CRISPR/Cas9 genome editing for comparative genetic analysis related to soy sauce brewing in Aspergillus sojae industrial strains.

Aspergillus sojae has traditionally been used in soy sauce brewing. Genetic modification techniques have been established in A. sojae, but it is difficult to apply them to various industrial strains. Although we have previously developed a CRISPR/Cpf1 system for genetic modification of A. sojae, another genome editing system was required for versatile modification. In addition, repetitive genetic modification using the CRISPR system has not been established in A. sojae. In this study, we demonstrated mutagenesis, gene deletion/integration, and large deletion of a chromosomal region in A. sojae using the CRISPR/Cas9 system. We also successfully performed repetitive genetic modification using a method that involved forced recycling of genome-editing plasmids. Moreover, we demonstrated that the effects of genetic modification related to soy sauce brewing differed among A. sojae industrial strains. These results showed that our technique of using the CRISPR/Cas9 system is a powerful tool for genetic modification in A. sojae.

Trace copper-mediated asexual development via a superoxide dismutase and induction of AobrlA in Aspergillus oryzae.

The filamentous fungus Aspergillus oryzae, in which sexual reproduction remains to be discovered, proliferates mainly via asexual spores (conidia). Therefore, despite its industrial importance in food fermentation and recombinant protein production, breeding beneficial strains by genetic crosses is difficult. In Aspergillus flavus, which is genetically close to A. oryzae, structures known as sclerotia are formed asexually, but they are also related to sexual development. Sclerotia are observed in some A. oryzae strains, although no sclerotia formation has been reported in most strains. A better understanding of the regulatory mechanisms underlying sclerotia formation in A. oryzae may contribute to discover its sexual development. Some factors involved in sclerotia formation have been previously identified, but their regulatory mechanisms have not been well studied in A. oryzae. In this study, we found that copper strongly inhibited sclerotia formation and induced conidiation. Deletion of AobrlA encoding a core regulator of conidiation and ecdR involved in transcriptional induction of AobrlA suppressed the copper-mediated inhibition of sclerotia formation, suggesting that AobrlA induction in response to copper leads not only to conidiation but also to inhibition of sclerotia formation. In addition, deletion of the copper-dependent superoxide dismutase (SOD) gene and its copper chaperone gene partially suppressed such copper-mediated induction of conidiation and inhibition of sclerotia formation, indicating that copper regulates asexual development via the copper-dependent SOD. Taken together, our results demonstrate that copper regulates asexual development, such as sclerotia formation and conidiation, via the copper-dependent SOD and transcriptional induction of AobrlA in A. oryzae.

CRISPR/Cpf1-mediated mutagenesis and gene deletion in industrial filamentous fungi Aspergillus oryzae and Aspergillus sojae.

In industrial applications such as fermentation and heterologous protein production, various Aspergillus oryzae and A. sojae strains are used. Although genetic engineering techniques have been developed for these filamentous fungi, applying such classical techniques to many strains is difficult. Therefore, the establishment of innovative technologies applicable to various industrial strains is required. We previously developed a genome editing technology using the CRISPR/Cas9 system for the efficient genetic engineering of A. oryzae; however, this system is limited by its protospacer adjacent motif sequence. In A. sojae, no genetic engineering using genome editing has been developed. In this study, we aimed to develop a genome editing technology using the Cpf1 nuclease for the genetic engineering of A. oryzae and A. sojae. AMA1-based genome editing vectors bearing codon-optimized cpf1 expression cassettes were constructed, and guide RNA expression cassettes were inserted into the Cpf1 genome editing vectors. Using the resultant plasmids, we performed mutagenesis of the AowA and sC genes in A. oryzae and the AswA gene in A. sojae. We deleted these genes by co-introducing the Cpf1 genome editing plasmid and the donor plasmid. Our study demonstrates that the CRISPR/Cpf1 system can be used as an efficient alternative to the CRISPR/Cas9 system to genetically engineer A. oryzae and as a new approach for efficient genetic engineering of A. sojae.

Novel Fus3- and Ste12-interacting protein FsiA activates cell fusion-related genes in both Ste12-dependent and -independent manners in Ascomycete filamentous fungi.

Filamentous fungal cells, unlike yeasts, fuse during vegetative growth. The orthologs of mitogen-activated protein (MAP) kinase Fus3 and transcription factor Ste12 are commonly involved in the regulation of cell fusion. However, the specific regulatory mechanisms underlying cell fusion in filamentous fungi have not been revealed. In the present study, we identified the novel protein FsiA as an AoFus3- and AoSte12-interacting protein in the filamentous fungus Aspergillus oryzae. The expression of AonosA and cell fusion-related genes decreased upon fsiA deletion and increased with fsiA overexpression, indicating that FsiA is a positive regulator of cell fusion. In addition, the induction of cell fusion-related genes by fsiA overexpression was also observed in the Aoste12 deletion mutant, indicating that FsiA can induce the cell fusion-related genes in an AoSte12-independent manner. Surprisingly, the fsiA and Aoste12 double deletion mutant exhibited higher cell fusion efficiency and increased mRNA levels of the cell fusion-related genes as compared to the fsiA single deletion mutant, which revealed that AoSte12 represses the cell fusion-related genes in the fsiA deletion mutant. Taken together, our data demonstrate that FsiA activates the cell fusion-related genes by suppressing the negative function of AoSte12 as well as by an AoSte12-independent mechanism.

A CRISPR/Cas9-mediated gene knockout system in Aspergillus luchuensis mut. kawachii.

We developed an approach to genome editing of the white koji fungus, Aspergillus luchuensis mut. kawachii using clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system. Co-transformation of AMA1-based Cas9 and gRNA expression plasmids achieved efficient gene knockout in A. kawachii. The plasmids were easily lost when selective pressure was removed, allowing for successive rounds of genome editing.

Functional production of human antibody by the filamentous fungus Aspergillus oryzae.

BACKGROUND: Monoclonal antibodies (mAbs) as biopharmaceuticals take a pivotal role in the current therapeutic applications. Generally mammalian cell lines, such as those derived from Chinese hamster ovaries (CHO), are used to produce the recombinant antibody. However, there are still concerns about the high cost and the risk of pathogenic contamination when using mammalian cells. Aspergillus oryzae, a filamentous fungus recognized as a GRAS (Generally Regarded As Safe) organism, has an ability to secrete a large amount of proteins into the culture supernatant, and thus the fungus has been used as one of the cost-effective microbial hosts for heterologous protein production. Pursuing this strategy the human anti-TNFα antibody adalimumab, one of the world's best-selling antibodies for the treatment of immune-mediated inflammatory diseases including rheumatoid arthritis, was chosen to produce the full length of mAbs by A. oryzae. Generally, N-glycosylation of the antibody affects immune effector functions such as antibody-dependent cell-mediated cytotoxicity (ADCC) via binding to the Fc receptor (FcγR) on immune cells. The CRISPR/Cas9 system was used to first delete the Aooch1 gene encoding a key enzyme for the hyper-mannosylation process in fungi to investigate the binding ability of antibody with FcγRIIIa. RESULTS: Adalimumab was expressed in A. oryzae by the fusion protein system with α-amylase AmyB. The full-length adalimumab consisting of two heavy and two light chains was successfully produced in the culture supernatants. Among the producing strains, the highest amount of antibody was obtained from the ten-protease deletion strain (39.7 mg/L). Two-step purifications by Protein A and size-exclusion chromatography were applied to obtain the high purity sample for further analysis. The antigen-binding and TNFα neutralizing activities of the adalimumab produced by A. oryzae were comparable with those of a commercial product Humira®. No apparent binding with the FcγRIIIa was detected with the recombinant adalimumab even by altering the N-glycan structure using the Aooch1 deletion strain, which suggests only a little additional activity of immune effector functions. CONCLUSION: These results demonstrated an alternative low-cost platform for human antibody production by using A. oryzae, possibly offering a reasonable expenditure for patient's welfare.

Invasive growth of Aspergillus oryzae in rice koji and increase of nuclear number.

BACKGROUND: 'Rice koji' is a solid culture of Aspergillus oryzae on steamed rice grains. Multiple parallel fermentation, wherein saccharification of rice by A. oryzae and alcohol fermentation by the budding yeast occur simultaneously, leads to the formation of a variety of ingredients of Japanese sake. In sake brewing, the degree of mycelial invasive growth into the steamed rice, called 'haze-komi', highly correlates with the digestibility and quality of rice koji, since the hyphae growing into the rice secrete amylases and digest starch. RESULTS: In this study, we investigated mycelial distribution of GFP-tagged A. oryzae in rice koji made with different types of rice, such as sake rice and eating rice, with 50 or 90% polishing rate to remove abundant proteins and lipids near the surface. In addition, we compared transcriptomes of A. oryzae in the different types of rice koji. Finally, we found that A. oryzae increases the nuclear number and hyphal width in the course of 1-3 days cultivation. CONCLUSIONS: Our imaging analyses indicate that A. oryzae hyphae grew more deeply into 50% polished rice than 90% polished rice. The increases of nuclear number may be a selectively acquired characteristic for the high secretory capacity during the long history of cultivation of this species.

A novel Pezizomycotina-specific protein with gelsolin domains regulates contractile actin ring assembly and constriction in perforated septum formation.

Septum formation in fungi is equivalent to cytokinesis. It differs mechanistically in filamentous ascomycetes (Pezizomycotina) from that of ascomycete yeasts by the retention of a central septal pore in the former group. However, septum formation in both groups is accomplished by contractile actin ring (CAR) assembly and constriction. The specific components regulating septal pore organization during septum formation are poorly understood. In this study, a novel Pezizomycotina-specific actin regulatory protein GlpA containing gelsolin domains was identified using bioinformatics. A glpA deletion mutant exhibited increased distances between septa, abnormal septum morphology and defective regulation of septal pore closure. In glpA deletion mutant hyphae, overaccumulation of actin filament (F-actin) was observed, and the CAR was abnormal with improper assembly and failure in constriction. In wild-type cells, GlpA was found at the septum formation site similarly to the CAR. The N-terminal 329 residues of GlpA are required for its localization to the septum formation site and essential for proper septum formation, while its C-terminal gelsolin domains are required for the regular CAR dynamics during septum formation. Finally, in this study we elucidated a novel Pezizomycotina-specific actin modulating component, which participates in septum formation by regulating the CAR dynamics.

Author Correction: BiFC-based visualisation system reveals cell fusion morphology and heterokaryon incompatibility in the filamentous fungus Aspergillus oryzae.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Novel Antifungal Compound Z-705 Specifically Inhibits Protein Kinase C of Filamentous Fungi.

The cell wall integrity signaling (CWIS) pathway is involved in fungal cell wall biogenesis. This pathway is composed of sensor proteins, protein kinase C (PKC), and the mitogen-activated protein kinase (MAPK) pathway, and it controls the transcription of many cell wall-related genes. PKC plays a pivotal role in this pathway; deficiencies in PkcA in the model filamentous fungus Aspergillus nidulans and in MgPkc1p in the rice blast fungus Magnaporthe grisea are lethal. This suggests that PKC in filamentous fungi is a potential target for antifungal agents. In the present study, to search for MgPkc1p inhibitors, we carried out in silico screening by three-dimensional (3D) structural modeling and performed growth inhibition tests for M. grisea on agar plates. From approximately 800,000 candidate compounds, we selected Z-705 and evaluated its inhibitory activity against chimeric PKC expressed in Saccharomyces cerevisiae cells in which the kinase domain of native S. cerevisiae PKC was replaced with those of PKCs of filamentous fungi. Transcriptional analysis of MLP1, which encodes a downstream factor of PKC in S. cerevisiae, and phosphorylation analysis of the mitogen-activated protein kinase (MAPK) Mpk1p, which is activated downstream of PKC, revealed that Z-705 specifically inhibited PKCs of filamentous fungi. Moreover, the inhibitory activity of Z-705 was similar to that of a well-known PKC inhibitor, staurosporine. Interestingly, Z-705 inhibited melanization induced by cell wall stress in M. grisea We discuss the relationships between PKC and melanin biosynthesis.IMPORTANCE A candidate inhibitor of filamentous fungal protein kinase C (PKC), Z-705, was identified by in silico screening. A screening system to evaluate the effects of fungal PKC inhibitors was constructed in Saccharomyces cerevisiae Using this system, we found that Z-705 is highly selective for filamentous fungal PKC in comparison with S. cerevisiae PKC. Analysis of the AGS1 mRNA level, which is regulated by Mps1p mitogen-activated protein kinase (MAPK) via PKC, in the rice blast fungus Magnaporthe grisea revealed that Z-705 had a PKC inhibitory effect comparable to that of staurosporine. Micafungin induced hyphal melanization in M. grisea, and this melanization, which is required for pathogenicity of M. grisea, was inhibited by PKC inhibition by both Z-705 and staurosporine. The mRNA levels of 4HNR, 3HNR, and SCD1, which are essential for melanization in M. grisea, were suppressed by both PKC inhibitors.

Inter-strain expression of sequence-diverse HET domain genes severely inhibits growth of Aspergillus oryzae.

In the Pezizomycotina (filamentous ascomycete) species, genes that encode proteins with an HET domain (Pfam: PF06985) are reportedly involved in heterokaryon incompatibility (HI) in which cell death or growth defects are induced after fusion of cells that are genetically incompatible owing to diversities in their nucleotide sequence. HET domain genes are commonly found in Pezizomycotina genomes and are functionally characterized in only a few species. Here, we compared 44 HET domain genes between an incompatible strain pair of Aspergillus oryzae RIB40 and RIB128 and performed inter-strain expression of 37 sequence-diverse genes for mimicking HI. Four HET domain genes were identified to cause severe growth inhibition in a strain- or sequence-specific manner. Furthermore, SNPs responsible for the inhibition of cell growth were identified. This study provides an important insight into the physiological significance of sequence diversity of HET domain genes and their potential functions in HI of A. oryzae.

Forced Recycling of an AMA1-Based Genome-Editing Plasmid Allows for Efficient Multiple Gene Deletion/Integration in the Industrial Filamentous Fungus Aspergillus oryzae.

Filamentous fungi are used for food fermentation and industrial production of recombinant proteins. They also serve as a source of secondary metabolites and are recently expected as hosts for heterologous production of useful secondary metabolites. Multiple-step genetic engineering is required to enhance industrial production involving these fungi, but traditional sequential modification of multiple genes using a limited number of selection markers is laborious. Moreover, efficient genetic engineering techniques for industrial strains have not yet been established. We have previously developed a clustered regulatory interspaced short palindromic repeats (CRISPR)/Cas9-based mutagenesis technique for the industrial filamentous fungus Aspergillus oryzae, enabling mutation efficiency of 10 to 20%. Here, we improved the CRISPR/Cas9 approach by including an AMA1-based autonomously replicating plasmid harboring the drug resistance marker ptrA By using the improved mutagenesis technique, we successfully modified A. oryzae wild and industrial strains, with a mutation efficiency of 50 to 100%. Conditional expression of the Aoace2 gene from the AMA1-based plasmid severely inhibited fungal growth. This enabled forced recycling of the plasmid, allowing repeated genome editing. Further, double mutant strains were successfully obtained with high efficiency by expressing two guide RNA molecules from the genome-editing plasmid. Cotransformation of fungal cells with the genome-editing plasmid together with a circular donor DNA enabled marker-free multiplex gene deletion/integration in A. oryzae The presented repeatable marker-free genetic engineering approach for mutagenesis and gene deletion/integration will allow for efficient modification of multiple genes in industrial fungal strains, increasing their applicability.IMPORTANCE Multiple gene modifications of specific fungal strains are required for achieving industrial-scale production of enzymes and secondary metabolites. In the present study, we developed an efficient multiple genetic engineering technique for the filamentous fungus Aspergillus oryzae The approach is based on a clustered regulatory interspaced short palindromic repeats (CRISPR)/Cas9 system and recycling of an AMA1-based autonomous replicating plasmid. Because the plasmid harbors a drug resistance marker (ptrA), the approach does not require the construction of auxotrophic industrial strains prior to genome editing and allows for forced recycling of the gene-editing plasmid. The established plasmid-recycling technique involves an Aoace2-conditional expression cassette, whose induction severely impairs fungal growth. We used the developed genetic engineering techniques for highly efficient marker-free multiple gene deletion/integration in A. oryzae The genome-editing approaches established in the present study, which enable unlimited repeatable genetic engineering, will facilitate multiple gene modification of industrially important fungal strains.

BiFC-based visualisation system reveals cell fusion morphology and heterokaryon incompatibility in the filamentous fungus Aspergillus oryzae.

Aspergillus oryzae is an industrially important filamentous fungus used for Japanese traditional food fermentation and heterologous protein production. Although cell fusion is important for heterokaryon formation and sexual/parasexual reproduction required for cross breeding, knowledge on cell fusion and heterokaryon incompatibility in A. oryzae is limited because of low cell fusion frequency. Therefore, we aimed to develop a BiFC system to specifically visualise fused cells and facilitate the analysis of cell fusion in A. oryzae. The cell fusion ability and morphology of 15 A. oryzae strains were investigated using heterodimerising proteins LZA and LZB fused with split green fluorescence protein. Morphological investigation of fused cells revealed that cell fusion occurred mainly as conidial anastomosis during the early growth stage. Self-fusion abilities were detected in most industrial A. oryzae strains, but only a few strain pairs showed non-self fusion. Protoplast fusion assay demonstrated that almost all the pairs capable of non-self fusion were capable of heterokaryon formation and vice versa, thus providing the first evidence of heterokaryon incompatibility in A. oryzae. The BiFC system developed in this study provides an effective method in studying morphology of fused cells and heterokaryon incompatibility in the filamentous fungal species with low cell fusion efficiency.

Highly efficient gene targeting in Aspergillus oryzae industrial strains under ligD mutation introduced by genome editing: Strain-specific differences in the effects of deleting EcdR, the negative regulator of sclerotia formation.

Numerous strains of Aspergillus oryzae are industrially used for Japanese traditional fermentation and for the production of enzymes and heterologous proteins. In A. oryzae, deletion of the ku70 or ligD genes involved in non-homologous end joining (NHEJ) has allowed high gene targeting efficiency. However, this strategy has been mainly applied under the genetic background of the A. oryzae wild strain RIB40, and it would be laborious to delete the NHEJ genes in many A. oryzae industrial strains, probably due to their low gene targeting efficiency. In the present study, we generated ligD mutants from the A. oryzae industrial strains by employing the CRISPR/Cas9 system, which we previously developed as a genome editing method. Uridine/uracil auxotrophic strains were generated by deletion of the pyrG gene, which was subsequently used as a selective marker. We examined the gene targeting efficiency with the ecdR gene, of which deletion was reported to induce sclerotia formation under the genetic background of the strain RIB40. As expected, the deletion efficiencies were high, around 60~80%, in the ligD mutants of industrial strains. Intriguingly, the effects of the ecdR deletion on sclerotia formation varied depending on the strains, and we found sclerotia-like structures under the background of the industrial strains, which have never been reported to form sclerotia. The present study demonstrates that introducing ligD mutation by genome editing is an effective method allowing high gene targeting efficiency in A. oryzae industrial strains.

Comparative genomic analysis identified a mutation related to enhanced heterologous protein production in the filamentous fungus Aspergillus oryzae.

Genomic mapping of mutations using next-generation sequencing technologies has facilitated the identification of genes contributing to fundamental biological processes, including human diseases. However, few studies have used this approach to identify mutations contributing to heterologous protein production in industrial strains of filamentous fungi, such as Aspergillus oryzae. In a screening of A. oryzae strains that hyper-produce human lysozyme (HLY), we previously isolated an AUT1 mutant that showed higher production of various heterologous proteins; however, the underlying factors contributing to the increased heterologous protein production remained unclear. Here, using a comparative genomic approach performed with whole-genome sequences, we attempted to identify the genes responsible for the high-level production of heterologous proteins in the AUT1 mutant. The comparative sequence analysis led to the detection of a gene (AO090120000003), designated autA, which was predicted to encode an unknown cytoplasmic protein containing an alpha/beta-hydrolase fold domain. Mutation or deletion of autA was associated with higher production levels of HLY. Specifically, the HLY yields of the autA mutant and deletion strains were twofold higher than that of the control strain during the early stages of cultivation. Taken together, these results indicate that combining classical mutagenesis approaches with comparative genomic analysis facilitates the identification of novel genes involved in heterologous protein production in filamentous fungi.

Molecular dissection of SO (SOFT) protein in stress-induced aggregation and cell-to-cell interactive functions in filamentous fungal multicellularity.

Filamentous fungi grow by organizing multicellularity through hyphal compartmentalization and cell fusion. SO (SOFT) protein, which was originally identified in Neurospora crassa, plays distinct functional roles in cell-to-cell interactions, such as septal plugging and cell fusion. We previously reported that AoSO, an Aspergillus oryzae SO homologue, forms aggregates at the septal pore in response to stress, as well as upon hyphal wounding. However, the functional regions that mediate the multicellular functions of AoSO, which is a large protein composed of 1195 amino acids, have not been elucidated. Here, we divided AoSO protein into regions according to amino acid sequence conservation among other fungal SO homologues. By heterologous expression of full-length and truncated forms of AoSO in the yeast Saccharomyces cerevisiae, the region responsible for the stress-induced aggregation of AoSO was identified to be between amino acids 556 and 1146. In A. oryzae, however, septal localization of AoSO aggregates required the 49 C-terminal amino acids. Thus, expression of only the C-terminal half of AoSO was sufficient for septal plugging and prevention of excessive cytoplasmic loss upon hyphal wounding. In contrast, the N-terminal half of AoSO, from amino acids 1 to 555, together with the C-terminal end, was revealed to be indispensable for cell fusion. Collectively, these findings suggest that the C-terminal half of AoSO, which mediates stress-induced aggregation, is required for both septal plugging and cell fusion, whereas the N-terminal half confers an additional functionality that is essential for cell fusion.

Development of a genome editing technique using the CRISPR/Cas9 system in the industrial filamentous fungus Aspergillus oryzae.

OBJECTIVES: To develop a genome editing method using the CRISPR/Cas9 system in Aspergillus oryzae, the industrial filamentous fungus used in Japanese traditional fermentation and for the production of enzymes and heterologous proteins. RESULTS: To develop the CRISPR/Cas9 system as a genome editing technique for A. oryzae, we constructed plasmids expressing the gene encoding Cas9 nuclease and single guide RNAs for the mutagenesis of target genes. We introduced these into an A. oryzae strain and obtained transformants containing mutations within each target gene that exhibited expected phenotypes. The mutational rates ranged from 10 to 20 %, and 1 bp deletions or insertions were the most commonly induced mutations. CONCLUSIONS: We developed a functional and versatile genome editing method using the CRISPR/Cas9 system in A. oryzae. This technique will contribute to the use of efficient targeted mutagenesis in many A. oryzae industrial strains.

Protein kinase C regulates the expression of cell wall-related genes in RlmA-dependent and independent manners in Aspergillus nidulans.

A protein kinase C of Aspergillus nidulans, PkcA, is required for cell wall integrity (CWI) and is considered a major component of the regulating pathway. To investigate whether PkcA regulates the transcription of cell wall-related genes, we constructed strains expressing pkcA(R429A) that encodes an activated form of PkcA. The mRNA levels of most chitin synthase genes and an α-glucan synthase gene, agsB, were increased when pkcA(R429A) expression was induced. These mRNA increases were not observed or were only partially observed, in a deletion mutant of rlmA, an ortholog of RLM1 that encodes a transcription factor in the CWI pathway in Saccharomyces cerevisiae. In addition, in a pkcA temperature-sensitive mutant under heat stress, the mRNA levels of some chitin synthase genes and agsB did not increase. These results suggest that PkcA is involved in CWI maintenance through the transcriptional regulation of cell wall-related genes.

Base composition and nucleosome density in exonic and intronic regions in genes of the filamentous ascomycetes Aspergillus nidulans and Aspergillus oryzae.

We sequenced nucleosomal DNA fragments of the filamentous ascomycetes Aspergillus nidulans and Aspergillus oryzae and then mapped those sequences on their genomes. We compared the GC content and nucleosome density in the exonic and intronic regions in the genes of A. nidulans and A. oryzae. Although the GC content and nucleosome density in the exonic regions tended to be higher than those in the intronic regions, the difference in the distribution of the GC content was more notable than that of the nucleosome density. Next, we compared the GC content and nucleosome density in the exonic regions of 9616 orthologous gene pairs. In both Aspergillus species, the GC content did not correlate with the nucleosome density. In addition, the Spearman's rank correlation coefficient (ρ=0.51) between the GC content of the exonic regions of the 9616 orthologous gene pairs was higher than that (ρ=0.31) of the nucleosome densities of A. nidulans and A. oryzae. These results strongly suggest that the GC content in the exons of the orthologous gene pairs has been conserved during evolution but the nucleosome density has varied throughout.

Involvement of protein kinase C in the suppression of apoptosis and in polarity establishment in Aspergillus nidulans under conditions of heat stress.

The pkcA gene, which encodes a protein kinase C (PKC) in the filamentous fungus Aspergillus nidulans, is essential for its viability. However, little is known about its functions. To address this issue, we constructed and characterized temperature-sensitive mutants of pkcA. The conidia of these mutants swelled slightly and exhibited apoptotic phenotypes at 42°C. The apoptotic phenotypes were suppressed by an osmotic stabilizer. Under these conditions, the conidia swelled extensively and did not form germ tubes. Moreover, polarized distribution of F-actin was not observed. We then utilized deletion mutants of bckA, an ortholog of Saccharomyces cerevisiae bck1 that encodes a mitogen-activated protein (MAP) kinase kinase kinase and functions downstream of PKC in the cell wall integrity pathway. These mutants exhibited apoptotic phenotypes at 42°C, but they did not show defects in polarity establishment under osmotically stabilized conditions. These results suggest that PkcA plays multiple roles during germination under conditions of heat stress. The first of these roles is the suppression of apoptosis induction, while the other involves polarity establishment. The former depends on the MAP kinase cascade, whereas the latter does not. In addition, repolarization, which was observed after depolarization in the wild-type strain and the bckA deletion mutant under conditions of heat stress, was not observed in the pkcA-ts mutant. This suggests that PkcA also plays role in polarity establishment during hyphal growth independent of the MAP kinase cascade under these conditions.