Nucleotide sequences coding an endopolygalacturonase inhibitor

BACKGROUND OF THE INVENTION

This invention relates to nucleotide sequences coding a endopolygalacturonase inhibitor.

More particularly, this invention relates to a gene coding a new protein having an inhibiting activity against the fungal endopolygalacturonase enzyme, or fractions thereof, and to the protein itself or fractions thereof. The invention relates also to recombinant vectors comprising such sequences, to cells and plants transformed by means of such recombinant vectors and to the use of such vectors and cells to produce the protein.

References are listed at the end of the specification in the order cited.

The fungal endo alpha-1,4-D-polygalactorunase enzyme (EC 3.21.15) is an important pathogenic factor for plants (1,2). The enzyme catalyzes the scission reaction of polygalacturonic acid and induces the subsequent solubilization of plant cell wall homo-alpha-1,4-D-galactorunanes, thus facilitating the penetration of fungi into plant tissues and then supplying the same fungi with nutritional substrates.

Moreover, the activity of such an enzyme could, if controlled, produce non-virulence factors, i.e. factors able to activate some plant defence mechanisms by converting plant cell wall homogalactorunanes into oligogalacturonides, which are known to stimulate phytoalexin synthesis and other defence mechanisms as well (2).

An inhibiting activity of fungal endopolygalacturonase, associated with the cell wall, named PGIP, has been detected in dicot plants.

The authors of the present invention have already detected such activity in different plant tissues of Phaseolus vulgaris L. (3), showing that it is specific for fungal endopolygalacturonases, with no activity against either bacterial or plant enzymes, or other pectic enzymes of microbial origin (4), Moreover, the authors found that the PGIP is able to stabilize in vitro and oligogalacturonide mixture which stimulates phytoalexins (5).

Therefore it is evident the need to purify and isolate the molecule responsible for such activity by means of cloning coding PGIP sequences. Said sequences may be inserted in suitable vectors to transform plants that are sensitive to fungi or other microorganism pathogens, into plants producing the PGIP protein on large scale. The protein could be then used with many advantages in the food industry.

SUMMARY OF THE INVENTION

Prior attempts to isolate the PGIP gene have been unsuccessful, essentially due to low levels of the protein in plant tissues.

The authors of the invention have now isolated and sequenced the PGIP protein coding DNA, both from a genomic and cDNA library, and have also identified the amino acid sequence of the PGIP protein.

An object of the present invention is a DNA fragment comprising a sequence coding a PGIP protein having an inhibiting activity of the fungal endo-alpha-1,4-D-polygalacturonase enzyme (PG), or parts thereof. Preferably said DNA fragment is of plant origin, more preferably isolated either from plants, or parts of plants, or in vitro cultured plant cells of the Phaseolus genus, most preferably of the Phaseolus vulgaris species.

According to a preferred embodiment said DNA fragment codes for a PGIP protein having the amino acid sequence of SEQ ID N15, or parts thereof. Alternatively said amino acid sequence SEQ ID N15 lacks or is substituted of one or more amino acids, preferably is a variant of a PGIP protein, more preferably said variant of a PGIP protein comprises the amino acid sequence SEQ ID N17, or parts thereof.

According to another embodiment said DNA fragment comprises the nucleotide sequence of SEQ ID N14, consisting of:

a coding region from nucleotide 1 to nucleotide 1026;

a 3'-end untranslated sequence from nucleotide 1027 to nucleotide 1116.

Alternatively said nucleotide sequence lacks or is substituted of one or more nucleotides, preferably comprising the nucleotide sequence of SEQ ID N16.

Another object of the invention is a DNA fragment hybridizing to at least one of DNA fragments having the sequences above described, preferably complementary to them.

Another object of the invention is a DNA fragment comprising an allele of either SEQ ID N14 or SEQ ID N16 sequences.

All of the DNA fragments according to the invention may be derived from either a genomic library, or a cDNA library or in vitro synthesis.

Another object of the invention is a substantially purified PGIP protein having an inhibiting activity for the fungine PG enzyme and being of natural or recombinant origin, or parts thereof. Preferably said substantially purified PGIP protein derives from plants, parts thereof or cultured plant cells, more preferably of the Phaseolus genus, most preferably of the Phaseolus vulgaris species.

According to a preferred embodiment said protein comprises the amino acid sequence of SEQ ID N15, or parts thereof, alternatively said protein lacks or is substituted of one or more amino acids, preferably said protein comprises the amino acid sequence of SEQ ID N17.

Another object of the invention is a recombinant vector comprising any one of the nucleotide sequences of the invention, preferably of plasmid or phage origin, more preferably said recombinant vector is the pAD-1 plasmid (DSM N.6821).

In a preferred embodiment said recombinant vector comprises a promoter able to express said coding PGIP nucleotide sequence, preferably said promoter is active either in bacteria, or in yeasts, or in higher plants.

Other objects of the invention are cells transformed by one of the vectors above described, preferably said cells are bacterial cells, more preferably belonging to the Escherichia coli species. Alternatively said cells are eukaryotic cells, preferably yeast cells, alternatively plant cells.

A further object of the invention is a process for isolating and cloning PGIP protein coding sequences, or parts thereof, comprising:

partial sequencing of the amino acid sequence of the PGIP protein;

in vitro synthesing oligonucleotides, the sequence of which is derived from said amino acid sequence;

probing a genomic or a cDNA library from plant cells with said oligonucleotides, previously labelled;

isolating positive clones, extracting the recombinant vector from a positive clone and identifying the recombinant insert from said recombinant vector;

sequencing said recombinant insert.

The invention shall be illustrated in the following examples by making references to some preferred embodiments, completed with the following figures where ;

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the PGIP elution profile on Mono S column;

FIG. 2 shows an agarose gel electrophorosis of amplified DNA, using the following primers: A: mol weight markers; B: control; C: N-A and Int; D: N-B and Int; E: N-A; F: N-B;

FIG. 3 shows a map of primers and of amplified DNAs;

FIG. 4 shows a restriction map of lambda PGIP-3.3 and of pPT-2;

FIG. 5 shows a hydrophobicity plot of the PGIP protein;

FIG. 6 shows a Northern blot by using RNAs from different plant tissues, probed with a radiolabelled pPT-1 fragment;

FIG. 7 shows a comparative analysis of PGIP coding sequences isolated from two different cultivars of Phaseolus vulgaris species.

DETAILED DESCRIPTION OF THE INVENTION

EXAMPLES

Example 1

Purification and Sequencing of PGIP Protein

Plants of Phaseolus vulgaris L., Pinto cultivar, are grown on vermiculite under 18 hour light exposure conditions. The callus and cell suspension cultures from Pinto cultivar are obtained as described in (3).

The PGIP protein is purified from hypocotyls (30 .mu.g/kg of fresh weight) by affinity chromatography on Sepharose coupled to endopolygalacturonase, as described in (6). One unit of PGIP protein is the amount able to reduce of 50% the activity of one unit of endopolygalacturonase, isolated from Aspergillus nidulans.

PGIP is then diluted at 1:4 ratio in 30 mM. Hepes pH 7.5 buffer, and loaded on Mono S column (Pharmacia), equilibrated with the same buffer. The protein is eluted with a linear gradient 0-0.3M NaCl in 20 mM Hepes pH 7.5 buffer. Approximately 90% of the loaded material having a 280 nm absorbance elutes with the excluded volume, showing no PGIP activity at all. The remaining 10% elutes with a single peak at a 0.1M NaCl and shows PGIP activity (FIG. 1). Fractions containing PGIP activity are collected and loaded again on Mono S column to concentrate the protein to 35 .mu.g/ml.

5 .mu.g of the protein are used to determine the N-terminal sequence, according to standard methodology. The remaining amount is digested by 1 mg/ml of TPCK (Sigma) treated trypsin in 1.0M ammonium bicarbonate pH 7.8 buffer, at 37.degree. C. for 16 hours. The resulting peptides are separated using a binary gradient (solvent A: 0.1% TFA in water; solvent B: 0.085% TFA in 80% aqueous CH.sub.3 CN) at a flow rate of 0.2 ml/min. Single peaks are collected, dried, dissolved in 50 .mu.l of solvent A and chromatographed again on the same column.

Peptides, as well as the undigested PGIP protein, are sequenced by a ABI 470A or 477A sequencer. Aminoacid sequence of the N-terminal fragment and of four tryptic fragments of the PGIP protein are shown in the Table 1 hereinbelow.

TABLE 1 ______________________________________ Aminoacid sequence of the N-terminal fragment and of four tryptic peptides of PGIP protein Residue number N-terminal P-1 P-2 P-3 P-4 ______________________________________ 1 Glu Ile Leu Ile Ile 2 Leu His Phe Tyr Ser 3 X Leu Thr X Gly 4 Asn Ala Ser Thr Ala 5 Pro Lys Met Leu Ile 6 Glu(n) Pro Pro 7 Asp Gln Asp 8 Lys Gly Ser 9 Glu(n) Leu Tyr 10 Ala Thr Gly 11 Leu Gln Ser 12 Leu Leu Phe 13 X Lys 14 Ile 15 Lys 16 Lys 17 Asp 18 Leu 19 Gly 20 Asn 21 Pro ______________________________________ X means presence of undefined residues; aa. 6 and 9 of the Nterminal sequence may be either Glu or Gln. .sup.1 SEQ ID N1; .sup.2 SEQ ID N2; .sup.3 SEQ ID N3; .sup.4 SEQ ID N4; .sup.5 SEQ ID N5

Example 2

Nucleotide Amplification, Sequencing and Cloning

Two independent oligonucleotides are synthesized on the basis of the N-terminal sequence (N-A and N-B). A third oligonucleotide (Int) is sythesized on the basis of the peptide P-3 sequence, starting from the first nucleotide coding for aa. 6 (Pro) to the second nucleotide coding for aa. 11 (Gln). The nucleotide sequences are shown in the following Table 2.

Table 2

Primer oligonucleotides for amplification reaction and corresponding amino acid sequence

4-10 N-terminal sequence (N-A) ##STR1##

14-21 N-terminal sequence (N-B) ##STR2##

6-11 P-3 peptide sequence (Int) ##STR3## N means one of the four nucleotides.

N-A, N-B and Int oligonucleotides are used as primers of a polymerase chain reaction (PCR) using genomic DNA, extracted from Phaseolus vulgaris, Pinto cultivar, as template, by conventional methods (7). Polymerizing reactions are carried out on Perkin Elmer/Cetus equipment according to manufacturer's instructions. The primer hybridization is carried out at 45.degree. C. or 50.degree. C., depending upon using respectively degenerate or nondegenerate nucleotides. The polymerization enzyme (Amplitaq, Perkin Elmer) is used at 1 U/40 .mu.l of reaction mix. PCR products are fractionated on a 0.8% agarose gel and dyed with ethidium bromide.

A clear DNA band of appr. 0.79 kb, defined as PGIP-1, is observed (FIG. 2, line c), when using the N-A oligo. The band is missing in control reactions (FIG. 2, lines b, e, q). By using N-D and Int oligos as primers, the DNA product (PGIP-2) is of appr. 0.76 kb, i.e. 30 bp shorter than PGIP-1 (FIG. 2, line d).

The agarose gel is transferred to nitrocellulose membrane and probed with PGIP-2, previously eluted from the agarose gel and amplified with 6 PCR cycles in presence of P.sup.32 ATP. PGIP-1 and PGIP-2 sequences hybridized with the same intensity to the radiolabelled probe. FIG. 3 shows a scheme of oligonucleotides and PCR obtained products.

The PGIP-2 fragment, eluted from the agarose gel and further amplified by PCR using N-B and Int oligonucleotides, is purified by removing the low melting point agarose and inserted into the pGEM7zf(+) vector (Promega) by conventional methods. The recombinant vector, containing the whole DNA fragment of PGIP-2, is defined as pPT-1.

The pPt-1 insert is sequenced and an open reading frame of 252 amino acids is deduced. As expected the amino acid sequence comprises sequences corresponding to the 14-21 N-terminal and to the P-3 peptide as well. Moreover it comprises sequences of the P-1, P-2 and P-4 peptides.

Example 3

Isolation of a Genomic PGIP Clone

A genomic DNA library from the Saxa cultivar of Phaseolus vulgaris, obtained by partial digestion with restriction enzyme MboI, into the EMBL3 lambda vector, has been supplied by Clontech (Ca, USA). The library is probed by PCR and plaque-hybridization combination. 5.times.10.sup.5 recombinant phages are probed by conventional plaque-hybridization, as described in (7), by using the radiolabelled p-PT1 insert. Positive clones are grouped in three different classes, to be further analyzed with PCR. PCR screening is performed using crude phage DNA as template and two nondegenerate primers (P21 and P22), synthesized on the basis of the p-PT1 insert sequence, whose sequences are as follows: P21: AAGGCTCTGTTCAGTTGCTT (SEQ ID N12); P22:CTATACATTGGCGGCATCAAT (SEQ ID N13). Positive clones have an expected 378 bp amplification fragment. The first class shows strong hybridization signals and is able to amplify the 378 bp fragment. The second class also shows strong hybridization signals, but amplification products from PCR are not observed. The third class shows a weaker hybridization, but gives rise to specific amplification products from PCR, other than the 378 bp fragment. Among positive clones of the first class, one clone (lambda PGIP-3.3. insert=17 kb) is selected and isolated for further analysis. A 3.3 kb SalI--SalI fragment is shown to contain the PGIP gene, as demonstrated by Southern blot analysis and PCR experiments. The fragments is subcloned in the SalI site of the Bluescript SK.sup.+ plasmid (Stratagene), resulting in a recombinant plasmid named pPt-2. The orientation and the position of the PGIP gene in the 3.3 kb cloned fragment is defined by PCR (FIG. 4), using different combination of both plasmid (T3 and T7) and PTGP specific primers (FIG. 4). It is calculated that the 3.3 kb SalI--SalI fragment comprises appr. 1860 nt upstream the first ATG, 1026 nt of coding region and 97 nt of the 3'-untranslated region, with appr. 450 nt adjacent sequences. A partial restriction map of the 17 kb genomic insert, the orientation and the PGIP position in the pPT-2 subclone are shown in FIG. 4.

Both DNA strands of the PGIP gene are sequenced; the sequence strategy is shown in FIG. 4. The whole nucleotide sequence of the PGIP coding region and of the untranslated 3' region (1116 nt) is reported in SEQ ID N14. The polyadenylation site is 97 bp downstream of the TAA stop codon. Three sequences, corresponding to putative eukaryotic polyadenylation signals, are at 42, 22 and 16 nt upstream of the polyadonylation site respectively. A comparative analysis between the genomic sequence and the relevant cDNA sequence above that no introns are present in the 345 nucleotide 3'-end gene region. Further analysis shows that no introns are present for the whole PGIP gene, as confirmed by PCR experiments. The PGIP gene DNA sequence defines a 342 amino acid PGIP protein (see SEQ ID N14 and N15). Neither the nucleotide or amino acid sequences show any significant homology with any known sequence.

A hydrophobicity plot of the PGIP protein is shown in FIG. 5. The sequence includes four potential glycosylation sites.

Example 4

Northern Blot Analysis

PGIP specific mRNA levels are analysed in different plant tissues. The pPT1 purified insert is used to probe polyA.sup.+ RNA, isolated from either hypocotyls, or leaves, or flowers or suspension cultured cells of Phaseolus vulgaris, Pinto cultivar. A single 1,2 kb transcript is identified in mRNA from suspension cultured cells. The fragment is also present in other tissues, even if minor amount.

PCR experiments, using single-strand cDNA from suspension cultured cells and P-21 and P-22 as templates, shows the expected amplification of the 370 bp fragment, thus indicating that at least a fraction of the PGIP gene expression in cultured cells may be ascribed to the cloned and characterized gene.

Example 5

Analysis of PGIP Genes From Two Different Phaseolus vulgaris cultivars

A cDNA library is made in the lambda gt11 vector from polyA.sup.30 RNA, purified from suspension cultured cells of Phaseolus vulgaris, Pinto cultivar. By probing the cDNA library wit pPT1, a cDNA recombinant clone (lambda AD-1) containing PGIP coding sequences is isolated. The lambda AD-1 insert (842 bp) is subcloned in the Bluescript SK+ vector, named pAD-1, deposited at DSM, Germany, with the n. 6821, and sequenced. The insert represents a whole PGIP cDNA fragment and includes the polyA tail. The sequence of the pAD-1 insert is shown in SEQ ID N16. When compared to SEQ ID N14, the pAD-1 insert refers to sequence from 345 to 1116 nt of SEQ ID N14. A comparative analysis between the genomic clone from Saxa cultivar and the cDNA clone from Pinto cultivar shows a 96.4% homology of nucleotide sequence and a 97.4% homology of amino acid sequence (FIG. 7).

This invention has been described with reference to some preferred embodiments; however it is understood that any changes and/or modification could be made by anyone skilled in the art, remaining within the scope of the claims.

REFERENCES

1) Cooper, R. M. In: Plant Disease: Infection, Damage and Loss. Wood, R. K. S. & Jellis, G. J. eds, Oxford, Blackwell Scientific Publications, pp. 13-27 (1984).

2) Hahn, M. G. Bucheli, P., Cervone F., Doares, S. H., Darvill, A. G. and Albersheim, P. In: Plant-Microbe Interaction, Vol. 3, Nester, E. and Kosuge, T., eds, New York, MacMillan Press, pp. 131-181 (1989).

3) Salvi, G., Giarizzo, F., De Lorenzo, G. and Cervone, F. J. Plant Physiol, 136, 513-518 (1990).

4) Cervone, F., De Lorenzo, G. Pressey, R., Darvill, A., and Albersheim, P. Phytochemistry 29, 447-449 (1990).

5) Cervone F., Hahn, N. G., De Lorenzo, G., Darvill, A. and Albersheim, P. Plant Physiology, 90, 542-548 (1989).

6) Degra, L, Salvi, G., Mariotti, D., De Lorenzo, G. and Cervone, F. J. Plant Physiol., 133, 364-366 (1988).

7) Sambrook, D., Fritsch, E. F., Naniatis, T. In: Molecular Cloning: A Laboratory Manual, 2nd ed., vols.1, 2, 3, CSH Lab Press (1989).

__________________________________________________________________________ SEQUENCE LISTING (1) GENERAL INFORMATION: (iii) NUMBER OF SEQUENCES: 17 (2) INFORMATION FOR SEQ ID NO:1: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Phaseolus vulgaris (B) STRAIN: Pinto (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: GluLeuXaaAsnProGlxAspLysGlxAlaLeuLeuXaaIleLysLys 151015 AspLeuGlyAsnPro 20 (2) INFORMATION FOR SEQ ID NO:2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 5 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Phaseolus vulgaris (B) STRAIN: Pinto (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: IleHisLeuAlaLys 15 (2) INFORMATION FOR SEQ ID NO:3: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 5 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Phaseolus vulgaris (B) STRAIN: Pinto (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: LeuPheThrSerMet 15 (2) INFORMATION FOR SEQ ID NO:4: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 13 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Phaseolus vulgaris (B) STRAIN: Pinto (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: IleTrpXaaThrLeuProGlnGlyLeuThrGlnLeuLys 1510 (2) INFORMATION FOR SEQ ID NO:5: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 12 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Phaseolus vulgaris (B) STRAIN: Pinto (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: IleSerGlyAlaIleProAspSerTyrGlySerPhe 1510 (2) INFORMATION FOR SEQ ID NO:6: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 7 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Phaseolus vulgaris (B) STRAIN: Pinto (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: AsnProGlxAspLysGlxAla 15 (2) INFORMATION FOR SEQ ID NO:7: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: synthetic primer/probe (iii) HYPOTHETICAL: YES (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: AAYCCNSARGAYAARSARCC20 (2) INFORMATION FOR SEQ ID NO:8: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 8 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Phaseolus vulgaris (B) STRAIN: Pinto (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: IleLysLysAspLeuGlyAsnPro 15 (2) INFORMATION FOR SEQ ID NO:9: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 23 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: synthetic primer/probe (iii) HYPOTHETICAL: YES (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: ATHAARAARGAYYTNGGNAAYCC23 (2) INFORMATION FOR SEQ ID NO:10: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 6 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Phaseolus vulgaris (B) STRAIN: Pinto (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10: ProGlnGlyLeuThrGln 15 (2) INFORMATION FOR SEQ ID NO:11: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 17 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: synthetic primer/probe (iii) HYPOTHETICAL: YES (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: GGNGTYCCNRANTGNGT17 (2) INFORMATION FOR SEQ ID NO:12: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: synthetic primer/probe (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Phaseolus vulgaris (B) STRAIN: Pinto (vii) IMMEDIATE SOURCE: (B) CLONE: P21 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: AAGGCTCTGTTCAGTTGCTT20 (2) INFORMATION FOR SEQ ID NO:13: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: synthetic primer/probe (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Phaseolus vulgaris (B) STRAIN: Pinto (vii) IMMEDIATE SOURCE: (B) CLONE: P22 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13: CTATACATTGGCGGCATCAAT21 (2) INFORMATION FOR SEQ ID NO:14: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1116 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Phaseolus vulgaris (B) STRAIN: Saxa (vii) IMMEDIATE SOURCE: (B) CLONE: lambda PGIP-3.3 (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 1..1026 (ix) FEATURE: (A) NAME/KEY: 3'UTR (B) LOCATION: 1027..1116 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14: ATGACTCAATTCAATATCCCAGTAACCATGTCTTCAAGCTTAAGCATA48 MetThrGlnPheAsnIleProValThrMetSerSerSerLeuSerIle 151015 ATTTTGGTCATTCTTGTATCTTTGAGAACTGCACTCTCAGAGCTATGC96 IleLeuValIleLeuValSerLeuArgThrAlaLeuSerGluLeuCys 202530 AACCCACAAGATAAGCAAGCCCTTCTCCAAATCAAGAAAGACCTTGGC144 AsnProGlnAspLysGlnAlaLeuLeuGlnIleLysLysAspLeuGly 354045 AACCCAACCACTCTCTCTTCATGGCTTCCAACCACCGACTGTTGTAAC192 AsnProThrThrLeuSerSerTrpLeuProThrThrAspCysCysAsn 505560 AGAACCTGGCTAGGTGTTTTATGCGACACCGACACCCAAACATATCGC240 ArgThrTrpLeuGlyValLeuCysAspThrAspThrGlnThrTyrArg 65707580 GTCAACAACCTCGACCTCTCCGGCCATAACCTCCCAAAACCCTACCCT288 ValAsnAsnLeuAspLeuSerGlyHisAsnLeuProLysProTyrPro 859095 ATCCCTTCCTCCCTCGCCAACCTCCCCTACCTCAATTTTCTATACATT336 IleProSerSerLeuAlaAsnLeuProTyrLeuAsnPheLeuTyrIle 100105110 GGCGGCATCAATAACCTCGTCGGTCCAATCCCCCCCGCCATCGCTAAA384 GlyGlyIleAsnAsnLeuValGlyProIleProProAlaIleAlaLys 115120125 CTCACCCAACTCCACTATCTCTATATCACTCACACCAATGTCTCCGGC432 LeuThrGlnLeuHisTyrLeuTyrIleThrHisThrAsnValSerGly 130135140 GCAATACCCGATTTCTTGTCACAGATCAAAACCCTCGTCACCCTCGAC480 AlaIleProAspPheLeuSerGlnIleLysThrLeuValThrLeuAsp 145150155160 TTCTCCTACAACGCCCTCTCCGGCACCCTCCCTCCCTCCATCTCTTCT528 PheSerTyrAsnAlaLeuSerGlyThrLeuProProSerIleSerSer 165170175 CTCCCCAACCTCGGAGGAATCACATTCGACGGCAACCGAATCTCCGGC576 LeuProAsnLeuGlyGlyIleThrPheAspGlyAsnArgIleSerGly 180185190 GCCATCCCCGACTCCTACGGCTCGTTTTCGAAGCTGTTTACGGCGATG624 AlaIleProAspSerTyrGlySerPheSerLysLeuPheThrAlaMet 195200205

ACCATCTCCCGCAACCGCCTCACCGGGAAGATTCCACCGACGTTTGCG672 ThrIleSerArgAsnArgLeuThrGlyLysIleProProThrPheAla 210215220 AATCTGAACCTGGCGTTCGTTGACTTGTCTCGGAACATGCTGGAGGGT720 AsnLeuAsnLeuAlaPheValAspLeuSerArgAsnMetLeuGluGly 225230235240 GACGCGTCGGTGTTGTTCGGGTCAGATAAGAACACGAAGAAGATACAT768 AspAlaSerValLeuPheGlySerAspLysAsnThrLysLysIleHis 245250255 CTGGCGAAGAACTCTCTTGCTTTTGATTTGGGGAAAGTGGGGTTGTCA816 LeuAlaLysAsnSerLeuAlaPheAspLeuGlyLysValGlyLeuSer 260265270 AAGAACTTGAACGGGTTGGATCTGAGGAACAACCGTATCTATGGAACG864 LysAsnLeuAsnGlyLeuAspLeuArgAsnAsnArgIleTyrGlyThr 275280285 CTACCTCAGGGACTAACGCAGCTAAAGTTTCTGCAAAGTTTAAATGTG912 LeuProGlnGlyLeuThrGlnLeuLysPheLeuGlnSerLeuAsnVal 290295300 AGCTTCAACAATCTGTGCGGTGAGATTCCTCAAGGTGGGAACTTGAAA960 SerPheAsnAsnLeuCysGlyGluIleProGlnGlyGlyAsnLeuLys 305310315320 AGGTTTGACGTTTCTTCTTATGCCAACAACAAGTGCTTGTGTGGTTCT1008 ArgPheAspValSerSerTyrAlaAsnAsnLysCysLeuCysGlySer 325330335 CCTCTTCCTTCCTGCACTTAACCATTTCCAGATTCGGTAATTATGGAT1056 ProLeuProSerCysThr 340 GCATCATGTTTGCCTTTCTATGAACATCAATAATGATACAAGTGTAAAAATAAAAAATTA1116 (2) INFORMATION FOR SEQ ID NO:15: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 342 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15: MetThrGlnPheAsnIleProValThrMetSerSerSerLeuSerIle 151015 IleLeuValIleLeuValSerLeuArgThrAlaLeuSerGluLeuCys 202530 AsnProGlnAspLysGlnAlaLeuLeuGlnIleLysLysAspLeuGly 354045 AsnProThrThrLeuSerSerTrpLeuProThrThrAspCysCysAsn 505560 ArgThrTrpLeuGlyValLeuCysAspThrAspThrGlnThrTyrArg 65707580 ValAsnAsnLeuAspLeuSerGlyHisAsnLeuProLysProTyrPro 859095 IleProSerSerLeuAlaAsnLeuProTyrLeuAsnPheLeuTyrIle 100105110 GlyGlyIleAsnAsnLeuValGlyProIleProProAlaIleAlaLys 115120125 LeuThrGlnLeuHisTyrLeuTyrIleThrHisThrAsnValSerGly 130135140 AlaIleProAspPheLeuSerGlnIleLysThrLeuValThrLeuAsp 145150155160 PheSerTyrAsnAlaLeuSerGlyThrLeuProProSerIleSerSer 165170175 LeuProAsnLeuGlyGlyIleThrPheAspGlyAsnArgIleSerGly 180185190 AlaIleProAspSerTyrGlySerPheSerLysLeuPheThrAlaMet 195200205 ThrIleSerArgAsnArgLeuThrGlyLysIleProProThrPheAla 210215220 AsnLeuAsnLeuAlaPheValAspLeuSerArgAsnMetLeuGluGly 225230235240 AspAlaSerValLeuPheGlySerAspLysAsnThrLysLysIleHis 245250255 LeuAlaLysAsnSerLeuAlaPheAspLeuGlyLysValGlyLeuSer 260265270 LysAsnLeuAsnGlyLeuAspLeuArgAsnAsnArgIleTyrGlyThr 275280285 LeuProGlnGlyLeuThrGlnLeuLysPheLeuGlnSerLeuAsnVal 290295300 SerPheAsnAsnLeuCysGlyGluIleProGlnGlyGlyAsnLeuLys 305310315320 ArgPheAspValSerSerTyrAlaAsnAsnLysCysLeuCysGlySer 325330335 ProLeuProSerCysThr 340 (2) INFORMATION FOR SEQ ID NO:16: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 792 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Phaseolus vulgaris (B) STRAIN: Pinto (vii) IMMEDIATE SOURCE: (B) CLONE: pAD-1 (DSM No.6821) (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 2..685 (ix) FEATURE: (A) NAME/KEY: 3'UTR (B) LOCATION: 686..792 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16: CAATAACCTCGTCGGTCCAATCCCCCCCGCCATCGCTAAACTCACC46 AsnAsnLeuValGlyProIleProProAlaIleAlaLysLeuThr 151015 CAACTCCACTATCTCTATATCACCCACACCAATGTCTCCGGCGCAATA94 GlnLeuHisTyrLeuTyrIleThrHisThrAsnValSerGlyAlaIle 202530 CCCGATTTCTTGTCACAGATCAAAACCCTCGTCACCCTCGACTTCTCC142 ProAspPheLeuSerGlnIleLysThrLeuValThrLeuAspPheSer 354045 TACAACGCCCTCTCCGGCACCCTACCTCCCTCCATCTCTTCTCTCCCC190 TyrAsnAlaLeuSerGlyThrLeuProProSerIleSerSerLeuPro 505560 AACCTCGTAGGAATCACATTCGACGGCAACCGAATCTCCGGCGCCATC238 AsnLeuValGlyIleThrPheAspGlyAsnArgIleSerGlyAlaIle 657075 CCCGACTCCTACGGCTCATTTTCGAAGCTGTTCACGTCGATGACCATC286 ProAspSerTyrGlySerPheSerLysLeuPheThrSerMetThrIle 80859095 TCCCGCAACCGCCTCACCGGGAAGATTCCGCCGACGTTTGCGAATCTG334 SerArgAsnArgLeuThrGlyLysIleProProThrPheAlaAsnLeu 100105110 AACCTGGCGTTCGTTGACTTGTCTCGAAACATGCTGCAGGGTGACGCG382 AsnLeuAlaPheValAspLeuSerArgAsnMetLeuGlnGlyAspAla 115120125 TCGGTGTTGTTCGGATCAGATAAGAACACGCAGAAGATACATCTGGCG430 SerValLeuPheGlySerAspLysAsnThrGlnLysIleHisLeuAla 130135140 AAGAACTCTCTTGCTTTTGATTTGGAGAAAGTGGGGTTGTCAAAGAAC478 LysAsnSerLeuAlaPheAspLeuGluLysValGlyLeuSerLysAsn 145150155 TTGAACGGGTTGGATCTGAGGAACAACCGTATCTATGGGACGCTACCG526 LeuAsnGlyLeuAspLeuArgAsnAsnArgIleTyrGlyThrLeuPro 160165170175 CAGGGACTGACGCAGCTAAAGTTTCTGCACAGTTTAAATGTGAGCTTC574 GlnGlyLeuThrGlnLeuLysPheLeuHisSerLeuAsnValSerPhe 180185190 AACAATCTGTGCGGTGAGATTCCTCAAGGTGGGAACTTGCAAACATTT622 AsnAsnLeuCysGlyGluIleProGlnGlyGlyAsnLeuGlnThrPhe 195200205 CACGTTTCTGCTTATGCCAACAACAAGTGCTTGTGTGGTTCTCCTCTT670 HisValSerAlaTyrAlaAsnAsnLysCysLeuCysGlySerProLeu 210215220 CCTGCCTGCACTTAATCATTTCCAGATTCGGTAATTATGGATGCATCATGTT722 ProAlaCysThr 225 TGCCTTTCTATGAACATCAATAATGATACAAGTTTAAATAAAAATAAATTTATGAAATAA782 AAAAAAAAAA792 (2) INFORMATION FOR SEQ ID NO:17: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 227 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17: AsnAsnLeuValGlyProIleProProAlaIleAlaLysLeuThrGln 151015 LeuHisTyrLeuTyrIleThrHisThrAsnValSerGlyAlaIlePro 202530 AspPheLeuSerGlnIleLysThrLeuValThrLeuAspPheSerTyr 354045 AsnAlaLeuSerGlyThrLeuProProSerIleSerSerLeuProAsn 505560 LeuValGlyIleThrPheAspGlyAsnArgIleSerGlyAlaIlePro 65707580 AspSerTyrGlySerPheSerLysLeuPheThrSerMetThrIleSer 859095 ArgAsnArgLeuThrGlyLysIleProProThrPheAlaAsnLeuAsn 100105110 LeuAlaPheValAspLeuSerArgAsnMetLeuGlnGlyAspAlaSer 115120125 ValLeuPheGlySerAspLysAsnThrGlnLysIleHisLeuAlaLys 130135140 AsnSerLeuAlaPheAspLeuGluLysValGlyLeuSerLysAsnLeu 145150155160 AsnGlyLeuAspLeuArgAsnAsnArgIleTyrGlyThrLeuProGln 165170175 GlyLeuThrGlnLeuLysPheLeuHisSerLeuAsnValSerPheAsn 180185190 AsnLeuCysGlyGluIleProGlnGlyGlyAsnLeuGlnThrPheHis 195200205 ValSerAlaTyrAlaAsnAsnLysCysLeuCysGlySerProLeuPro 210215220 AlaCysThr 225 __________________________________________________________________________