The Experts below are selected from a list of 1299 Experts worldwide ranked by ideXlab platform
Sheng Yuan - One of the best experts on this subject based on the ideXlab platform.
-
Glucanase-Induced Stipe Wall Extension Shows Distinct Differences from Chitinase-Induced Stipe Wall Extension of Coprinopsis Cinerea.
Applied and environmental microbiology, 2019Co-Authors: Liqin Kang, Zhonghua Liu, Jiangsheng Zhou, Cuicui Liu, Rui Wang, Xingwei Zhang, Sheng YuanAbstract:ABSTRACT This study reports that a high concentration of the endo-β-1,3-glucanase ENG (200 μg ml−1) induced heat-inactivated stipe wall extension of Coprinopsis Cinerea, whereas a high concentration of the extracellular β-glucosidase BGL2 (1,000 μg ml−1) did not; however, in combination, low concentrations of ENG (25 μg ml−1) and BGL2 (260 μg ml−1) induced heat-inactivated stipe cell wall extension. In contrast to the previously reported chitinase-reconstituted stipe wall extension, β-1,3-glucanase-reconstituted heat-inactivated stipe cell wall extension initially exhibited a fast extension rate that quickly decreased to zero after approximately 60 min; the stipe cell wall extension induced by a high concentration of β-1,3-glucanase did not result in stipe breakage during measurement, and the inner surfaces of glucanase-reconstituted extended cell walls still remained as amorphous matrices that did not appear to have been damaged. These distinctive features of the β-1,3-glucanase-reconstituted wall extension may be because chitin chains are cross-linked not only to the nonreducing termini of the side chains and the backbones of β-1,6 branched β-1,3-glucans but also to other polysaccharides. Remarkably, a low concentration of either the β-1,3-glucanase ENG or of chitinase ChiE1 did not induce heat-inactivated stipe wall extension, but a combination of these two enzymes, each at a low concentration, showed stipe cell wall extension activity that exhibited a steady and continuous wall extension profile. Therefore, we concluded that the stipe cell wall extension is the result of the synergistic actions of glucanases and chitinases. IMPORTANCE We previously reported that the chitinase could induce stipe wall extension and was involved in stipe elongation growth of the mushroom Coprinopsis Cinerea. In this study, we explored that β-1,3-glucanase also induced stipe cell wall extension. Interestingly, the extension profile and extended ultra-architecture of β-1,3-glucanase-reconstituted stipe wall were different from those of chitinase-reconstituted stipe wall. However, β-1,3-glucanase cooperated with chitinase to induce stipe cell wall extension. The significance of this synergy between glucanases and chitinases is that it enables a low concentration of active enzymes to induce wall extension, and the involvement of β-1,3-glucanases is necessary for the cell wall remodeling and the addition of new β-glucans during stipe elongation growth.
-
Chitinases Play a Key Role in Stipe Cell Wall Extension in the Mushroom Coprinopsis Cinerea.
Applied and environmental microbiology, 2019Co-Authors: Jiangsheng Zhou, Wenming Zhang, Zhonghua Liu, Xin Niu, Liqin Kang, Cuicui Liu, Xiaojun Wang, Hailong Liu, Jean-paul Latgé, Sheng YuanAbstract:The elongation growth of the mushroom stipe is a characteristic but not well-understood morphogenetic event of basidiomycetes. We found that extending native stipe cell walls of Coprinopsis Cinerea were associated with the release of N-acetylglucosamine and chitinbiose and with chitinase activity. Two chitinases among all detected chitinases from C. Cinerea, ChiE1 and ChiIII, reconstituted heat-inactivated stipe wall extension and released N-acetylglucosamine and chitinbiose. Interestingly, both ChiE1 and ChiIII hydrolyze insoluble crystalline chitin powder, while other C. Cinerea chitinases do not, suggesting that crystalline chitin components of the stipe cell wall are the target of action for ChiE1 and ChiIII. ChiE1- or ChiIII-reconstituted heat-inactivated stipe walls showed maximal extension activity at pH 4.5, consistent with the optimal pH for native stipe wall extension in vitro; ChiE1- or ChiIII-reconstituted heat-inactivated stipe wall extension activities were associated with stipe elongation growth regions; and the combination of ChiE1 and ChiIII showed a synergism to reconstitute heat-inactivated stipe wall extension at a low action concentration. Field emission scanning electron microscopy (FESEM) images showed that the inner surface of acid-induced extended native stipe cell walls and ChiE1- or ChiIII-reconstituted extended heat-inactivated stipe cell walls exhibited a partially broken parallel microfibril architecture; however, these broken transversely arranged microfibrils were not observed in the unextended stipe cell walls that were induced by neutral pH buffer or heat inactivation. Double knockdown of ChiE1 and ChiIII resulted in the reduction of stipe elongation, mycelium growth, and heat-sensitive cell wall extension of native stipes. These results indicate a chitinase-hydrolyzing mechanism for stipe cell wall extension.IMPORTANCE A remarkable feature in the development of basidiomycete fruiting bodies is stipe elongation growth that results primarily from manifold cell elongation. Some scientists have suggested that stipe elongation is the result of enzymatic hydrolysis of cell wall polysaccharides, while other scientists have proposed the possibility that stipe elongation results from nonhydrolytic disruption of the hydrogen bonds between cell wall polysaccharides. Here, we show direct evidence for a chitinase-hydrolyzing mechanism of stipe cell wall elongation in the model mushroom Coprinopsis Cinerea that is different from the expansin nonhydrolysis mechanism of plant cell wall extension. We presumed that in the growing stipe cell walls, parallel chitin microfibrils are tethered by β-1,6-branched β-1,3-glucans, and that the breaking of the tether by chitinases leads to separation of these microfibrils to increase their spacing for insertion of new synthesized chitin and β-1,3-glucans under turgor pressure in vivo.
-
Characteristics, transcriptional patterns and possible physiological significance of glycoside hydrolase family 16 members in Coprinopsis Cinerea.
FEMS microbiology letters, 2019Co-Authors: Liqin Kang, Yang Bai, Yiting Zhu, Sheng YuanAbstract:The glycoside hydrolase (GH) 16 family of Coprinopsis Cinerea includes 15 members distributed in four subgroups (A1, A2, B and D) by phylogenetic analysis. The expression patterns match well with the requirement of wall-softening in the germination of basidiospores, hyphal growth and branching, primordium formation, stipe elongation, pileus expansion and autolysis. Remarkably, expression levels of different GH16 members varied with different morphogenetic events. Like orthologs of Aspergillus fumigatus GH16 glucanases (ENG2-5), which were expressed in the dormant conidia and conidiogenesis, and essential for segregation of conidia, some members such as ENG in the subgroup A1 in C. Cinerea were also predominantly expressed in dormant basidiospores, primordia and maturing pilei during basidiosporogenesis. In contrast, other members in subgroup A2, subgroup B or D were dominantly expressed in the germinating basidiospores, the growing mycelia, and the elongating stipes. We did not find the members of the GH81 or GH55 family in C. Cinerea genome, which was different from A. fumigatus. However, C. Cinerea contains an extra three subgroups (A2, B and D) compared with A. fumigatus. These extra subgroups of GH16 family members may function as those endo-β-1,3-glucanases belonging to other GH families in the development and growth of C. Cinerea.
-
ChiE1 from Coprinopsis Cinerea is Characterized as a Processive Exochitinase and Revealed to Have a Significant Synergistic Action with Endochitinase ChiIII on Chitin Degradation.
Journal of agricultural and food chemistry, 2018Co-Authors: Jiangsheng Zhou, Zhonghua Liu, Liqin Kang, Yang Bai, Lingling Chen, Yao Yang, Sheng YuanAbstract:Fruiting bodies that exhibit strong autolysis of Coprinopsis Cinerea are a good resource for the chitinolytic system. In this study, a new Chitinase ChiE1 from C. Cinerea was cloned, heterologously expressed, and characterized. Biochemical analysis demonstrated that ChiE1 is an exochitinase with a processive mode of action. Although ChiE1 contains only a single catalytic domain without a binding domain, it can bind to and degrade insoluble chitin powder and colloidal chitin. The combination of ChiE1 and C. Cinerea endochitinase ChiIII could increase the amount of reducing sugar released from chitin powder by approximately 120% compared to using ChiE1 and ChiIII alone. The synergistic action of ChiE1 and ChiIII on degradation of chitin powder is higher than all previously reported synergism of chitinases. The recombinant Chitinase ChiE1 expressed in Pichia pastoris may be used as a synergistic chitinase for a reconstituted chitinolytic system for agricultural, biological, and environmental applications.
-
improved polysaccharide production by homologous co overexpression of phosphoglucomutase and udp glucose pyrophosphorylase genes in the mushroom Coprinopsis Cinerea
Journal of Agricultural and Food Chemistry, 2018Co-Authors: Jiangsheng Zhou, Zhonghua Liu, Yang Bai, Rujuan Dai, Xiaoli Guo, Sheng YuanAbstract:Coprinopsis polysaccharides exhibit hypoglycemic and antioxidant activities. In this report, increases in polysaccharide production by homologous co-overexpression or individual homologous overexpression of phosphoglucomutase and UDP glucose pyrophosphorylase gene in Coprinopsis Cinerea, which participate in polysaccharide biosynthesis. The transcription levels of the target genes were upregulated significantly in the oePGM-UGP strain when compared with the oePGM or oeUGP strain. The maximum intracellular polysaccharide content obtained in the oePGM-UGP strain was 1.49-fold higher than that of the WT strain, whereas a slight improvement in polysaccharide production was obtained in the oePGM and oeUGP strains. Extracellular polysaccharide production was enhanced by 75% in the oePGM-UGP strain when compared with that of the WT strain, whereas improvements of 30% and 16% were observed for the oePGM and oeUGP strains, respectively. These results show that multiple interventions in polysaccharide biosynthesis ...
Zhonghua Liu - One of the best experts on this subject based on the ideXlab platform.
-
Glucanase-Induced Stipe Wall Extension Shows Distinct Differences from Chitinase-Induced Stipe Wall Extension of Coprinopsis Cinerea.
Applied and environmental microbiology, 2019Co-Authors: Liqin Kang, Zhonghua Liu, Jiangsheng Zhou, Cuicui Liu, Rui Wang, Xingwei Zhang, Sheng YuanAbstract:ABSTRACT This study reports that a high concentration of the endo-β-1,3-glucanase ENG (200 μg ml−1) induced heat-inactivated stipe wall extension of Coprinopsis Cinerea, whereas a high concentration of the extracellular β-glucosidase BGL2 (1,000 μg ml−1) did not; however, in combination, low concentrations of ENG (25 μg ml−1) and BGL2 (260 μg ml−1) induced heat-inactivated stipe cell wall extension. In contrast to the previously reported chitinase-reconstituted stipe wall extension, β-1,3-glucanase-reconstituted heat-inactivated stipe cell wall extension initially exhibited a fast extension rate that quickly decreased to zero after approximately 60 min; the stipe cell wall extension induced by a high concentration of β-1,3-glucanase did not result in stipe breakage during measurement, and the inner surfaces of glucanase-reconstituted extended cell walls still remained as amorphous matrices that did not appear to have been damaged. These distinctive features of the β-1,3-glucanase-reconstituted wall extension may be because chitin chains are cross-linked not only to the nonreducing termini of the side chains and the backbones of β-1,6 branched β-1,3-glucans but also to other polysaccharides. Remarkably, a low concentration of either the β-1,3-glucanase ENG or of chitinase ChiE1 did not induce heat-inactivated stipe wall extension, but a combination of these two enzymes, each at a low concentration, showed stipe cell wall extension activity that exhibited a steady and continuous wall extension profile. Therefore, we concluded that the stipe cell wall extension is the result of the synergistic actions of glucanases and chitinases. IMPORTANCE We previously reported that the chitinase could induce stipe wall extension and was involved in stipe elongation growth of the mushroom Coprinopsis Cinerea. In this study, we explored that β-1,3-glucanase also induced stipe cell wall extension. Interestingly, the extension profile and extended ultra-architecture of β-1,3-glucanase-reconstituted stipe wall were different from those of chitinase-reconstituted stipe wall. However, β-1,3-glucanase cooperated with chitinase to induce stipe cell wall extension. The significance of this synergy between glucanases and chitinases is that it enables a low concentration of active enzymes to induce wall extension, and the involvement of β-1,3-glucanases is necessary for the cell wall remodeling and the addition of new β-glucans during stipe elongation growth.
-
Chitinases Play a Key Role in Stipe Cell Wall Extension in the Mushroom Coprinopsis Cinerea.
Applied and environmental microbiology, 2019Co-Authors: Jiangsheng Zhou, Wenming Zhang, Zhonghua Liu, Xin Niu, Liqin Kang, Cuicui Liu, Xiaojun Wang, Hailong Liu, Jean-paul Latgé, Sheng YuanAbstract:The elongation growth of the mushroom stipe is a characteristic but not well-understood morphogenetic event of basidiomycetes. We found that extending native stipe cell walls of Coprinopsis Cinerea were associated with the release of N-acetylglucosamine and chitinbiose and with chitinase activity. Two chitinases among all detected chitinases from C. Cinerea, ChiE1 and ChiIII, reconstituted heat-inactivated stipe wall extension and released N-acetylglucosamine and chitinbiose. Interestingly, both ChiE1 and ChiIII hydrolyze insoluble crystalline chitin powder, while other C. Cinerea chitinases do not, suggesting that crystalline chitin components of the stipe cell wall are the target of action for ChiE1 and ChiIII. ChiE1- or ChiIII-reconstituted heat-inactivated stipe walls showed maximal extension activity at pH 4.5, consistent with the optimal pH for native stipe wall extension in vitro; ChiE1- or ChiIII-reconstituted heat-inactivated stipe wall extension activities were associated with stipe elongation growth regions; and the combination of ChiE1 and ChiIII showed a synergism to reconstitute heat-inactivated stipe wall extension at a low action concentration. Field emission scanning electron microscopy (FESEM) images showed that the inner surface of acid-induced extended native stipe cell walls and ChiE1- or ChiIII-reconstituted extended heat-inactivated stipe cell walls exhibited a partially broken parallel microfibril architecture; however, these broken transversely arranged microfibrils were not observed in the unextended stipe cell walls that were induced by neutral pH buffer or heat inactivation. Double knockdown of ChiE1 and ChiIII resulted in the reduction of stipe elongation, mycelium growth, and heat-sensitive cell wall extension of native stipes. These results indicate a chitinase-hydrolyzing mechanism for stipe cell wall extension.IMPORTANCE A remarkable feature in the development of basidiomycete fruiting bodies is stipe elongation growth that results primarily from manifold cell elongation. Some scientists have suggested that stipe elongation is the result of enzymatic hydrolysis of cell wall polysaccharides, while other scientists have proposed the possibility that stipe elongation results from nonhydrolytic disruption of the hydrogen bonds between cell wall polysaccharides. Here, we show direct evidence for a chitinase-hydrolyzing mechanism of stipe cell wall elongation in the model mushroom Coprinopsis Cinerea that is different from the expansin nonhydrolysis mechanism of plant cell wall extension. We presumed that in the growing stipe cell walls, parallel chitin microfibrils are tethered by β-1,6-branched β-1,3-glucans, and that the breaking of the tether by chitinases leads to separation of these microfibrils to increase their spacing for insertion of new synthesized chitin and β-1,3-glucans under turgor pressure in vivo.
-
ChiE1 from Coprinopsis Cinerea is Characterized as a Processive Exochitinase and Revealed to Have a Significant Synergistic Action with Endochitinase ChiIII on Chitin Degradation.
Journal of agricultural and food chemistry, 2018Co-Authors: Jiangsheng Zhou, Zhonghua Liu, Liqin Kang, Yang Bai, Lingling Chen, Yao Yang, Sheng YuanAbstract:Fruiting bodies that exhibit strong autolysis of Coprinopsis Cinerea are a good resource for the chitinolytic system. In this study, a new Chitinase ChiE1 from C. Cinerea was cloned, heterologously expressed, and characterized. Biochemical analysis demonstrated that ChiE1 is an exochitinase with a processive mode of action. Although ChiE1 contains only a single catalytic domain without a binding domain, it can bind to and degrade insoluble chitin powder and colloidal chitin. The combination of ChiE1 and C. Cinerea endochitinase ChiIII could increase the amount of reducing sugar released from chitin powder by approximately 120% compared to using ChiE1 and ChiIII alone. The synergistic action of ChiE1 and ChiIII on degradation of chitin powder is higher than all previously reported synergism of chitinases. The recombinant Chitinase ChiE1 expressed in Pichia pastoris may be used as a synergistic chitinase for a reconstituted chitinolytic system for agricultural, biological, and environmental applications.
-
improved polysaccharide production by homologous co overexpression of phosphoglucomutase and udp glucose pyrophosphorylase genes in the mushroom Coprinopsis Cinerea
Journal of Agricultural and Food Chemistry, 2018Co-Authors: Jiangsheng Zhou, Zhonghua Liu, Yang Bai, Rujuan Dai, Xiaoli Guo, Sheng YuanAbstract:Coprinopsis polysaccharides exhibit hypoglycemic and antioxidant activities. In this report, increases in polysaccharide production by homologous co-overexpression or individual homologous overexpression of phosphoglucomutase and UDP glucose pyrophosphorylase gene in Coprinopsis Cinerea, which participate in polysaccharide biosynthesis. The transcription levels of the target genes were upregulated significantly in the oePGM-UGP strain when compared with the oePGM or oeUGP strain. The maximum intracellular polysaccharide content obtained in the oePGM-UGP strain was 1.49-fold higher than that of the WT strain, whereas a slight improvement in polysaccharide production was obtained in the oePGM and oeUGP strains. Extracellular polysaccharide production was enhanced by 75% in the oePGM-UGP strain when compared with that of the WT strain, whereas improvements of 30% and 16% were observed for the oePGM and oeUGP strains, respectively. These results show that multiple interventions in polysaccharide biosynthesis ...
-
Improved Polysaccharide Production by Homologous Co-overexpression of Phosphoglucomutase and UDP Glucose Pyrophosphorylase Genes in the Mushroom Coprinopsis Cinerea
2018Co-Authors: Jiangsheng Zhou, Zhonghua Liu, Yang Bai, Rujuan Dai, Xiaoli Guo, Sheng YuanAbstract:Coprinopsis polysaccharides exhibit hypoglycemic and antioxidant activities. In this report, increases in polysaccharide production by homologous co-overexpression or individual homologous overexpression of phosphoglucomutase and UDP glucose pyrophosphorylase gene in Coprinopsis Cinerea, which participate in polysaccharide biosynthesis. The transcription levels of the target genes were upregulated significantly in the oePGM-UGP strain when compared with the oePGM or oeUGP strain. The maximum intracellular polysaccharide content obtained in the oePGM-UGP strain was 1.49-fold higher than that of the WT strain, whereas a slight improvement in polysaccharide production was obtained in the oePGM and oeUGP strains. Extracellular polysaccharide production was enhanced by 75% in the oePGM-UGP strain when compared with that of the WT strain, whereas improvements of 30% and 16% were observed for the oePGM and oeUGP strains, respectively. These results show that multiple interventions in polysaccharide biosynthesis pathways of Basidiomycetes might improve polysaccharide yields when compared with that of single interventions
Hajime Muraguchi - One of the best experts on this subject based on the ideXlab platform.
-
Identification of a substrate of the methyltransferase Ich1 involved in cap differentiation in Coprinopsis Cinerea
Impact, 2020Co-Authors: Hajime MuraguchiAbstract:The ichijiku1 (ich1) gene is an essential controller of the formation of the 'cap', or pileus in the fruiting of Coprinopsis Cinerea, an edible mushroom more commonly known as grey shag. The ich1 gene encodes for the Ich1 protein, a methyltransferase enzyme with a winged helix-like DNA-binding domain as well as an o-methyltransferase domain. These structural features contribute towards its rather unique molecular mechanisms. Formation of the pileus represents a crucial part of the reproductive process of C. Cinerea. Thus, mutations or environmental conditions that affect pileus formation are likely to have a dramatic impact on the survival of such strains of the fungus. Dr Hajime Muraguchi, from Akita Prefectural University is leading a team that is seeking to elucidate the molecular mechanisms of the ich1 gene.
-
The Coprinopsis Cinerea Tup1 homologue Cag1 is required for gill formation during fruiting body morphogenesis
Biology open, 2016Co-Authors: Ryo Masuda, Naoki Iguchi, Kooki Tukuta, Takahiro Nagoshi, Kazuki Kemuriyama, Hajime MuraguchiAbstract:ABSTRACT The pileus (cap) of the fruiting body in homobasidiomycete fungi bears the hymenium, a layer of cells that includes the basidia where nuclear fusion, meiosis and sporulation occur. Coprinopsis Cinerea is a model system for studying fruiting body development. The hymenium of C. Cinerea forms at the surface of the gills in the pileus. In a previous study, we identified a mutation called cap-growthless1-1 (cag1-1) that blocks gill formation, which yields primordia that never mature. In this study, we found that the cag1 gene encodes a homologue of Saccharomyces cerevisiae Tup1. The C. Cinerea genome contains another Tup1 homologue gene called Cc.tupA. Reciprocal tagging of Cag1 and Cc.TupA with green and red fluorescent proteins revealed that the relative ratios of the amounts of the two Tup1 paralogues varied among tissues. Compared with Cc.TupA, Cag1 was preferentially expressed in the gill trama tissue cells, suggesting that the function of Cag1 is required for gill trama tissue differentiation and maintenance. Yeast two-hybrid analysis and co-localisation of Cag1 and Cc.TupA suggested that Cag1 interacts with Cc.TupA in the nuclei of certain cells.
-
RESEARCH ARTICLE Strand-Specific RNA-Seq Analyses of Fruiting Body Development in Coprinopsis Cinerea
2016Co-Authors: Hajime Muraguchi, Mai Niikura, Toshinori Kozaki, Kiyota Sakai, Kiyoshi Nakahori, Motoyuki Shimizu, Kiwamu Umezawa, Kazuo Ishii, Makoto Yoshida, Yuichi SakamotoAbstract:The basidiomycete fungus Coprinopsis Cinerea is an important model system for multicellu-lar development. Fruiting bodies of C. Cinerea are typical mushrooms, which can be pro-duced synchronously on defined media in the laboratory. To investigate the transcriptome in detail during fruiting body development, high-throughput sequencing (RNA-seq) was per-formed using cDNA libraries strand-specifically constructed from 13 points (stages/tissues) with two biological replicates. The reads were aligned to 14,245 predicted transcripts, and counted for forward and reverse transcripts. Differentially expressed genes (DEGs) between two adjacent points and between vegetative mycelium and each point were detected by Tag Count Comparison (TCC). To validate RNA-seq data, expression levels of selected genes were compared using RPKM values in RNA-seq data and qRT-PCR data, and DEGs detected in microarray data were examined in MA plots of RNA-seq data by TCC. We discuss events deduced from GO analysis of DEGs. In addition, we uncovere
-
strand specific rna seq analyses of fruiting body development in Coprinopsis Cinerea
PLOS ONE, 2015Co-Authors: Hajime Muraguchi, Mai Niikura, Toshinori Kozaki, Kiyota Sakai, Kiyoshi Nakahori, Motoyuki Shimizu, Kiwamu Umezawa, Kazuo Ishii, Makoto Yoshida, Yuichi SakamotoAbstract:The basidiomycete fungus Coprinopsis Cinerea is an important model system for multicellular development. Fruiting bodies of C. Cinerea are typical mushrooms, which can be produced synchronously on defined media in the laboratory. To investigate the transcriptome in detail during fruiting body development, high-throughput sequencing (RNA-seq) was performed using cDNA libraries strand-specifically constructed from 13 points (stages/tissues) with two biological replicates. The reads were aligned to 14,245 predicted transcripts, and counted for forward and reverse transcripts. Differentially expressed genes (DEGs) between two adjacent points and between vegetative mycelium and each point were detected by Tag Count Comparison (TCC). To validate RNA-seq data, expression levels of selected genes were compared using RPKM values in RNA-seq data and qRT-PCR data, and DEGs detected in microarray data were examined in MA plots of RNA-seq data by TCC. We discuss events deduced from GO analysis of DEGs. In addition, we uncovered both transcription factor candidates and antisense transcripts that are likely to be involved in developmental regulation for fruiting.
-
The Coprinopsis Cinerea septin Cc.Cdc3 is involved in stipe cell elongation.
Fungal genetics and biology : FG & B, 2013Co-Authors: Tatsuhiro Shioya, Takashi Kamada, Yuichi Sakamoto, Hiroe Nakamura, Noriyoshi Ishii, Naoki Takahashi, Noriaki Ozaki, Masayuki Kobayashi, Keiju Okano, Hajime MuraguchiAbstract:We have identified and characterized a Coprinopsis Cinerea mutant defective in stipe elongation during fruiting body development. In the wild-type, stipe cells elongate at the maturation stage of fruiting, resulting in very slender cells. In the mutant, the stipe cells fail to elongate, but become rather globular at the maturation stage. We found that the mutant phenotype is rescued by a gene encoding a homolog of Saccharomyces cerevisiae CDC3 septin, Cc.Cdc3. The C. Cinerea genome includes 6 septin genes, 5 of which, including Cc.cdc3, are highly transcribed during stipe elongation in the wild type. In the mutant, the level of Cc.cdc3 transcription in the stipe cells remains the same as that in the mycelium, and the level of Cc.cdc10 transcription is approximately 100 times lower than that in the wild-type stipe cells. No increase in transcription of Cc.cdc3 in the mutant may be due to the fact that the Cc.cdc3 gene has a 4-base pair insertion in its promoter and/or that the promoter region is methylated in the mutant. Overexpressed EGFP-Cc.Cdc3 fusion protein rescues the stipe elongation in the transformants, localizes to the cell cortex and assembles into abundant thin filaments in the elongating stipe cells. In contrast, in vegetative hyphae, EGFP-Cc.Cdc3 is localized to the hyphal tips of the apical cells of hyphae. Cellular defects in the mutant, combined with the localization of EGFP-Cc.Cdc3, suggest that septin filaments in the cell cortex provide the localized rigidity to the plasma membrane and allow cells to elongate cylindrically.
Liqin Kang - One of the best experts on this subject based on the ideXlab platform.
-
Glucanase-Induced Stipe Wall Extension Shows Distinct Differences from Chitinase-Induced Stipe Wall Extension of Coprinopsis Cinerea.
Applied and environmental microbiology, 2019Co-Authors: Liqin Kang, Zhonghua Liu, Jiangsheng Zhou, Cuicui Liu, Rui Wang, Xingwei Zhang, Sheng YuanAbstract:ABSTRACT This study reports that a high concentration of the endo-β-1,3-glucanase ENG (200 μg ml−1) induced heat-inactivated stipe wall extension of Coprinopsis Cinerea, whereas a high concentration of the extracellular β-glucosidase BGL2 (1,000 μg ml−1) did not; however, in combination, low concentrations of ENG (25 μg ml−1) and BGL2 (260 μg ml−1) induced heat-inactivated stipe cell wall extension. In contrast to the previously reported chitinase-reconstituted stipe wall extension, β-1,3-glucanase-reconstituted heat-inactivated stipe cell wall extension initially exhibited a fast extension rate that quickly decreased to zero after approximately 60 min; the stipe cell wall extension induced by a high concentration of β-1,3-glucanase did not result in stipe breakage during measurement, and the inner surfaces of glucanase-reconstituted extended cell walls still remained as amorphous matrices that did not appear to have been damaged. These distinctive features of the β-1,3-glucanase-reconstituted wall extension may be because chitin chains are cross-linked not only to the nonreducing termini of the side chains and the backbones of β-1,6 branched β-1,3-glucans but also to other polysaccharides. Remarkably, a low concentration of either the β-1,3-glucanase ENG or of chitinase ChiE1 did not induce heat-inactivated stipe wall extension, but a combination of these two enzymes, each at a low concentration, showed stipe cell wall extension activity that exhibited a steady and continuous wall extension profile. Therefore, we concluded that the stipe cell wall extension is the result of the synergistic actions of glucanases and chitinases. IMPORTANCE We previously reported that the chitinase could induce stipe wall extension and was involved in stipe elongation growth of the mushroom Coprinopsis Cinerea. In this study, we explored that β-1,3-glucanase also induced stipe cell wall extension. Interestingly, the extension profile and extended ultra-architecture of β-1,3-glucanase-reconstituted stipe wall were different from those of chitinase-reconstituted stipe wall. However, β-1,3-glucanase cooperated with chitinase to induce stipe cell wall extension. The significance of this synergy between glucanases and chitinases is that it enables a low concentration of active enzymes to induce wall extension, and the involvement of β-1,3-glucanases is necessary for the cell wall remodeling and the addition of new β-glucans during stipe elongation growth.
-
Chitinases Play a Key Role in Stipe Cell Wall Extension in the Mushroom Coprinopsis Cinerea.
Applied and environmental microbiology, 2019Co-Authors: Jiangsheng Zhou, Wenming Zhang, Zhonghua Liu, Xin Niu, Liqin Kang, Cuicui Liu, Xiaojun Wang, Hailong Liu, Jean-paul Latgé, Sheng YuanAbstract:The elongation growth of the mushroom stipe is a characteristic but not well-understood morphogenetic event of basidiomycetes. We found that extending native stipe cell walls of Coprinopsis Cinerea were associated with the release of N-acetylglucosamine and chitinbiose and with chitinase activity. Two chitinases among all detected chitinases from C. Cinerea, ChiE1 and ChiIII, reconstituted heat-inactivated stipe wall extension and released N-acetylglucosamine and chitinbiose. Interestingly, both ChiE1 and ChiIII hydrolyze insoluble crystalline chitin powder, while other C. Cinerea chitinases do not, suggesting that crystalline chitin components of the stipe cell wall are the target of action for ChiE1 and ChiIII. ChiE1- or ChiIII-reconstituted heat-inactivated stipe walls showed maximal extension activity at pH 4.5, consistent with the optimal pH for native stipe wall extension in vitro; ChiE1- or ChiIII-reconstituted heat-inactivated stipe wall extension activities were associated with stipe elongation growth regions; and the combination of ChiE1 and ChiIII showed a synergism to reconstitute heat-inactivated stipe wall extension at a low action concentration. Field emission scanning electron microscopy (FESEM) images showed that the inner surface of acid-induced extended native stipe cell walls and ChiE1- or ChiIII-reconstituted extended heat-inactivated stipe cell walls exhibited a partially broken parallel microfibril architecture; however, these broken transversely arranged microfibrils were not observed in the unextended stipe cell walls that were induced by neutral pH buffer or heat inactivation. Double knockdown of ChiE1 and ChiIII resulted in the reduction of stipe elongation, mycelium growth, and heat-sensitive cell wall extension of native stipes. These results indicate a chitinase-hydrolyzing mechanism for stipe cell wall extension.IMPORTANCE A remarkable feature in the development of basidiomycete fruiting bodies is stipe elongation growth that results primarily from manifold cell elongation. Some scientists have suggested that stipe elongation is the result of enzymatic hydrolysis of cell wall polysaccharides, while other scientists have proposed the possibility that stipe elongation results from nonhydrolytic disruption of the hydrogen bonds between cell wall polysaccharides. Here, we show direct evidence for a chitinase-hydrolyzing mechanism of stipe cell wall elongation in the model mushroom Coprinopsis Cinerea that is different from the expansin nonhydrolysis mechanism of plant cell wall extension. We presumed that in the growing stipe cell walls, parallel chitin microfibrils are tethered by β-1,6-branched β-1,3-glucans, and that the breaking of the tether by chitinases leads to separation of these microfibrils to increase their spacing for insertion of new synthesized chitin and β-1,3-glucans under turgor pressure in vivo.
-
Characteristics, transcriptional patterns and possible physiological significance of glycoside hydrolase family 16 members in Coprinopsis Cinerea.
FEMS microbiology letters, 2019Co-Authors: Liqin Kang, Yang Bai, Yiting Zhu, Sheng YuanAbstract:The glycoside hydrolase (GH) 16 family of Coprinopsis Cinerea includes 15 members distributed in four subgroups (A1, A2, B and D) by phylogenetic analysis. The expression patterns match well with the requirement of wall-softening in the germination of basidiospores, hyphal growth and branching, primordium formation, stipe elongation, pileus expansion and autolysis. Remarkably, expression levels of different GH16 members varied with different morphogenetic events. Like orthologs of Aspergillus fumigatus GH16 glucanases (ENG2-5), which were expressed in the dormant conidia and conidiogenesis, and essential for segregation of conidia, some members such as ENG in the subgroup A1 in C. Cinerea were also predominantly expressed in dormant basidiospores, primordia and maturing pilei during basidiosporogenesis. In contrast, other members in subgroup A2, subgroup B or D were dominantly expressed in the germinating basidiospores, the growing mycelia, and the elongating stipes. We did not find the members of the GH81 or GH55 family in C. Cinerea genome, which was different from A. fumigatus. However, C. Cinerea contains an extra three subgroups (A2, B and D) compared with A. fumigatus. These extra subgroups of GH16 family members may function as those endo-β-1,3-glucanases belonging to other GH families in the development and growth of C. Cinerea.
-
ChiE1 from Coprinopsis Cinerea is Characterized as a Processive Exochitinase and Revealed to Have a Significant Synergistic Action with Endochitinase ChiIII on Chitin Degradation.
Journal of agricultural and food chemistry, 2018Co-Authors: Jiangsheng Zhou, Zhonghua Liu, Liqin Kang, Yang Bai, Lingling Chen, Yao Yang, Sheng YuanAbstract:Fruiting bodies that exhibit strong autolysis of Coprinopsis Cinerea are a good resource for the chitinolytic system. In this study, a new Chitinase ChiE1 from C. Cinerea was cloned, heterologously expressed, and characterized. Biochemical analysis demonstrated that ChiE1 is an exochitinase with a processive mode of action. Although ChiE1 contains only a single catalytic domain without a binding domain, it can bind to and degrade insoluble chitin powder and colloidal chitin. The combination of ChiE1 and C. Cinerea endochitinase ChiIII could increase the amount of reducing sugar released from chitin powder by approximately 120% compared to using ChiE1 and ChiIII alone. The synergistic action of ChiE1 and ChiIII on degradation of chitin powder is higher than all previously reported synergism of chitinases. The recombinant Chitinase ChiE1 expressed in Pichia pastoris may be used as a synergistic chitinase for a reconstituted chitinolytic system for agricultural, biological, and environmental applications.
-
ChiE1 from Coprinopsis Cinerea is Characterized as a Processive Exochitinase and Revealed to Have a Significant Synergistic Action with Endochitinase ChiIII on Chitin Degradation
2018Co-Authors: Jiangsheng Zhou, Zhonghua Liu, Liqin Kang, Yang Bai, Lingling Chen, Yao Yang, Sheng YuanAbstract:Fruiting bodies that exhibit strong autolysis of Coprinopsis Cinerea are a good resource for the chitinolytic system. In this study, a new Chitinase ChiE1 from C. Cinerea was cloned, heterologously expressed, and characterized. Biochemical analysis demonstrated that ChiE1 is an exochitinase with a processive mode of action. Although ChiE1 contains only a single catalytic domain without a binding domain, it can bind to and degrade insoluble chitin powder and colloidal chitin. The combination of ChiE1 and C. Cinerea endochitinase ChiIII could increase the amount of reducing sugar released from chitin powder by approximately 120% compared to using ChiE1 and ChiIII alone. The synergistic action of ChiE1 and ChiIII on degradation of chitin powder is higher than all previously reported synergism of chitinases. The recombinant Chitinase ChiE1 expressed in Pichia pastoris may be used as a synergistic chitinase for a reconstituted chitinolytic system for agricultural, biological, and environmental applications
Yuichi Sakamoto - One of the best experts on this subject based on the ideXlab platform.
-
RESEARCH ARTICLE Strand-Specific RNA-Seq Analyses of Fruiting Body Development in Coprinopsis Cinerea
2016Co-Authors: Hajime Muraguchi, Mai Niikura, Toshinori Kozaki, Kiyota Sakai, Kiyoshi Nakahori, Motoyuki Shimizu, Kiwamu Umezawa, Kazuo Ishii, Makoto Yoshida, Yuichi SakamotoAbstract:The basidiomycete fungus Coprinopsis Cinerea is an important model system for multicellu-lar development. Fruiting bodies of C. Cinerea are typical mushrooms, which can be pro-duced synchronously on defined media in the laboratory. To investigate the transcriptome in detail during fruiting body development, high-throughput sequencing (RNA-seq) was per-formed using cDNA libraries strand-specifically constructed from 13 points (stages/tissues) with two biological replicates. The reads were aligned to 14,245 predicted transcripts, and counted for forward and reverse transcripts. Differentially expressed genes (DEGs) between two adjacent points and between vegetative mycelium and each point were detected by Tag Count Comparison (TCC). To validate RNA-seq data, expression levels of selected genes were compared using RPKM values in RNA-seq data and qRT-PCR data, and DEGs detected in microarray data were examined in MA plots of RNA-seq data by TCC. We discuss events deduced from GO analysis of DEGs. In addition, we uncovere
-
strand specific rna seq analyses of fruiting body development in Coprinopsis Cinerea
PLOS ONE, 2015Co-Authors: Hajime Muraguchi, Mai Niikura, Toshinori Kozaki, Kiyota Sakai, Kiyoshi Nakahori, Motoyuki Shimizu, Kiwamu Umezawa, Kazuo Ishii, Makoto Yoshida, Yuichi SakamotoAbstract:The basidiomycete fungus Coprinopsis Cinerea is an important model system for multicellular development. Fruiting bodies of C. Cinerea are typical mushrooms, which can be produced synchronously on defined media in the laboratory. To investigate the transcriptome in detail during fruiting body development, high-throughput sequencing (RNA-seq) was performed using cDNA libraries strand-specifically constructed from 13 points (stages/tissues) with two biological replicates. The reads were aligned to 14,245 predicted transcripts, and counted for forward and reverse transcripts. Differentially expressed genes (DEGs) between two adjacent points and between vegetative mycelium and each point were detected by Tag Count Comparison (TCC). To validate RNA-seq data, expression levels of selected genes were compared using RPKM values in RNA-seq data and qRT-PCR data, and DEGs detected in microarray data were examined in MA plots of RNA-seq data by TCC. We discuss events deduced from GO analysis of DEGs. In addition, we uncovered both transcription factor candidates and antisense transcripts that are likely to be involved in developmental regulation for fruiting.
-
The Coprinopsis Cinerea septin Cc.Cdc3 is involved in stipe cell elongation.
Fungal genetics and biology : FG & B, 2013Co-Authors: Tatsuhiro Shioya, Takashi Kamada, Yuichi Sakamoto, Hiroe Nakamura, Noriyoshi Ishii, Naoki Takahashi, Noriaki Ozaki, Masayuki Kobayashi, Keiju Okano, Hajime MuraguchiAbstract:We have identified and characterized a Coprinopsis Cinerea mutant defective in stipe elongation during fruiting body development. In the wild-type, stipe cells elongate at the maturation stage of fruiting, resulting in very slender cells. In the mutant, the stipe cells fail to elongate, but become rather globular at the maturation stage. We found that the mutant phenotype is rescued by a gene encoding a homolog of Saccharomyces cerevisiae CDC3 septin, Cc.Cdc3. The C. Cinerea genome includes 6 septin genes, 5 of which, including Cc.cdc3, are highly transcribed during stipe elongation in the wild type. In the mutant, the level of Cc.cdc3 transcription in the stipe cells remains the same as that in the mycelium, and the level of Cc.cdc10 transcription is approximately 100 times lower than that in the wild-type stipe cells. No increase in transcription of Cc.cdc3 in the mutant may be due to the fact that the Cc.cdc3 gene has a 4-base pair insertion in its promoter and/or that the promoter region is methylated in the mutant. Overexpressed EGFP-Cc.Cdc3 fusion protein rescues the stipe elongation in the transformants, localizes to the cell cortex and assembles into abundant thin filaments in the elongating stipe cells. In contrast, in vegetative hyphae, EGFP-Cc.Cdc3 is localized to the hyphal tips of the apical cells of hyphae. Cellular defects in the mutant, combined with the localization of EGFP-Cc.Cdc3, suggest that septin filaments in the cell cortex provide the localized rigidity to the plasma membrane and allow cells to elongate cylindrically.
-
Identification and characterization of CcCTR1, a copper uptake transporter-like gene, in Coprinopsis Cinerea.
Microbiological research, 2009Co-Authors: Yuko Nakagawa, Yuichi Sakamoto, Sayaka Kikuchi, Akira YanoAbstract:Copper (Cu) is an essential element for the physiological function of organisms. In basidiomycetes, Cu is necessary for the production of phenol oxidase enzymes such as laccase and tyrosinase. We isolated and characterized two genes, CcCTR1 and -2, from the model basidiomycete Coprinopsis Cinerea. CcCTR1 and -2 showed similarity to the Cu transporter CTR1 in Saccharomyces cerevisiae. Both CcCTRs had a MLxxM motif that is conserved in other CTR homologs. The addition of Cu to a liquid culture of C. Cinerea decreased the mRNA accumulation of CcCTR1 and -2. Heterologous expression of CcCTR1 in S. cerevisiae increased Cu sensitivity, suggesting that CcCTR1 is a Cu uptake transporter. Together, these results suggest that CcCTR1 plays an important role in Cu accumulation in C. Cinerea.