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.