Functional analysis of basidiomycete specific chitin synthase genes in the agaricomycete fungus Pleurotus ostreatus.

Chitin is an essential structural component of fungal cell walls composed of transmembrane proteins called chitin synthases (CHSs), which have a large range of reported effects in ascomycetes; however, are poorly understood in agaricomycetes. In this study, evolutionary and molecular genetic analyses of chs genes were conducted using genomic information from nine ascomycete and six basidiomycete species. The results support the existence of seven previously classified chs clades and the discovery of three novel basidiomycete-specific clades (BI-BIII). The agaricomycete fungus Pleurotus ostreatus was observed to have nine putative chs genes, four of which were basidiomycete-specific. Three of these basidiomycete specific genes were disrupted in the P. ostreatus 20b strain (ku80 disruptant) through homologous recombination and transformants were obtained (Δchsb2, Δchsb3, and Δchsb4). Despite numerous transformations Δchsb1 was unobtainable, suggesting disruption of this gene causes a crucial negative effect in P. ostreatus. Disruption of these chsb2-4 genes caused sparser mycelia with rougher surfaces and shorter aerial hyphae. They also caused increased sensitivity to cell wall and membrane stress, thinner cell walls, and overexpression of other chitin and glucan synthases. These genes have distinct roles in the structural formation of aerial hyphae and cell walls, which are important for understanding basidiomycete evolution in filamentous fungi.

Meiotic Silencing in Dothideomycetous Bipolaris maydis.

The filamentous ascomycete Bipolaris maydis is a plant pathogen that causes corn leaf blight and has been used in cytological studies of sexual reproduction. In this fungus, when null mutants of each septin are crossed with the wild-type strain, all ascospores derived from the same asci show abnormal morphology. The phenomenon was remarkably similar to the event known as "ascus dominance" in Neurospora crassa, which is known to be caused by MSUD (meiotic silencing by unpaired DNA). However, it is not clear whether B. maydis possesses functional MSUD. The object of this study is to elucidate whether this fungus carries a functional MSUD system that causes ascus dominance in the crosses of septin mutants and the wild-type strain. The results of homozygous and heterozygous crossing tests with mutants, having the insertional CDC10-septin gene sequence into the genome, suggested that the ascus dominance in B. maydis is triggered by the unpaired DNA as in N. crassa. To investigate whether MSUD is caused by the same mechanism as in N. crassa, an RNA-dependent RNA polymerase, one of the essential factors in MSUD, was identified and disrupted (Δrdr1) in B. maydis. When the Δrdr1 strain was crossed with each mutant of the septins, ascus dominance did not occur in all crosses. These results suggest that this ascus dominance is caused by RNA silencing triggered by an unpaired gene, as in N. crassa, and septin genes were affected by this silencing. To date, although MSUD has been found only in Fusarium graminearum and N. crassa, which are classified as Sordariomycetes, this study showed that MSUD is also functional in B. maydis, which is classified as a Dothideomycete. These results showed the possibility that this posttranscriptional regulation is extensively conserved among filamentous ascomycetes.

An exocyst component, Sec5, is essential for ascospore formation in Bipolaris maydis.

In this study, we identified Sec5 in Bipolaris maydis, a homologue of Sec5 in Saccharomyces cerevisiae and a possible exocyst component of the fungus. To examine how Sec5 affects the life cycle of B. maydis, we generated null mutant strains of the gene (Δsec5). The Δsec5 strains showed a strong reduction in hyphal growth and a slight reduction in pathogenicity. In sexual reproduction, they possessed the ability to develop pseudothecia. However, all ascospores were aborted in any of the asci obtained from crosses between Δsec5 and the wild-type. Our cytological study revealed that the abortion was caused by impairments of the post-meiotic stages in ascospore development, where ascospore delimitation and young spore elongation occur.