Biology subjects

Miwa, K.

Publications and source records attributed to Miwa, K..

3 recordsLinked to original sources

bioRxiv2025

Establishment of an antimetabolite-based transformation system for the wood-decaying basidiomycete Phanerochaete chrysosporium

The model wood-decaying basidiomycete Phanerochaete chrysosporium has been extensively studied to elucidate the molecular mechanisms of wood decomposition. However, genetic studies have been limited by the lack of adequate genetic tools. Here, we established an antimetabolite-based transformation system, originally developed for ascomycetes, for use in P. chrysosporium. The transformation system utilizes pyrithiamine (PT), a thiamine antimetabolite, in combination with the pPTRII vector that contains the PT resistance gene (ptrA). PT effectively inhibited the growth of P. chrysosporium, and the introduction of ptrA conferred resistance to transformant mycelia. The transformation efficiency was comparable to that in ascomycetes, suggesting that the transformation system is also applicable to basidiomycetes. To examine the suitability of the system for heterologous gene expression, four cassettes were constructed to express GFP under the promoters of the actin1, DED, and GAPDH genes. Promoter activities were assessed via fluorescent microscopy observation of transformant mycelia and GFP quantification in crude cell extracts, revealing that the actin1 promoter drove the highest level of expression. Furthermore, truncating repeat sequences of the autonomously replicating sequence in the vector backbone improved transformation efficiency, likely due to the reduction in vector size. The transformation efficiency of the gene cassette-inserted vector in P. chrysosporium was relatively higher than that reported with alternative transformation systems in other species of wood- decaying basidiomycetes. The present transformation system could provide a platform for protein expression and genetic engineering in P. chrysosporium and potentially in other wood-decaying basidiomycetes. ImportanceWood-decaying basidiomycetes are well-recognized for their exceptional capabilities to decompose lignocellulosic biomass and oxidize a broad range of complex organic compounds. These capabilities are essential for maintaining the forest ecosystem and hold potential in biotechnological applications such as transforming recalcitrant biomass into useful compounds and degrading toxic substances in industrial effluents. However, genetic manipulation in basidiomycetes remains challenging because of the inefficiency of transformation systems. In the model lignocellulose-degrading basidiomycete, P. chrysosporium, transformation methods using dominant markers are scarce and were reported over two decades ago, necessitating the re- establishment of a functional system compatible with modern genetic tools. In this study, an efficient genetic transformation system was achieved by using an antimetabolite-based selection strategy for P. chrysosporium. This transformation system would lay the foundation for advancing our understanding of the molecular mechanisms of wood decomposition and support the targeted optimization of basidiomycetes for various biotechnological applications.

microbiology↗

bioRxiv2023

Defense-related callose synthase PMR4 promotes root hair callose deposition and adaptation to phosphate deficiency in Arabidopsis thaliana

Plants acquire phosphorus (P) primarily as inorganic phosphate (Pi) from the soil. Under Pi deficiency, plants induce an array of physiological and morphological responses, termed phosphate starvation response (PSR), thereby increasing Pi acquisition and use efficiency. However, the mechanisms by which plants adapt to Pi deficiency remain to be elucidated. Here, we report that deposition of a {beta}-1,3-glucan polymer called callose is induced in Arabidopsis thaliana root hairs under Pi deficiency, in a manner independent of PSR-regulating PHR1/PHL1 transcription factors and LPR1/LPR2 ferroxidases. Genetic studies revealed PMR4 (GSL5) callose synthase being required for the callose deposition in Pi-depleted root hairs. Loss of PMR4 also reduces Pi acquisition in shoots and plant growth under low Pi conditions. The defects are not recovered by simultaneous disruption of SID2, mediating defense-associated salicylic acid (SA) biosynthesis, excluding SA defense activation from the cause of the observed pmr4 phenotypes. Grafting experiments and characterization of plants expressing PMR4 specifically in root hair cells suggest that a PMR4 pool in the cell type contributes to shoot growth under Pi deficiency. Our findings thus suggest an important role for PMR4 in plant adaptation to Pi deficiency. Significance statementWe reveal that PMR4 callose synthase mediates callose deposition in root hairs under phosphate (Pi) deficiency, without requiring Pi starvation response regulators PHR1/PHL1 or LPR1/LPR2. The loss of the callose deposition is accompanied by decreases in Pi acquisition and plant growth in pmr4. Root hair cell-specific PMR4 expression restores callose deposition in root hairs and shoot growth under Pi deficiency, indicating a critical role for root hair callose in plant adaptation to Pi deficiency.

plant biology↗

bioRxiv2020

Elucidation of the antiviral mechanism of cystine and theanine through transcriptome analysis of mice and comparison with COVID-19 gene set data

We previously showed that oral administration of cystine and theanine (CT) to mice confers resistance to influenza virus infection. In human studies, CT prevented colds in healthy subjects and enhanced antibody production after influenza vaccination in elderly individuals with a poor nutritional status. The mechanism of action of CT is thought to be glutathione (GSH)-mediated regulation of intracellular redox, which might affect innate immune systems such as macrophages to exert physiological effects. The effect of CT on influenza is independent of viral type, and this treatment has a broad range of antiviral activities. To explore the mechanisms of CT in viral infection, we performed transcriptome profiling of spleen tissues isolated from influenza A virus (IAV)-infected mice. We identified unique gene signatures in response to CT in the IAV-infected mice. Genes upregulated by CT included redox-regulated genes such as GCLC/GCLM (subunits of glutamate cysteine ligase, a rate-limiting enzyme of GSH biosynthesis), TXN1, TXN2, TXNRD2, and SOD1, suggesting that the intracellular redox environment is substantially altered by CT. However, genes downregulated in response to CT included chemokine/chemokine receptor genes (CCL5, CCL19, CXCL9, CXCL12, CXCR3, CXCR4, and ACKR3), some of which are related to cytokine storm. A comparison with public COVID-19-related gene set data showed that the upregulated gene signature was highly similar to the downregulated gene sets of SARS-CoV/SARS-CoV-2-infected cells and the upregulated gene set of attenuated SARS-CoV-infected cells. In conclusion, the unique gene signatures observed in response to orally administered CT in IAV-infected mouse spleen tissues suggested that CT may attenuate viral infection, replication and associated symptoms such as cytokine storm.

molecular biology↗