Chitin, a β-1,4-linked polysaccharide of N-acetylglucosamine, is a major structural component of fungal cell walls. Fungi have multiple classes of chitin synthases that catalyse N-acetylglucosamine polymerization. Here, we demonstrate the requirement for a class V chitin synthase during host infection by the vascular wilt pathogen Fusarium oxysporum. The chsV gene was identified in an insertional mutagenesis screen for pathogenicity mutants. ChsV has a putative myosin motor and a chitin synthase domain characteristic of class V chitin synthases. The chsV insertional mutant and a gene replacement mutant of F. oxysporum display morphological abnormalities such as hyphal swellings that are indicative of alterations in cell wall structure and can be partially restored by osmotic stabilizer. The mutants are unable to infect and colonize tomato plants or to grow invasively on tomato fruit tissue. They are also hypersensitive to plant antimicrobial defence compounds such as the tomato phytoanticipin α-tomatine or H2O2. Reintroduction of a functional chsV copy into the mutant restored the growth phenotype of the wild-type strain. These data suggest that F. oxysporum requires a specific class V chitin synthase for pathogenesis, most probably to protect itself against plant defence mechanisms.
Fungal cells are able to withstand adverse conditions in the natural environment by means of the cell wall, an external layer composed mostly of carbohydrates. One of the major structural components of the fungal wall is chitin, a β-1,4-linked polysaccharide made of N-acetylglucosamine (Bartnicki-García, 1968). Chitin metabolism includes synthesis, degradation and cross-linking of the polymer to other cell wall components and is an essential process for maintaining growth and shape of fungal hyphae. Polymerization of N-acetylglucosamine from the substrate UDP-N-acetylglucosamine is catalysed by chitin synthases (EC 220.127.116.11). Fungal chitin synthases have been divided into five classes on the basis of their structure in conserved regions (Bowen et al., 1992; Din et al., 1996; Specht et al., 1996; Munro and Gow, 2001), and the presence of multiple chitin synthase genes is common in a single fungal species (Munro and Gow, 2001; Roncero, 2002). Aspergillus nidulans, for example, contains members of all five chitin synthase classes (Horiuchi et al., 1999). Owing to this multiplicity, the deletion of a single chitin synthase gene generally has no or only minor effects on hyphal growth. In a few cases, however, the absence of a specific chitin synthase can result in severe morphological alterations or even lethality (Yarden and Yanofsky, 1991; Horiuchi et al., 1999; Munro et al., 2001). This strongly suggests that at least some of the multiple chitin synthases play unique and essential roles in the physiology of the fungal organism (Munro and Gow, 2001; Ruiz-Herrera et al., 2001; Roncero, 2002).
The genus Fusarium contains a number of soil-borne species with worldwide distribution, which have been known for a long time as important plant pathogens (Moss and Smith, 1984). More recently, Fusarium has also been reported as an emerging human pathogen in immunocompromised patients (Vartivarian et al., 1993). The most common species, F. oxysporum, causes vascular wilt disease in a wide variety of economically important crops (Beckman, 1987). We are studying the interaction between F. oxysporum f. sp. lycopersici and the tomato plant as a model to understand the molecular mechanisms controlling fungal pathogenesis. In the present work, we have performed an insertional mutagenesis screen for genes involved in host infection. Changes were introduced into the F. oxysporum genome via DNA-mediated transformation, and transformants were screened for defects in virulence. This strategy has been used previously to identify novel genes involved in fungal pathogenesis (Lu et al., 1994; Bölker et al., 1995; Urban et al., 1999; Dufresne et al., 2000). We report here the characterization of an insertional mutant of F. oxysporum with a dramatic reduction in virulence. The affected gene, chsV, encodes a protein homologous to class V chitin synthases with a characteristic myosin motor domain. F. oxysporum mutants in chsV show morphological abnormalities such as hyphal swellings that are indicative of alterations in cell wall stability. We demonstrate that strains lacking chsV are unable to survive within living host tissue and are hypersensitive to tomato defence compounds such as the phytoanticipin α-tomatine or H2O2. Our findings provide the first evidence that class V chitin synthase plays an essential role in fungal pathogenicity to plants, most probably as part of a protection mechanism against antimicrobial compounds produced by the host.
Isolation and characterization of F. oxysporum chsV
In an insertional mutagenesis screen, 155 hygromycin-resistant transformants of the highly virulent F. oxysporum isolate 4287 obtained with the vector pPgx4::hyg (García-Maceira et al., 2000) were tested for their ability to produce vascular wilt symptoms on tomato plants using a standardized root infection assay in minipots. One of the transformants, Tr5, was unable to cause disease symptoms on tomato plants. A 320 bp fragment containing 110 bp of the genomic sequence flanking the transformation vector was isolated by thermal asymmetric interlaced (TAIL) polymerase chain reaction (PCR) (Liu and Whittier, 1995) on total genomic DNA of strain Tr5. Sequencing of the short fragment revealed high similarity to fungal chitin synthases. Southern analysis with the fragment as a probe detected a restriction polymorphism between 4287 and Tr5 DNA digested with the enzyme HindIII (Fig. 1A). Stripping of the filter and reprobing with the hph gene produced the same hybridizing band in Tr5, demonstrating that the transformation vector had integrated at a single site in the genome, adjacent to the isolated DNA fragment. The fragment was used as a probe to isolate clones from a genomic λ EMBL3 library and a λ ZAP cDNA library. DNA sequence analysis of these clones revealed a large open reading frame (ORF) of 1863 amino acids, interrupted by two introns of 61 and 56 bp, respectively, which encodes a predicted polypeptide with a molecular mass of 207 kDa. The gene was designated chsV, and the sequence data have been submitted to the DDBJ/EMBL/GenBank databases under accession number AF484941. The plasmid insertion in strain Tr5 was located 2692 bp downstream of the putative start codon of chsV (Fig. 1B).
Comparison of the ChsV amino acid sequence with sequences in the databases revealed significant similarity to fungal class V chitin synthases. The highest degrees of identity (67% and 66% respectively) were detected with Csm1 from Pyricularia oryzae (Park et al., 1999) and Chs2 from Blumeria graminis (Zhang and Gurr, 2000). Amino acid alignment shows that ChsV contains an N-terminal domain with significant similarity to myosin motors. The ChsV sequence carries a putative P-loop, putative switches I and II and eight putative transmembrane helices. Similar to other members of the class, the C-terminal region of ChsV contains a class V chitin synthase domain with the highly conserved motifs QXXEY, EDRXL and QXRRW (Ruiz-Herrera et al., 2001).
Southern analysis of F. oxysporum genomic DNA digested with different restriction enzymes showed that chsV is present as a single copy. Using a fragment from the chitin synthase domain as a probe, an additional, faintly hybridizing band was detected, suggesting the presence of a second, structurally related gene in the F. oxysporum genome (see Fig. 3B).
To study the transcriptional regulation of the chsV gene, total RNAs from mycelia of strains 4287 and Tr5 grown in different media were hybridized to the chsV probe. Higher levels of chsV transcript were detected in the wild-type strain grown in 1.2 M sorbitol compared with the same medium with 1% glucose as the carbon source (Fig. 2). Increased chsV transcript levels were also detected in the presence of 50 mM tomato phytoanticipin α-tomatine. No transcript was detected in strain Tr5. To check whether expression of chsV depends on the Fmk1 mitogen-activated protein kinase (MAPK) signalling pathway, total RNAs from strain 4287 and an fmk1 knock-out mutant (Di Pietro et al., 2001) were hybridized to chsV. Both strains showed equally intense hybridization signals, suggesting that expression of chsV does not require a functional Fmk1 signalling pathway (data not shown).
ChsV is essential for vascular colonization and pathogenicity
To check whether the phenotype on the Tr5 mutant resulted from the vector insertion into the chsV gene, a 7 kb DNA fragment encompassing the chsV coding region with 0.9 kb of 5′-flanking and 0.4 kb of 3′-flanking sequence was amplified and introduced into strain Tr5 by co-transformation with the vector pAN8-1 bestowing phleomycin resistance. Seventeen phleomycin-resistant transformants were analysed by PCR with specific primers flanking the insertion point of the original plasmid in Tr5. In three of the transformants, a 2.0 kb fragment was amplified that had the same size as the fragment amplified from the wild-type strain (results not shown). We concluded that these transformants, denominated C1 to C3, had integrated an intact copy of chsV into the genome.
Targeted disruption of chsV was performed using a one-step gene replacement. Vector pDchsV was constructed by replacing a 1.2 kb HindIII–XhoI fragment of the chsV coding region with the hygromycin resistance cassette. A linearized fragment containing the disrupted chsV allele (Fig. 3A) was amplified by PCR and used to transform F. oxysporum strain 4287. Thirty-two hygromycin-resistant transformants were analysed for homologous integration of the construct at the chsV locus, using PCR with different combinations of primers specific for chsV and hph. Transformant D1 gave an amplification pattern consistent with targeted chsV replacement. Southern analysis of genomic DNA of transformant D1 digested with EcoRI showed that the 6 kb fragment corresponding to the wild-type chsV allele had been replaced by a 2.5 kb fragment, suggesting homologous integration of the replacement vector (Fig. 3B). Consistent with gene replacement, the 2.5 kb fragment also hybridized to the hph probe (results not shown). In contrast, another transformant, ND1, still contained the wild-type 6 kb fragment together with an additional hybridizing 2.5 kb fragment, indicative of ectopic insertion of the replacement vector. As expected, in the insertional mutant Tr5, the wild-type 6 kb fragment was replaced by a 4 kb fragment. The faintly hybridizing 4.5 kb fragment showing the same pattern in all four strains most probably corresponds to an additional, structurally related gene.
Root infection assays with tomato plants were performed by immersing roots of 2-week-old tomato plants in a microconidial suspension of the following strains: wild-type strain 4287, chsV knock-out strain D1, the ectopic transformant ND1 and the original insertional mutant Tr5. Plants were scored for vascular wilt symptoms at different time intervals. The development of disease is shown in Fig. 4. Plants inoculated with the wild-type strain showed characteristic wilt symptoms starting 7 days after inoculation. Disease severity increased steadily throughout the experiment, and all the plants were dead 18 days after inoculation. Plants inoculated with the ectopic transformant ND1 showed a very similar disease profile. In contrast, plants inoculated with the chsV knock-out strain D1 and the insertional transformant Tr5 failed to show any visible disease symptoms and remained as healthy as the water controls throughout the whole duration of the experiment. To determine colonization of the plant vascular tissue by the fungus, plants were removed from the vermiculite at different times after inoculation, roots and stems were cut into 2 cm fragments, surface sterilized and transferred to potato dextrose agar plates with or without hygromycin. F. oxysporum was readily isolated from roots and stems of plants that had been inoculated with the wild-type strain or the ectopic insertion transformant, whereas no fungal growth was detected from any of the plants inoculated with strains D1 or Tr5 (results not shown). The identity of the isolated strains, i.e. the presence of the functional chsV allele, was confirmed by PCR analysis of their genomic DNA. We conclude that ChsV is required for pathogenicity on tomato and for survival of the fungus within the host plant.
The ability of F. oxysporum to infect its host and cause disease symptoms is a complex phenotype. Fungal hyphae must adhere to the roots, penetrate them and invade the root cortex, spread upwards through the vascular system and, finally, produce wilt symptoms (Beckman, 1987). To determine whether ChsV plays a role in the initial stages of root adhesion (Di Pietro et al., 2001), roots of 3-week-old tomato plants were immersed in Erlenmeyer flasks containing a microconidial suspension of wild-type strain 4287, chsV knock-out strain D1, insertional mutant Tr5 or ectopic transformant ND1. After 48 h incubation under gentle shaking, germlings of all the strains had become visibly attached to the tomato roots (results not shown). We conclude that ChsV is not required for microconidial germination and initial attachment to tomato roots.
To assess the ability of the chsV mutants to colonize the vascular tissue independently of root penetration, the roots of tomato plants were cut off just below the hypocotyls, and plants were immersed in sterile glass tubes containing a microconidial suspension of wild-type strain 4287, chsV knock-out strain D1 or insertional mutant Tr5. This procedure allows the fungal propagules to enter the tomato xylem vessels directly without the need for previous root penetration. After 10 days, the control plants immersed in water were completely healthy and had formed new adventive roots, whereas those immersed in a spore suspension of the wild-type strain showed severe wilt symptoms and died rapidly. Plants immersed in spore suspensions of the chsV knock-out strain D1 or the insertional transformant Tr5 remained as healthy as the water controls in spite of the fungal mycelium visibly growing in the liquid surrounding the plant. We conclude that ChsV is required for efficient colonization of the tomato vascular system independently of root penetration.
To study the effect of the chsV mutation on the capacity to proliferate on living host tissue further, tomato fruits at different stages of ripening were injected with a microconidial suspension of the different strains. Figure 5A shows an inoculated tomato fruit after 6 days of incubation at 100% relative humidity. The wild-type strain, the ectopic transformant ND1 and the complemented transformant C2 colonized and macerated the fruit tissue surrounding the site of inoculation, forming a dense mat of mycelium and conidia on the surface of the fruit. In contrast, chsV knock-out strain D1 and insertion mutant Tr5 had a strongly reduced capacity to invade and macerate the fruit tissue and failed to produce mycelium on the fruit surface. In both mutants, a dark necrotic region was visible around the site of inoculation. Microscopic analysis revealed that hyphal growth of the mutants in the fruit tissue was greatly reduced compared with that of the wild-type strain. The dark region around the site of inoculation with the chsV mutants was apparently caused by localized necrosis of the tomato epidermis cells and was not detected around inoculation sites injected with water or with the wild-type strain (Fig. 5B). We conclude that ChsV is required for efficient invasive growth of F. oxysporum on living host plant tissue.
ChsV determines cell wall integrity and resistance to antimicrobial plant defence compounds
To address the role of ChsV in cell wall integrity and hyphal growth, the effect of the chsV mutation on growth and conidiation of F. oxysporum was determined. Mycelial growth rate of the chsV mutants on solid synthetic medium supplemented with either 1% glucose or 1.2 M sorbitol was only slightly slower than that of the wild-type strain at 28°C, although the appearance of the colony was clearly different (Fig. 6A and B). At 37°C, however, the chsV mutants were significantly reduced in growth rate compared with the wild-type strain, although this reduction was partially relieved in the presence of 1.2 M sorbitol (Fig. 6C and D). Staining of the chsV mutant colonies grown without osmotic stabilizer with the vital stain BCIP produced a light blue zone around the colony, suggesting that hyphal lysis was occurring at the colony margin. No such blue zone was observed around wild-type colonies or around mutant colonies grown with 1.2 M sorbitol (data not shown). When grown in potato dextrose broth, the chsV mutants produced 30% less microconidia than the wild-type strain, and many of these had an abnormal lemon-like shape compared with the typical oval shape of the wild-type conidia (results not shown). Along the hyphae of the mutants, swollen, balloon-like structures were frequently observed that stained intensely with the chitin-binding dye Calcofluor white (Fig. 6G). These aberrant structures were not detected in the wild-type strain (Fig. 6E and F) or in the complemented strain C2 (data not shown). They were also absent in the chsV mutants when these were grown in the presence of 1.2 M sorbitol (Fig. 6H). The chitin content in mycelia of the chsV mutants, as determined by the Morgan–Elson assay (Wheat, 1966), was 294 ± 40 nmol N-acetylglucosamine mg−1 protein compared with 332 ± 39 nmol mg−1 in the wild-type strain.
To determine whether ChsV plays a role in resistance to antifungal compounds present in the host plant tissue, we first tested the effects of crude aqueous extracts from tomato roots, vascular tissue or fruits on the wild-type and the chsV mutant strains. All types of extract significantly inhibited hyphal growth of the chsV mutants, whereas growth of the wild-type strain was only slightly affected. The strongest differential effect was observed with extracts from tomato vascular tissue (Fig. 7A). This effect was reproduced in an even more dramatic manner by adding 50 µg ml−1 tomato saponin α-tomatine to the medium (Fig. 7B). Differential growth inhibition was also observed with 5 µg ml−1 Congo red, a compound that interferes with fungal cell wall assembly (Fig. 7C), and with 3.25 mM H2O2 (Fig. 7D). Growing the chsV mutants in the presence of 1.2 M sorbitol did not alleviate growth inhibition by α-tomatine, Congo red or H2O2 (data not shown). In contrast, growth inhibition was reversed in the transformant C2 that had been complemented with a functional chsV gene (data not shown). We conclude that ChsV is important for maintaining the integrity of the F. oxysporum cell wall and for mediating resistance to plant antifungal compounds.
Molecular analysis of an insertional mutant of the vascular wilt fungus F. oxysporum identified the chitin synthase-encoding gene, chsV, as an essential pathogenicity determinant. Fungal chitin synthases are integral membrane proteins that participate in the biosynthesis of the cell wall and are important for hyphal growth and differentiation (Cabib et al., 1996; Roncero, 2002). Several lines of evidence suggest that the chsV gene encodes a functional class V chitin synthase from F. oxysporum. First, the N-terminal region of ChsV carries a myosin domain, which has been reported as a characteristic feature of class V chitin synthases (Fujiwara et al., 1997). Myosins are motor proteins with ATP hydrolytic activity that produce mechanical force to move along actin cables (Sellers, 2000). Because in filamentous fungi, actin fibres are concentrated at hyphal tips, septa and branching sites (Torralba and Heath, 2001), the fusion of a myosin motor to a cell wall biosynthetic enzyme may serve primarily for transport and localization of the chitin synthase at the cellular locations where cell wall biosynthesis is most active (Horiuchi et al., 1999).
Secondly, the phenotype of F. oxysporum mutants lacking a functional copy of chsV is highly similar to that of mutants in the class V chitin synthase gene csmA/chsD of A. nidulans (Horiuchi et al., 1999) and in the chsE gene of A. fumigatus (Aufauvre-Brown et al., 1997). As reported for the Aspergillus mutants, the F. oxysporum chsV mutants show hyphal lysis at the colony margins, increased sensitivity to the chitin-binding dye Congo red and balloon-like swellings along the hyphae that stain intensely with Calcofluor white. In both F. oxysporum and Aspergillus, the morphological abnormalities of the chitin synthase mutants can be partially restored by supplementing the growth medium with osmotic stabilizers. The similarity of the mutant phenotypes strongly suggests that the role of class V chitin synthases in the maintenance of cell wall rigidity is highly conserved between Fusarium and Aspergillus.
The role of ChsV in pathogenesis
Owing to the importance and specificity of chitin synthases in fungal growth and differentiation, it has long been speculated that they may play a role in fungal pathogenesis and represent potential targets for antifungal intervention (Munro and Gow, 2001). So far, however, the analysis of mutants in single chitin synthase genes has failed to provide conclusive evidence for their specific role in fungal infection. In Candida albicans, a class I chitin synthase is required for virulence but is also essential for cell viability and, therefore, cannot be considered a specific pathogenicity factor (Munro et al., 2001). In the same organism, two independent studies produced conflicting results on the role in virulence of chs3 encoding a class IV chitin synthase (Bulawa et al., 1995; Mio et al., 1996). In another human pathogen, A. fumigatus, the class V chitin synthase ChsE was found to be important for hyphal growth, but not for host infection (Aufauvre-Brown et al., 1997). The role of chitin synthases in pathogenicity to plants has been examined primarily in the corn smut fungus Ustilago maydis, in which disruption of chs1 and chs2 encoding class III and class I enzymes, respectively, had no effect on pathogenicity (Gold and Kronstad, 1994). In contrast, inactivation of a class IV chitin synthase gene, Umchs5, did result in a reduction in virulence (Xoconostle-Cazares et al., 1997).
To our knowledge, the data on ChsV provide the first evidence for an essential and specific role of a class V chitin synthase in fungal pathogenesis. F. oxysporum strains lacking this chitin synthase are viable but dramatically reduced in virulence. A number of hypotheses can be formulated for the function of ChsV during host infection. First, as an important player in cell wall biosynthesis, ChsV could contribute to efficient adhesion of fungal propagules to the host surface. Our data demonstrate that this is not the case, as chsV mutants adhere to tomato roots as avidly as the wild-type strain. In contrast, signalling mutants lacking the MAPK Fmk1 were shown previously to be strongly impaired in root adhesion (Di Pietro et al., 2001). Secondly, ChsV may be important during host penetration by ensuring the increased cell wall rigidity required for infection-related morphogenesis (Mendgen et al., 1996). Although this hypothesis cannot be completely ruled out, our data suggest that host penetration is probably not the primary function of ChsV in pathogenesis, because the chsV mutants are still unable to colonize the vascular tissue efficiently after the structural barrier of the root cortex and endodermis has been removed to allow for direct entry of the fungus into the vascular tissue. Even upon injection into tomato fruits, the chsV mutants are still unable to proliferate within the host tissue. A third hypothesis is that ChsV contributes in an essential way to the structural defence function of the cell wall by preventing the access of antifungal plant compounds to their cellular targets. This hypothesis is supported by at least two lines of evidence. First, the incapacity of the chsV mutants to grow on living plant tissue and the fact that we failed to recover the mutants from the tomato vascular system suggest that they are unable to survive in the host environment. Secondly, chsV mutants show reduced growth on plates containing aqueous extracts from tomato vascular tissue and are hypersensitive to two different classes of antimicrobial compounds implicated in plant defence, H2O2 and the tomato phytoanticipin α-tomatine. H2O2 is produced by plants during the pathogen-induced oxidative burst and has been suggested to play a direct antimicrobial role in plant defence (Lamb and Dixon, 1997). On the other hand, tomatine and other saponins are naturally present at high concentrations in different parts of tomato plants (Roddick, 1974). These compounds exert their antifungal activity by binding to sterols of the fungal membrane, thereby altering membrane permeability (Roddick, 1974; Keukens et al., 1992; Ruiz-Rubio et al., 2001). F. oxysporum can tolerate high levels of α-tomatine through a number of mechanisms, including secretion of a tomatine-degrading enzyme (Lairini et al., 1996; Roldán-Arjona et al., 1999) and modification of the membrane sterol content (Défago et al., 1983). Our results suggest that integrity of the fungal cell wall is a key factor in mediating resistance to α-tomatine, most likely by preventing diffusion of the saponin to the plasma membrane, and that ChsV plays a crucial role in this resistance mechanism. In support of this view, we found that chsV transcript levels are upregulated in response to α-tomatine or to hyperosmotic stress. It is worth noting that the 5′ non-coding region of chsV contains five copies of the STRE consensus motif (CCCCT) that mediates transcriptional activation in response to a variety of stresses in yeast, including heat, hyperosmotic shock or oxidative damage (Marchler et al., 1993). Intriguingly, a recent study established that compounds that produce an increase in plasma membrane permeability, including α-tomatine, also induce STRE-dependent transcription in yeast (Moskvina et al., 1999). We therefore speculate that upregulation of chsV transcription in F. oxysporum in response to stress-inducing compounds of plant origin may be mediated by a signalling mechanism analogous to the one acting via STRE elements in yeast. We propose that a natural role of ChsV in the Fusarium–tomato interaction is to ensure survival of the pathogen within the host by protecting it from toxic host compounds. As production of antifungals is a general defence strategy in plants (Lamb and Dixon, 1997; Dixon, 2001) and resistance to these compounds appears to be a prerequisite for pathogenesis (Morrissey and Osbourn, 1999), it seems likely that this role can be extended to class V chitin synthases from other invasive fungal plant pathogens.
Fungal isolates and culture conditions
Fusarium oxysporum f. sp. lycopersici strain 4287 (race 2) was obtained from J. Tello, Universidad de Almería, Spain, and stored at −80°C with glycerol as a microconidial suspension. The pathotype of the isolate was periodically confirmed by plant infection assays. For extraction of DNA and microconidia production, cultures were grown in potato dextrose broth (Difco) at 28°C as described previously (Di Pietro and Roncero, 1998).
For phenotypic analysis of colony growth, a drop with freshly obtained microconidia was transferred on 1.5% (w/v) agar plates of synthetic medium (Di Pietro and Roncero, 1998) containing either 1% (w/v) glucose or 1.2 M sorbitol as the carbon source and 0.1% NaNO3 as the nitrogen source. When needed, Congo red, α-tomatine, 5-bromo-4-chloro-3-indolylphosphate (BCIP) (all from Sigma) were dissolved in water, sterile filtered and added to the medium at the indicated concentrations after autoclaving. For determining growth inhibition by H2O2, microconidia were allowed to germinate for 12 h before applying them to the plates to which H2O2 had been added after autoclaving. Vascular tissue was obtained from tomato plants as described previously (Di Pietro and Roncero, 1998), ground in a mortar with liquid nitrogen and resuspended in water (5 g of tissue in 20 ml of water). The extract was sterile filtered and added to the medium at a proportion of 1:1 (v/v). After inoculation, plates were maintained in the dark at 28°C.
For microscopic analysis of chsV mutants, strains were grown in Erlenmeyer flasks containing liquid synthetic medium as described above without agar. For analysis of gene expression, freshly obtained microconidia were germinated for 14 h in potato dextrose broth, germlings were washed twice in sterile water and transferred for 12 h to liquid synthetic medium. Chitin content in fungal hyphae grown for 24 h in potato dextrose broth was determined in three separate experiments following a previously reported protocol (Aufauvre-Brown et al., 1997). N-acetylglucosamine content was measured by the Morgan–Elson assay (Wheat, 1966).
Nucleic acid manipulations and cloning of the chsV gene
Total RNA and genomic DNA were extracted from F. oxysporum mycelium according to previously reported protocols (Raeder and Broda, 1985; Chomzcynski and Sacchi, 1987). Southern and Northern analyses and probe labelling were carried out as described previously (Di Pietro and Roncero, 1998) using the non-isotopic digoxigenin labelling kit (Roche Applied Science). All experiments were carried out at least twice with similar results.
For TAIL PCR, genomic DNA from F. oxysporum insertional mutant Tr5 was amplified on a PTC-150 Minicycler (MJ Research) using the specific primers olihph2 (5′-AAACC GACGCCCCAGCACTC-3′), olitrpter4 (5′-CTGGGTTCGCA AAGATAATT-3′) and olipgx5 (5′-TGGTTGGGGATACATAC-3′) in three consecutive PCRs in combination with the arbitrary degenerate primer AD1 (5′-NTCGASTWTSGWGTT-3′) following the conditions reported previously (Liu and Whittier, 1995). The amplified DNA fragment was cloned into pGEM-T (Promega), and the genomic sequence flanking the transformation vector was used as a probe to screen a λ EMBL3 genomic library and a λ ZAP cDNA library (Roldán-Arjona et al., 1999) of F. oxysporum f. sp. lycopersici isolate 4287. Library screening, subcloning and other routine procedures were performed as described in standard protocols. Sequencing of both DNA strands was performed at the Servicio de Secuenciación, Universidad de Córdoba, using the Dyedeoxy Terminator cycle sequencing kit (PE Biosystems) on an ABI Prism 377 genetic analyser apparatus. DNA and protein sequence databases were searched using the blast algorithm (Altschul et al., 1990) at the National Center for Biotechnology Information (Bethesda, MD, USA).
Construction of plasmid vectors and fungal transformation
Gene replacement vector pDchsV was constructed as follows: a 2.3 kb fragment containing the F. oxysporum chsV gene was amplified from genomic DNA by PCR with primers olichsV6 (5′-CCGAGTTTCTGGGTATGACA-3′) and olichsV25 (5′-GTGGAGTAGTCTTCAACGATC-3′) and cloned in pGEM-T. A 1.2 kb XhoI–HindIII fragment encompassing part of the chsV coding region was excised and replaced with a XhoI–HindIII fragment from the plasmid pAN7-1 containing the hygromycin B resistance gene under control of the A. nidulans gpdA promoter (Punt et al., 1987). A linear fragment containing the interrupted chsV allele was generated by amplifying the entire construct with primers olichsV6 and olichsV25. The amplified fragment was used to transform protoplasts of F. oxysporum strain 4287 to hygromycin resistance according to a protocol described previously (Di Pietro and Roncero, 1998). For complementation experiments, a 7 kb DNA fragment encompassing the chsV coding region with 0.9 kb of 5′-flanking and 0.4 kb of 3′-flanking sequence was amplified by PCR using the primers olichsV32 (5′-GGTTGAGGTCATCATAGCAC-3′) and olichsV21 (5′-TCTCCTGTTAGGCACTCAAAT-3′) and introduced into strain Tr5 by co-transformation with the vector pAN8-1 conferring phleomycin resistance (Mattern et al., 1988). Transformants were selected, purified by monoco-nidial isolation and stored as microconidial suspensions at −80°C as described previously.
Root inoculation assays were performed as described previously (Di Pietro and Roncero, 1998). Briefly, 10-day-old tomato seedlings (cultivar Vemar; seeds kindly provided by Novartis) were inoculated with F. oxysporum strains by immersing the roots in a microconidial suspension for 30 min, planted in vermiculite and maintained in a growth chamber. At different times after inoculation, severity of disease symptoms was recorded using an index from 1 (healthy plant) to 5 (dead plant). Twenty plants were used for each treatment. To assay the virulence of F. oxysporum strains under conditions allowing direct access of the fungus to the tomato vascular tissue, the roots of the tomato seedlings were cut off just below the hypocotyls by means of a sterile scalpel, and plants were immersed in sterile glass tubes containing a suspension of 107 microconidia ml−1 in synthetic medium lacking any nitrogen or carbon source. Assays for root adhesion and invasive growth on tomato fruits were carried out as described previously (Di Pietro et al., 2001). All experiments were performed at least twice with similar results.
The authors gratefully acknowledge Fe Isabel García Maceira for valuable help in obtaining the insertional transformants, and Isabel Caballero for technical assistance. This research was supported by the Ministerio de Ciencia y Tecnología (BIO2001-2601) of Spain and Junta de Andalucia (CVI-138). M.P.M. was supported by a PhD fellowship from the Ministerio de Ciencia y Tecnología.