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.

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.