1 UMR 5546 CNRS-Université Paul Sabatier
2 IFR 40 Pôle de Biotechnologie Végétale, 24 Chemin de Borde Rouge, BP17 Auzeville, 31326 Castanet-Tolosan, France
* Author for correspondence (e-mail: dumas{at}scsv.ups-tlse.fr)
Accepted 27 October 2004
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Summary |
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Key words: Fungal pathogenicity, Colletotrichum lindemuthianum, Secretion
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Introduction |
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We have studied the regulation of the production of extracellular pathogenicity factors in the plant pathogen Colletotrichum lindemuthianum. The genus Colletotrichum comprises several hundred species, mostly plant pathogens. Although only rarely pathogenic to humans, Colletotrichum spp. have been reported as almost exclusively causing keratis (Fernandez et al., 2002) but cutaneous infection has also been described (Guarro et al., 1998
; O'Quinn et al., 2001
). C. lindemuthianum is a hemibiotrophic fungus that causes anthracnose disease on bean (Perfect et al., 1999
). During pathogenic development, the fungus produces a series of infection structures including germ tubes, appressoria, intracellular hyphae and secondary necrotrophic hyphae. Each stage of infection is characterized by the production of specific cell-wall-localised glycoproteins and extracellular enzymes (Centis et al., 1997
; Herbert et al., 2004
; Hutchinson et al., 2002
) illustrating the importance of the secretory pathway in this system. Recently, we identified a Rab/GTPase gene, CLPT1, from C. lindemuthianum. CLPT1 is able to complement the yeast thermosensitive mutation sec4-8 (Dumas et al., 2001
) suggesting that it is involved in the last step of exocytosis, in the transport of post-Golgi vesicles to the plasma membrane. Interestingly, expression of CLPT1 was up-regulated on pectin medium, which also induces the production of extracellular pectinases. This suggests that activation of the secretory pathway can sustain the production of extracellular enzymes (Dumas et al., 2001
). In the present study, we characterized C. lindemuthianum transgenic strains expressing the wild-type sequence of CLPT1 or a trans-dominant inhibitor of CLPT1, which was placed under the control of a pectinase gene promoter, induced during growth on pectin medium and pathogenesis, and repressed on glucose medium. This strategy allowed us to study precisely the function of CLPT1 in protein secretion and pathogenicity.
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Materials and Methods |
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Plasmid constructions and mutagenesis
The CLPT1 coding sequence (Dumas et al., 2001) was amplified by PCR and the two restriction sites NcoI and PstI were added at the 5' and 3' ends, respectively. The PCR fragment was introduced between the NcoI and PstI site of the PG2-90 plasmid (Herbert et al., 2002
) to replace the GFP nucleotide sequence. A mutated version of CLPT1, in which the ATC/N123 codon was replaced by AAC/I123, was produced by site-directed mutagenesis using the QuickChange site directed mutagenesis kit (Stratagene, La Jolla, CA) and following the supplier's instructions.
Northern blotting
Total RNA was isolated from the mycelium of C. lindemuthianum using the Extract-all kit (Eurobio, Les Ullis, France) following the supplier's instructions. Samples containing 10 µg of total RNA were denatured with glyoxal and submitted to electrophoresis in a 1.2% (w/v) agarose gel in 10 mM phosphate buffer (pH 7). The gels were transferred to Hybond N+ membranes (Amersham Pharmacia Biotech, France) and fixed by baking for 2 hours at 80°C. The membranes were then prehybridized for 2 hours at 42°C in 50% formamide, 0.1% SDS, 1x Denhardt's solution, 2x SSC and 50 ng/ml denatured salmon sperm DNA. Hybridization was carried out overnight under the same conditions, after addition of the 32P-labeled DNA probe.
Production of antibodies directed against CLPT1 expressed in Escherichia coli
The CLPT1 cDNA (Dumas et al., 2001) was cloned into the pMAL-cR1 vector (New England Biolabs, Beverly, MA) in the same translational reading frame as the malE gene, which encodes the maltose-binding protein (MBP). The resulting plasmid was introduced in BL21 cells by electroporation. The bacterial culture (1 l) was induced with IPTG (10 mM) and the MBP-CLPT1 fusion protein was purified by affinity chromatography on an amylose column (New England Biolabs) according to the manufacturer's instructions. A rabbit was given four intramuscular injections of the purified fusion protein (100 µg each). Ten days after the last boost, the serum was collected, clarified by centrifugation and stored at -20°C.
Western blotting
The mycelium was ground in liquid nitrogen. The powder (200 mg fresh weight; FW) was suspended in 0.5 ml of 0.05 M acetate buffer, pH 5.2. After centrifugation at 4°C for 15 minutes at 10,000 g, the soluble extract was recovered and the protein content was determined by the method of Bradford (1976). Culture filtrates (25 µl) and mycelium soluble extracts (20 µg protein) were subjected to SDS-PAGE in a 12% polyacrylamide gel (Laemmli, 1970). After migration, the proteins were transferred to a nitrocellulose membrane using a Biorad semi-dry apparatus at a constant current (1.8 mA/cm2). The membrane was soaked for 30 minutes in a Tris-buffered saline solution (Tris-HCl pH 7.5, NaCl 150 mM) containing 3% non-fat milk before being incubated overnight in the same buffer containing the MBP-CLPT1 antiserum which was used at a 1000x dilution (Hugouvieux et al., 1995). The antigen-antibody complex was visualized by colorimetric detection using alkaline phosphatase-conjugated goat anti-rabbit immunoglobulin G (Sigma, France).
Polygalacturonase activity
The extracellular medium was dialyzed against acetate buffer 50 mM, pH 5.2 overnight at 4°C. Three flasks were sampled for each assay. The enzymatic assay consisted of a sample (50 µl) of the dialysed medium and 1 ml of polygalacturonic acid 0.1% (w/v) in acetate buffer (50 mM pH 5.2). After incubation for 30 minutes at 30°C, total reducing sugars were assayed by the colourimetric method of Somogyi (Somogyi, 1952). Controls in which either the dialyzed growth medium, or the substrate were omitted were simultaneously performed. The enzymic activity was expressed in nanokatals (nkat), 1 nkat corresponding to the release of 1 nmol of reducing group equivalent per second.
-D-galacturonic acid (Sigma) was used as a standard.
Fluorescence confocal microscopy and TEM
For confocal laser scanning microscopy, samples were stained with Congo Red (0.5% in water) for 5-10 minutes and washed in distilled water. They were mounted on microscope slides and observed using a Leica SP-2 confocal spectral microscope (Germany) with a 40x (1.25 NA) oil immersion objective. The 543 nm ray line of a helium laser was used for excitation and emitted light was collected between 560-630 nm. Pictures were computed by projection of 10-15 plan-confocal images acquired in z-dimension. There were finally treated by image analysis using Image Pro-Plus (Media Cybernetics, Silver Spring, MD) for best look-up-table adjustment.
For transmission electron microscopy, the samples were fixed for 2 hours at room temperature in 50 mM sodium cacodylate buffer (pH 7.0) containing 2% (w/v) glutaraldehyde (Oxford Agar, Oxford, UK) and then washed in the same buffer without glutaraldehyde. They were post-fixed with osmium tetroxide (1%, w/v) in the same cacodylate buffer for 1 hour at room temperature. The samples were washed in water and dehydrated in a series of aqueous solutions of increasing ethanol concentration (20, 40, 60, 70, 80, 90, 100% v/v; 30 minutes each). Progressive infiltration with Spurr's epoxy resin (Oxford Agar, Oxford, UK) was carried out by serial incubation in ethanolic solutions of increasing concentration of Spurr's resin (30, 50, 80%, v/v; 12 hours each) and two incubations in undiluted resin. Infiltrated samples were then embedded in capsules and allowed to polymerize for 24 hours at 70°C. Ultrathin sections (80-90 nm thickness) were prepared using an UltraCut E ultramicrotome (Reichert-Leica, Germany) and collected on gold grids. They were either submitted to the periodic acid-thiocarbohydrazide-silver proteinate reaction (PATAg) according to the method of Thiéry (Thiéry, 1967) or uranyl acetate, lead citrate staining. For PATAg staining, sections were floated on a 1% (w/v) aqueous solution of periodic acid for 30 minutes at room temperature and rinsed twice in distilled water for 15 minutes. They were treated overnight at 4°C with a 20% aqueous solution of acetic acid containing 0.2% thiocarbohydrazide, washed in solutions of decreasing acetic acid concentration and finally in water. They were floated on a 1% (w/v) aqueous solution of silver proteinate for 30 minutes in the dark, washed in water and air dried before examination with a transmission electron microscope (Hitachi, H-600, Japan) operating at 75 kV. Photographs were taken using Kodak-Electron films (Kodak, France). For uranyl acetate and lead citrate staining, sections were floated for 5 minutes in the dark on an aqueous solution of 5% (w/v) uranyl acetate containing 50% (v/v) ethanol, washed in water and then treated with an aqueous solution of 0.4% (w/v) lead citrate for 2 minutes in the dark.
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Results |
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The strategy that was used to construct the dominant-negative mutation, was based on the data reported for the yeast SEC4 gene by Walworth et al. (Walworth et al., 1989). Thus, the Asn123 AAC codon present in the coding sequence of CLPT1 was mutagenized to an ATC Ile codon, by site-directed mutagenesis. The resulting sequence was fused to the PG2-90 promoter fragment as well as a control construct in which the wild-type CLPT1 sequence was also fused to the PG2-90 promoter fragment. The two constructs, named PG2::CLPT1(N123I) and PG2::CLPT1, were introduced into the C. lindemuthianum genome by protoplast transformation along with the selection plasmid pAN7, harbouring hygromycin resistance (Punt et al., 1987
). Resistant strains were regenerated and analysed by PCR for the presence of the PG2-90::CLPT1 or PG2-90::CLPT1(N123I) constructs (data not shown). For each construct, two transformants (CLPT1.4, CLPT1.9 and N123I.6, N123I.9) were selected for further studies.
Expression of CLPT1 and CLPT1(N123I) in the selected transgenic strains was followed by northern blot analysis using RNA extracted from the mycelium grown on pectin. A signal corresponding to the wild-type CLPT1 mRNA was visible in all tested samples. A second faster migrating band corresponding to the additional copy of CLPT1 or CLPT1(N123I), was detected only in the transgenic strains (Fig. 2A). Different levels of expression were obtained between the different strains, probably reflecting a position effect of the transgene insertion site.
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In order to obtain specific antibodies directed against CLPT1, the protein was produced in E. coli as a fusion protein with the maltose binding protein (MBP). The fusion protein was purified on an amylose column and subsequently used to raise antibodies in rabbits. The antibodies were purified with the CLPT1-MBP fusion protein and used for western blots experiments. Protein extracts were prepared from the mycelium grown on glucose or pectin medium. A protein with a molecular mass of approximately 23 kDa was detected by the CLPT1 antibodies, and was shown to accumulate preferentially on pectin (Fig. 2B). However, we did not detect a stronger accumulation in the strains expressing additional copies of CLPT1 compare to the wild type.
Effect of CLPT1(N123I) on protein secretion
The ability of the CLPT1 and CLPT1(N123I) transgenic strains to secrete extracellular polygalacturonases was examined. To use the same mycelium biomass for each strain, the mycelium was first grown on glucose for 3 days before transfer to pectin. Extracellular polygalacturonase (PG) activity was determined using polygalacturonic acid as substrate. In the case of the wild-type strain, the production of PG was transient, reaching a peak about 2 days after the transfer to pectin medium (Fig. 3). Expression of CLPT1 under the control of the PG2-90 promoter fragment had no significant effect on the production of extracellular PGs. However, induction of CLPT1(N123I) led to a dramatic reduction of PG activity. Interestingly, after 3 days in culture, by which time there is a decrease in the activity of the CLPG2 promoter (Fig. 1), the level of PG increased. This showed that expression of CLPT1(N123I) is tightly correlated with an inhibition of PG production.
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Effect of CLPT1(N123I) on ultrastructural morphology
The cell wall of living hyphae from various strains were stained with Congo Red, a dye with high affinity for ß-1,4 polysaccharides, and observed with a confocal laser scanning microscope. The hyphal walls of WT and CLPT1 strains grown on glucose medium were labelled homogeneously along the germ tube. In contrast, strongly stained patches were detected along the hyphae of the CLPT1(N123I) strain grown on pectin, indicating abnormal accumulation and location of Congo Red stained material (Fig. 4).
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Ultra-thin sections of hyphae were obtained from wild-type cultures and from the two transgenic CLPT1 or CLPT1(N123I) strains. The three samples were indistinguishable if they had been grown on glucose (Fig. 5A-C). Each had some vesicle-like material (arrowheads) appressed along the cell wall. However, in CLPT1(N123I) strains grown on pectin a dramatic accumulation of vesicle-like material (arrowheads) within the hyphae was observed (Fig. 5F). Further investigations were performed on ultra-thin sections submitted to the PATAg reaction (Fig. 5G-J). This staining mainly revealed polysaccharidic components with high amounts of vicinal hydroxyl groups (Roland and Vian, 1991). Large accumulation of PATAg-stained material was visible only within the CLPT1(N123I) hyphae grown on pectin (Fig. 5J). The PATAg-stained material appeared as vesicle-like structures (arrowheads) and also as large and highly reactive spots (white arrows).
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Effect of CLPT1(N123I) on appressoria development an pathogenesis
Production of appressoria was monitored for the wild-type and mutant strains by placing the conidia on a glass slide in water. It was shown previously that the PG2-90 promoter fragment allowed the expression of the GFP marker gene during appressorium formation (Herbert et al., 2002). The wild-type strain and the two strains expressing the CLPT1(N123I) gene developed melanized appresoria. However, it was noted occasionally that the two CLPT1(N123I) strains produced abnormal appressorial cells with multiple germ tubes, which were never observed for the wild-type strain (Fig. 6). This suggested that expression of the negative-dominant mutation can impair appressorial differentiation.
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To test the pathogenicity of the strains expressing CLPT1(N123I), conidia suspensions were used to inoculate bean leaves and symptoms were recorded 7 days post-inoculation. The strain N123I.6 produced some necrotic spots with a very limited maceration of plant tissues, whereas the strain N123I.9 did not produce any visible symptoms (Fig. 6). This might reflect different levels of expression of the dominant-negative CLPT1 allele in the two transgenic strains. Microscopic examination of the infected tissues revealed that N123I mutants failed to penetrate the host cells. Whereas the wild-type strain formed an appressorium and differentiated an infection vesicle inside a host cell, N123I strains grew saprophytically on the plant surface (Fig. 6).
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Discussion |
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The regulated expression of a dominant-negative allele is particularly useful when one wants to study the function of genes that might play an essential role in development. In this case, gene disruption can lead to a lethal phenotype or pleiotropic phenotypes that could hide the primary function of the gene. Alternative approaches to study the functions of essential genes that are commonly used in S. cerevisiae and other model fungi such as C. albicans include the generation of conditional lethal mutants, expression of an antisense gene or overexpression of a dominant-negative allele under the control of a tightly regulated promoter. Proteins belonging to the small Ras-like GTPases family are well suited for this latter strategy since they contain conserved domains required for guanine nucleotide binding, GTP-GDP exchange and GTP hydrolysis, whose site-directed mutagenesis might lead to dominant-negative mutations. In CLPT1, the N123I substitution corresponds to the N133I substitution in Sec4p (Walworth et al., 1989), N121I substitution in YPT1 (Schmitt et al., 1986
) and to the N124I substitution in the human Rab1 (Pind et al., 1994
). This amino acid is located in the G4 region found in Ras (Lazar et al., 1997
) and is involved in guanine nucleotide binding (Pind et al., 1994
). In the case of Ras, mutations in the consensus guanine nucleotide binding sequence reduced guanine nucleotide binding affinity, leading to an enhancement of GDP/GTP exchange and to a dominant-negative phenotype probably through the sequestering of regulatory factors. A similar substitution in Sec4p resulted in a mutant protein that does not bind GTP and produces dominant lethal phenotypes and secretory defects when expressed in yeast (Walworth et al., 1989
). Therefore, we reasoned that it might be possible to define the functions of CLPT1 by expressing, in a conditional manner, a N123I allele of CLPT1 in a wild-type strain of C. lindemuthianum.
To express the N123I allele, the CLPG2 promoter was fused to the mutated CLPT1 gene. This promoter is not active on glucose medium and is highly, but transiently, induced on pectin medium and during pathogenesis (Dumas et al., 1999; Herbert et al., 2002
). This strategy allowed us to study the effect of the dominant-negative mutation on pectinase secretion and pathogenicity. Induction of the N123I allele on pectin medium led to a nearly complete inhibition of PG secretion. This showed that CLPT1 is essential for the intracellular vesicular transport of pectinases. Similarly, overexpression of a SEC4 allele harbouring a dominant-negative mutation inhibited aspartyl protease secretion in the human pathogen C. albicans (Mao et al., 1999
). Ultrastructural examination of the strains expressing the CLPT1(N123I) allele revealed the abnormal accumulation of vesicles that were heavily stained by the PATAg reagent and by Congo Red. Thus, it is clear that the presence of the N123I mutation blocks the transport of vesicles in a dominant fashion by preventing interaction of the wild-type CLPT1 with a transport component required for targeting and fusion of the vesicles with the plasma membrane. No morphological differences were noticed between the different strains during saprophytic growth, indicating that CLPT1 is not essential for hyphal elongation. This is in contrast to the results obtained on the role of SRGA, a putative SEC4 homologue from the filamentous fungus Aspergillus niger. SRGA mutants displayed a twofold increase in their hyphal diameter and unusual apical branching (Punt et al., 2001
). While comparison of SRGA and CLPT1 amino acid sequences revealed a high percentage of identity [82.6% (Dumas et al., 2001
)] these two proteins could have different functional roles since CLPT1 is able to complement a yeast sec4 mutant (Dumas et al., 2001
) whereas SRGA cannot (Punt et al., 2001
).
Pathogenicity was strongly reduced in the strains expressing CLPT1(N123I). This suggests that the inhibition of protein secretion is an essential process at this stage of infection. It has been showed in a number of plant and animal systems that extracellular proteins play an essential role in pathogenic development. These include several plant cell wall degrading enzymes from phytopathogens (Rogers et al., 2000; Oeser et al., 2002
; Isshiki et al., 2001
), aspartyl proteases from the animal pathogen C. albicans and an extracellular phospholipase from Cryptococcus neoformans (Cox et al., 2001
). While our data suggest that the reduced virulence of CLPT1 dominant negative mutants could arise from a strong decrease of cell wall degrading enzyme secretion, other extracellular or cell surface proteins are known to also play a major role in pathogenesis. For example it has been reported that the synthesis of hydrophobin is necessary for efficient appressorial differentiation in the rice pathogen Magnaporthe grisea (Talbot et al., 1996
). In C. lindemuthianum, pathogenic development is accompanied by a highly coordinated secretion of cell-surface proteins, which could have different roles in cell adhesion or biotrophic development (Hutchinson et al., 2002
). Alteration of the secretory pathway through the expression of the dominant negative CLPT1 allele could have a detrimental effect on all these processes leading to a non-pathogenic phenotype. Future work will aim at obtaining a more global view of the modification of the cell-surface proteome of the CLPT1(N123I)-expressing strains to identify essential secreted components of pathogenesis.
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Acknowledgments |
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