A class V chitin synthase, chsA, and its roles in cell wall integrity in the fungus Colletotrichum graminicola

The Colletotrichum graminicola mutant T30 was generated by transformation of the wild type strain M1 with pUC-ATPH in the presence of SfuI. The mutant has conidia that burst and hyphal tips that swell in media with low osmotic pressure. The disrupted gene in T30 was identified as a class V chitin synthase gene (CSV) “ chsA.” The gene chsA is a single copy gene that has an open reading frame of 1,783 amino acids and two introns of 52 and 54 bp. Cloning and sequencing of the 5′ and 3 ′ borders of the insertion site in T30 genome showed that pUC-ATPH was inserted into a SfuI site in the wild type chsA , which is located at the 1,046th bp of the 5,458 bp chsA ORF. Reverse transcriptase PCR indicated that T30 does not express the chsA transcript fragment in the conserved region in CSVs. Complementation of the mutant with chsA indicates that the encoded protein is responsible for 29% of the chitin in conidial walls and is essential for wall strength of conidia and hyphal tips in media with high water potential. Analysis of the aligned deduced amino acid sequences of ten CSVs suggests they are in two subgroups.; Conidial wall composition of M1 and T30 was determined using gas chromatography and colorimetric methods. In T30, the conidia had significant decreases in chitin and in a trifluoroacetic acid (TFA)-insoluble hexose (P = 0.011 and 0.024 respectively) compared to M1. Both strains had similar percentages of two TFA-soluble sugars (mannose, and galactose) and of covalently bound protein. Three tests of wall strength indicated that T30 conidia were dramatically weaker than those of the wild type. After shaking with glass beads or extraction with NaOH, half of T30 conidia fragmented or collapsed, respectively, whereas few of M1 conidia did. After incubation in 1M PEG-600, 32% of the T30 versus 8% of M1conidial walls collapsed (cytorrhyzed). The high propensity of the chsA-disrupted mutant T30 to wall breakage and collapse supports that the chitin fraction synthesized by ChsA is critical for conidial wall strength and consequently for maintenance of cell wall integrity.