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A gene encoding a sixth chitin synthase (Umchs6, sequence GenBank accession No. ) from the plant pathogenic hemibasidiomycete Ustilago maydis (DC.) Cda. was isolated and characterized. The predicted protein is 1103 amino acids in length with a calculated molecular mass of 123.5 kDa. a2b2 null mutants were obtained by substitution of a central fragment of the Umchs6 gene with the hygromycin resistance cassette, and a1b1 null mutants were obtained by genetic recombination in plants of an a2b2deltach6 and a wild-type a1b1 strain. The mutation had no effect on the dimorphic transition in vitro or on mating, and growth rate of the mutants was only slightly reduced. On the other hand, they displayed important alterations in cell morphology, particularly at the mycelial stage, and in the staining pattern with calcofluor white. Levels of chitin synthase activity in vitro and chitin content were reduced. The most noticeable characteristic of the mutants was their almost complete loss of virulence to maize (Zea mays L.). This was a recessive character. Microscopic observations during the infectious process suggest that chitin synthase 6 activity is very important for growth of the fungus into the plant. Transformation of a2b2deltach6 mutants with an autonomous replicating plasmid carrying the full Umchs6 gene restored their normal morphological phenotype and virulence. These results are evidence that the mutation in the Umchs6 gene was solely responsible for the phenotypic alterations observed.

A fragment corresponding to a conserved region of a fifth gene coding for chitin synthase in the plant pathogenic fungus Ustilago maydis was amplified by means of the polymerase chain reaction (PCR). The amplified fragment was utilized as a probe for the identification of the whole gene in a genomic library of the fungus. The predicted gene product of Umchs5 has highest similarity with class IV chitin synthases encoded by the CHS3 genes from Saccharomyces cerevisiae and Candida albicans, chs-4 from Neurospora crassa, and chsE from Aspergillus nidulans. Umchs5 null mutants were constructed by substitution of most of the coding sequence with the hygromycin B resistance cassette. Mutants displayed significant reduction in growth rate, chitin content, and chitin synthase activity, specially in the mycelial form. Virulence to corn plantules was also reduced in the mutants. PCR was also used to obtain a fragment of a sixth chitin synthase, Umchs6. It is suggested that multigenic control of chitin synthesis in U. maydis operates as a protection mechanism for fungal viability in which the loss of one activity is partially compensated by the remaining enzymes., (Copyright 1997 Academic Press.)

The gene encoding ornithine decarboxylase (ODC) from Ustilago maydis was cloned. A conserved PCR product amplified from U. maydis DNA was synthesized and used to screen a genomic library of the fungus. Alignment of its deduced protein sequence with those of other cloned ODCs showed a high degree of homology. Gene replacement was obtained by removal of a central part of the gene and insertion of the hygromycin resistance cassette. The null mutant thus obtained displayed no ODC activity and behaved as a polyamine auxotroph. This result is evidence that a single ODC gene exists in the fungus, and that U. maydis utilizes the ODC pathway as the only mechanism for polyamine biosynthesis. When grown in polyamine-containing media, the null mutant accumulated a polyamine pool which further sustained its normal rate of growth in polyamine-free media for approximately 12-16 h. When putrescine concentrations lower than 0.5 mM were employed, the mutant grew at a normal rate but was unable to engage in the dimorphic transition. Under conditions favourable for mycelial growth, the mutant grew with a yeast-like morphology in liquid media, and formed smooth colonies consisting of yeast cells on solid media. Reversion to normal dimorphic phenotype required high concentrations of putrescine or spermidine. These results are evidence that concentrations of polyamines higher than those necessary to sustain vegetative growth are required for the dimorphic transition in U. maydis.

PCR was used to amplify fragments corresponding to CHS genes from Ustilago maydis, utilizing as primers oligonucleotides devised according to the conserved regions of fungal CHS genes. The PCR product was employed as a probe to screen a genomic library of the fungus. Two different CHS genes (Umchs1 and Umchs2) were thus identified in the positive clones recovered. Their sequence revealed high similarity with the CHS genes previously cloned from other fungi, especially in their central region. Alignment with the deduced protein sequences of all CHS genes reported up to date showed the existence of seven conserved domains. Transcripts from both genes were detected in the yeast and mycelial forms. In general, the transcripts from the Umchs1 gene appeared to be present at a higher level than the transcripts from the Umchs2 gene; the transcripts from both genes appeared to be more abundant in the mycelial form. Gene replacement of either gene and analysis of the resulting phenotype demonstrated that they are non-essential. Nevertheless, growth, chitin synthase activity levels, and chitin content of mycelial cells induced by cultivation in acidic media were all reduced in chs1 and chs2 mutants. However, mating, virulence and dimorphic behaviour were unaffected. Overall, the results indicate that the CHS1 and CHS2 genes encode products with redundant functions in U. maydis.