Development of a gene-targeting system using CRISPR/Cas9 and utilization of pyrG as a novel selectable marker in Lentinula edodes.

First, we attempted to recombine the Shiitake (Lentinula edodes) pyrG (ura3) gene homologously by introducing a donor vector containing a carboxin resistance gene (lecbxR) flanked by homologous sequences of pyrG into protoplasts of the fungus. However, all the carboxin-resistant transformants only contained ectopic insertions of the exogenous gene and no homologous insertions. Agaricomycetes are generally known for their low efficiency of homologous recombination, and a similar result was shown for L. edodes. We then co-introduced a Cas9 plasmid vector containing a CRISPR/Cas9 expression cassette targeting pyrG and donor plasmid vector. As a result, ∆pyrG strains containing the expected homologous recombination were obtained. However, only two of the seven ∆pyrG strains had the Cas9 sequence; the others did not. Our results suggest that genome editing occurred via the transient expression of the CRISPR/Cas9 cassette in the Cas9 plasmid vector introduced into the fungal cell. Transforming pyrG into a ∆pyrG strain (strain I8) resulted in prototrophic strains with an efficiency of 6.5 strains/experiment.

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.

Draft Genome Sequence of Dicyma pulvinata Strain 414-3, a Mycoparasite of Cladosporium fulvum, Causal Agent of Tomato Leaf Mold.

Dicyma pulvinata strain 414-3, isolated from the surface of a tomato leaf, is a mycoparasitic fungus of Cladosporium fulvum, which causes leaf mold of tomato. We report here the draft genome sequence of strain 414-3, which will contribute to elucidating the molecular mechanisms involved in the mycoparasitism.

Cla4 PAK-like kinase is required for pathogenesis, asexual/sexual development and polarized growth in Bipolaris maydis.

PAK (p21-activated protein kinases)-like kinases are master regulators of development and morphogenesis, which were conserved among eukaryotes, including fungi. In budding yeast, two types of PAK-like kinases, Ste20 and Cla4 have distinct but shared roles in the regulation of pseudohyphal development, budding and mating. In this study, to examine the broad functions of PAK-like kinases in growth, pathogenicity and asexual/sexual reproduction in filamentous fungi, we identified and characterized two PAK-like kinases, Ste20 and Cla4 in Bipolaris maydis. A single mutant of both Ste20 and Cla4 gene was viable, while the double mutant was not available, possibly because of lethality. In growth, conidiation, and pathogenicity, Δste20 strains showed phenotypes similar to those of the wild-type, while Δcla4 strains showed severely defected phenotypes. In this study, we also clarified that Ste20 is partially involved in pseudothecium development but is dispensable for maternity, while Cla4 is essential for maternal pseudothecium development and also involve in ascospore development in paternal pseudothecium. Fluorescent microscopy visualized the disorder in cell polarity at the hyphal tip in Δcla4. These results suggested that not Ste20 but Cla4 is a master regulator of growth, pathogenicity and asexual/sexual development in B. maydis. In addition, we successfully visualized alternation of branching pattern and distribution of Spitzenkörper at the hyphal tip in Δcla4 strains.

Characterization of the autophagy-related gene BmATG8 in Bipolaris maydis.

Autophagy is involved in cellular development and the maintenance of viability under nutrient deprivation in a wide range of eukaryotes. A filamentous ascomycete Bipolaris maydis, responsible for southern corn leaf blight, is also studied as a model fungus for sexual reproduction in filamentous ascomycetes that form filiform ascospores. In order to clarify the roles of autophagy in various stages of the life cycle of B. maydis, we constructed null mutants of BmATG8, an orthologue of the Saccharomyces cerevisiae autophagy gene ATG8 in B. maydis. Deletion of BmATG8 impaired localization of cytosolic components to the vacuole under nitrogen starvation, suggesting that autophagy was deficient in the null mutants. Additionally, fluorescent microscopic observations on a eGFP-fused BmATG8 expressing strain showed that BmATG8 is associated with autophagy-related structures. In vegetative growth, ΔBmATG8 strains showed a reduction in conidiation and aerial mycelial growth. Interestingly, the mutant conidia indicated loss of the germination rate under starvation conditions and affected longevity. However, germinated mutant conidia were still capable of infecting the host plant via appressoria. In sexual reproduction, ascospores with ΔBmATG8 genetic background were aborted. Our results revealed that autophagy plays a crucial role in the function of conidia, not in host infection via appressoria in B. maydis. In addition, conservation of the importance of autophagy in ascospore development is suggested among ascomycetes including species that form bitunicate ascus.