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A cis-Acting Sequence Homologous to the Yeast Filamentation and Invasion Response Element Regulates Expression of a Pectinase Gene from the Bean PathogenColletotrichum lindemuthianum

2002; Elsevier BV; Volume: 277; Issue: 32 Linguagem: Inglês

10.1074/jbc.m201489200

1083-351X

Corentin Herbert, Christophe Jacquet, Charlotte Borel, Marie‐Thérèse Esquerré‐Tugayé, Bernard Dumas,

Fungal and yeast genetics research

Phytopathogenic fungi secrete hydrolytic enzymes that degrade plant cell walls, notably pectinases. The signaling pathway(s) that control pectinase gene expression are currently unknown in filamentous fungi. Recently, the green fluorescent protein coding sequence was used as a reporter gene to study the expression ofCLPG2, a gene encoding an endopolygalacturonase of the bean pathogen Colletotrichum lindemuthianum. CLPG2is transcriptionally induced by pectin in the axenic culture of the fungus and during formation of the appressorium, an infection structure specialized in plant tissue penetration. In the present study, promoter deletion and mutagenesis, as well as gel shift mobility assays, allowed for the first time identification of cis-acting elements that bind protein factors and are essential for the regulation of a pectinase gene. We found that two different adjacent DNA motifs are combined to form an active element that shows a strong sequence homology with the yeast filamentation and invasion response element. The same element is required for the transcriptional activation ofCLPG2 by pectin and during appressorium development. This study strongly suggests that the control of virulence genes of fungal plant pathogens, such as pectinases, involves the formation of a complex of transcriptional activators similar to those regulating the invasive growth in yeast. Phytopathogenic fungi secrete hydrolytic enzymes that degrade plant cell walls, notably pectinases. The signaling pathway(s) that control pectinase gene expression are currently unknown in filamentous fungi. Recently, the green fluorescent protein coding sequence was used as a reporter gene to study the expression ofCLPG2, a gene encoding an endopolygalacturonase of the bean pathogen Colletotrichum lindemuthianum. CLPG2is transcriptionally induced by pectin in the axenic culture of the fungus and during formation of the appressorium, an infection structure specialized in plant tissue penetration. In the present study, promoter deletion and mutagenesis, as well as gel shift mobility assays, allowed for the first time identification of cis-acting elements that bind protein factors and are essential for the regulation of a pectinase gene. We found that two different adjacent DNA motifs are combined to form an active element that shows a strong sequence homology with the yeast filamentation and invasion response element. The same element is required for the transcriptional activation ofCLPG2 by pectin and during appressorium development. This study strongly suggests that the control of virulence genes of fungal plant pathogens, such as pectinases, involves the formation of a complex of transcriptional activators similar to those regulating the invasive growth in yeast. green fluorescent protein filamentation and invasion response element pheromone response element TEA/ATTS consensus sequence(s) PRE-like elements TCS-like element gel mobility shift assay mitogen-activated protein kinase endopolygalacturonase Saprophytic and phytopathogenic filamentous fungi secrete extracellular enzymes that degrade plant cell wall polymers. Among them, pectinases are the subject of intense research, because pectin degradation contributes to fungal pathogenicity in several host-pathogen systems (1Ten Have A. Mulder W. Visser J. Van Kan J.A. Mol. Plant-Microbe Interact. 1998; 11: 1009-1016Crossref PubMed Google Scholar, 2Shieh M.T. Brown R.L. Whitehead M.P. Cary J.W. Cotty P.J. Cleveland T.E. Dean R.A. Appl. Environ. Microbiol. 1997; 63: 3548-3552Crossref PubMed Google Scholar, 3Rogers L.M. Kim Y.K. Guo W. Gonzalez-Candelas L. Li D. Kolattukudy P.E. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 9813-9818Crossref PubMed Scopus (102) Google Scholar, 4Yakoby N. Beno-Moualem D. Keen N.T. Dinoor A. Pines O. Prusky D. Mol. Plant-Microbe Interact. 2001; 14: 988-995Crossref PubMed Scopus (112) Google Scholar) and is of considerable interest for various biotechnological processes. Pectinase gene expression is regulated at the transcriptional level by environmental conditions such as the pH of the medium (5Prusky D. McEvoy J.L. Leverentz B. Conway W.S. Mol. Plant-Microbe Interact. 2001; 14: 1105-1113Crossref PubMed Scopus (152) Google Scholar, 6Wubben J.P. ten Have A. van Kan J.A. Visser J. Curr. Genet. 2000; 37: 152-157Crossref PubMed Scopus (116) Google Scholar) and by carbon sources, being induced by pectin and pectic components (polygalacturonic acid, galacturonic acid, arabinose, and rhamnose) and repressed by glucose (6Wubben J.P. ten Have A. van Kan J.A. Visser J. Curr. Genet. 2000; 37: 152-157Crossref PubMed Scopus (116) Google Scholar, 7Fraissinet-tachet L. Fèvre M. Curr. Microbiol. 1996; 33: 49-53Crossref PubMed Scopus (33) Google Scholar, 8Hugouvieux V. Centis S. Lafitte C. Esquerré-Tugayé M.-T. Appl. Environ. Microbiol. 1997; 63: 2287-2292Crossref PubMed Google Scholar). Whereas the regulatory pathways that control pectinase gene expression are well documented in phytopathogenic bacteria (9Hugouvieux-Cotte-Pattat N. Condemine G. Nasser W. Reverchon S. Annu. Rev. Microbiol. 1996; 50: 213-257Crossref PubMed Scopus (337) Google Scholar), little is known about the regulation of fungal pectinases. Recently, ccSNF1, a gene encoding a protein homologous to the yeast protein kinase SNF1 required for expression of glucose-repressed genes, was isolated from the maize pathogen Cochliobolus carbonum (10Tonukari N.J. Scott-Craig J.S. Walton J.D. Plant Cell. 2000; 12: 237-248PubMed Google Scholar). Mutants disrupted in this gene showed a reduced pathogenicity, and genes coding for hydrolytic enzymes were down-regulated. Colletotrichum lindemuthianum is a pathogenic fungus that is the causal agent of bean anthracnose. Conidia germinate on the surface of the aerial part of the plant and differentiate a specialized cell called appressorium, which allows the parasite to penetrate plant tissues (11Perfect S.E. Hughes H.B. O'Connell R.J. Green J. Fungal. Genet. Biol. 1999; 27: 186-198Crossref PubMed Scopus (281) Google Scholar). During the first stages of infection, C. lindemuthianum establishes a biotrophic interaction with the host plant. 3–4 days post-inoculation, the parasite develops secondary hyphae and becomes necrotrophic, causing tissue necrosis. In a previous work, we characterized two endoPG genes, CLPG1 andCLPG2, from C. lindemuthianum (12Centis S. Dumas B. Fournier J. Marolda M. Esquerré-Tugayé M.-T. Gene (Amst.). 1996; 170: 125-129Crossref PubMed Scopus (38) Google Scholar, 13Centis S. Guillas I. Séjalon N. Esquerré-Tugayé M.-T. Dumas B. Mol. Plant-Microbe Interact. 1997; 10: 769-775Crossref PubMed Scopus (55) Google Scholar).CLPG1 encodes the major endoPG isoform that is produced during axenic culture of the fungus on pectin and during the necrotrophic stage of infection. CLPG2 is early and only transiently expressed at the onset of plant infection and on pectin. Recently we developed the use of GFP1 as a reporter gene to study the transcriptional regulation of CLPG2 (14Dumas B. Centis S. Sarrazin N. Esquerré-Tugayé M.T. Appl. Environ. Microbiol. 1999; 65: 1769-1771Crossref PubMed Google Scholar). The promoter of CLPG2 allowed expression of the reporter gene during the germination of conidia on pectin medium and during appressorium formation both on a glass slide and during pathogenesis, which indicates that diverse signals can induce endoPG gene transcription. The main goal of the present study was to look forcis-acting elements in the promoter of CLPG2involved in the induction of this gene under various situations. Deletions of the promoter delineated a 27-bp fragment required forCLPG2 induction on pectin and during appressorium formation. It is shown that this DNA fragment binds protein factors and contains two essential elements that are highly homologous to the yeast filamentation and invasion response element (FRE). C. lindemuthianum race β was maintained on synthetic agar medium as described (15Bannerot H. Ann. Amélior. Plantes. 1965; 15: 201-222Google Scholar) or grown on 9-cm cellophane disks laid down onto the surface of 20 ml of solid ANM medium (2% malt extract, 0.1% bactopeptone, 2% d(+)-glucose, 2% agar) in Petri dishes. After inoculation with 105 conidia/dish, the mycelium was allowed to develop for 48 h at 24 °C before being transferred for 10 h on solid medium supplemented with apple pectin (16Barthe J.P. Cantenys D. Touzé A. Phytopathology. 1981; 100: 162-171Crossref Scopus (29) Google Scholar) or for 24 h on bean cotyledonary leaves in a highly humid atmosphere. Transformation of C. lindemuthianum was done according to Ref. 17Hargreaves J. Turner G. Gurr S.J. MacPherson M.J. Bowles D.J. Molecular Plant Pathology: A Practical Approach. Oxford University Press, Oxford1992: 79-97Google Scholar except that the protoplasts were purified by the flotation method (18Yelton M.M. Hamer J.E. Timberlake W.E. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 1470-1474Crossref PubMed Google Scholar). Transformants were regenerated on a medium containing hygromycin at a final concentration of 50 μg·ml−1. The 5′ end of theCLPG2 mRNA was determined by 5′ rapid amplification of cDNA ends. Total RNA from pectin-induced C. lindemuthianum mycelium was prepared and submitted to first strand cDNA synthesis using a gene-specific reverse primer pg2+1207 (TableI) and SuperScriptTM II (Invitrogen). The original mRNA template was removed by treatment with RNase H. cDNA was purified with GlassMAX® Spin Cartridge, and a homopolymeric tail was added to the 3′ end using terminal transferase and dCTP. PCR amplification was accomplished usingTaq DNA polymerase, the nested gene-specific reverse primer pg2+178 (Table I), and a C-tailing annealing primer. 5′ rapid amplification of cDNA ends products were cloned in pGEM®-T vector (Promega) and sequenced.Table ISequence and position of primers used for PCR, 5′ end mapping, and GMSA Open table in a new tab To fuse the CLPG2 promoter to the coding sequence of the GFP,NcoI and BamHI restriction sites were introduced, respectively, in the 5′ and 3′ ends of the promoter fragment by PCR. The construct PG2-490 (see Fig. 1 A) was obtained by PCR amplification using the CLPG2 genomic clone (13Centis S. Guillas I. Séjalon N. Esquerré-Tugayé M.-T. Dumas B. Mol. Plant-Microbe Interact. 1997; 10: 769-775Crossref PubMed Scopus (55) Google Scholar) as a template and pg2-490 and pg2+178 (Table I) as 5′ and 3′ primers, respectively. The PCR product was digested by BamHI andNcoI and cloned in the blue SGFP-TYG-nos SK plasmid (19Sheen J. Hwang S. Niwa Y. Kobayashi H. Galbraith D.W. Plant J. 1995; 8: 777-784Crossref PubMed Scopus (328) Google Scholar) restricted with BamHI and NcoI, resulting in a translational fusion of the CLPG2 promoter with the GFP gene, which included six codons of the CLPG2 coding sequence. Constructs PG2-90, PG2-63, andPG2-90:TLE were obtained through the same strategy using, respectively, pg2-90, pg2-63, and pg2-90:TLE as 5′ end primers (TableI). Deletion PG2-490Δ27 was generated by the GenEditorTM in vitro site-directed mutagenesis system (Promega) using the 5′-phosphorylated primer pg2-490Δ27 according to the recommendations of the supplier. To constitutively express GFP, the GFP coding sequence was fused to a 405-bp fragment of the GPDA promoter from Aspergillus nidulans, which was obtained by amplification with primers gpd-S and gpd-R (Table I) and pAN7–1 vector (20Punt P. Oliver R.P. Dingemanse M.A. Pouwels P.H. Van Den Hondel C.A.M.J.J. Gene (Amst.). 1987; 56: 117-124Crossref PubMed Scopus (784) Google Scholar) as template. The construct containing 3×TLE-90 was prepared by annealing primers 3×TLE-90-S and 3×TLE-90-R. The resulting DNA fragment, which contained the 5′-SstII and 3′-BamHI restriction sites, was cloned in front of the 405-bp GPDA promoter of the constitutive GFP-expressing vector restricted with SstII andBamHI. Wells of enzyme-linked immunosorbent assay microplates were filled with 100 μl of liquid medium supplemented with d(+)-glucose or apple pectin as above, inoculated with 103 conidia, and incubated for 24 h at 24 °C in the dark. For appressorium differentiation, 100 μl of droplets containing 103 conidia were laid down onto glass slides and incubated for 48 h at 24 °C in a highly humid atmosphere in plastic boxes. For infection experiments, hypocotyls of 7-day-old bean seedlings were excised and inoculated with 5 μl of a suspension containing 5 × 102 conidia. After 48 h at 24 °C, strips of epidermal tissue were harvested and mounted in distilled water for microscopy. All of the samples were observed under fluorescence microscopy with an inverted microscope Leitz DM IRBE and a computer monitored digital color camera (Photonic Science). The figures were prepared from digitized images, and fluorescence was quantified using ImageProPlus (Media Cybernetic). The mycelium that was grown on glucose or pectin or recovered from inoculated cotyledonary leaves was washed with water and ground to a fine powder under liquid nitrogen. A crude protein extract was prepared by stirring the powder in extraction buffer (25 mm Hepes, pH 7.8, 12% glycerol, 5 mm EDTA, 1 mmphenylmethylsulfonyl fluoride, 50 mm KCl, 1 mmdithiothreitol, 10 μm ZnCl2) containing a fungal protease and phosphatase inhibitor mixture (Sigma). After incubation on ice for 1 h, the extract was centrifuged at 10,000 × g for 15 min at 4 °C. The recovered supernatant was retained at the source of soluble protein (1–2 mg protein/ml). All of the DNA fragments used for GMSA were generated by annealing two complementary primers. The probe used for DNA binding reactions consisted of two annealed complementary primers, gmsa-S and gmsa-R (Table I), labeled by end filling with the Klenow fragment of DNA polymerase using [α-32P]dCTP. DNA-protein binding reactions were performed by incubating 15 μg of total protein and 10 fmol of the labeled probe at 22 °C for 20 min in 30 μl of 25 mmHepes, pH 7.8, 12% (v/v) glycerol, 5 mm EDTA, 1 mm phenylmethylsulfonyl fluoride, 50 mm KCl, 1 mm dithiothreitol, 10 μm ZnCl2, acetylated bovine serum albumin (0.25 mg/ml), 2 μg of poly(dI-dC). In competition experiments, unlabeled DNA was added to the reaction medium in a 50–1000-fold excess over the labeled probe. The DNA-protein complexes were separated on a 10% nondenaturing polyacrylamide gel in 1× Tris-borate-EDTA buffer. Previous experiments using transgenic strains containing a CLPG2-GFP construct showed that a promoter fragment of 668 bp was sufficient to induce expression of the reporter gene in the mycelium grown on pectin and during appressorium formation both in vitro on glass slides and in planta at early stages of pathogenesis (14Dumas B. Centis S. Sarrazin N. Esquerré-Tugayé M.T. Appl. Environ. Microbiol. 1999; 65: 1769-1771Crossref PubMed Google Scholar). The general organization of the CLPG2 promoter (PG2-490) is depicted on Fig.1 A. Two transcription start sites were identified 178 and 166 bp upstream from the initiation codon by 5′ rapid amplification of cDNA ends-PCR, corresponding to positions +1 and +12, respectively. A putative TATA (TATAA) box was located at position −40. To identify cis-acting elements involved in transcriptional control in vitro and in planta,CLPG2-GFP constructs harboring sequential 5′ deletions of the promoter region of CLPG2 were introduced in the genome of C. lindemuthianum (Fig. 1 A). For each construct, at least four independent transformants were isolated. The presence of GFP was verified by PCR, and the number of GFP copies integrated into the fungal genome was evaluated by Southern blotting. Accumulation of GFP in conidia was detected by fluorescence microscopy. Fig. 1 B shows that induction of GFP expression by pectin and appressorium formation was of the same magnitude in transformants containing the full-length promoter (PG2-490) or the promoter deleted to −90 (PG2-90). However, deletion of a further 27 bp (construct PG2-63) abolished induction of the reporter gene during the three growth conditions. Removal of this 27-bp sequence only from the full-length promoter (constructPG2-490Δ27) did not modify the promoter activity, showing that additional regulatory elements were present upstream on the promoter (Fig. 1 B). To confirm that the construct PG2-90 induced accumulation of GFP transcript on pectin medium, a Northern blot experiment was performed on RNA extracted from the mycelium grown on glucose and transferred on glucose or pectin. Hybridization with probes corresponding to CLPG2 DNA or to the GFP coding sequence showed the simultaneous accumulation of CLPG2 and GFP RNAs on pectin medium (data not shown). Taken together these results indicated that the 27-bp DNA fragment contained regulatory elements allowing GFP transcription in the fungus grown on pectin medium and during appressorium formation in vitro and in planta at the very first stages of pathogenesis. The 27-bp fragment corresponding to nucleotides −90 to −63 was chosen as a probe for GMSA (Fig.2). Protein extracts were prepared from the mycelium, which was grown on glucose and subsequently transferred either on glucose or pectin or on bean leaves. In the latter case, the fungus was allowed to grow on a cellophane sheet laid down onto the surface of cotyledonary leaves of the susceptible bean cultivar P12S. Microscopy analysis showed that in these conditions, 24–48 h after inoculation, appressoria were fully differentiated and started to develop primary hyphae through the cellophane sheet, thereby mimicking the first stages of pathogenesis. Removal of the cellophane sheet allowed recovery of the mycelium. In preliminary experiments, we compared the formation of protein-DNA complexes obtained with protein prepared from isolated nuclei or from mycelium. In each case, the same major protein-DNA complex was observed on the gel as shown in Fig. 2 (A andB) with proteins extracted from the mycelium. Accordingly, total protein extracts were used for further experiments. The signal was more intense when the probe was incubated with protein extracts prepared from the mycelium grown on pectin or during pathogenesis, but a signal was also observable with proteins extracted from the mycelium grown on glucose. Competition experiments carried out with an excess of the unlabeled DNA probe efficiently eliminated appearance of the signal, whereas nonspecific competitor DNA did not affect the binding (Fig. 2, A and B). The addition of deoxycholic acid, a chaotropic agent that dissociates protein complexes, led to disappearance of the signal (Fig. 2 C), suggesting that protein-protein interactions are required for the formation of the protein-DNA complex. Analysis of the 27-bp sequence revealed the presence of two different putative regulatory elements (Fig. 3 A). The first element showed a strong homology with the eukaryotic TCS motif containing the TEA/ATTS consensus sequence CATTCY, which binds transcriptional factors belonging to the TEA/ATTS family. Therefore, this element was designated TLE because it contains the sequence GATTCY. A second class of element containing the consensus sequence WN(1, 2)AAN(1, 2)A was called the PRE-like element (PLE) according to its homology with the yeast DNA regulatory sequence WGAAACA called the pheromone response element (PRE), which interacts with the STE12 trans-acting factor. Four TLEs and PLEs were detected along the CLPG2 promoter (Fig.3 A). In yeast, a combination of TCS and PRE has been shown to play a major role in the regulation of genes during the filamentation and invasive response through the binding of TEC1 and STE12 transcription factors (21Madhani H.D. Fink G.R. Trends Cell Biol. 1998; 8: 348-353Abstract Full Text Full Text PDF PubMed Scopus (235) Google Scholar). Remarkably, TLEs and PLEs are also arranged in tandem in the promoter of CLPG2. To determine whether the binding of protein factors on the 27-bp fragment was due to the presence of TLEs and PLEs, GMSA was performed with the 27-bp fragment as the probe and double-stranded oligonucleotides corresponding to three repetitions of either TLE-90 (3×TLE-90), PLE-79 (3×PLE-79), or PLE-71 (3×PLE-71) as cold competitors (Fig. 3 B). A 50 molar excess of 3×PLE-79 and 3×PLE-71 was sufficient to compete very efficiently with the formation of the complex (Fig. 3 B), whereas a 1000-fold excess of 3×TLE-90 was necessary to disrupt the binding (data not shown). Thus, the proteins binding to the 27-bp DNA fragment appeared to have more affinity for a combination of TLE and PLE than for three TLEs. To further identify mutations in PLEs that alter the binding of nuclear proteins, a competition experiment was performed in the presence of different double-stranded mutated PLE-79 oligonucleotides containing one or two base substitutions compared with the wild type sequence (Fig. 3 C). Oligonucleotides M1, M2, and M4, which contained PLE mutated in the first nucleotide, the central adenine doublet, and the terminal adenine residue, respectively, failed to inhibit the formation of the complex. Thus, the nucleotides residues that are totally conserved between the yeast STE12 binding site and the C. lindemuthianum PLEs are essential for the binding of protein factors. In yeast, TEC1 and STE12 bind as heterodimers on FRE. To determine whether a combination of PLE and TLE can also bind protein factors, GMSA was performed with double-strand DNA fragments corresponding to the 27-bp fragment mutagenized in each of the elements (Fig.4 A). It was found that mutagenesis of TLE-90 alone did not significantly modify the binding of proteins, whereas mutation of PLE-79 and to a lesser extent of PLE-71 strongly increased the formation of the protein-DNA complex. Mutation of TLE-90 and PLE-71 eliminated the binding of protein factors (Fig.4 A). The same oligonucleotides were used as competitors with the wild type sequence as probe (Fig. 4 B). Mutations in TLE-90 or PLE-71 strongly reduced the competition, showing that these mutations decreased the affinity for nuclear factors. Altogether, these experiments showed that a combination of TLE-90 and PLE-71 binds nuclear factors with higher efficiency than two PLEs. In a further experiment, it was determined that transformants harboring GFP under the control of a CLPG2 promoter mutated in the TLE-90 and PLE-71 expressed only a basal level of fluorescence (Fig.4 C). Thus, the ability of DNA fragments to form a complex with proteins in vitro is correlated with the promoter activity in vivo. In yeast, KSS1 is a negative regulator of invasive growth and binds in its unphosphorylated form to STE12, thereby repressing the transcription of target genes containing FRE (22Bardwell L. Cook J.G. Voora D. Baggott D.M. Martinez A.R. Thorner J. Genes Dev. 1998; 12: 2887-2898Crossref PubMed Scopus (142) Google Scholar). Because mutation of one PLE increased the formation of the protein-DNA complex observed in inducing conditions, we tested the hypothesis that PLEs could be involved in the repression of CLPG2 in the fungus grown on glucose. A synthetic promoter was constructed by fusing three copies of PLE-71 to a constitutive promoter from the A. nidulans GPDA gene (23Punt P.J. Kuyvenhoven A. van den Hondel C.A. Gene (Amst.). 1995; 158: 119-123Crossref PubMed Scopus (13) Google Scholar). This DNA fragment was fused to GFP, and C. lindemuthianumstrains harboring this construct were analyzed by quantitative fluorescence microscopy. The level of fluorescence exhibited by the strains expressing the GFP gene under the control of theGPDA promoter was of the same magnitude when grown on glucose or pectin. However, the addition of 3×PLE-71 strongly reduced the expression on glucose, whereas accumulation of GFP was unchanged on pectin medium (Fig. 5). Thus, a combination of PLEs is sufficient to confer glucose repression on a constitutive promoter. Despite the long lasting interest of plant pathology and biotechnology in fungal pectinases, the molecular mechanisms governing their expression are still unknown. In the present study, we report on the identification of regulatory elements involved in the transcriptional control of CLPG2, a pectinase gene ofC. lindemuthianum. These elements bind protein factors and are essential for expression of a reporter gene during saprophytic growth of the fungus on pectin and during interaction with the host plant. A search for homology with previously described nuclear factor-binding sites revealed the presence of putative TCS. TCS have been reported to bind transcription factors of the TEA/ATTS family mainly involved in developmental processes in fungi and higher eukaryotes (24Madhani H.D. Fink G.R. Science. 1997; 275: 1314-1317Crossref PubMed Scopus (319) Google Scholar). A second sequence was identified by homology with the yeast PRE found in promoters of genes involved in the mating response (25Hagen D.C. McCaffrey G. Sprague Jr., G.F. Mol. Cell. Biol. 1991; 11: 2952-2961Crossref PubMed Scopus (106) Google Scholar). A combination of the CLPG2 elements, called TLE and PLE, was sufficient to bind protein factors and ensure promoter activity. These effects were totally lost upon mutagenesis of these elements. Moreover, a construct comprising three PLEs fused to a constitutive promoter was able to repress the constitutive expression of a reporter gene in the fungus grown on glucose medium. Taken together, these results show that the regulation of CLPG2 requires the binding of transcription factors to a DNA sequence comprising TLE and PLE. Up to now, such combinations of elements were not reported in true filamentous fungi. In yeast, a combination of TCS and PRE, also called the FRE, mediates the binding of an heterodimer formed by the association of the transcriptional activators TEC1 and STE12 (21Madhani H.D. Fink G.R. Trends Cell Biol. 1998; 8: 348-353Abstract Full Text Full Text PDF PubMed Scopus (235) Google Scholar, 26Gancedo J.M. FEMS Microbiol. Rev. 2001; 25: 107-123Crossref PubMed Google Scholar). The MAPK KSS1 plays a key role in the transcriptional control of genes regulated by FRE both by derepression and activation. Indeed, the unphosphorylated form of KSS1 is part of a protein complex that also contains STE12, TEC1, and the inhibitory proteins DIG1 or DIG2. Upon phosphorylation through a MAPK cascade, KSS1 dissociates from the complex, thereby destabilizing the STE12-DIG association leading to derepression of the target genes. Simultaneously, phosphorylation of STE12 by KSS1 activates the STE12-driven transcription. The transcriptional repression activity mediated by C. lindemuthianum PLEs strongly suggests that similar mechanisms operate in this fungus. The hypothesis that CLPG2 is regulated by transcription factors related to the yeast STE12 and TEC1 proteins is strengthened by the recent finding that expression of a polygalacturonase gene,PGU1, is induced in yeast during the haploid-invasive growth and diploid pseudohyphal development (27Madhani H.D. Galitski T. Lander E.S. Fink G.R. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 12530-12535Crossref PubMed Scopus (158) Google Scholar). This induction requires a functional filamentation MAPK pathway, including the presence of TEC1 and STE12. Thus, during the yeast invasive response, the same molecular mechanisms activate genes involved in cell elongation and differentiation and genes that encode extracellular proteins, allowing the successful colonization of their natural substrates. Related results have been obtained for two animal pathogens, Candida albicans and Cryptoccocus neoformans. In C. albicans, the TEA/ATTS transcription factor CaTEC1 controls hyphal development and expression of genes encoding extracellular proteinases (28Schweizer A. Rupp S. Taylor B.N. Rollinghoff M. Schroppel K. Mol. Microbiol. 2000; 38: 435-445Crossref PubMed Scopus (187) Google Scholar). Similarly, a STE12 homologue from C. neoformansregulates expression of virulence genes, notably encoding an extracellular phospholipase (29Chang Y.C. Penoyer L.A. Kwon-Chung K.J. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 3258-3263Crossref PubMed Scopus (66) Google Scholar). Recently, a STE12-like gene that plays an essential role in sexual reproduction,STEA, was isolated from a true filamentous fungi, A. nidulans (30Vallim M.A. Miller K.Y. Miller B.L. Mol. Microbiol. 2000; 36: 290-301Crossref PubMed Scopus (130) Google Scholar). However, the role of this factor in the expression of pectinase genes was not investigated. The likely involvement of transcriptional activators homologous to STE12 in the regulation of the C. lindemuthianum pectinase gene CLPG2 could be related to the presence of MAPKs belonging to the FUS3/KSS1 family. Interestingly, MAPKs homologous to the yeast FUS3/KSS1 have been identified in a number of phytopathogenic fungi (31Tucker S. Talbot N. Annu. Rev. Phytopathol. 2001; 39: 385-417Crossref PubMed Scopus (349) Google Scholar) where they play essential roles in pathogenicity. Thus, disruption of the MAPK FMK1 in the tomato root pathogenFusarium oxysporum greatly reduced the expression of the endopectate lyase gene pl1 (32Di Pietro A. Garcia-MacEira F.I. Meglecz E. Roncero M.I. Mol. Microbiol. 2001; 39: 1140-1152Crossref PubMed Google Scholar). According to the results presented in this paper, homologues of the yeast STE12 and TEC1 factors are likely to play a key role in pectinase gene expression, suggesting that mechanisms regulating invasive growth share striking similarities between saprophytic and pathogenic microorganisms. Such similarities were already pointed out for dimorphic fungi able to switch between a yeast and a multicellular invasive filamentous form (21Madhani H.D. Fink G.R. Trends Cell Biol. 1998; 8: 348-353Abstract Full Text Full Text PDF PubMed Scopus (235) Google Scholar). From our data and the above report, it emerges that the regulation ofCLPG2 might comply with the model proposed in Fig.6. Thus, in repressive medium (glucose medium), the MAPK pathway would not be activated, and binding of protein factors related to the yeast STE12 would repress expression of the gene. In inductive medium (pectin medium) or at early stages of pathogenesis (appressorium development), activation of the MAPK pathway would induce a rearrangement of the protein-DNA complex comprising a STE12-like factor and a protein belonging to TEA/ATTS family of transcription factors. Isolation and functional analysis of these transcription factors are currently underway. 2C. Herbert and B. Dumas, unpublished observations. Their characterization will help unravel the signaling pathways leading to induction of fungal pathogenicity. We thank Philippe Rech for advice in GMSA experiments and Alain Jauneau for help with fluorescence microscopy.