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Isolation and sequence analysis of Clpgl, a gene coding for an endopolygalacturonase of the phytopathogenic fungus Colletotrichum lindemuthianum
Gene, 170 (1996) 125 129 © 1996 Elsevier Science B.V. All rights reserved. 0378-1119/96/$15.00
125
G E N E 09569
Isolation and sequence analysis of Clpgl, a gene coding for an endopolygalacturonase of the phytopathogenic fungus Colletotrichum
lindemuthianum (Pectin degradation; pectinase; polymerase chain reaction; nucleotide sequence; sequence comparison; Southern blot analysis)
Sylvie Centis, B e r n a r d D u m a s , Jo~lle F o u r n i e r , Mich61e M a r o l d a a n d M a r i e - T h 6 r 6 s e Esquerr6-Tugay6 UniversiM Paul Sabatier, Centre de Biologie et Physiologie Vdgktale URA 1941 CNRS, 31062 Toulouse c~dex, France
Received by J.R. Kinghorn: 31 July 1995; Revised/Accepted 21 October 1995; Received at publishers 11 December 1995
SUMMARY
Oligodeoxyribonucleotide primers designed from the N-terminal amino acid (aa) sequence of the endopolygalacturonase (EndoPG) of Colletotrichum lindemuthianum (Cl) race ~ and from an internal sequence conserved among different fungal EndoPG were used in a polymerase chain reaction (PCR) to amplify genomic related sequences of the fungus. A 542-bp fragment, designated pgA, was obtained and used as a probe to screen a partial genomic library of C1. Among the positive clones, one was further analyzed. Nucleotide sequencing of this clone revealed an ORF encoding a 363-amino-acid (aa) polypeptide begining with a signal peptide of 26 aa interrupted by an intron of 70 bp, and showing a high degree of homology to ten fungal EndoPG sequences. Consensus sequences were identified in the 5' non-coding region. This genomic clone was thereafter designated Clpgl. Southern analysis, performed with a Clpgl-specific probe, showed that this gene is present as a single copy in the Cl genome.
INTRODUCTION
Pectin, a methylated heteropolymer containing ~-l,4-1inked galacturonic acid, is one of the predominant polysaccharides of plant cell walls (Carpita et al., 1993). Since this material constitutes a mechanical barrier, as Correspondence to: Dr. M.-T. Esquerr6-Tugay6, Centre de Biologie et Physiologie V6g6tale, URA 1941 CNRS, 118 route de Narbonne, 31062 Toulouse c6dex, France. Tel. (33) 61556758; Fax (33) 61558378; e-mail: [email protected]
Abbreviations: A., Aspergillus; aa, amino acid(s); bp, base pair(s); Cl, Colletotrichum lindemuthianum; Clpgl, gene coding for an EndoPG of Cl; EndoPG, endopolygalacturonase(s); kb, kilobase(s) or 1000 bp; M, A or C; N, A or C or G or T; nt, nucleotide(s); oligo, oligodeoxyribonucleotide; ORF, open reading frame; PCR, polymerase chain reaction; PG, polygalacturonase(s); pg, gene encoding PG; PGIP, PG inhibiting protein; PR, pathogenesis related; S, C or G; SDS, sodium dodecyl sulfate; SP, signal peptide; W, A or T; Y, C or T. SSDI 0378-1119(95)00867-5
well as a carbon source, many phytopathogenic and saprophytic microorganisms secrete pectolytic enzymes such as polygalacturonase, pectin methyl esterase, pectate lyase or pectin lyase. These enzymes are important in pathogenicity for not only do they cause cell-wall degradation (Cooper and Wood 1975; Collmer and Keen, 1986), but also they act as indirect elicitors of plant defence responses through the oligosaccharides they release (Hahn et al., 1981). Endopolygalacturonase (or EndoPG, EC 3.2.1.15) catalyses the hydrolytic cleavage of glycosidic a-1,4 linkages in polygalacturonide chains of pectic substances. Colletotrichum lindemuthianum (Cl) is a fungal pathogen causing anthracnose on bean seedlings. When grown on pectin-containing liquid medium, this fungus secretes an EndoPG which has been thoroughly characterized with molecular probes (Hugouvieux et al., 1995).
126 EndoPG activity is partly inhibited during the parasitic stage of the fungus by the bean cell-wall polygalacturonase inhibiting protein (PGIP) (Albersheim and Anderson, 1971; Lafitte et al., 1984; Wijesundera et al., 1989). The use of gold-labeled P G I P allowed EndoPG and associated cell-wall degradation to be visualised in planta in infected bean tissues (Benhamou et al., 1991). The enzyme is a glycoprotein of around 38 kDa whose sugar moiety accounts for 4% of the molecule and is composed of galactose, glucosamine and mannose (Keon et al., 1990). We recently reported that the EndoPG purified from C1 race 13elicits the biosynthesis of PR (pathogenesis related) proteins in bean cuttings, notably 13-1,3-glucanase, in a cultivar-specific manner (Lafitte et al., 1993), suggesting an important role for this fungal protein in triggering the expression of the host defence genes. A better understanding of this dual role requires further studies of EndoPG at the gene level. PG-encoding genes have been isolated from a few fungi, notably Cochliobolus carbonum (Scott-Graig et al., 1990), Aspergillus niger (Bussink et al., 1992), Aspergillus orizae (Kitamoto et al., 1993), Fusarium moniliforme (Caprari et al., 1993) and Sclerotinia sclerotorium (Reymond et al., 1994). As a prerequisite to evaluating the role played by Clpg gene expression, we have cloned and subsequently characterized a Clpg gene from Cl.
EXPERIMENTALAND DISCUSSION
(a) Amplification ofpg genomic DNA sequences by PCR and isolation of genomic clones An oligo primer (5'-GTYCTIAACAACATYCC) based on the N-terminal aa sequence of Cl race ~ EndoPG (Keon et al., 1990), and an antisense primer (5'-GCRASRCAGTCRTCYTGGTT) designed from an internal aa sequence well conserved among fungal EndoPG (Fig. 1), were synthesized (R = A/G; S = C/G; Y = C / T ) . These two primers were used to amplify by PCR a genomic DNA fragment encoding a portion of C1PG. An amplified 542-bp product, named pgA, was ligated in pGEM-T (Promega, Madison, WI, USA) and sequenced. Sequence analysis showed a high degree of homology to fungal pg genes. This fragment was used to screen a partial genomic library of Cl constructed in LZap (Stratagene, La Jolla, CA, USA). Among the positive clones, some showed high hybridization to the pgA probe, while others gave much weaker signals. Clones of both types were purified and submitted to in vivo excision. Restriction patterns led us to the conclusion that we had isolated two different classes of clones. One clone of the
CCLPGN I SCEEPGA SCEEPGB AFPECA ANPGAI ANPGA2 ATPGA2 ANPG
ANPGAC FSOWPGA
.... SAG-AHNTDAFDIGSSSGI T ISNANI KI~DDCVAINSGSDIHVTNC .... SLG- -HNTDAFDVGS S TDI T ISGANVQ~aDCLKIN SGTG ITF TGG .... SLG- -HNTDAFDVGSSTDITISGANVQm~)DCLKINSGTGI TFTGG .... AEG--HNTDAFDVGSSTY INIDGA~NSGSHITFTNG .... DNG-GHNTDGFDI SESTGVYISGA~DCIAINSGES ISFTGG TQG -GHW/DAFDVGN SVGVN I I~ V N S G E N I W F T G G .... TLG-GHNTDAFDVGNSVGVNI I ~ N S G E N I W F T S G .... TLG-GHNTDAFDVGNSVGVNI I~ I N S G E N I W F TSG . . . . T D D L A A N T D G F D I G E S T Y I T I TC.AE I Y N ~ t R D G ' V A I N S G E N I Y F S A S AKSGSLPAAHNTDGFDISSSDHVTLD~DG~/AVTSGTNTVVSNM ***.**...* . . . *****.*..**. * .. .
.
.
.
219 238 238 218 223 217 217 217 237 228
Fig. 1. Comparisonof a portion of the deducedaa sequencesof different fungal EndoPG corresponding to: CCLPGN1, C. carbonum; SCEEPGA and SCEEPGB, S. scleriotorium, AFPECA, A. flavus; ANPGA1, ANPGA2 and ANPG, A. niger; ATPGA2, A. tubigensis; ANPGAC, A. nidulansand FSOWPGA,F. moniliforme.Sequencealignments were performed using the CLUSTAL software program (IntelliGenetics).Identical(*) and conserved(.) aa substitutionsbetween the fungiare shown.The antisenseprimerwas deducedfrom the homology region indicatedin bold letters.
class which hybridized strongly to the probe, designated Clpgl, was retained for further analysis.
(b) Characterization of the genomic clone Cipgl From the cloned genomic DNA, 1700 nt overlapping the region corresponding to pgA were sequenced according to standard procedures (Sanger et al., 1977), using both universal and specific primers. Analysis of this sequence with the Blast sequence comparison program revealed the occurrence of an ORF of l152bp. The coding region is interrupted by one intron of 70 bp (Fig. 2). The encoded 363 aa (36685 Da) is in good agreement with 37 kDa obtained by SDS-PAGE (Lafitte et al., 1984), and the 38.5 kDa obtained by gel filtration (Keon et al., 1990). A potential N-glycosylation site (AsnGly-Ser) was found at Asn TM. The presence of this site is correlated with the glycoproteic nature of EndoPG (Keon et al., 1990). The N-terminal aa sequence of Cl EndoPG published by Keon et al., (1990) and confirmed in our laboratory, corresponded to aa 27 to 62, thereby indicating that the first 26-aa peptide, rich in hydrophobic residues, corresponded to the signal peptide (SP) of EndoPG. This SP might have been eliminated by a trypsin-like protease cleavage (Benoist et al., 1987) since an Arg 26 occurs in the Cl aa sequence, as in the case of the PG of F. moniliforme and of the PGII of A. niger (Bussink et al., 1991a; Caprari et al., 1993). The 5'-noncoding region of the clone, representing 160 bp, did not contain the typical eukaryotic TATA box (TATAAAT), but a CCAAT sequence, which is often observed in fungal promoters, was found 55 nt upstream the ATG start codon. This sequence has been shown to be important in setting both basal and derepressed expression of the amdS gene in A. nidulans (Littlejohn et al., 1992), and could be involved in the repression of the EndoPG of C1 by glucose (Hugouvieux et al., 1995). Furthermore, pyrimidine-rich sequences which are also
127 CTCGAGTCTTGGCTCATTCTTCTTTCACTA~CTG~CTGTTCCTTTTGCGTTACTTCT
T
60
GTCTCATACATTCTGATACTGCGTATC~GTCACTCTCTAAAGAAAC~CC~TCA
120
TCACCACCACC~CAAACCAGCAAACATCTCTGCC~GAT~TCTCTTACCTCTTCGTGC M V S Y L F V TC~C~CCTGGCTTCTGTT~CATCGCCTCCCCCGTCCCCGA~TC~G~CCGC~CA L G A L A S V A I A S P V P E L K A ~ A GCT~ACCTTCACCGATGCT~CTCT~CATC~GC~GGCCA~T~ACCACCATCG S C T F T D A A S A I K G N A S C T T I TCCTC~C~TATT~TCCCTGCCGGCACCACCCTCGACAT~CCGGCCTC~GTCTG V L N N I A V p A G T T L D M T G L K S GCACTCACGT~GTTTCTCTCTCTCTCTCAGTCTCATTATCTGTTCCTAGAGACATCCAC G T H V S F S C~CACACCCTTC~CAGGTCACCTTCC~CAAAACCACCTTCGGCTAC~GGAGTGG G K T T F G Y K E W GA~GCCCCCTCATCTCCTTCTCCGGCTCC~CGTCGTCATCGACGGCGCCTCCG~CAC E G P L I S F S G S N V V I D G A S G H I 0 4 TCCATCGACTGCCAGG~TCCCGCTGGTG~ACTCC~C~CGGCG~GACC S I D C Q G S R W W D S K G G N G G K T ~GCCC~GTTCTTCTACGCCCACAGCCTC~GGACTCGACCATCA~CTCCACACG K P K F F Y A H S L K D S T I R G L H T I 4 4 CTC~CACCCCCGTCCAG~CTTCTCCATC~CGGC~CGCC~CCTCGGGTCTTACGAC L N T ~ V Q A F S I N G A A N L G V Y D I 6 4 GTCTCCGTCGAC~CTCG~CG~GACTCCGCCG~GGCCAC~CACCGAC~CTTCGAC V S V D M S A G D S A G G H N T D A F D I 8 4 GTC~CTCCTCCACCGGCGTCTACATCTCTGGCGCCGACGTC~G~CCAGGACGACTGC V G S S T G V Y I S G A D V K N Q D D C 2 0 4 CTC~CGTC~CTCGGGCACC~TATTACCTTCACCGGCGGCACCT~TCCGGCGGCCAC L A V N S G T N I T F T G G T C S G G H 2 2 4 GGTCTTTCCATCGGCTCCGTTGGCGGCCGC~GGAT~TGTCGTC~GAGCGTCA~ATC G L S I G S V G G R K D ~ V V K S V S I 2 4 4 ACC~CTCC~GATTATC~CTCGGAT~CGGCGTGCGTATC~GACCGTGGCCG~GCT T N S K I I N S D N G V B I K T V A G A 2 6 4 ACTGGTCCCGTTTCCGACATCACCTACTCGGGCATCACCCTCTCC~CATCGCC~GTAC T G P V S D I T Y S G I T L S N I A K Y G~ATCGTCATCG~CA~ACTACGAG~CGGTTCTCCCACCGGC~GCCCACCTCCGGT G I V I E Q D Y E N _ G S P T G K P T S G 3 0 4 GTCCCTATTTCCGGCCTCACCCTCAGC~GATCAGC~TTCCGTCTCCTCCTCCGCCACC V P I S G L T L S K I S G S V S S S A T CCCGTTTACATCCTCT~GCCTCCT~ACC~CTGG~GTGGTCCGGCGTCAGCGTCACC P V Y I L C A S C T N W K W S G V S V T G~GGC~G~GTCCTCC~GTGCACC~TATCCCCAGCGGGAGCGGTGCTGCTTGCT~ G G K K S S K C T G I P S G S G A A C * ~GGCTGAGTCTTTACGCGC~ACTCGGC~TGAGC~TCA%/%~TCTCACACCTATATA~
180 7 240 27 300 47 360 67 420 74 480 84 540 600 124 660 720 780 840 900 960 1020 1080 284 1140 1200 324 1260 344 1320 363 1380
TACACACACAAACATACTGCGGACTT~TCGGTCGACACACGGTCTCCGGGGCTAGTTTT
1440
CTCCCTTGAGGAGCAC~GACACCGCTC~AGTG~GCT~ACTGTCTTTCTTGT~T
1500
T~TT~GTCCTGTCTGAGGTGTCCGTCCATCCCTCCCTGGGGATGCAAATATTTCCTGT
1560
ATATATTTCCTTTACTCCCCTCGTCTCCTTCTTCGACGAG~TGGT~CAAACATAGC
1620
TTGATCCAAATTGATGT~TTTGGACGC~TTTCGCTGCCGTCTTTCCCGTC~CGGT~T
1680
CTACAAGGAACC GTGGTCTG
1700
Fig. 2. Nucleotide sequence of the Clpgl gene and deduced aa sequence. The putative CCAAT box and sites of polyadenylation are underlined. The CAATC consensus sequence and conserved aa between fungi are in bold letters. C + T-rich regions are indicated by broken lines. The stop codon is marked by an asterisk. A putative glycosylation site is double underlined. The intron covers the region between nt 381 and 451. The upward arrow indicates a possible signal peptide cleavagesite. Nucleotide and aa sequences were analysed using the PC/GENE sequence analysis software (IntelliGenetics). The nt sequence data for Clpgl have been assigned GenBank accession No. X89370. Methods: DNA isolated from Cl race 13as described by Dellaporta et al. (1983), was digested with EcoRI and XhoI. Southern experiments revealed that the PCR fragment pgA probe hybridized with a 3-kb genomicfragment; accordingly DNA fragments of 3 kb were purified from the gel, ligated to )~Zaparms (Stratagene), and in vitro packaged phages were used to infect E. coil XL1 Blue cells. This partial genomic library was screened with the PCR-amplifiedpg genomic fragment (pgA). After two rounds of screening, the cloned DNA fragment was recovered by in vivo excision of pBLUESCRIPT from )~ZAP and sequenced by the dideoxy chain-termination method (Sanger et al, 1977). considered as important fungal promoter elements were identified. C + T - r i c h regions were detected at positions -140, - 1 0 4 and - 6 5 . The sequence surrounding the ATG start codon, 5'-CCAAGATGGT, closely resembles the K o z a k sequence (CAMMATGNC) identified in filamentous fungi and higher eukaryote's genes (Kozak, 1986; Ballance, 1990).
The 3'-noncoding region (340 nt) did not contain the typical polyadenylation signal, AATAAA, as already reported in the case of other fungal genes (Ballance, 1986; Benoist et a1.,1980). However, similar sequences situated 177 nt and 228 nt downstream the stop codon AATTAA and AAATA, respectively, were found. The CAATC sequence wich is present 38 nt downstream from the stop codon is in agreement with the consensus sequence CAWTS involved in the termination of translation (Berget, 1984) and is proposed as a site of polyadenylation (Mullaney et al., 1985). T A G T and T T T motifs situated at nt 1434 have also been considered as important functional components of polyadenylation or transcription termination in yeasts (Zaret and Sherman, 1982). Analysis of the codon usage of the Clpgl sequence showed that 44 out of the 61 sense codons are used. Codons ending in C are preferred and represent 74.4% of the codons used.
(c) Comparison of the deduced aa sequence of EndoPG with different fungal P G The CIPG1 sequence was compared to eleven fungal E n d o P G aa sequences: P G N 1 from C. carbonum (ScottGraig et al., 1990), S C E E P G (Reymond et al., 1994), S C E E P G A and S C E E P G B from S. sclerotiorium (GenBank accession No. L29040 and L29041 respectively), AFPECA from A.flavus (GenBank accession No. U05015), A N P G , A N P G A 1 and A N P G A 2 from A. niger (Ruttkowski et al., 1991; Bussink et al., 1991a,b), P G A C from A. nidulans (Bussink et al., 1992), P G A from F. moniliforme (Caprari et al., 1993) and ATPGA2 from A. tubigensis (Bussink et al., 1991b). The different aa sequences showed similar sizes with an overall similarity of 33.3%. The highest degree of homology was observed between the Cl EndoPG, CIPG1 and S C E E P G of S. sclerotiorum (66%; Table I). Most of the P G contain one or two putative glycosylation sites. The CIPG1 sequence shows only one glycosylation site, located in the same region as the glycosylation site of four of the above mentionned EndoPG. In addition, CIPG1 exhibits the 231CXGGHGXSIGSVG 243 sequence, which is specific to fungal pectinases and the sequence 2S6RIK258 found in all fungal E n d o P G and plant ExoPG, which is considered to be a specific signature of the P G (Reymond et al., 1994).
(d) Two genes coding for EndoPG are present in the C! genome The previous analysis of Cl genomic DNA, using the e D N A coding for the E n d o P G of C. carbonum as a probe, suggested that at least two genes are present in the genome of this fungus (Hugouvieux et al., 1995). Two major fragments of 9 and 5 kb, hybridizing with pgA,
128 TABLE 1 Comparison of the Cl EndoPG aa sequence with those of other fungal EndoPG
Cl PG1 homology (%)"
EndoPG of various fungi
Cochliobolus carbonum: Sclerotinia sclerotiorium:
Aspergillus flavus: Aspergillus niger:
Aspergillus tubigensis: Aspergillus nidulans: Fusarium moniliforme:
CCLPGN1 SCEEPG SCEEPGA SCEEPGB AFPECA ANPGA2 ANPGA1 ANPG ATPGA2 ANPGAC FSOWPGA
63 66 58 57 57 54 53 53 53 42 37
A
B 1
2
3
4
5
6
7
kb
kb
9.4
9.4
6.6
5.6
4.4
~.4
2.3
2.3
C XhoI
BamHl
EcoRI
a Numbers indicate the percent aa identity determined for the most favorable alignment in each case, by using the CLUSTAL software program (IntelliGenetics).
I were generated by XhoI cleavage of fungal DNA (Fig. 3A). To determine which of these fragments corresponded to Clpgl, a specific probe was prepared (Fig. 3C), corresponding to an adjacent DNA sequence at the 3'-noncoding region of the gene. Fig. 3B shows that this probe hybridized with only one of the XhoI fragments, suggesting that the Clpgl gene is contained in the 9-kb fragment and is present as a single copy in the Cl genome. Northern blot analysis with the pgA probe showed only one transcript size (data not shown). Further investigations on the regulation of transcription of the Clpgl gene will involve the use of a specific probe for Northern experiments and promoter studies.
(e) Conclusions (I) Oligos derived from EndoPG aa sequences were used to amplify a DNA fragment from the plant pathogenic Cl fungus. A 542-bp fragment was obtained and used to probe a partial genomic library of this fungus. Two genomic clones were isolated and one of them, Clpgl, was sequenced. (2) The Clpgl coding sequence is interrupted by one intron and codes for a 363-aa protein which has a putative 26-aa signal peptide. The deduced aa sequence shows homology to other fungal EndoPG and contains one Nglycosylation site. Regulatory sequences were detected such as a CCAAT motif found in the Y-non-coding region which is likely to represent a binding site for a regulatory protein as was shown in the case of the amdS gene of A.
nidulans. (3) Southern analysis using a Clpgl-specific probe shows the presence of Clpgl gene as a single copy in the Cl genome.
pgA Specificprobeof
C/pgl
~_~ 350 bp
Fig. 3. Southern blot analysis of CI DNA to the PCR-obtained pgA clone. Genomic DNA (5 gg) of the fungus was digested with the following enzymes: (A) Lanes: 1, ApaI, 2, KpnI, 3, XbaI, 4, Sinai, 5, EcoRI, 6, XhoI, 7, EcoRI + XhoI. After electrophoresis on a 1% agarose gel, DNA was transferred to a nitrocellulose membrane and hybridized with pgA. Hybridization was carried out overnight at 65°C in 6 x SSC/0.5% SDS/5 x Denhardt's solution/0.1% denatured calf thymus DNA. The membrane was washed twice at 65°C with 0.1% SDS/2 × SSC (0.16 M NaC1/0.015 M Na3"citrate pH 7.6). (B) CI DNA (5 gg) was digested with XhoI and fractionated as in A. A 1500-bp specific sequence recovered from the 3' region of Clpgl was used as a probe. (C) Restriction map of Clpgl and the regions used as probes for Southern experiments.
REFERENCES Albersheim, P. and Anderson, A.J.: Proteins from plant cell walls inhibit polygalacturonases secreted by plant pathogens. Proc. Natl. Acad. Sci. USA 68 (1971) 1815-1819. Ballance, D.J.: Sequences important in gene expression in filamentous fungi. Yeast 2 (1986) 224-236. Benhamou, N., Lafitte, C., Barthe, J.P. and Esquerr6-Tugay6, M.T.: Cell surface interactions between bean leaf cells and Colletotrichum lindemuthianum. Cytochemical aspects of pectin breakdown and fungal endopolygalacturonase accumulation. Plant. Physiol. 97 (1991) 234-244. Benoist. C., O'Hare, K., Breathnach, R. and Chambon, P.: The ovalbumin gene sequence of putative control regions. Nucleic Acids Res. 8 (1980) 127-142. Berget, S.M.: Are U4 small nuclear ribonucleoproteins involved in polyadenylation? Nature 309 (1984) 179-182. Bussink, H.J.D., Brouwer, K., De Graaf, L.H., Kester, H.C.M. and Visser, J.: Identification and characterization of a second polygalacturonase gene of Aspergillus niger. Curr. Genet. 20 (1991a) 301-307. Bussink, H.J.D., Buxton, F.P. and Visser, J.: Expression and sequence comparison of the Aspergillus niger and Aspergillus tubigensis genes encoding polygalacturonase II. Curr. Genet. 19 (1991b) 467-474. Bussink, H.J.D., Buxton, F.P., Fraage, B.A., De Graaf, L.H. and Visser,
129 J.: The polygalacturonases of Aspergillus niger are encoded by a family of diverged genes. Eur. J. Biochem. 208 (1992) 83-90. Caprari, C., Richter, A., Bergmann, C., Lo Cicero, S., Salvi, G., Cervonne, F. and De Lorenzo, G.: Cloning and characterization of a gene encoding the endopolygalacturonase of Fusarium moniliforme. Myeol. Res. 97 (1993)497-505. Carpita, N.C. and Gibeaut, D.M.: Structural models of primary cell wails in flowering plants consistency of molecular structure with the physical properties of the walls during growth. Plant J. 31 (1993) 1-30. Collmer, A. and Keen, N.T.,: The role of pectic enzymes in plant pathogenesis. Annu. Rev. Phytopathol. 24 (1986) 1389-1409. Cooper, R.M. and Wood, R.K.S.: Regulation of synthesis of cell wall degrading enzymes by Verticillum alboatrum and Fusarium oxysporum f. sp. lycopersici. Physiol. Plant Pathol. 5 (1975) 135-156. Dellaporta, S.L., Wood, J. and Hicks, J.B.: A plant DNA minipreparation, version II. Plant Mol. Biol. Rep. 1 (1983) 19-21. Hahn, M.G., Darvill, A.G. and Albersheim, P.: Host pathogen interactions, XIX. The endogenous elicitor, a fragment of a plant cell wall polysaccharide that elicits phytoalexins accumulation in soybeans. Plant Physiol. 68 (198i) 1161-1169. Hugouvieux, V., Centis, S., Lafitte, C. and Esquerr6-Tugay6, M.T.: Characterization of Colletotrichum lindemuthianum endopolygalacturonase with molecular probes. C. R. Acad. Sci. Paris 318 (1995) 113 120. Keon, J.P.R., Waksman, G. and Bailey, J.A.: A comparison of the biochemical and physiological properties of a polygalacturonase from two races of Colletotrichum lindemuthianum. Physiol. Mol. Plant Pathol. 37 (1990) 193-206. Kitamoto, N., Kimura, T., Kito, Y., Ohmiya, K. and Tsukagoshi, N.: Structural features of a polygalacturonase gene cloned from Aspergillus oryzae KBN616. FEMS Microbiol. Lett. 111 (1993) 37-41. Kozak, M.: Point mutations close to the AUG initiator eodon affect the efficiency of translation of rat preproinsulin in vivo. Nature 308 (1984) 241-247.
Lafitte, C., Barthe, J.P., Montillet, J.L. and Touz6, A.: Glycoprotein inhibitors of Colletotrichum lindemuthianum endopolygalacturonase in near isogenic lines of Phaseolus vulgaris resistant and susceptible to anthracnose. Physiol. Plant Pathol. 25 (1984) 39-53. Lafitte, C., Barthe, J.P., Gansel, X., Dechamp-Guillaume, G., Faucher, C., Mazau, D. and Esquerr6-Tugay6, M.T.: Differential induction by endopolygalacturonase of 13-1,3-glucanases in Phaseolus vulgaris isolines susceptible and resistant to Colletotrichum lindemuthianum race 13. Mol. Plant-Microb. Interact. 6 (1993) 628-634. Littlejohn, T.G. and Hynes, M.J.: Analysis of the site of action of the amdR product for regulation of the amdS gene of Aspergillus nidulans. Mol. Gen. Genet. 235 (1992) 81-88. Mullaney, E.J., Hamer, J.E., Yelton, M.M. and Timberlake, W.H.: Primary structure of the trpC gene from Aspergillus nidulans. Mol. Gen. Genet. 199 (1985) 37-45. Ruttkowski, E., Khanh, N.Q., Wientjes, F.J. and Gottschalk, M.: Characterization of a polygalacturonase gene from Aspergillus niger RH5344. Mol. Microbiol. 5 (1991) 1353-1361. Sanger, F., Nicklen, S. and Coulson, A.R.: DNA sequencing with chainterminating inhibitors. Proc. Natl. Acad. Sci. USA 74 (1977) 5463-5467. Scott-Craig, J.S., Panaccione, D.G., Cervone, F. and Walton, D.J.: Endopolygalacturonase is not required for pathogenicity of Cochliobolus carbonum on maize. Plant Cell 2 (1990) 1191-1200. Reymond, P., Del6age, G., Rascle, C. and F~vre, M.: Cloning and sequence analysis of a polygalacturonase-encoding gene from the phytopathogenic fungus Sclerotinia sclerotiorum. Gene 146 (1994) 233 237. Wijesundera, R.L.C., Bailey, J.A., Byrde, R.J.W. and Fielding, A.H.: Cell wall degrading enzymes of Colletotrichum lindemuthianum: their role in the development of bean anthracnose. Physiol. Mol. Plant Pathol. 34 (1989) 403 413. Zaret, K.S. and Sherman, F.: DNA sequence required for efficient transcription termination in yeast. Cell 28 (1982) 563-573.
125
G E N E 09569
Isolation and sequence analysis of Clpgl, a gene coding for an endopolygalacturonase of the phytopathogenic fungus Colletotrichum
lindemuthianum (Pectin degradation; pectinase; polymerase chain reaction; nucleotide sequence; sequence comparison; Southern blot analysis)
Sylvie Centis, B e r n a r d D u m a s , Jo~lle F o u r n i e r , Mich61e M a r o l d a a n d M a r i e - T h 6 r 6 s e Esquerr6-Tugay6 UniversiM Paul Sabatier, Centre de Biologie et Physiologie Vdgktale URA 1941 CNRS, 31062 Toulouse c~dex, France
Received by J.R. Kinghorn: 31 July 1995; Revised/Accepted 21 October 1995; Received at publishers 11 December 1995
SUMMARY
Oligodeoxyribonucleotide primers designed from the N-terminal amino acid (aa) sequence of the endopolygalacturonase (EndoPG) of Colletotrichum lindemuthianum (Cl) race ~ and from an internal sequence conserved among different fungal EndoPG were used in a polymerase chain reaction (PCR) to amplify genomic related sequences of the fungus. A 542-bp fragment, designated pgA, was obtained and used as a probe to screen a partial genomic library of C1. Among the positive clones, one was further analyzed. Nucleotide sequencing of this clone revealed an ORF encoding a 363-amino-acid (aa) polypeptide begining with a signal peptide of 26 aa interrupted by an intron of 70 bp, and showing a high degree of homology to ten fungal EndoPG sequences. Consensus sequences were identified in the 5' non-coding region. This genomic clone was thereafter designated Clpgl. Southern analysis, performed with a Clpgl-specific probe, showed that this gene is present as a single copy in the Cl genome.
INTRODUCTION
Pectin, a methylated heteropolymer containing ~-l,4-1inked galacturonic acid, is one of the predominant polysaccharides of plant cell walls (Carpita et al., 1993). Since this material constitutes a mechanical barrier, as Correspondence to: Dr. M.-T. Esquerr6-Tugay6, Centre de Biologie et Physiologie V6g6tale, URA 1941 CNRS, 118 route de Narbonne, 31062 Toulouse c6dex, France. Tel. (33) 61556758; Fax (33) 61558378; e-mail: [email protected]
Abbreviations: A., Aspergillus; aa, amino acid(s); bp, base pair(s); Cl, Colletotrichum lindemuthianum; Clpgl, gene coding for an EndoPG of Cl; EndoPG, endopolygalacturonase(s); kb, kilobase(s) or 1000 bp; M, A or C; N, A or C or G or T; nt, nucleotide(s); oligo, oligodeoxyribonucleotide; ORF, open reading frame; PCR, polymerase chain reaction; PG, polygalacturonase(s); pg, gene encoding PG; PGIP, PG inhibiting protein; PR, pathogenesis related; S, C or G; SDS, sodium dodecyl sulfate; SP, signal peptide; W, A or T; Y, C or T. SSDI 0378-1119(95)00867-5
well as a carbon source, many phytopathogenic and saprophytic microorganisms secrete pectolytic enzymes such as polygalacturonase, pectin methyl esterase, pectate lyase or pectin lyase. These enzymes are important in pathogenicity for not only do they cause cell-wall degradation (Cooper and Wood 1975; Collmer and Keen, 1986), but also they act as indirect elicitors of plant defence responses through the oligosaccharides they release (Hahn et al., 1981). Endopolygalacturonase (or EndoPG, EC 3.2.1.15) catalyses the hydrolytic cleavage of glycosidic a-1,4 linkages in polygalacturonide chains of pectic substances. Colletotrichum lindemuthianum (Cl) is a fungal pathogen causing anthracnose on bean seedlings. When grown on pectin-containing liquid medium, this fungus secretes an EndoPG which has been thoroughly characterized with molecular probes (Hugouvieux et al., 1995).
126 EndoPG activity is partly inhibited during the parasitic stage of the fungus by the bean cell-wall polygalacturonase inhibiting protein (PGIP) (Albersheim and Anderson, 1971; Lafitte et al., 1984; Wijesundera et al., 1989). The use of gold-labeled P G I P allowed EndoPG and associated cell-wall degradation to be visualised in planta in infected bean tissues (Benhamou et al., 1991). The enzyme is a glycoprotein of around 38 kDa whose sugar moiety accounts for 4% of the molecule and is composed of galactose, glucosamine and mannose (Keon et al., 1990). We recently reported that the EndoPG purified from C1 race 13elicits the biosynthesis of PR (pathogenesis related) proteins in bean cuttings, notably 13-1,3-glucanase, in a cultivar-specific manner (Lafitte et al., 1993), suggesting an important role for this fungal protein in triggering the expression of the host defence genes. A better understanding of this dual role requires further studies of EndoPG at the gene level. PG-encoding genes have been isolated from a few fungi, notably Cochliobolus carbonum (Scott-Graig et al., 1990), Aspergillus niger (Bussink et al., 1992), Aspergillus orizae (Kitamoto et al., 1993), Fusarium moniliforme (Caprari et al., 1993) and Sclerotinia sclerotorium (Reymond et al., 1994). As a prerequisite to evaluating the role played by Clpg gene expression, we have cloned and subsequently characterized a Clpg gene from Cl.
EXPERIMENTALAND DISCUSSION
(a) Amplification ofpg genomic DNA sequences by PCR and isolation of genomic clones An oligo primer (5'-GTYCTIAACAACATYCC) based on the N-terminal aa sequence of Cl race ~ EndoPG (Keon et al., 1990), and an antisense primer (5'-GCRASRCAGTCRTCYTGGTT) designed from an internal aa sequence well conserved among fungal EndoPG (Fig. 1), were synthesized (R = A/G; S = C/G; Y = C / T ) . These two primers were used to amplify by PCR a genomic DNA fragment encoding a portion of C1PG. An amplified 542-bp product, named pgA, was ligated in pGEM-T (Promega, Madison, WI, USA) and sequenced. Sequence analysis showed a high degree of homology to fungal pg genes. This fragment was used to screen a partial genomic library of Cl constructed in LZap (Stratagene, La Jolla, CA, USA). Among the positive clones, some showed high hybridization to the pgA probe, while others gave much weaker signals. Clones of both types were purified and submitted to in vivo excision. Restriction patterns led us to the conclusion that we had isolated two different classes of clones. One clone of the
CCLPGN I SCEEPGA SCEEPGB AFPECA ANPGAI ANPGA2 ATPGA2 ANPG
ANPGAC FSOWPGA
.... SAG-AHNTDAFDIGSSSGI T ISNANI KI~DDCVAINSGSDIHVTNC .... SLG- -HNTDAFDVGS S TDI T ISGANVQ~aDCLKIN SGTG ITF TGG .... SLG- -HNTDAFDVGSSTDITISGANVQm~)DCLKINSGTGI TFTGG .... AEG--HNTDAFDVGSSTY INIDGA~NSGSHITFTNG .... DNG-GHNTDGFDI SESTGVYISGA~DCIAINSGES ISFTGG TQG -GHW/DAFDVGN SVGVN I I~ V N S G E N I W F T G G .... TLG-GHNTDAFDVGNSVGVNI I ~ N S G E N I W F T S G .... TLG-GHNTDAFDVGNSVGVNI I~ I N S G E N I W F TSG . . . . T D D L A A N T D G F D I G E S T Y I T I TC.AE I Y N ~ t R D G ' V A I N S G E N I Y F S A S AKSGSLPAAHNTDGFDISSSDHVTLD~DG~/AVTSGTNTVVSNM ***.**...* . . . *****.*..**. * .. .
.
.
.
219 238 238 218 223 217 217 217 237 228
Fig. 1. Comparisonof a portion of the deducedaa sequencesof different fungal EndoPG corresponding to: CCLPGN1, C. carbonum; SCEEPGA and SCEEPGB, S. scleriotorium, AFPECA, A. flavus; ANPGA1, ANPGA2 and ANPG, A. niger; ATPGA2, A. tubigensis; ANPGAC, A. nidulansand FSOWPGA,F. moniliforme.Sequencealignments were performed using the CLUSTAL software program (IntelliGenetics).Identical(*) and conserved(.) aa substitutionsbetween the fungiare shown.The antisenseprimerwas deducedfrom the homology region indicatedin bold letters.
class which hybridized strongly to the probe, designated Clpgl, was retained for further analysis.
(b) Characterization of the genomic clone Cipgl From the cloned genomic DNA, 1700 nt overlapping the region corresponding to pgA were sequenced according to standard procedures (Sanger et al., 1977), using both universal and specific primers. Analysis of this sequence with the Blast sequence comparison program revealed the occurrence of an ORF of l152bp. The coding region is interrupted by one intron of 70 bp (Fig. 2). The encoded 363 aa (36685 Da) is in good agreement with 37 kDa obtained by SDS-PAGE (Lafitte et al., 1984), and the 38.5 kDa obtained by gel filtration (Keon et al., 1990). A potential N-glycosylation site (AsnGly-Ser) was found at Asn TM. The presence of this site is correlated with the glycoproteic nature of EndoPG (Keon et al., 1990). The N-terminal aa sequence of Cl EndoPG published by Keon et al., (1990) and confirmed in our laboratory, corresponded to aa 27 to 62, thereby indicating that the first 26-aa peptide, rich in hydrophobic residues, corresponded to the signal peptide (SP) of EndoPG. This SP might have been eliminated by a trypsin-like protease cleavage (Benoist et al., 1987) since an Arg 26 occurs in the Cl aa sequence, as in the case of the PG of F. moniliforme and of the PGII of A. niger (Bussink et al., 1991a; Caprari et al., 1993). The 5'-noncoding region of the clone, representing 160 bp, did not contain the typical eukaryotic TATA box (TATAAAT), but a CCAAT sequence, which is often observed in fungal promoters, was found 55 nt upstream the ATG start codon. This sequence has been shown to be important in setting both basal and derepressed expression of the amdS gene in A. nidulans (Littlejohn et al., 1992), and could be involved in the repression of the EndoPG of C1 by glucose (Hugouvieux et al., 1995). Furthermore, pyrimidine-rich sequences which are also
127 CTCGAGTCTTGGCTCATTCTTCTTTCACTA~CTG~CTGTTCCTTTTGCGTTACTTCT
T
60
GTCTCATACATTCTGATACTGCGTATC~GTCACTCTCTAAAGAAAC~CC~TCA
120
TCACCACCACC~CAAACCAGCAAACATCTCTGCC~GAT~TCTCTTACCTCTTCGTGC M V S Y L F V TC~C~CCTGGCTTCTGTT~CATCGCCTCCCCCGTCCCCGA~TC~G~CCGC~CA L G A L A S V A I A S P V P E L K A ~ A GCT~ACCTTCACCGATGCT~CTCT~CATC~GC~GGCCA~T~ACCACCATCG S C T F T D A A S A I K G N A S C T T I TCCTC~C~TATT~TCCCTGCCGGCACCACCCTCGACAT~CCGGCCTC~GTCTG V L N N I A V p A G T T L D M T G L K S GCACTCACGT~GTTTCTCTCTCTCTCTCAGTCTCATTATCTGTTCCTAGAGACATCCAC G T H V S F S C~CACACCCTTC~CAGGTCACCTTCC~CAAAACCACCTTCGGCTAC~GGAGTGG G K T T F G Y K E W GA~GCCCCCTCATCTCCTTCTCCGGCTCC~CGTCGTCATCGACGGCGCCTCCG~CAC E G P L I S F S G S N V V I D G A S G H I 0 4 TCCATCGACTGCCAGG~TCCCGCTGGTG~ACTCC~C~CGGCG~GACC S I D C Q G S R W W D S K G G N G G K T ~GCCC~GTTCTTCTACGCCCACAGCCTC~GGACTCGACCATCA~CTCCACACG K P K F F Y A H S L K D S T I R G L H T I 4 4 CTC~CACCCCCGTCCAG~CTTCTCCATC~CGGC~CGCC~CCTCGGGTCTTACGAC L N T ~ V Q A F S I N G A A N L G V Y D I 6 4 GTCTCCGTCGAC~CTCG~CG~GACTCCGCCG~GGCCAC~CACCGAC~CTTCGAC V S V D M S A G D S A G G H N T D A F D I 8 4 GTC~CTCCTCCACCGGCGTCTACATCTCTGGCGCCGACGTC~G~CCAGGACGACTGC V G S S T G V Y I S G A D V K N Q D D C 2 0 4 CTC~CGTC~CTCGGGCACC~TATTACCTTCACCGGCGGCACCT~TCCGGCGGCCAC L A V N S G T N I T F T G G T C S G G H 2 2 4 GGTCTTTCCATCGGCTCCGTTGGCGGCCGC~GGAT~TGTCGTC~GAGCGTCA~ATC G L S I G S V G G R K D ~ V V K S V S I 2 4 4 ACC~CTCC~GATTATC~CTCGGAT~CGGCGTGCGTATC~GACCGTGGCCG~GCT T N S K I I N S D N G V B I K T V A G A 2 6 4 ACTGGTCCCGTTTCCGACATCACCTACTCGGGCATCACCCTCTCC~CATCGCC~GTAC T G P V S D I T Y S G I T L S N I A K Y G~ATCGTCATCG~CA~ACTACGAG~CGGTTCTCCCACCGGC~GCCCACCTCCGGT G I V I E Q D Y E N _ G S P T G K P T S G 3 0 4 GTCCCTATTTCCGGCCTCACCCTCAGC~GATCAGC~TTCCGTCTCCTCCTCCGCCACC V P I S G L T L S K I S G S V S S S A T CCCGTTTACATCCTCT~GCCTCCT~ACC~CTGG~GTGGTCCGGCGTCAGCGTCACC P V Y I L C A S C T N W K W S G V S V T G~GGC~G~GTCCTCC~GTGCACC~TATCCCCAGCGGGAGCGGTGCTGCTTGCT~ G G K K S S K C T G I P S G S G A A C * ~GGCTGAGTCTTTACGCGC~ACTCGGC~TGAGC~TCA%/%~TCTCACACCTATATA~
180 7 240 27 300 47 360 67 420 74 480 84 540 600 124 660 720 780 840 900 960 1020 1080 284 1140 1200 324 1260 344 1320 363 1380
TACACACACAAACATACTGCGGACTT~TCGGTCGACACACGGTCTCCGGGGCTAGTTTT
1440
CTCCCTTGAGGAGCAC~GACACCGCTC~AGTG~GCT~ACTGTCTTTCTTGT~T
1500
T~TT~GTCCTGTCTGAGGTGTCCGTCCATCCCTCCCTGGGGATGCAAATATTTCCTGT
1560
ATATATTTCCTTTACTCCCCTCGTCTCCTTCTTCGACGAG~TGGT~CAAACATAGC
1620
TTGATCCAAATTGATGT~TTTGGACGC~TTTCGCTGCCGTCTTTCCCGTC~CGGT~T
1680
CTACAAGGAACC GTGGTCTG
1700
Fig. 2. Nucleotide sequence of the Clpgl gene and deduced aa sequence. The putative CCAAT box and sites of polyadenylation are underlined. The CAATC consensus sequence and conserved aa between fungi are in bold letters. C + T-rich regions are indicated by broken lines. The stop codon is marked by an asterisk. A putative glycosylation site is double underlined. The intron covers the region between nt 381 and 451. The upward arrow indicates a possible signal peptide cleavagesite. Nucleotide and aa sequences were analysed using the PC/GENE sequence analysis software (IntelliGenetics). The nt sequence data for Clpgl have been assigned GenBank accession No. X89370. Methods: DNA isolated from Cl race 13as described by Dellaporta et al. (1983), was digested with EcoRI and XhoI. Southern experiments revealed that the PCR fragment pgA probe hybridized with a 3-kb genomicfragment; accordingly DNA fragments of 3 kb were purified from the gel, ligated to )~Zaparms (Stratagene), and in vitro packaged phages were used to infect E. coil XL1 Blue cells. This partial genomic library was screened with the PCR-amplifiedpg genomic fragment (pgA). After two rounds of screening, the cloned DNA fragment was recovered by in vivo excision of pBLUESCRIPT from )~ZAP and sequenced by the dideoxy chain-termination method (Sanger et al, 1977). considered as important fungal promoter elements were identified. C + T - r i c h regions were detected at positions -140, - 1 0 4 and - 6 5 . The sequence surrounding the ATG start codon, 5'-CCAAGATGGT, closely resembles the K o z a k sequence (CAMMATGNC) identified in filamentous fungi and higher eukaryote's genes (Kozak, 1986; Ballance, 1990).
The 3'-noncoding region (340 nt) did not contain the typical polyadenylation signal, AATAAA, as already reported in the case of other fungal genes (Ballance, 1986; Benoist et a1.,1980). However, similar sequences situated 177 nt and 228 nt downstream the stop codon AATTAA and AAATA, respectively, were found. The CAATC sequence wich is present 38 nt downstream from the stop codon is in agreement with the consensus sequence CAWTS involved in the termination of translation (Berget, 1984) and is proposed as a site of polyadenylation (Mullaney et al., 1985). T A G T and T T T motifs situated at nt 1434 have also been considered as important functional components of polyadenylation or transcription termination in yeasts (Zaret and Sherman, 1982). Analysis of the codon usage of the Clpgl sequence showed that 44 out of the 61 sense codons are used. Codons ending in C are preferred and represent 74.4% of the codons used.
(c) Comparison of the deduced aa sequence of EndoPG with different fungal P G The CIPG1 sequence was compared to eleven fungal E n d o P G aa sequences: P G N 1 from C. carbonum (ScottGraig et al., 1990), S C E E P G (Reymond et al., 1994), S C E E P G A and S C E E P G B from S. sclerotiorium (GenBank accession No. L29040 and L29041 respectively), AFPECA from A.flavus (GenBank accession No. U05015), A N P G , A N P G A 1 and A N P G A 2 from A. niger (Ruttkowski et al., 1991; Bussink et al., 1991a,b), P G A C from A. nidulans (Bussink et al., 1992), P G A from F. moniliforme (Caprari et al., 1993) and ATPGA2 from A. tubigensis (Bussink et al., 1991b). The different aa sequences showed similar sizes with an overall similarity of 33.3%. The highest degree of homology was observed between the Cl EndoPG, CIPG1 and S C E E P G of S. sclerotiorum (66%; Table I). Most of the P G contain one or two putative glycosylation sites. The CIPG1 sequence shows only one glycosylation site, located in the same region as the glycosylation site of four of the above mentionned EndoPG. In addition, CIPG1 exhibits the 231CXGGHGXSIGSVG 243 sequence, which is specific to fungal pectinases and the sequence 2S6RIK258 found in all fungal E n d o P G and plant ExoPG, which is considered to be a specific signature of the P G (Reymond et al., 1994).
(d) Two genes coding for EndoPG are present in the C! genome The previous analysis of Cl genomic DNA, using the e D N A coding for the E n d o P G of C. carbonum as a probe, suggested that at least two genes are present in the genome of this fungus (Hugouvieux et al., 1995). Two major fragments of 9 and 5 kb, hybridizing with pgA,
128 TABLE 1 Comparison of the Cl EndoPG aa sequence with those of other fungal EndoPG
Cl PG1 homology (%)"
EndoPG of various fungi
Cochliobolus carbonum: Sclerotinia sclerotiorium:
Aspergillus flavus: Aspergillus niger:
Aspergillus tubigensis: Aspergillus nidulans: Fusarium moniliforme:
CCLPGN1 SCEEPG SCEEPGA SCEEPGB AFPECA ANPGA2 ANPGA1 ANPG ATPGA2 ANPGAC FSOWPGA
63 66 58 57 57 54 53 53 53 42 37
A
B 1
2
3
4
5
6
7
kb
kb
9.4
9.4
6.6
5.6
4.4
~.4
2.3
2.3
C XhoI
BamHl
EcoRI
a Numbers indicate the percent aa identity determined for the most favorable alignment in each case, by using the CLUSTAL software program (IntelliGenetics).
I were generated by XhoI cleavage of fungal DNA (Fig. 3A). To determine which of these fragments corresponded to Clpgl, a specific probe was prepared (Fig. 3C), corresponding to an adjacent DNA sequence at the 3'-noncoding region of the gene. Fig. 3B shows that this probe hybridized with only one of the XhoI fragments, suggesting that the Clpgl gene is contained in the 9-kb fragment and is present as a single copy in the Cl genome. Northern blot analysis with the pgA probe showed only one transcript size (data not shown). Further investigations on the regulation of transcription of the Clpgl gene will involve the use of a specific probe for Northern experiments and promoter studies.
(e) Conclusions (I) Oligos derived from EndoPG aa sequences were used to amplify a DNA fragment from the plant pathogenic Cl fungus. A 542-bp fragment was obtained and used to probe a partial genomic library of this fungus. Two genomic clones were isolated and one of them, Clpgl, was sequenced. (2) The Clpgl coding sequence is interrupted by one intron and codes for a 363-aa protein which has a putative 26-aa signal peptide. The deduced aa sequence shows homology to other fungal EndoPG and contains one Nglycosylation site. Regulatory sequences were detected such as a CCAAT motif found in the Y-non-coding region which is likely to represent a binding site for a regulatory protein as was shown in the case of the amdS gene of A.
nidulans. (3) Southern analysis using a Clpgl-specific probe shows the presence of Clpgl gene as a single copy in the Cl genome.
pgA Specificprobeof
C/pgl
~_~ 350 bp
Fig. 3. Southern blot analysis of CI DNA to the PCR-obtained pgA clone. Genomic DNA (5 gg) of the fungus was digested with the following enzymes: (A) Lanes: 1, ApaI, 2, KpnI, 3, XbaI, 4, Sinai, 5, EcoRI, 6, XhoI, 7, EcoRI + XhoI. After electrophoresis on a 1% agarose gel, DNA was transferred to a nitrocellulose membrane and hybridized with pgA. Hybridization was carried out overnight at 65°C in 6 x SSC/0.5% SDS/5 x Denhardt's solution/0.1% denatured calf thymus DNA. The membrane was washed twice at 65°C with 0.1% SDS/2 × SSC (0.16 M NaC1/0.015 M Na3"citrate pH 7.6). (B) CI DNA (5 gg) was digested with XhoI and fractionated as in A. A 1500-bp specific sequence recovered from the 3' region of Clpgl was used as a probe. (C) Restriction map of Clpgl and the regions used as probes for Southern experiments.
REFERENCES Albersheim, P. and Anderson, A.J.: Proteins from plant cell walls inhibit polygalacturonases secreted by plant pathogens. Proc. Natl. Acad. Sci. USA 68 (1971) 1815-1819. Ballance, D.J.: Sequences important in gene expression in filamentous fungi. Yeast 2 (1986) 224-236. Benhamou, N., Lafitte, C., Barthe, J.P. and Esquerr6-Tugay6, M.T.: Cell surface interactions between bean leaf cells and Colletotrichum lindemuthianum. Cytochemical aspects of pectin breakdown and fungal endopolygalacturonase accumulation. Plant. Physiol. 97 (1991) 234-244. Benoist. C., O'Hare, K., Breathnach, R. and Chambon, P.: The ovalbumin gene sequence of putative control regions. Nucleic Acids Res. 8 (1980) 127-142. Berget, S.M.: Are U4 small nuclear ribonucleoproteins involved in polyadenylation? Nature 309 (1984) 179-182. Bussink, H.J.D., Brouwer, K., De Graaf, L.H., Kester, H.C.M. and Visser, J.: Identification and characterization of a second polygalacturonase gene of Aspergillus niger. Curr. Genet. 20 (1991a) 301-307. Bussink, H.J.D., Buxton, F.P. and Visser, J.: Expression and sequence comparison of the Aspergillus niger and Aspergillus tubigensis genes encoding polygalacturonase II. Curr. Genet. 19 (1991b) 467-474. Bussink, H.J.D., Buxton, F.P., Fraage, B.A., De Graaf, L.H. and Visser,
129 J.: The polygalacturonases of Aspergillus niger are encoded by a family of diverged genes. Eur. J. Biochem. 208 (1992) 83-90. Caprari, C., Richter, A., Bergmann, C., Lo Cicero, S., Salvi, G., Cervonne, F. and De Lorenzo, G.: Cloning and characterization of a gene encoding the endopolygalacturonase of Fusarium moniliforme. Myeol. Res. 97 (1993)497-505. Carpita, N.C. and Gibeaut, D.M.: Structural models of primary cell wails in flowering plants consistency of molecular structure with the physical properties of the walls during growth. Plant J. 31 (1993) 1-30. Collmer, A. and Keen, N.T.,: The role of pectic enzymes in plant pathogenesis. Annu. Rev. Phytopathol. 24 (1986) 1389-1409. Cooper, R.M. and Wood, R.K.S.: Regulation of synthesis of cell wall degrading enzymes by Verticillum alboatrum and Fusarium oxysporum f. sp. lycopersici. Physiol. Plant Pathol. 5 (1975) 135-156. Dellaporta, S.L., Wood, J. and Hicks, J.B.: A plant DNA minipreparation, version II. Plant Mol. Biol. Rep. 1 (1983) 19-21. Hahn, M.G., Darvill, A.G. and Albersheim, P.: Host pathogen interactions, XIX. The endogenous elicitor, a fragment of a plant cell wall polysaccharide that elicits phytoalexins accumulation in soybeans. Plant Physiol. 68 (198i) 1161-1169. Hugouvieux, V., Centis, S., Lafitte, C. and Esquerr6-Tugay6, M.T.: Characterization of Colletotrichum lindemuthianum endopolygalacturonase with molecular probes. C. R. Acad. Sci. Paris 318 (1995) 113 120. Keon, J.P.R., Waksman, G. and Bailey, J.A.: A comparison of the biochemical and physiological properties of a polygalacturonase from two races of Colletotrichum lindemuthianum. Physiol. Mol. Plant Pathol. 37 (1990) 193-206. Kitamoto, N., Kimura, T., Kito, Y., Ohmiya, K. and Tsukagoshi, N.: Structural features of a polygalacturonase gene cloned from Aspergillus oryzae KBN616. FEMS Microbiol. Lett. 111 (1993) 37-41. Kozak, M.: Point mutations close to the AUG initiator eodon affect the efficiency of translation of rat preproinsulin in vivo. Nature 308 (1984) 241-247.
Lafitte, C., Barthe, J.P., Montillet, J.L. and Touz6, A.: Glycoprotein inhibitors of Colletotrichum lindemuthianum endopolygalacturonase in near isogenic lines of Phaseolus vulgaris resistant and susceptible to anthracnose. Physiol. Plant Pathol. 25 (1984) 39-53. Lafitte, C., Barthe, J.P., Gansel, X., Dechamp-Guillaume, G., Faucher, C., Mazau, D. and Esquerr6-Tugay6, M.T.: Differential induction by endopolygalacturonase of 13-1,3-glucanases in Phaseolus vulgaris isolines susceptible and resistant to Colletotrichum lindemuthianum race 13. Mol. Plant-Microb. Interact. 6 (1993) 628-634. Littlejohn, T.G. and Hynes, M.J.: Analysis of the site of action of the amdR product for regulation of the amdS gene of Aspergillus nidulans. Mol. Gen. Genet. 235 (1992) 81-88. Mullaney, E.J., Hamer, J.E., Yelton, M.M. and Timberlake, W.H.: Primary structure of the trpC gene from Aspergillus nidulans. Mol. Gen. Genet. 199 (1985) 37-45. Ruttkowski, E., Khanh, N.Q., Wientjes, F.J. and Gottschalk, M.: Characterization of a polygalacturonase gene from Aspergillus niger RH5344. Mol. Microbiol. 5 (1991) 1353-1361. Sanger, F., Nicklen, S. and Coulson, A.R.: DNA sequencing with chainterminating inhibitors. Proc. Natl. Acad. Sci. USA 74 (1977) 5463-5467. Scott-Craig, J.S., Panaccione, D.G., Cervone, F. and Walton, D.J.: Endopolygalacturonase is not required for pathogenicity of Cochliobolus carbonum on maize. Plant Cell 2 (1990) 1191-1200. Reymond, P., Del6age, G., Rascle, C. and F~vre, M.: Cloning and sequence analysis of a polygalacturonase-encoding gene from the phytopathogenic fungus Sclerotinia sclerotiorum. Gene 146 (1994) 233 237. Wijesundera, R.L.C., Bailey, J.A., Byrde, R.J.W. and Fielding, A.H.: Cell wall degrading enzymes of Colletotrichum lindemuthianum: their role in the development of bean anthracnose. Physiol. Mol. Plant Pathol. 34 (1989) 403 413. Zaret, K.S. and Sherman, F.: DNA sequence required for efficient transcription termination in yeast. Cell 28 (1982) 563-573.