The Plant Journal 6(2):271-282). Several methods of plant transformation, collectively referred to as direct gene transfer, have been developed as an alternative to Agrobacterium-mediated transformation.
A generally applicable method of plant transformation is microprojectile-mediated transformation, where DNA is carried on the surface of microprojectiles measuring about 1 to 4 pm. The expression vector is introduced into plant tissues with a biolistic device that accelerates the microprojectiles to speeds of 300 to 600 mls which is sufficient to penetrate the plant cell walls and membranes. (Sanford et al., 1987, Part.Sci.
Technol 5:27; Sanford, 1988, Trends Biotech 6:299; Sanford, 1990, Physiol.
Plant 79:206; Klein et al., 1992, Biotechnology 10:268) Another method for physical delivery of DNA to plants is sonication of target cells as described in Zhang et al., 1991, BiolTechnology 9:996.
Alternatively, Applicant Ref. 0561-PCT

liposome or spheroplast fusions have been used to introduce expression vectors into plants. See, for example, Deshayes et al., 1985, EMBO J. 4:2731-2737; and Christou, et - al., 1987, PNAS USA 84:3962-3966. Direct uptake of DNA into protoplasts using CaCl2 precipitation, polyvinyl alcohol or poly-L-ornithine have also been reported.
See, for example, Hain et al., 1985, Mol. Gen.Genet. 199:161; and Draper, et al., 1982, Plant &
Cell Physiol. 23:451.
Electroporation of protoplasts and whole cells and tissues has also been described. See, for example, D'Halluin, et al., 1992, Plant Cell 4:1495-1505;
and Spencer, et al., 1994, PlantMol.Biol. 24:51-61.
Particle Woundin~lA~robacterium Delivery Another useful basic transformation protocol involves a combination of wounding by particle bombardment, followed by use of Agrobacterium for DNA
delivery, as described by Bidney, et al. 1992, Plant Mol. Biol. 18:301-313. Useful plasmids for plant transformation include pPHP9762 shown in Figure 5. The binary backbone for pPHP9762 is pPHP6333. See Bevan, 1984, Nucleic Acids Research 12:8711-8721.
This protocol is preferred for transformation of sunflower plants, and employs either the "intact meristem" method or the "split meristem" method.
In general, the intact meristem transformation method (Bidney, et al., SupYa) involves imbibing seed for 24 hours in the dark, removing the cotyledons and root radical, followed by culturing of the meristem explants. Twenty-four hours later, the primary leaves are removed to expose the apical meristem. The explants are placed apical dome side up and bombarded, e.g., twice with particles, followed by co-cultivation with Agrobacterium. To start the co-cultivation for intact meristems, Agrobacterium is placed on the meristem. After about a 3-day co-cultivation period the meristems are transferred to culture medium with cefotaxime (plus kanamycin for the NPTII selection).
Selection can also be done using kanamycin.
The split meristem method involves imbibing seed, breaking of the cotyledons to produce a clean fracture at the plane of the embryonic axis, excising the root tip and then bisecting the explants longitudinally between the primordial leaves (Malone-Schoneberg et al., 1994, Plant Science 103:199-207). The two halves are placed cut surface up on the medium then bombarded twice with particles, followed by co-cultivation with Agrobacterium. For split meristems, after bombardment the meristems Applicant Ref. 0561-PCT

are placed in an Agrobacterium suspension for 30 minutes. They are then removed from the suspension onto solid culture medium for three day co-cultivation. After this period, - the meristems are transferred to fresh medium with cefotaxime (plus kanamycin for selection).
Transfer by Plant Breeding Alternatively, once a single transformed plant has been obtained by the foregoing recombinant DNA method, conventional plant breeding methods can be used to transfer the structural gene and associated regulatory sequences via crossing and backcrossing. Such intermediate methods will comprise the further steps of:
(1) sexually crossing the disease-resistant plant with a plant from the disease-susceptible taxon; (2) recovering reproductive material from the progeny of the cross; and (3) growing disease-resistant plants from the reproductive material. Where desirable or necessary, the agronomic characteristics of the susceptible taxon can be substantially preserved by expanding this method to include the further steps of repetitively: (1) backcrossing the disease-resistant progeny with disease-susceptible plants from the susceptible taxon; and (2) selecting for expression of APOXD activity (or an associated marker gene) among the progeny of the backcross, until the desired percentage of the characteristics of the susceptible taxon are present in the progeny along with the gene imparting APOXD
activity.
By the term "taxon" herein is meant a unit of botanical classification of genus or lower. It thus includes genus, species, cultivars, varieties, variants and other minor taxonomic groups which lack a consistent nomenclature.
Assay Methods Transgenic plant cells, callus, tissues, shoots, and transgenic plants are tested for the presence of the APOXD gene by DNA analysis (Southern blot or PCR) and for expression of the gene by immunoassay or by assay of oxalate decarboxylase activity.
Tolerance to exogenous oxalic acid can also be used as a functional test of enzyme expression in transformed plants.
APOXD ELISA
Transgenic cells, callus, plants and the like are screened for the expression of APOXD protein by immunological assays, including ELISA. Anti-APOXD
antibodies are generated against APOXD preparations by known methods and are used in typical Applicant Ref. 0561-PCT

ELISA reactions. Polyclonal anti-APOXD can, for example, detect a range of about 10-100 pg APOXD protein in transgenic plant tissues.
In a suitable method for an APOXD-ELISA assay, fresh leaf or callus tissue is homogenized and centrifuged. An aliquot of the supernatant is added to a microtiter plate with a first anti-APOXD antibody and incubated for sufficient time for antibody-antigen reaction. The bound antibody is then reacted with a second antibody linked to a marker, which marker is developed or otherwise converted to a detectable signal correlated to the amount of APOXD protein in the sample. Any of the known methods for producing antibodies and utilizing such antibodies in an immunoassay can be used to determine the amount of APOXD expressed in transgenic plant cells and tissues of the invention.
Oxalate Decarboxylase Assay Transgenic cells, tissue, or plants expressing the APOXD gene are assayed for enzyme activity to verify expression of the gene. In general, the cells or tissue is frozen in liquid nitrogen, placed on a lyophilizer overnight to dehydrate, then crushed into a fme powder for use in the assay reaction. Leaf tissue is homogenized as fresh tissue in the reaction mixture, or dehydrated and treated as described above.
A typical assay reaction is begun by adding 0.75 mg of powdered tissue, such as callus, to 1 ml of oxalate decarboxylase reaction mixture: 900 ~1 0.2 M sodium phosphate buffer, pH 5.0, and 100 ~l of 10 mM sodium oxalate, pH 5Ø The reaction is incubated at room temperature for 3 hours with gentle mixing, and is stopped by the addition of 150 p1 of 1 M Tris-HCI, pH 7Ø The mixture is centrifuged, and an aliquot is placed in a cuvette with NAD (600 fig) and formate dehydrogenase (200 pg). The absorbance at 340 nm is correlated to the activity of the APOXD enzyme.
Use of Oxalate Decarboxylase as a Selectable Marker Oxalate decarboxylase is useful in selecting successful transformants, e.g., as a selectable marker. Growth of plant cells in the presence of oxalic acid favors the survival of plant cells that have been transformed with a gene encoding an oxalate-degrading enzyme, such as APOXD. In published PCT application WO 94/13790, herein incorporated by reference, plant cells grown on a selection medium containing oxalic acid (and all of the elements necessary for multiplication and differentiation of plant cells) demonstrated selection of only those cells transformed with and expressing oxalate Applicant Ref. 0561-PCT

oxidase. In like manner, transformation. and expression of the gene encoding APOXD in plant cells is used to degrade oxalic acid present in the media and allow the growth of - only APOXD-gene transformed cells.
Production of APOXD in Plants Trangenic plants of the present invention, expressing the APOXD gene, are used to produce oxalate decarboxylase in commercial quantities. The gene transformation and assay selection techniques described above yield a plurality of transgenic plants which are grown and harvested in a conventional manner.
Oxalate decarboxylase is extracted from the plant tissue or from total plant biomass.
Oxalate decarboxylase extraction from biomass is accomplished by known methods. See for example, Heney and Orr, 1981, Ahal. Biochem. 114:92-96.
In any extraction methodology, losses of material are expected and costs of the procedure are also considered. Accordingly, a minimum level of expression of oxalate decarboxylase is required for the process to be deemed economically worthwhile.
The terms "commercial" and "commercial quantities" here denote a level of expression where at least 0.1% of the total extracted protein is oxalate decarboxylase.
Higher levels of oxalate decarboxylase expression are preferred.
Diagnostic Oxalate Assay Clinical measurement of oxalic acid in urine is important, for example, in the diagnosis and treatment of patients with urinary tract disorders or hyperoxaluric syndromes. The recombinant APOXD enzyme of the invention is preferably immobilized onto beads or solid support, or added in aqueous solution to a sample for quantitation of oxalate. As discussed above, oxalate decarboxylase catalyzes the conversion of oxalate to C02 and formic acid. A variety of detection systems can be utilized to quantify this enzyme catalyzed conversion, including methods for detecting an increase in CO2, or for detecting an increase in formic acid.
For example, the conversion of oxalate to formic acid and COZ is assayed by determining formate production via the reduction of NAD in the presence of formate dehydrogenase. This method is described in Lung, et al., 1994, J.
Bacteriology, 176:2468-2472 and Johnson, et al., 1964, Biochem. Biophys. Acta 89:35.
A calibration curve is generated using known amounts of oxalic acid. The amount of oxalate in a specimen is extrapolated from the standard curve.

Applicant Ref. 0561-PCT

Other enzymatic assays and the like are adapted by known methods to utilize the APOXD enzyme to detect conversion of oxalate.
EXAMPLES
The invention is described more fully below in the following Examples, which are exemplary in nature and are not intended to limit the scope of the invention in any way.
Example 1 Cloning of the Gene Encoding APOXD
Protein Seguence A commercial preparation of A. phoenices oxalate decarboxylase enzyme was obtained from Boehringer Mannheim. (Catalog #479 586) SDS polyacrylamide gel electrophoresis was used to determine the purity of the enzyme. Only one dark band appeared following Coomassie blue staining of the polyacrylamide gel (12.5%).
This band was about 49 kd in size, as determined by comparison to molecular weight markers.
Aliquots of the preparation were sent to the University of Michigan for sequence analysis by Edman degradation on an automated protein sequencer. Preparative polyacrylamide gels were run and the APOXD band was isolated from the gel prior to sequencing. The protein was first sequenced at the amino terminus. Proteins were chemically cleaved into fragments by cyanogen bromide, size separated on polyacrylamide gels, and isolated as bands on the gel for further preparation and sequencing. The results of the sequencing are shown below in Table 2.

Peptide Seq uence* Seq. ID
No.

ammo termlnLiSG1n AspLys Pro Phe ThrPro Asp HisArg 6 Asp ProTyr Asp His LysVal Asp AlaIle Gly GluX Pro Leu His Glu fragment Val IleArg Glu Leu HisTrp His ArgGlu 7 Ala Gly fragment Arg LeuAsp Glu Gly ValIle Arg GluLeu His CysHis Arg 1u G Ala Glu fragment Ser TyrPhe Lys Arg GlyArg Ala ArgTyr Thr IlePhe Ala la a g A Glu Ar Gly Asn Al fragment Ser AlaHis Thr Pro ProSer Val LeuAla Gly Asn * X = Unknown.

a Applicant Ref. 0561-PCT

PCR Amplification of Genomic A. phoenices Genomic DNA was used as the PCR template to amplify the APOXD
- sequence. Aspergillus phoenices was obtained from the American Type Culture Collection (ATCC), Rockville, MD. Cultures were established on solid potato dextrose agar medium (Difco formulation). Liquid stationary cultures were started from culture plates by innoculatory spores in a minimal growth medium previously described for the culture of Aspergillus strains (Emiliani, et al., 1964, Arch. Biochem. Biophys 105:488-493, cited above).
To isolate DNA, mycelial mats were recovered from 4-day liquid stationary cultures, washed in cold water, and blotted dry. The tissue was then frozen in liquid nitrogen, ground by mortar and pestle, and stored frozen at -80°C. DNA was extracted by the method described for fungal mycelium in Sunis et al. (eds.), 1990, PCR
protocols, pages 282-287.
PCR Strategy As diagrammed in Figure 1, primers were designed for both the N-terminal protein sequence and for an internal peptide fragment. One set of primers (PHN

[Seq ID No. 11] and PHN 11339 [Seq ID No. 12]) was designed with nearly full degeneracy. A second set of primers (PHN 11471 [Seq. ID No. 13] and PHN 11476 [Seq ID No. 14]) was designed with no degeneracy. These were based on a codon usage table for Aspergillus niger generated using the Wisconsin Sequence Analysis Package (GCG) (Genetics Computer Group, Inc., Madison, WI). The sequences of these primers is shown in Table 3, below, and diagrammatically in Figure 1. Table 3 shows the degenerate primer mixtures using ICTPAC designations, as described in Cornish-Bowden, 1985, Nucleic Acids Res. 13:3021-3030. The lUPAC nucleic acid symbols include: Y=C
or T;
N=A, T, C, or G; R=A or G; D=A, T, or G; and V=A, C, or G. Both of these PCR
strategies were successful in amplifying a DNA fragment, shown in Table 4, having homology to the protein sequence data shown in Table 2.

Primer Sets-3') Seq. ID
(5' #

CAU TA

CUA VA

CTT

Applicant Ref. 0561-PCT

PCR reactions were set up containing increasing quantities of A. phoenices genomic DNA, in the range of 1-10 nanograms, and various oligonucleotide primer sets.
Degenerate primers were added at a ten-fold higher concentration than that standardly used, due to their degeneracy. All other conditions for PCR were standard, essentially as described in Innis, et aL, 1990, PCR Protocols, pages 282-287, except for the annealing temperatures for the primers. These temperatures were determined on an individual basis using the Oligo 4.0 computer program for analysis as described in Rychlik et al., 1989, Nuc.Acids Res. 17:8543-8551. Specifically, the primers and annealing temperatures were primer first 5 cycles next 30 cycles PHN 11471 50 C 58~ C

PHN 11476 50 C 58~ C

Transformation and Seauencin~
PCR amplification products were ligated into pCR II using the TA Cloning Kit (InVitrogen, San Diego, CA), and transformed into E.coli strain DHSa competent cells 1 S (Life Technologies, Gathersburg, MD) according to the protocol provided with the strain, for cloning and sequencing. Transformed bacteria with plasmid insertions were selected on medium 34Z (LB agar plates containing 100 mg/I carbenicillin) using standard X-GAL
selection protocols (Ausubel, et al., eds, 1989, Current Protocols in Molecular Biology, pages 1Ø3-1.15.8). Briefly, white colonies were picked with an inoculating loop and inoculated directly into a PCR reaction mixture containing primers specific to the universal and reverse promoter regions just outside the multiple cloning site.
The remaining innoculum on the loop was used to streak a plate of 34Z medium and numbered to correspond to the PCR reaction. Successful amplification of an inserted PCR fragment resulted in a band on an ethidium bromide stained agaraose gel which was slightly larger than the size of the insert. Bacterial isolates with an insert of the correct Applicant Ref. 0561-PCT

size were inoculated into shaking liquid cultures and subsequently used for plasmid isolation protocols, followed by sequencing of the insert of interest.
Sequence quality plasmid was prepared by using the Nucleobond P-100 plasmid isolation kit (Machery-Nagle GmBH & Co., Cat.No. BP 101352m distributed by the Nest Group, Southboro, MA). This kit uses an alkaline lysis step and is followed by an ion exchange silica column purification step. Plasmid and gene specific primers were sent to Iowa State University to be sequenced on an automated, ABI DNA
Sequencing machine.
The degenerate primer PCR experiment resulted in the amplification of a 0.4 kb band, which was sequenced and determined to have a deduced amino acid sequence matching the protein data in Table 2. The non-degenerate primer experiment resulted in DNA fragments of various sizes. One fragment was about 0.4 kb in length and encoded a protein having homology to the protein sequence data of Table 2. The region of the APOXD gene that was amplified by both primer sets was nearly the same, so DNA
sequence data for the amplified fragments was compiled, and the sequence of the compiled APOXD genomic fragment is shown in Table 4 [Seq ID No. 15] together with its deduced amino acid sequence [Seq ID Nos. 16 and 29]. The underlined amino acid sequences were represented in the original protein sequence analysis data (Table 2).

Applicant Ref. 0561-PCT

APOXD FRAGMENT

ACG ATC ACA AGG TGG ATG CGA TCG GGG AAG GCC ATG AGC CCT TGC CCT
Asp His Lys Val Asp Ala Ile Gly Glu Gly His Glu Pro Leu Pro GGC GCA TGG GAG ATG GAG CCA CCA TCA TGG GAC CCC GCA ACA AGG ACC
Trp Arg Met Gly Asp Gly Ala Thr Ile Met Gly Pro Arg Asn Lys Asp GTG AGC GCC AGA ACC CCG ACA TGC TCC GTC CTC CGA GCA CCG ACC ATG
Arg Glu Arg Gln Asn Pro Asp Met Leu Arg Pro Pro Ser Thr Asp His GCA ACA TGC CGA ACA TGC GGT GGA GCT TTG CTG ACT CCC ACA TTC GCA
Gly Asn Met Pro Asn Met Arg Trp Ser Phe Ala Asp Ser His Ile Arg TCG AGG TAA GCC CTT CGA GGG TTT TGT GTA CGA CAA GCA AAA TAG GCT
Ile Glu AAT GCA CTG CAG GAG GGC GGC TGG ACA CGC CAG ACT ACC GTA CGC GAG
Gly Trp Thr Arg Gln Thr Thr Val Arg Glu CTG CCA ACG AGC AAG GAG CTT GCG GGT GTA AAC ATG CGC CTC GAT GAG
Leu Pro Thr Ser Lys Glu Leu Ala Gly Val Asn Met Arg Leu Asp Glu GGT GTC ATC CGC GAG TTG CAC TGG CAA GGG CTG AAG GCG AAT TCC AGC
Gly Val Ile Arg Glu Leu His Trp ACA CTG GCG GCC GTT ACT AGT GGA TCC GAG CTC GGT ACC AAG CTT GAT
GC ATAGCT

Applicant Ref. 0561-PCT

3' RACE
Nested oligonucleotide primers were designed based on the genomic DNA
_ sequence fragment which was previously amplified (Table 4) and used for 3' RACE to enhance gene specific amplification. The nested primer design is diagrammatically shown in Figure 2 and the nucleic acid sequences of the primers is shown below in Table 5. Arrows represent the gene specific primers (from top to bottom) PHN 11811, PHN
11810, and the oligo dT based 3' primer from a commercially supplied 3' RACE
kit (Life Technologies, Gaithersburg, MD, Cat. No. 18373-019) TABLE S
3' RACE Primers (5'-3') seq 1D
No.

GGT AAG

The first round of PCR amplification using the outside gene specific primer (GSP) PHN11810 and the oligo dT based 3' primer resulted in no visible DNA
bands. The inside GSP PHNI 1811 and the oligo dT based 3' primer were then used for a second round of amplification on the same sample. A large number of bands appeared, some of which stained intensely with ethidium bromide and some which did not.
The prominent bands were 0.4, 0.8 and 1.3 kb in size. This experiment was set up using 5' and 3' primers with custom ends which only allow ligation of DNA fragments amplified by both. This method permitted the reaction to be used in the ligation protocol without further purification or characterization of the DNA fragments. All three of the prominent bands described above were ligated into pAMPl (Life Technologies, Cat. No., 016), transformed into DHSa cells (Life Technologies, Cat. No. 18263-12), cloned and sequenced. The 0.4 kb band was found to encode an amino acid sequence having homology to the APOXD sequence data of Table 1.
5' RACE
Total RNA was reverse transcribed with commercially available components and a set of oligo dT-based primers ending in G, C or A which are collectively termed Bam T17V (5' CGC GGA TCC GT1~ V) 3') [Seq >D No. 19] These Applicant Ref. 0561-PCT

primers are disclosed in published PCT Application No. U596/08582. First strand cDNA
was oligo dC-tailed and then column purified using commercially available components.
- (Life Technologies, Gaithersburg). The product of this reaction was then used in PCR
with primer set Bam G13H, an equimolar mixture of oligo dG primers ending in A, C, or T (5' TAA GGA TCC TG~3 H 3') [Seq. ID NO: 20], and a second gene specific primer, PHN 11813 [Seq ID No. f1 ]. Amplified products were characterized by Southern analysis using the protocol as described in Ausubel, et al. (eds.), 1989, Current Protocols in Molecular Biology, pages 2Ø1 - 2.12.5.
Hybridization of the 5' RACE product was done using the PCR amplified genomic DNA fragment (Table 4) as a radiolabeled probe. A 0.6 kb band was amplified by this reaction and was strongly labeled with the probe. No other bands appeared. This 0.6 kb band was ligated into the PCR II vector using the TA-cloning procedure, transformed into DHSI, cloned and sequenced. The DNA sequence analysis of the 0.6 kb PCR fragment showed it was homologous to the APOXD sequence data shown in Table 2.

5' RACE Primers SEQ. ID
No.

Bam T17V 5' CGC GGA TCC GT1,V 3' 19 Bam G13H 5' TAA GGA TCC TG13H 3' 2Q

PHN 118135' CAU CAU CAU CAU TAC CTC GAT GCG AAT 21 GTG 3' IUPAC Symbols: V=G,C, or A; H=A, T, or C.
PCR For Full Length The 5' and 3' RACE products were sequenced to their ends as determined by the initiating methionine and the poly-A tail respectively. DNA sequence at each end was analyzed by Oligo 4.0 for oligonucleotide primer design in preparation for PCR to obtain the complete gene.
Primer PHN 12566 designed to the 3' end of the sequence, was used to reverse transcribe total RNA. Primers PHN 12565 and PHN 12567 were used to amplify first strand cDNA. The PCR amplified band was ligated into PCR II using the TA

a Applicant Ref. 0561-PCT

cloning kit (In Vitrogen; San Diego, CA) then transformed into DHSI, cloned, and sequenced.

Full Length cDNA Primers (5'-~3') sEQ: ID
No.

GTA

CC

CCG

A 1.4 kb band was amplified which stained very intensely with ethidium bromide. Other, smaller bands were present, but clearly, the 1.4 kb band was prominent.
This band was sequenced and subjected to open reading frame analysis. All of the protein fragments originally sequenced (Table 2), were found in the deduced amino acid sequence of this PCR product.
Southern analysis was performed on genomic DNA using the 1.4 kb cDNA
as a radiolabeled probe. Only one band hybridized, suggesting that the gene is a single copy and unique in the A. phoenices genome.
Table 1 (pages 4-7) shows the full length cDNA sequence [Seq ID No:l]
and deduced amino acid sequence [Seq ID No:2] of the A. phoehices oxalate decarboxylase gene as amplified, using PCR primers PHN 12565 and PHN 12567.
The underlined amino acid sequences were represented in the original protein sequence analysis data (Table 2). The protein sequence encoded by the full length cDNA
includes a pre-protein, amino acid residues 27-458 [Seq ID No:4], and a mature protein, amino acid residues 50-458 [Seq ID No:S].
Example 2 Transformed plant tissue degrades oxalate CaMV35S/0'/APOXD
The insert ofpPHP9685 (1.4 kb APOXD cDNA in pCR II) was placed into a cloning vector intermediate (pLitmus 28, New England Biolabs) between a plant Applicant Ref. 0561-PCT

expressible promoter and 3' region as shown in the construction diagrams of Figure 3.
The upstream region consists of a cauliflower mosaic virus 35S promoter with a - duplicated enhancer region (2X35S; bases -421 to -90 and -421 to +2, Gardner, et al., 1985, Nucleic Acids Res. 9:2871-2888) with a flanking 5' Notl site and a 3' Pst site, and S2' RNA leader sequence. The 3' region is from potato proteinase inhibitor II.
These are described in Bidney, et al., 1992, Plant Mol. Biol. 18:301-313. The 2X CaMV

promoter is described in Odell, et al., Nature 313:810-812.
The plant-expressible APOXD gene cassette was then isolated from the cloning intermediate and ligated into the ALS::NPT II:: PIN II-containing pBINl9 construct, pPHP8110. Plasmid pPHP8110 was created from pBIN 19 (Bevan, 1984, Nucleic Acids Res. 12:8711-8721) by replacing the NOS::NPTII::NOS gene cassette in pBINI9 with an ALS::NPTII::PINII cassette. As shown in Figure 3, pPHP8110 is a derivative of pBINl9 containing the NPT II gene, the aminoglycoside-3'-O-phosphotransferase coding sequence, bases 1551 to 2345 from E.coli transposon (Genbank Accession Number V00004, Beck, et al., 1982, Gene 19:327-336). The second amino acid was modified from an isoleucine to a valine in order to create a Nco I
restriction site which was used to make a translational fusion with the ALS
promoter (see copending U.S. Patent Application Serial No. 08/409,297). pPHP8110 further contains the potato proteinase inhibitor II terminator (PIN II) bases 2-310, as described in An, et al., 1989, Plant Cell 1:115-122.
As shown in Figure 4, the resultant plasmid, pPHP9723, carries the APOXD gene construct, together with the NPTII gene for selection of transgenic plant cells, positioned between Agrobacterium T-DNA borders.
Germin/APOXD
A second APOXD cDNA containing plasmid was constructed using the methods described above for producing pPHP 9723. In the second construct, the APOXD
fungal signal and presequence (49 amino acids) were replaced with a plant signal sequence obtained from the 5' end of an enzyme subunit of wheat oxalate oxidase. (Lane, et al., 1991, J. Biol. Chem. 266:10461.) This was accomplished by designing primers that were homologous to the Germin signal sequence, and having extensions to provide the addition of a Sal I restriction site at the 5' end and APOXD 5' sequence followed by a Applicant Ref. 0561-PCT

Nru I site at the 3' end. The primers were used to amplify the Germin signal sequence and are shown below in Table 8.
. Table 8 Germin Signal Sequence Primers (5'-3') Seq ID
No.

GAC

CCT TC

AT CCT

GGG TAG CC AAAA CAG CT GGAG

The amplified Germin signal sequence product [Seq ID N0:27] shown below in Table 9, and a vector containing the full length APOXD cDNA
(pPHP9648) were each digested with Sal I and Nru I. A ligation reaction was set up with the digested fragments to form a Germin signal sequence - APOXD coding sequence fusion construct.
Clones of the correct size were sequenced to verify correct results.
As shown in Table 9, the SaIIlNruI cut Germin SS - containing sequence also contained modif ed APOXD codons matched to fill in the NruI-cut APOXD
sequence. The Germin signal sequence [Seq. m No: 28] is shown in lower case.
Table 9 Amplified Germin Signal/APOXD Sequence*

Sa.ZI start 51 TACAATTACT ATTTACAATT ACAGTCGACC CGGGATCC atg ggt tac 98 tca aag acc ttg gtt get ggt ttg ttc get atg ttg ttg 137 ttg get cca get gtt ttg get acc ICAG GAT AAG CCT TTC
NruI

Applicant Ref. 0561-PCT

*The SaII (GTCGAC) and NruI (TCGCGA) restriction sites are underlined, the Germin signal sequence is in lower case, with the Germin start site in bold. APOXD
sequences modified in the PCR primer design are shown in bold.
This fusion gene was placed in the binary T-DNA plasmid to produce plasmid pPHP9762 carrying the fusion gene and the plant expressible NPTII gene positioned between Agrobacterium T-DNA borders, as described above.
Agrobacterium tumefaciens strain EHA105 (as described in Hood, et al., 1993, Transgen. Res. 2:208-218) was transformed with kanamycin resistant binary T-DNA vectors carrying the different versions of APOXD. Transformation was accomplished by the freeze-thaw method of Holsters, et al., 1978, Mol. Gen.
Genetics 1:181-7. The transformed isolates were selected on solidified 60A (YEP; 10 g/1 yeast extract, 10 g/1 bactopeptone, 5 g/1 NaCI, pH 7.0) medium with 50 mg/1 kanamycin.
Transformed bacteria were cultured in liquid culture of YEP medium containing 50 mg/1 I5 kanamycin, to log phase growth (O.D.6o~ 0.5-1.0) for use in plant transformations. Binary plasmids were re-isolated from transformed Agrobacterium to verify that integrity was maintained throughout the transformation procedures.
Sunflower leaf discs were obtained by harvesting leaves which were not fully expanded, sterilizing the surface in 20% bleach with TWEEN 20, and punching discs out of the leaf with a paper punch. Agrobacterium suspensions were centrifuged and resuspended in inoculation medium (12.5 ~M MES buffer, pH 5.7, 1 g/1 NH4C1, 0.3 g/1 MgS04) to a calculated OD6oo of 0.75 as described in Malone-Schoneberg, et al., 1994, Plant Science 103:199-207. Leaf discs were inoculated in the resuspended Agrobacterium for 10 minutes then blotted on sterile filter paper.
The tissue and bacteria were co-cultivated on 527 for 3 days, then transferred to 527E medium for the selection of transgenic plant cells. After 2 weeks of Applicant Ref. 0561-PCT

culture, the transgenic callus nodes were removed from the leaf disc and subcultured on fresh 527E medium. A number of subcultures were repeated prior to the assay of the callus tissue for enzyme activity.
To assay for enzyme activity, callus was harvested, snap frozen in liquid nitrogen, lyophilized to dryness and powdered. A quantity of 0.75 mg of powder from each prepared callus line was added to 1.0 ml reaction mixture (900 p1 200 mM
NaP04, pH 5.0, 100 p1 10 mM Na-oxalate pH 5.0). The reaction proceeded for 3 hours at room temperature and was stopped by the addition of 150 p,1 of 1M TRIS-HCI, pH 7Ø
Each sample was spun at 14,000 rpm for one minute and 1 ml was removed to a cuvette. One hundred (100) ~l of (3-NAD (6.6 mg/ml stock) and 50 p1 formate dehydrogenase (4.0 mg/ml stock) were added and the increase in absorbance was measured at 340 nm. A
slope was generated for each sample as well as for a formate standard curve.
Assay results were reported as pM oxalate metabolized /mg powder.
The results of the leaf disk assay are shown below in Table 10, and demonstrate that the APOXD gene sequence produces enzyme that is active in transgenic callus. No activity was seen in control callus, or callus transformed with the native APOXD gene (pPHP 9723).
Table 10 Oxalate Decarboxylase Activity in Trans~enic Sunflower Tissue Callus Line Binary Vector Activity pM oxalate/min/mg SMF3 None 0 9723 -1 pPHP 9723 0 -2 pPHP 9723 0 -3 pPHP 9723 0 9762-1 pPHP 9762 1.35 -2 pPHP 9762 1.40 -3 pPHP 9762 0.87 -4 pPHP 9762 0.81 -5 pPHP 9762 0.81 -6 pPHP 9762 0.90 Applicant Ref. 0561-PCT

Example 3 Transgenic Sunflower Plants Expressing APOXD
Sunflower plants were transformed using a basic transformation protocol involving a combination of wounding by particle bombardment, followed by use of Agrobacterium for DNA delivery, as described by Bidney, et al. Plant Mol.
Biol. 18:301 313. The plasmid pPHP9762, as described above for Example 2 and shown in Figure 5, was used in these experiments. pPHP9762 contains the APOXD gene with the fungal signal and presequence replaced with the Germin signal sequence and a plant expressible NPTII gene which provides kanamycin resistance to transgenic plant tissues.
Procedures for preparation of Agrobacterium and preparation of particles for wounding are described in Bidney, et al., 1992, Plant Mol. Biol. 18:301-313. The Pioneer sunflower line SMF3, used in these experiments, is described in Burros, et al., 1991, Plant Cell Rep. 10:161-166. The Agrobacterium strain used in these experiments, EHA 105. Procedures for use of the helium gun, intact meristem preparation, tissue culture and co-cultivation conditions, as well as recovery of transgenic plants, are described in Bidney, et al., 1992, Plant Mol. Biol. 18:301-313.
Sunflower explants were prepared by imbibing seed overnight, removing the cotyledons and radical tip, then culturing overnight on medium containing plant growth regulators. Primary leaves were then removed and explants arranged in the center of a petri plate for bombardment. The PDS 1000 helium-driven particle bombardment device (Bio-Rad) was used with 600 psi rupture discs and a vacuum of 26 inches, Hg to bombard meristem explants twice on the highest shelf position. Following bombardment, log phase Agrobacterium cultures transformed with the APOXD-plasmid pPHP 9762, as described for Example 2, were centrifuged and resuspended at a calculated OD600 (vis) of 4.0 in inoculation buffer. Agrobacterium was then dropped onto the meristem explants using a fine tipped pipettor. Inoculated explants were co-cultured for three days then transferred to medium containing 50 mg/1 kanamycin and 250 mg/1 cefotaxime for selection. Explants were cultured on this medium for two weeks then transferred to the same medium, but lacking kanamycin. Green, kanamycin-resistant shoots were recovered to the greenhouse and assayed by an NPTII ELISA assay to verify transformation.
Oxalate decarboxylase enzyme assays are performed on these plants and/or progeny to confirm the expression of APOXD.

Applicant Ref. 0561-PCT

The invention has been described with reference to various specific and preferred embodiments and techniques. However, it should be understood that many variations and modifications may be made while remaining within the spirit and scope of the invention.

SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: PIONEER HI-BRED INTERNATIONAL, INC.
(ii) TITLE OF INVENTION: GENE ENCODING OXALATE DECARBOXYLASE FROM
ASPERGILLUS PHOENICES
(iii) NUMBER OF SEQUENCES: 30 (iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: SMART & BIGGAR
(B) STREET: P.O. BOX 2999, STATION D
(C) CITY: OTTAWA
(D) STATE: ONT
(E) COUNTRY: CANADA
(F) ZIP: K1P 5Y6 (v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: ASCII (text) (vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER: CA 2,350,328 (B) FILING DATE: 26-JUN-2001 (C) CLASSIFICATION:
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE:
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: SMART & BIGGAR
(B) REGISTRATION NUMBER:
(C) REFERENCE/DOCKET NUMBER: 75529-56 (ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (613)-232-2486 (B) TELEFAX: (613)-232-8440 (2) INFORMATION FOR SEQ ID NO.: 1:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 1438 (B) TYPE: nucleic acid (C) STRANDEDNESS:
(D) TOPOLOGY:
(ii) MOLECULE TYPE: DNA
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Aspergillus phoenices (ix) FEATURE
(A) NAME/KEY: CDS
(B) LOCATION: (24)...(1394) (ix) FEATURE
(A) NAME/KEY: mat_peptide (B) LOCATION: (171)...(1394) (ix) FEATURE
(A) NAME/KEY: sig~eptide (B) LOCATION: (24)...(101) (xi) SEQUENCE DESCRIPTION: SEQ ID NO.: 1:

Met Gln Leu Thr Leu Pro Pro Arg Gln Leu Leu Leu Ser Phe Ala Thr Val Ala Ala Leu Leu Asp Pro Ser His Gly Gly Pro Val Pro Asn Glu Ala Tyr Gln Gln Leu Leu Gln Ile Pro Ala Ser Ser Pro Ser Ile Phe Phe Gln Asp Lys Pro Phe Thr Pro Asp His Arg Asp Pro Tyr Asp His Lys Val Asp Ala Ile Gly Glu Gly His Glu Pro Leu Pro Trp Arg Met Gly Asp Gly Ala Thr Ile Met Gly Pro Arg Asn Lys Asp Arg Glu Arg Gln Asn Pro Asp Met Leu Arg Pro Pro Ser Thr Asp His Gly Asn Met Pro Asn Met Arg Trp Ser Phe Ala Asp Ser His Ile Arg Ile Glu Glu Gly Gly Trp Thr Arg Gln Thr Thr Val Arg Glu Leu Pro Thr Ser Lys Glu Leu Ala Gly Val Asn Met Arg Leu Asp Glu Gly Val Ile Arg Glu Leu His Trp His Arg Glu Ala Glu Trp Ala Tyr Val Leu Ala Gly Arg Val Arg Val Thr Gly Leu Asp Leu Glu Gly Gly Ser Phe Ile Asp Asp Leu Glu Glu Gly Asp Leu Trp Tyr Phe Pro Ser Gly His Pro His Ser Leu Gln Gly Leu Ser Pro Asn Gly Thr Glu Phe Leu Leu Ile Phe Asp Asp Gly Asn Phe Ser Glu Glu Ser Thr Phe Leu Leu Thr Asp Trp Ile Ala His Thr Pro Lys Ser Val Leu Ala Gly Asn Phe Arg Met Arg Pro Gln Thr Phe Lys Asn Ile Pro Pro Ser Glu Lys Tyr Ile Phe Gln Gly Ser Val Pro Asp Ser Ile Pro Lys Glu Leu Pro Arg Asn Phe Lys Ala Ser Lys Gln Arg Phe Thr His Lys Met Leu Ala Gln Lys Pro Glu His Thr Ser Gly Gly Glu Val Arg Ile Thr Asp Ser Ser Asn Phe Pro Ile Ser Lys Thr Val Ala Ala Ala His Leu Thr Ile Asn Pro Gly Ala Ile Arg Glu Met His Trp His Pro Asn Ala Asp Glu Trp Ser Tyr Phe Lys Arg Gly Arg Ala Arg Val Thr Ile Phe Ala Ala Glu Gly Asn Ala Arg Thr Phe Asp Tyr Val Ala Gly Asp Val Gly Ile Val Pro Arg Asn Met Gly His Phe Ile Glu Asn Leu Ser Asp Asp Glu Glu Val Glu Val Leu Glu Ile Phe Arg Ala Asp Arg Phe Arg Asp Phe Ser Leu Phe Gln Trp Met Gly Glu Thr Pro Gln Arg Met Val Ala Glu His Val Phe Lys Asp Asp Pro Asp Ala Ala Arg Glu Phe Leu Lys Ser Val Glu Ser Gly Glu Lys Asp Pro Ile Arg Ser Pro Ser Glu (2) INFORMATION FOR SEQ ID NO.: 2:
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Met Gln Leu Thr Leu Pro Pro Arg Gln Leu Leu Leu Ser Phe Ala Thr Val Ala Ala Leu Leu Asp Pro Ser His Gly Gly Pro Val Pro Asn Glu Ala Tyr Gln Gln Leu Leu Gln Ile Pro Ala Ser Ser Pro Ser Ile Phe Phe Gln Asp Lys Pro Phe Thr Pro Asp His Arg Asp Pro Tyr Asp His Lys Val Asp Ala Ile Gly Glu Gly His Glu Pro Leu Pro Trp Arg Met Gly Asp Gly Ala Thr Ile Met Gly Pro Arg Asn Lys Asp Arg Glu Arg Gln Asn Pro Asp Met Leu Arg Pro Pro Ser Thr Asp His Gly Asn Met Pro Asn Met Arg Trp Ser Phe Ala Asp Ser His Ile Arg Ile Glu Glu Gly Gly Trp Thr Arg Gln Thr Thr Val Arg Glu Leu Pro Thr Ser Lys Glu Leu Ala Gly Val Asn Met Arg Leu Asp Glu Gly Val Ile Arg Glu Leu His Trp His Arg Glu Ala Glu Trp Ala Tyr Val Leu Ala Gly Arg Val Arg Val Thr Gly Leu Asp Leu Glu Gly Gly Ser Phe Ile Asp Asp Leu Glu Glu Gly Asp Leu Trp Tyr Phe Pro Ser Gly His Pro His Ser Leu Gln Gly Leu Ser Pro Asn Gly Thr Glu Phe Leu Leu Ile Phe Asp Asp Gly Asn Phe Ser Glu Glu Ser Thr Phe Leu Leu Thr Asp Trp Ile Ala His Thr Pro Lys Ser Val Leu Ala Gly Asn Phe Arg Met Arg Pro Gln Thr Phe Lys Asn Ile Pro Pro Ser Glu Lys Tyr Ile Phe Gln Gly Ser Val Pro Asp Ser Ile Pro Lys Glu Leu Pro Arg Asn Phe Lys Ala Ser Lys Gln Arg Phe Thr His Lys Met Leu Ala Gln Lys Pro Glu His Thr Ser Gly Gly Glu Val Arg Ile Thr Asp Ser Ser Asn Phe Pro Ile Ser Lys Thr Val Ala Ala Ala His Leu Thr Ile Asn Pro Gly Ala Ile Arg Glu Met His Trp His Pro Asn Ala Asp Glu Trp Ser Tyr Phe Lys Arg Gly Arg Ala Arg Val Thr Ile Phe Ala Ala Glu Gly Asn Ala Arg Thr Phe Asp Tyr Val Ala Gly Asp Val Gly Ile Val Pro Arg Asn Met Gly His Phe Ile Glu Asn Leu Ser Asp Asp Glu Glu Val Glu Val Leu Glu Ile Phe Arg Ala Asp Arg Phe Arg Asp Phe Ser Leu Phe Gln Trp Met Gly Glu Thr Pro Gln Arg Met Val Ala Glu His Val Phe Lys Asp Asp Pro Asp Ala Ala Arg Glu Phe Leu Lys Ser Val Glu Ser Gly Glu Lys Asp Pro Ile Arg Ser Pro Ser Glu (2) INFORMATION FOR SEQ ID NO.: 3:
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Met Gln Leu Thr Leu Pro Pro Arg Gln Leu Leu Leu Ser Phe Ala Thr Val Ala Ala Leu Leu Asp Pro Ser His Gly (2) INFORMATION FOR SEQ ID NO.: 4:
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Gly Pro Val Pro Asn Glu Ala Tyr Gln Gln Leu Leu Gln Ile Pro Ala Ser Ser Pro Ser Ile Phe Phe Gln Asp Lys Pro Phe Thr Pro Asp His Arg Asp Pro Tyr Asp His Lys Val Asp Ala Ile Gly Glu Gly His Glu Pro Leu Pro Trp Arg Met Gly Asp Gly Ala Thr Ile Met Gly Pro Arg Asn Lys Asp Arg Glu Arg Gln Asn Pro Asp Met Leu Arg Pro Pro Ser Thr Asp His Gly Asn Met Pro Asn Met Arg Trp Ser Phe Ala Asp Ser His Ile Arg Ile Glu Glu Gly Gly Trp Thr Arg Gln Thr Thr Val Arg Glu Leu Pro Thr Ser Lys Glu Leu Ala Gly Val Asn Met Arg Leu Asp Glu Gly Val Ile Arg Glu Leu His Trp His Arg Glu Ala Glu Trp Ala Tyr Val Leu Ala Gly Arg Val Arg Val Thr Gly Leu Asp Leu Glu Gly Gly Ser Phe Ile Asp Asp Leu Glu Glu Gly Asp Leu Trp Tyr Phe Pro Ser Gly His Pro His Ser Leu Gln Gly Leu Ser Pro Asn Gly Thr Glu Phe Leu Leu Ile Phe Asp Asp Gly Asn Phe Ser Glu Glu Ser Thr Phe Leu Leu Thr Asp Trp Ile Ala His Thr Pro Lys Ser Val Leu Ala Gly Asn Phe Arg Met Arg Pro Gln Thr Phe Lys Asn Ile Pro Pro Ser Glu Lys Tyr Ile Phe Gln Gly Ser Val Pro Asp Ser Ile Pro Lys Glu Leu Pro Arg Asn Phe Lys Ala Ser Lys Gln Arg Phe Thr His Lys Met Leu Ala Gln Lys Pro Glu His Thr Ser Gly Gly Glu Val Arg Ile Thr Asp Ser Ser Asn Phe Pro Ile Ser Lys Thr Val Ala Ala Ala His Leu Thr Ile Asn Pro Gly Ala Ile Arg Glu Met His Trp His Pro Asn Ala Asp Glu Trp Ser Tyr Phe Lys Arg Gly Arg Ala Arg Val Thr Ile Phe Ala Ala Glu Gly Asn Ala Arg Thr Phe Asp Tyr Val Ala Gly Asp Val Gly Ile Val Pro Arg Asn Met Gly His Phe Ile Glu Asn Leu Ser Asp Asp Glu Glu Val Glu Val Leu Glu Ile Phe Arg Ala Asp Arg Phe Arg Asp Phe Ser Leu Phe Gln Trp Met Gly Glu Thr Pro Gln Arg Met Val Ala Glu His Val Phe Lys Asp Asp Pro Asp Ala Ala Arg Glu Phe Leu Lys Ser Val Glu Ser Gly Glu Lys Asp Pro Ile Arg Ser Pro Ser Glu (2) INFORMATION FOR SEQ ID NO.: 5:
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Gln Asp Lys Pro Phe Thr Pro Asp His Arg Asp Pro Tyr Asp His Lys Val Asp Ala Ile Gly Glu Gly His Glu Pro Leu Pro Trp Arg Met Gly Asp Gly Ala Thr Ile Met Gly Pro Arg Asn Lys Asp Arg Glu Arg Gln 20 Asn Pro Asp Met Leu Arg Pro Pro Ser Thr Asp His Gly Asn Met Pro Asn Met Arg Trp Ser Phe Ala Asp Ser His Ile Arg Ile Glu Glu Gly Gly Trp Thr Arg Gln Thr Thr Val Arg Glu Leu Pro Thr Ser Lys Glu Leu Ala Gly Val Asn Met Arg Leu Asp Glu Gly Val Ile Arg Glu Leu His Trp His Arg Glu Ala Glu Trp Ala Tyr Val Leu Ala Gly Arg Val Arg Val Thr Gly Leu Asp Leu Glu Gly Gly Ser Phe Ile Asp Asp Leu Glu Glu Gly Asp Leu Trp Tyr Phe Pro Ser Gly His Pro His Ser Leu Gln Gly Leu Ser Pro Asn Gly Thr Glu Phe Leu Leu Ile Phe Asp Asp Gly Asn Phe Ser Glu Glu Ser Thr Phe Leu Leu Thr Asp Trp Ile Ala His Thr Pro Lys Ser Val Leu Ala Gly Asn Phe Arg Met Arg Pro Gln Thr Phe Lys Asn Ile Pro Pro Ser Glu Lys Tyr Ile Phe Gln Gly Ser Val Pro Asp Ser Ile Pro Lys Glu Leu Pro Arg Asn Phe Lys Ala Ser Lys Gln Arg Phe Thr His Lys Met Leu Ala Gln Lys Pro Glu His Thr Ser Gly Gly Glu Val Arg Ile Thr Asp Ser Ser Asn Phe Pro Ile Ser Lys Thr Val Ala Ala Ala His Leu Thr Ile Asn Pro Gly Ala Ile Arg Glu Met His Trp His Pro Asn Ala Asp Glu Trp Ser Tyr Phe Lys Arg Gly Arg Ala Arg Val Thr Ile Phe Ala Ala Glu Gly Asn Ala Arg Thr Phe Asp Tyr Val Ala Gly Asp Val Gly Ile Val Pro Arg Asn Met Gly His Phe Ile Glu Asn Leu Ser Asp Asp Glu Glu Val Glu Val Leu Glu Ile Phe Arg Ala Asp Arg Phe Arg Asp Phe Ser Leu Phe Gln Trp Met Gly Glu Thr Pro Gln Arg Met Val Ala Glu His Val Phe Lys Asp Asp Pro Asp Ala Ala Arg Glu Phe Leu Lys Ser Val Glu Ser Gly Glu Lys Asp Pro Ile Arg Ser Pro Ser Glu (2) INFORMATION FOR SEQ ID NO.: 6:
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Gln Asp Lys Pro Phe Thr Pro Asp His Arg Asp Pro Tyr Asp His Lys Val Asp Ala Ile Gly Glu Xaa His Glu Pro Leu (2) INFORMATION FOR SEQ ID NO.: 7:
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40 Ser Ala His Thr Pro Pro Ser Val Leu Ala Gly Asn (2) INFORMATION FOR SEQ ID NO.: 11:
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Asp His Lys Val Asp Ala Ile Gly Glu Gly His Glu Pro Leu Pro Trp Arg Met Gly Asp Gly Ala Thr Ile Met Gly Pro Arg Asn Lys Asp Arg Glu Arg Gln Asn Pro Asp Met Leu Arg Pro Pro Ser Thr Asp His Gly Asn Met Pro Asn Met Arg Trp Ser Phe Ala Asp Ser His Ile Arg Ile Glu Gly Trp Thr Arg Gln Thr Thr Val Arg Glu Leu Pro Thr Ser Lys Glu Leu Ala Gly Val Asn Met Arg Leu Asp Glu Gly Val Ile Arg Glu Leu His Trp (2) INFORMATION FOR SEQ ID NO.: 16:
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Asp His Lys Val Asp Ala Ile Gly Glu Gly His Glu Pro Leu Pro Trp 50 Arg Met Gly Asp Gly Ala Thr Ile Met Gly Pro Arg Asn Lys Asp Arg Glu Arg Gln Asn Pro Asp Met Leu Arg Pro Pro Ser Thr Asp His Gly Asn Met Pro Asn Met Arg Trp Ser Phe Ala Asp Ser His Ile Arg Ile Glu Gly Trp Thr Arg Gln Thr Thr Val Arg Glu Leu Pro Thr Ser Lys Glu Leu Ala Gly Val Asn Met Arg Leu Asp Glu Gly Val Ile Arg Glu Leu His Trp (2) INFORMATION FOR SEQ ID NO.: 17:
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(ii) MOLECULE TYPE: DNA
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Triticum aestivum (xi) SEQUENCE DESCRIPTION: SEQ ID NO.: 27:

(2) INFORMATION FOR SEQ ID NO.: 28:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 72 (B) TYPE: nucleic acid (C) STRANDEDNESS:
(D) TOPOLOGY:
(ii) MOLECULE TYPE: DNA
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Triticum aestivum (xi) SEQUENCE DESCRIPTION: SEQ ID NO.: 28:

(2) INFORMATION FOR SEQ ID NO.: 29:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 16 (B) TYPE: nucleic acid (C) STRANDEDNESS:
(D) TOPOLOGY:
(ii) MOLECULE TYPE: DNA
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Artificial Sequence (ix) FEATURE
(C) OTHER INFORMATION: primer (ix) FEATURE
(A) NAME/KEY: misc_feature (B) LOCATION: (1). .(16) (C) OTHER INFORMATION: n = A,T,C or G
(xi) SEQUENCE DESCRIPTION: SEQ ID NO.: 29:

(2) INFORMATION FOR SEQ ID NO.: 30:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 17 (B) TYPE: nucleic acid (C) STRANDEDNESS:
(D) TOPOLOGY:
(ii) MOLECULE TYPE: DNA
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Artificial Sequence (ix) FEATURE
(C) OTHER INFORMATION: primer (ix) FEATURE
(A) NAME/KEY: misc_feature (B) LOCATION: (1). .(17) (C) OTHER INFORMATION: n = A,T,C or G
(xi) SEQUENCE DESCRIPTION: SEQ ID NO.: 30:

Claims (28)

1. An isolated nucleic acid, having the sequence of the Aspergillus phoenices oxalate decarboxylase insert in the plasmid ATCC No. 97959.

2. An isolated nucleic acid selected from the group consisting of:
a) the nucleic acid shown in SEQ ID NO: 1; and b) nucleotide 171 to 1437 of the nucleic acid shown in SEQ ID NO: 1.

3. An isolated nucleic acid comprising a nucleic acid encoding the polypeptide of SEQ ID NO: 2 or SEQ ID NO: 5.

4. The nucleic acid of any of the claims 1, 2, or 3, further comprising a plant signal sequence.

5. A vector for delivery of a nucleic acid to a host cell, the vector comprising the nucleic acid of any of the claims 1, 2, or 3.

6. A host cell containing the vector of claim 5.

7. A host cell transformed with the nucleic acid of any of the claims 1, 2, or 3.

8. The host cell of claim 7, wherein the cell is a plant cell.

9. The host cell of claim 8, wherein the nucleic acid further comprises a plant signal sequence.

10. The host cell of claim 9, wherein said plant signal sequence comprises the Germin signal sequence contained in SEQ ID NO: 28.

11. The host cell of claim 8, wherein the plant is selected from the group consisting of sunflower, bean, canola, alfalfa, soybean, flax, safflower, peanut and clover.

12. A plant cell transformed with a nucleic acid comprising the nucleic acid of any of the claims 1, 2, or 3.

13. A plant having stably incorporated within its genome a nucleic acid comprising the nucleic acid of any of the claims 1, 2, or 3.

14. The plant of claim 13, wherein said nucleic acid further comprises a plant signal sequence.

15. The plant of claim 14, wherein said plant signal sequence comprises the Germin signal sequence contained in SEQ ID NO: 28.

16. A method for degrading oxalic acid comprising the steps of:
a) transforming a plant with a nucleic acid, wherein the nucleic acid is the nucleic acid of any of the claims 1, 2, or 3; and b) expressing said nucleic acid for a time sufficient to allow for degradation of the oxalic acid.

17. The method of claim 16, wherein said nucleic acid further comprises a plant signal sequence.

18. The method of claim 17, wherein said plant signal sequence comprises the Germin signal sequence contained in SEQ ID NO: 28.

19. The method of claim 16, wherein said plant is selected from the group consisting of sunflower, bean, canola, alfalfa, soybean, flax, safflower, peanut and clove.

20. The method of claim 19, wherein said plant is sunflower.

21. A method of identifying transformed plant cells comprising the steps of:
a) culturing cells from a selected target plant in a culture medium;

b) transforming said cells with at least one copy of an expression cassette comprising the nucleic acid of any of the claims 1, 2, or 3;
c) introducing oxalic acid into the culture medium; and d) identifying transformed cells as the surviving cells in the oxalic acid-treated culture.

22. An isolated nucleic acid encoding a signal peptide comprising nucleotide 24 to 101 of SEQ ID NO: 1.

23. An isolated nucleic acid which hybridizes under highly stringent conditions to SEQ ID NO: 1.

24. An isolated nucleic acid which is at least 80% identical to SEQ ID NO: 1.

25. An isolated nucleic acid which is at least 90% identical to SEQ ID NO: 1.

26. A method of preventing pathogenic disease in plants where oxalic acid plays a role in pathogenesis, the method comprising:
a) transforming a plant cell with a nucleic acid of any of the claims 1, 2, or 3;
b) culturing the plant cell under plant growing conditions to produce a regenerated plant; and c) inducing expression of said polypeptide for a time sufficient to prevent pathogenic disease.

27. The method of claim 26 wherein the plant cell is selected from the group consisting of sunflower, bean, canola, alfalfa, soybean, flax, safflower, peanut and clove.

28. The method of claim 26- wherein the pathogenic disease is caused by Sclerotinia or Sclerotium.