Three structural chitin synthase genes, chs1, chs2 and chs3, were identified in the genome of Fusarium oxysporum f. sp. lycopersici, a soilborne pathogen causing vascular wilt disease in tomato plants. Based on amino acid identities with related fungal species, chs1, chs2 and chs3 encode structural chitin synthases (CSs) of class I, class II and class III, respectively. A gene (chs7) encoding a chaperone-like protein was identified by comparison of the deduced protein with Chs7p from Saccharomyces cerevisiae, an endoplasmic reticulum (ER) protein required for the export of ScChs3p (class IV) from the ER. So far no CS gene belonging to class IV has been isolated from F. oxysporum, although it probably contains more than one gene of this class, based on the genome data of the closely related species Fusarium graminearum. F. oxysporum chs1-, chs2-and chs7-deficient mutants were constructed through targeted gene disruption by homologous recombination. No compensatory mechanism seems to exist between the CS genes studied, since chitin content determination and expression analysis of the chs genes showed no differences between the disruption mutants and the wild-type strain. By fluorescence microscopy using Calcofluor white and DAPI staining, the wild-type strain and Dchs2 and Dchs7 mutants showed similar septation and even nuclear distribution, with each hyphal compartment containing only one nucleus, whereas the Dchs1 mutant showed compartments containing up to four nuclei. Pathogenicity assays on tomato plants indicated reduced virulence of Dchs2 and Dchs7 null mutants. Stress conditions affected normal development in Dchs2 but not in Dchs1 or Dchs7 disruptants, and the three chs-deficient mutants showed increased hyphal hydrophobicity compared to the wild-type strain when grown in sorbitol-containing medium. The chitin synthase mutants will be useful for elucidating cell wall biogenesis in F. oxysporum and the relationship between fungal cell wall integrity and pathogenicity. INTRODUCTIONChitin, an important structural cell wall component in many species of yeast and filamentous fungi but absent from plants and vertebrates, is a b(1,4)-linked polymer of N-acetylglucosamine which forms a fibrous polysaccharide. This taxonomic difference provides the rationale for considering chitin as a safe and largely selective target for developing antifungal control agents (Cohen, 1990). Chitin synthases (CSs) catalyse the transfer of N-acetylglucosamine from uridine diphosphate N-acetylglucosamine (UDPGlcNAc) to a growing chain of b(1,4)-linked Nacetylglucosamine residues (chitin) (Ruiz-Herrera et al., 1992). The specific mechanism by which this polymer is synthesized in vivo by the different species appears to have selective characteristics. Fungal CSs are integral membranebound proteins that participate in the biosynthesis of the cell wall and are important for hyphal growth and differentiation (reviewed by Cabib et al., 1996;Roncero, 2002). Comparative analysis of the amino acid sequences deduced from fungal chs genes revea...