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

Mamun, Md. Abdulla Al; Katayama, Takuya; Cao, Wei; Nakamura, Shugo; Maruyama, Jun‐ichi

doi:10.1111/mmi.14463

Cited by 14 publications

(72 citation statements)

References 68 publications

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“…2a). The expression cassettes were ectopically inserted into the niaD locus of A. oryzae strain NSlD1 10 by homologous recombination (Fig. 2a and Supplementary Data 3: Strain list).…”

Section: Resultsmentioning

confidence: 99%

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Multiple genes evolved for fungal septal pore plugging identified via large-scale localization and functional screenings

Mamun

Cao

Nakamura

et al. 2022

Preprint

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Multicellular organisms exhibit cytoplasmic exchange using porous structures for cooperation among cells. Fungal multicellular lineages have evolved septal pores for this function. Interconnected hyphal cells possess the risk of wound-related cytoplasmic loss unless the septal pores are plugged. However, the gene evolution of regulatory mechanisms underlying fungal septal pore plugging remains poorly understood. To identify novel septal components, 776 uncharacterized proteins were identified using genomic comparisons between septal pore-bearing and -lacking ascomycete species. We then determined their subcellular localizations, and in total 62 proteins localized to the septum or septal pore. We analyzed the effects of deleting the encoding genes on septal pore plugging upon hyphal wounding. Of the 62 proteins, 23 were involved in regulating septal pore plugging. Here, using orthologous group and phylogenetic analyses, this study suggests that septal pore regulation has evolved either by co-option of preexisting genes or by Pezizomycotina-specific gene acquisition.

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“…2a). The expression cassettes were ectopically inserted into the niaD locus of A. oryzae strain NSlD1 10 by homologous recombination (Fig. 2a and Supplementary Data 3: Strain list).…”

Section: Resultsmentioning

confidence: 99%

“…Cell-to-cell connectivity via gap junctions 54 , plasmodesmata 55 , and septal pores 10 can be blocked in response to both biotic and abiotic stresses. In this study, the status of cell-to-cell connectivity was quantitively analyzed in response to cold stress (Fig.…”

Section: Discussionmentioning

confidence: 99%

Multiple genes evolved for fungal septal pore plugging identified via large-scale localization and functional screenings

Mamun

Cao

Nakamura

et al. 2022

Preprint

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“…To construct strains expressing organelle marker fluorescence proteins, donor plasmids were constructed. To construct the donor plasmid pwAup-PH1-H1-mCherry, the DNA fragment containing the mCherry-encoding gene and the amyB terminator (TamyB) amplified from pisCIIA-mCherry (41) were inserted into the SmaI site of pisC ( 41 To construct the donor plasmid pwAup-Cit1-EGFP-amyBPT, the fragment of Aocit1 was amplified from the pgACEN plasmid (43) and ligated with the SmaI-digested pwAupEGFP plasmid (40).…”

Section: Plasmid Constructionmentioning

confidence: 99%

Mitochondrial fission dysfunction alleviates heterokaryon incompatibility-triggered cell death in the industrial filamentous fungus Aspergillus oryzae

Katayama

Mori

et al. 2021

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In filamentous fungi, cell-to-cell recognition is a fundamental requirement for the formation, development, and maintenance of complex hyphal networks. Basically, self/compatible individuals within the fungal species are capable of fusing together, a step important for crossbreeding, which results in the formation of viable vegetative heterokaryons. Conversely, the fusion of incompatible individuals does not result in the formation of viable hyphal networks, but it often leads to growth inhibition or cell death. Even though a number of studies have been conducted to investigate such incompatibility, the understanding of the associated molecular mechanism is still limited, and this restricts the possibility of crossbreeding incompatible individuals. Therefore, in this study, the characteristics of compatibility/incompatibility in the industrial filamentous fungus, Aspergillus oryzae, were comprehensively investigated. Protoplast fusion and co-culture assays indicated the existence of a correlation between strain phylogeny and compatibility/incompatibility features. Time-course fluorescence observations were employed to investigate the types of incompatible responses that are induced at different cellular levels upon incompatible cell fusion, which eventually lead to cell death. Propidium iodide-indicated cell death, ROS accumulation, and mitochondrial fragmentation were identified as the major responses, with mitochondrial fragmentation showing the most significant subcellular change immediately after incompatible cell fusion. Furthermore, the deletions of mitochondrial fission-related genes Aofis1 and Aodnm1 in incompatible pairing alleviated cell death, indicating that mitochondrial fission is an important mechanism by which incompatibility-triggered cell death occurs. Therefore, this study provides new insights about heterokaryon incompatibility.IMPORTANCEFor a long time, it was believed that as an asexual fungus, A. oryzae does not exhibit any sexual cycle. However, the fungus has two mating types, indicating the potential for sexual reproduction besides a known parasexual cycle. Therefore, given that viable heterokaryon formation following cell fusion is an important step required for genetic crossing, we explored the mechanism of incompatibility, which restricts the possibility of cell fusion in A. oryzae. Protoplast fusion and co-culture assays led to the identification of various vegetative compatible groups. Mitochondrial fragmentation was found to be the most significant incompatible cellular response that occurred in organelles during incompatible pairing, while the deletion of mitochondrial fission-related genes was identified as a strategy used to alleviate incompatibility-triggered cell death. Thus, this study revealed a novel mechanism by which mitochondrial fission regulates incompatible responses.

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“…The A. oryzae strains used in this study for antibody production are listed in the Table 1 for KRGGG (cleavage site for Kex2-like protease) and the mature heavy or light chain of adalimumab. Vectors pUtNAN [60] and pisCIIA [61] were used for the transformation of A. oryzae to introduce expression cassettes containing adalimumab's heavy chain or light chain into the niaD and sC loci, respectively. These vectors contain the dextrin-inducible amyB promoter and amyB terminator, and the expression cassette was inserted at the SmaI site located at the downstream of the amyB promoter.…”

Section: Strains and Growth Mediamentioning

confidence: 99%

Functional production of human antibody by the filamentous fungus Aspergillus oryzae

Huynh

Morita

Sakamoto

et al. 2020

Fungal Biol Biotechnol

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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.

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

Cited by 14 publications

References 68 publications

Multiple genes evolved for fungal septal pore plugging identified via large-scale localization and functional screenings

Multiple genes evolved for fungal septal pore plugging identified via large-scale localization and functional screenings

Mitochondrial fission dysfunction alleviates heterokaryon incompatibility-triggered cell death in the industrial filamentous fungus Aspergillus oryzae

Functional production of human antibody by the filamentous fungus Aspergillus oryzae

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