Three structural chitin synthase genes, , and , were identified in the genome of f. sp. , a soilborne pathogen causing vascular wilt disease in tomato plants. Based on amino acid identities with related fungal species, , and encode structural chitin synthases (CSs) of class I, class II and class III, respectively. A gene () encoding a chaperone-like protein was identified by comparison of the deduced protein with Chs7p from , 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 , although it probably contains more than one gene of this class, based on the genome data of the closely related species . -, - and -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 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 Δ and Δ mutants showed similar septation and even nuclear distribution, with each hyphal compartment containing only one nucleus, whereas the Δ mutant showed compartments containing up to four nuclei. Pathogenicity assays on tomato plants indicated reduced virulence of Δ and Δ null mutants. Stress conditions affected normal development in Δ but not in Δ or Δ disruptants, and the three -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 and the relationship between fungal cell wall integrity and pathogenicity.


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