The Experts below are selected from a list of 360 Experts worldwide ranked by ideXlab platform

Jianzhen Zhang - One of the best experts on this subject based on the ideXlab platform.

  • miR-71 and miR-263 Jointly Regulate Target Genes Chitin Synthase and Chitinase to Control Locust Molting
    2016
    Co-Authors: Meiling Yang, Jianzhen Zhang, Yanli Wang, Feng Jiang, Tianqi Song, Huimin Wang, Qing Liu, Jie Zhang, Le Kang
    Abstract:

    Chitin Synthase and Chitinase play crucial roles in Chitin biosynthesis and degradation during insect molting. Silencing of Dicer-1 results in reduced levels of mature miRNAs and severely blocks molting in the migratory locust. However, the regulatory mechanism of miRNAs in the molting process of locusts has remained elusive. In this study, we found that in Chitin metabolism, two crucial enzymes, Chitin Synthase (CHS) and Chitinase (CHT) were regulated by miR-71 and miR-263 during nymph molting. The coding sequence of CHS1 and the 3’-untranslated region of CHT10 contain functional binding sites for miR-71 and miR-263, respectively. miR-71/miR-263 displayed cellular co-localization with their target genes in epidermal cells and directly interacted with CHS1 and CHT10 in the locust integument, respectively. Injections of miR-71 and miR-263 agomirs suppressed the expression of CHS1 and CHT10, which consequently altered Chitin production of new and old cuticles and resulted in a molting-defective phenotype in locusts. Unexpectedly, reduced expression of miR-71 and miR-263 increased CHS1 and CHT10 mRNA expression and led to molting defects similar to those induced by miRNA delivery. This study reveals a novel function and balancing modulation pattern of two miRNAs in Chitin biosynthesis and degradation, and it provides insight into the underlying molecular mechanisms of the molting process in locusts.

  • characterization of a midgut specific Chitin Synthase gene lmchs2 responsible for biosynthesis of Chitin of peritrophic matrix in locusta migratoria
    Insect Biochemistry and Molecular Biology, 2012
    Co-Authors: Xiaojian Liu, Jianzhen Zhang, Kun Yan Zhu, Huanhuan Zhang
    Abstract:

    Chitin, an essential component of peritrophic matrix (PM), is produced by a series of biochemical reactions. Chitin Synthase plays a crucial role in Chitin polymerization in Chitin biosynthetic pathway. In this study, we identified and characterized a full-length cDNA of Chitin Synthase 2 gene (LmCHS2) from Locusta migratoria. The cDNA contains an open reading frame of 4569 nucleotides that encode 1523 amino acid residues, and 76- and 373-nucleotides for 5′- and 3′-noncoding regions, respectively. Analysis of LmCHS2 transcript in different tissues of the locust by using real-time quantitative PCR indicated that LmCHS2 was exclusively expressed in midgut and gastric caeca (a part of the midgut). The highest expression was found in the anterior midgut with a decline of the transcript level from the anterior to posterior regions. During growth and development of locusts, there was only a slight expression in eggs, but the expression gradually increased from nymphs to adults. In situ hybridization further revealed that LmCHS2 transcript mainly presented in the apical regions of brush border forming columnar cells of gastric caeca. LmCHS2 dsRNA was injected to fifth-instar nymphs to further explore biological functions of LmCHS2. Significantly down-regulated transcript of LmCHS2 resulted in a cessation of feeding and a high mortality of the insect. However, no visible abnormal morphological change of locusts was observed until insects molted to adults. After dissection, we found that the average length of midguts from the LmCHS2 dsRNA-injected locusts was shorter than that of the control insects that were injected with dsGFP. Furthermore, microsection of midguts showed that the PM of the LmCHS2 dsRNA-injected nymphs was amorphous and thin as compared with the controls. Our results demonstrate that LmCHS2 is responsible for the biosynthesis of Chitin associated with PM and plays an essential role in locust growth and development.

  • identification and characterization of two Chitin Synthase genes in african malaria mosquito anopheles gambiae
    Insect Biochemistry and Molecular Biology, 2012
    Co-Authors: Xin Zhang, Jianzhen Zhang, Yoonseong Park, Kun Yan Zhu
    Abstract:

    Chitin Synthase (CHS) represents an attractive target site for combating insect pests as insect growth and development are strictly dependent on precisely tuned Chitin biosynthesis and this pathway is absent in humans and other vertebrates. Current knowledge on CHS in insects, especially their structures, functions, and regulations is still very limited. We report the identification and characterization of two Chitin Synthase genes, AgCHS1 and AgCHS2, in African malaria mosquito, Anopheles gambiae. AgCHS1 and AgCHS2 were predicted to encode proteins of 1,578 and 1,586 amino acid residues, respectively. Their deduced amino acid sequences show high similarities to other insect Chitin Synthases. Transcriptional analysis indicated that AgCHS1 was expressed in egg, larval, pupal and adult stages whereas AgCHS2 appeared to be expressed at relatively low levels, particularly during the larval stages as examined by reverse transcription (RT)-PCR and real-time quantitative PCR. Relatively high expression was detected in the carcass followed by the foregut and hindgut for AgCHS1, and the foregut (cardia included) followed by the midgut for AgCHS2. Fluorescence in situ hybridization (FISH) and immunohistochemical analysis revealed new information including the localization of the two enzymes in the ommatidia of the compound eyes, and AgCHS2 in the thoracic and abdominal inter-segmental regions of pupal integument.

  • characterization of a Chitin Synthase cdna and its increased mrna level associated with decreased Chitin synthesis in anopheles quadrimaculatus exposed to diflubenzuron
    Insect Biochemistry and Molecular Biology, 2006
    Co-Authors: Jianzhen Zhang, Kun Yan Zhu
    Abstract:

    Chitin Synthase (EC 2.4.1.16) is a crucial enzyme responsible for Chitin biosynthesis in all Chitin-containing organisms. This paper reports a complete cDNA encoding Chitin Synthase 1 (AqCHS1), change of AqCHS1 mRNA level in response to diflubenzuron exposure, and concentration-dependent effect of diflubenzuron on Chitin synthesis in the common malaria mosquito (Anopheles quadrimaculatus). The cDNA consists of 5723 nucleotides, including an open reading frame (ORF) of 4734 nucleotides that encode 1578 amino acid residues and a non-translated region of 989 nucleotides. The deduced amino acid sequence contains all the Chitin Synthase signature motifs (EDR, QRRRW and SWGTR) and shows 97% identity to that of An. gambiae (AgCHS1, XM_321337). Northern blot and real-time quantitative PCR analyses revealed a significant increase of AqCHS1 mRNA level in the larvae exposed to diflubenzuron at 100 and 500 microg/L. As confirmed by real-time quantitative PCR, AqCHS1 mRNA level was enhanced by 2-fold in the larvae exposed to diflubenzuron at 500 microg/L for 24 h. In contrast, exposures of the larvae to diflubenzuron at 4.0, 20, 100 and 500 microg/L for 48 h resulted in decreases of Chitin content by 9.0%, 43%, 58% and 76%, respectively. Significantly increased AqCHS1 mRNA level associated with decreased Chitin synthesis may imply possible inhibition of Chitin Synthase, or abnormal Chitin Synthase translocation or Chitin microfibril assembly conferred by diflubenzuron. Increased AqCHS1 expression due to increased transcription and/or increased mRNA stability may serve as a feedback mechanism to compensate such an effect in the mosquitoes. Further studies are necessary to elucidate the relationship between reduced Chitin synthesis and increased expression of AqCHS1 in order to shed new light on trafficking and regulation of Chitin biosynthesis in the mosquito affected by diflubenzuron.

Paul J. Szaniszlo - One of the best experts on this subject based on the ideXlab platform.

  • immunoaffinity purification of the class v Chitin Synthase of wangiella exophiala dermatitidis
    Preparative Biochemistry & Biotechnology, 2009
    Co-Authors: Dariusz Abramczyk, Paul J. Szaniszlo
    Abstract:

    The class V Chitin Synthase is unique because it has a myosin motor-like domain fused to its catalytic domain. The biochemical properties of this enzyme and its function remain undefined beyond the knowledge that it is the only single Chitin Synthase required for sustained cell growth at elevated temperatures and, consequently, virulence. This report describes our successful efforts to isolate and purify an active and soluble form of the enzyme from the cell membranes of Wangiella by using a specific polyclonal antibody. To our knowledge, this is the first purification of a single Chitin Synthase of a filamentous fungus.

  • cytolocalization of the class v Chitin Synthase in the yeast hyphal and sclerotic morphotypes of wangiella exophiala dermatitidis
    Fungal Genetics and Biology, 2009
    Co-Authors: Dariusz Abramczyk, Changwon Park, Paul J. Szaniszlo
    Abstract:

    Abstract Wangiella (Exophiala) dermatitidis is a polymorphic fungus that produces polarized yeast and hyphae, as well as a number of non-polarized sclerotic morphotypes. The phenotypic malleability of this agent of human phaeohyphomycosis allows detailed study of its biology, virulence and the regulatory mechanisms responsible for the transitions among the morphotypes. Our prior studies have demonstrated the existence of seven Chitin Synthase structural genes in W. dermatitidis, each of which encodes an isoenzyme of a different class. Among them, the class V Chitin Synthase (WdChs5p) is most unique in terms of protein structure, because it has an N-terminal myosin motor-like domain with a P-loop (MMD) fused to its C-terminal Chitin Synthase catalytic domain (CSCD). However, the exact role played by WdChs5p in the different morphotypes remains undefined beyond the knowledge that it is the only single Chitin Synthase required for sustained cell growth at 37 °C and consequently virulence. This report describes the expression in Escherichia coli of a 12 kDa polypeptide (WdMyo12p) of WdChs5p, which was used to raise in rabbits a polyclonal antibody that recognized exclusively its MMD region. Results from the use of the antibody in immunocytolocalization studies supported our previous findings that WdChs5p is critically important at infection temperatures for maintaining the cell wall integrity of developing yeast buds, elongating tips of hyphae, and random sites of expansion in sclerotic forms. The results also suggested that WdChs5p localizes to the regions of cell wall growth in an actin-dependent fashion.

  • wdchs1p a class ii Chitin Synthase is more responsible than wdchs2p class i for normal yeast reproductive growth in the polymorphic pathogenic fungus wangiella exophiala dermatitidis
    Archives of Microbiology, 2006
    Co-Authors: Li Zheng, Changwon Park, Zheng Wang, Sarah Kauffman, Jeffrey M Becker, Leonel Mendoza, Paul J. Szaniszlo
    Abstract:

    The Chitin Synthase gene WdCHS1 was isolated from a partial genomic DNA library of the pathogenic polymorphic fungus Wangiella dermatitidis. Sequencing showed that WdCHS1 encoded a class II Chitin Synthase composed of 988 amino acids. Disruption of WdCHS1 produced strains that were hyperpigmented in rich media, grew as yeast at wild-type rates at both 25 and 37°C and were as virulent as the wild type in a mouse model. However, detailed morphological and cytological studies of the wdchs1Δ mutants showed that yeast cells often failed to separate, tended to be enriched with Chitin in septal regions and, sometimes, were enlarged with multiple nuclei, had broader mother cell–daughter bud regions and had other cell wall defects seen considerably less often than in the wild type or wdchs2Δ strains. Disruption of WdCHS1 and WdCHS2 in the same background revealed that WdChs1p had functions synergistic to those of WdChs2p, because mutants devoid of both isozymes produced growth that was very abnormal at 25°C and was not viable at 37°C unless osmotically stabilized. These results suggested that WdChs1p was more responsible than WdChs2p for normal yeast cell reproductive growth because strains with defects in the latter exhibited no morphological abnormalities, whereas those with defects in WdChs1p were frequently impaired in one or more yeast developmental processes.

  • wangiella exophiala dermatitidis wdchs5p a class v Chitin Synthase is essential for sustained cell growth at temperature of infection
    Eukaryotic Cell, 2004
    Co-Authors: Sarah Kauffman, Jeffrey M Becker, Paul J. Szaniszlo
    Abstract:

    The Chitin Synthase structural gene WdCHS5 was isolated from the black fungal pathogen of humans Wangiella dermatitidis. Sequence analysis revealed that the gene has a myosin motor-like-encoding region at its 5′ end and a Chitin Synthase (class V)-encoding region at its 3′ end. Northern blotting showed that WdCHS5 is expressed at high levels under conditions of stress. Analysis of the 5′ upstream region of WdCHS5 fused to a reporter gene indicated that one or more of the potential regulatory elements present may have contributed to the high expression levels. Disruption of WdCHS5 produced mutants that grow normally at 25°C but have severe growth and cellular abnormalities at 37°C. Osmotic stabilizers, such as sorbitol and sucrose, rescued the wild-type phenotype, which indicated that the loss of WdChs5p causes cell wall integrity defects. Animal survival tests with a mouse model of acute infection showed that all wdchs5Δ mutants are less virulent than the parental strain. Reintroduction of the WdCHS5 gene into the wdchs5Δ mutants abolished the temperature-sensitive phenotype and reestablished their virulence. We conclude that the product of WdCHS5 is required for the sustained growth of W. dermatitidis at 37°C and is of critical importance to its virulence.

  • compensatory expression of five Chitin Synthase genes a response to stress stimuli in wangiella exophiala dermatitidis a melanized fungal pathogen of humans
    Microbiology, 2002
    Co-Authors: Qingfeng Wang, Paul J. Szaniszlo
    Abstract:

    Numerous Chitin Synthase structural (CHS) genes have been identified in fungi, and usually there are several CHS genes per species. Compensatory expression of one CHS gene in response to defects in other CHS genes has not been reported. Five Chitin Synthase structural (WdCHS) genes have been identified in the melanized human pathogen Wangiella dermatitidis: WdCHS1, WdCHS2, WdCHS3, WdCHS4 and WdCHS5. This study showed that increased WdCHS expression existed as a compensatory mechanism in response to stress induced by Chitin Synthase gene disruptions, or by exposure of the wild-type or two temperature-sensitive morphological mutants, for short or long periods, to 37 °C. In general, the compensatory responses varied with each WdCHS gene, and in accordance with the hypothesized functions of the Chitin Synthase (WdChsp) encoded. It is suggested that these compensatory responses indicate that WdCHS gene transcription in W. dermatitidis functions as part of a cell-wall integrity pathway in a manner similar to that recently described for Saccharomyces cerevisiae.

Kun Yan Zhu - One of the best experts on this subject based on the ideXlab platform.

  • characterization of a midgut specific Chitin Synthase gene lmchs2 responsible for biosynthesis of Chitin of peritrophic matrix in locusta migratoria
    Insect Biochemistry and Molecular Biology, 2012
    Co-Authors: Xiaojian Liu, Jianzhen Zhang, Kun Yan Zhu, Huanhuan Zhang
    Abstract:

    Chitin, an essential component of peritrophic matrix (PM), is produced by a series of biochemical reactions. Chitin Synthase plays a crucial role in Chitin polymerization in Chitin biosynthetic pathway. In this study, we identified and characterized a full-length cDNA of Chitin Synthase 2 gene (LmCHS2) from Locusta migratoria. The cDNA contains an open reading frame of 4569 nucleotides that encode 1523 amino acid residues, and 76- and 373-nucleotides for 5′- and 3′-noncoding regions, respectively. Analysis of LmCHS2 transcript in different tissues of the locust by using real-time quantitative PCR indicated that LmCHS2 was exclusively expressed in midgut and gastric caeca (a part of the midgut). The highest expression was found in the anterior midgut with a decline of the transcript level from the anterior to posterior regions. During growth and development of locusts, there was only a slight expression in eggs, but the expression gradually increased from nymphs to adults. In situ hybridization further revealed that LmCHS2 transcript mainly presented in the apical regions of brush border forming columnar cells of gastric caeca. LmCHS2 dsRNA was injected to fifth-instar nymphs to further explore biological functions of LmCHS2. Significantly down-regulated transcript of LmCHS2 resulted in a cessation of feeding and a high mortality of the insect. However, no visible abnormal morphological change of locusts was observed until insects molted to adults. After dissection, we found that the average length of midguts from the LmCHS2 dsRNA-injected locusts was shorter than that of the control insects that were injected with dsGFP. Furthermore, microsection of midguts showed that the PM of the LmCHS2 dsRNA-injected nymphs was amorphous and thin as compared with the controls. Our results demonstrate that LmCHS2 is responsible for the biosynthesis of Chitin associated with PM and plays an essential role in locust growth and development.

  • identification and characterization of two Chitin Synthase genes in african malaria mosquito anopheles gambiae
    Insect Biochemistry and Molecular Biology, 2012
    Co-Authors: Xin Zhang, Jianzhen Zhang, Yoonseong Park, Kun Yan Zhu
    Abstract:

    Chitin Synthase (CHS) represents an attractive target site for combating insect pests as insect growth and development are strictly dependent on precisely tuned Chitin biosynthesis and this pathway is absent in humans and other vertebrates. Current knowledge on CHS in insects, especially their structures, functions, and regulations is still very limited. We report the identification and characterization of two Chitin Synthase genes, AgCHS1 and AgCHS2, in African malaria mosquito, Anopheles gambiae. AgCHS1 and AgCHS2 were predicted to encode proteins of 1,578 and 1,586 amino acid residues, respectively. Their deduced amino acid sequences show high similarities to other insect Chitin Synthases. Transcriptional analysis indicated that AgCHS1 was expressed in egg, larval, pupal and adult stages whereas AgCHS2 appeared to be expressed at relatively low levels, particularly during the larval stages as examined by reverse transcription (RT)-PCR and real-time quantitative PCR. Relatively high expression was detected in the carcass followed by the foregut and hindgut for AgCHS1, and the foregut (cardia included) followed by the midgut for AgCHS2. Fluorescence in situ hybridization (FISH) and immunohistochemical analysis revealed new information including the localization of the two enzymes in the ommatidia of the compound eyes, and AgCHS2 in the thoracic and abdominal inter-segmental regions of pupal integument.

  • characterization of a Chitin Synthase cdna and its increased mrna level associated with decreased Chitin synthesis in anopheles quadrimaculatus exposed to diflubenzuron
    Insect Biochemistry and Molecular Biology, 2006
    Co-Authors: Jianzhen Zhang, Kun Yan Zhu
    Abstract:

    Chitin Synthase (EC 2.4.1.16) is a crucial enzyme responsible for Chitin biosynthesis in all Chitin-containing organisms. This paper reports a complete cDNA encoding Chitin Synthase 1 (AqCHS1), change of AqCHS1 mRNA level in response to diflubenzuron exposure, and concentration-dependent effect of diflubenzuron on Chitin synthesis in the common malaria mosquito (Anopheles quadrimaculatus). The cDNA consists of 5723 nucleotides, including an open reading frame (ORF) of 4734 nucleotides that encode 1578 amino acid residues and a non-translated region of 989 nucleotides. The deduced amino acid sequence contains all the Chitin Synthase signature motifs (EDR, QRRRW and SWGTR) and shows 97% identity to that of An. gambiae (AgCHS1, XM_321337). Northern blot and real-time quantitative PCR analyses revealed a significant increase of AqCHS1 mRNA level in the larvae exposed to diflubenzuron at 100 and 500 microg/L. As confirmed by real-time quantitative PCR, AqCHS1 mRNA level was enhanced by 2-fold in the larvae exposed to diflubenzuron at 500 microg/L for 24 h. In contrast, exposures of the larvae to diflubenzuron at 4.0, 20, 100 and 500 microg/L for 48 h resulted in decreases of Chitin content by 9.0%, 43%, 58% and 76%, respectively. Significantly increased AqCHS1 mRNA level associated with decreased Chitin synthesis may imply possible inhibition of Chitin Synthase, or abnormal Chitin Synthase translocation or Chitin microfibril assembly conferred by diflubenzuron. Increased AqCHS1 expression due to increased transcription and/or increased mRNA stability may serve as a feedback mechanism to compensate such an effect in the mosquitoes. Further studies are necessary to elucidate the relationship between reduced Chitin synthesis and increased expression of AqCHS1 in order to shed new light on trafficking and regulation of Chitin biosynthesis in the mosquito affected by diflubenzuron.

Mariechristine Soulie - One of the best experts on this subject based on the ideXlab platform.

  • discovery of two new inhibitors of botrytis cinerea Chitin Synthase by a chemical library screening
    Bioorganic & Medicinal Chemistry, 2013
    Co-Authors: Herve Magellan, Martine Boccara, Thierry Drujon, Mariechristine Soulie, Catherine Guillou, Joelle Dubois, Hubert F Becker
    Abstract:

    : Chitin Synthases polymerize UDP-GlcNAC to form Chitin polymer, a key component of fungal cell wall biosynthesis. Furthermore, Chitin Synthases are desirable targets for fungicides since Chitin is absent in plants and mammals. Two potent Botrytis cinerea Chitin Synthase inhibitors, 2,3,5-tri-O-benzyl-d-ribose (compound 1) and a 2,5-functionalized imidazole (compound 2) were identified by screening a chemical library. We adapted the wheat germ agglutinin (WGA) test for Chitin Synthase activity detection to allow miniaturization and robotization of the screen. Both identified compounds inhibited Chitin Synthases in vitro with IC50 values of 1.8 and 10μM, respectively. Compounds 1 and 2 were evaluated for their antifungal activity and were found to be active against B. cinerea BD90 strain with MIC values of 190 and 100μM, respectively. Finally, we discovered that both compounds confer resistance to plant leaves against the attack of the fungus by reducing the propagation of lesions by 37% and 23%, respectively. Based on the inhibitory properties found in different assays, compounds 1 and 2 can be considered as antifungal hit inhibitors of Chitin Synthase, allowing further optimization of their pharmacological profile to improve their antifungal properties.

  • Disruption of Bcchs4, Bcchs6 or Bcchs7 Chitin Synthase genes in Botrytis cinerea and the essential role of class VI Chitin Synthase (Bcchs6)
    Fungal Genetics and Biology, 2013
    Co-Authors: Serena Morcx, Mathias Choquer, Caroline Kunz, Sébastien Assie, Eddy Blondet, Karina Gajek, Florence Chapeland-leclerc, Dominique Expert, Mariechristine Soulie
    Abstract:

    Chitin Synthases play critical roles in hyphal development and fungal pathogenicity. Previous studies on Botrytis cinerea, a model organism for necrotrophic pathogens, have shown that disruption of Bcchs1 and more particularly Bcchs3a genes have a drastic impact on virulence (Soulie et al., 2003, 2006). In this work, we investigate the role of other CHS including BcCHS4, BcCHS6 and BcCHS7 during the life cycle of B. cinerea. Single deletions of corresponding genes were carried out. Phenotypic analysis indicates that: (i) BcCHS4 enzyme is not essential for development and pathogenicity of the fungus; (ii) BcCHS7 is required for pathogenicity in a host dependant manner. For Bcchs6 gene disruption, we obtained only heterokaryotic strains. Indeed, sexual or asexual purification assays were unsuccessful. We concluded that class VI Chitin Synthase could be essential for B. cinerea and therefore BcCHS6 represents a valuable antifungal target.

  • botrytis cinerea virulence is drastically reduced after disruption of Chitin Synthase class iii gene bcchs3a
    Cellular Microbiology, 2006
    Co-Authors: Mariechristine Soulie, Martine Boccara, Annie Piffeteau, Mathias Choquer, Caroline Kunz, Claude Perino, Pierrette Malfatti, Agnes Cimerman, Anne Vidalcros
    Abstract:

    Botrytis cinerea is an important phytopathogenic fungus requiring new methods of control. Chitin biosynthesis, which involves seven classes of Chitin Synthases, could be an attractive target. A fragment encoding one of the class III enzymes was used to disrupt the corresponding Bcchs3a gene in the B. cinerea genome. The resulting mutant exhibited a 39% reduction in its Chitin content and an 89% reduction in its in vitro Chitin Synthase activity, compared with the wild-type strain. Bcchs3a mutant was not affected in its growth in liquid medium, neither in its production of sclerotia, micro- and macroconidia. In contrast, the mutant Bcchs3a was severely impaired in its growth on solid medium. Counterbalancing this defect in radial growth, Bcchs3a mutant presented a large increase in hyphal ramification, resulting in an enhanced aerial growth. Observations by different techniques of microscopy revealed a thick extracellular matrix around the hyphal tips. Moreover, Bcchs3a mutant had a largely reduced virulence on Vitis vinifera and Arabidopsis thaliana leaves.

  • disruption of botrytis cinerea class i Chitin Synthase gene bcchs1 results in cell wall weakening and reduced virulence
    Fungal Genetics and Biology, 2003
    Co-Authors: Mariechristine Soulie, Martine Boccara, Annie Piffeteau, Mathias Choquer, Anne Vidalcros
    Abstract:

    To get a better insight into the relationship between cell wall integrity and pathogenicity of the fungus Botrytis cinerea, we have constructed Chitin Synthase mutants. A 620 bp class I Chitin Synthase gene fragment (Bcchs1) obtained by PCR amplification was used to disrupt the corresponding gene in the genome. Disruption of Bcchs1 occurred at a frequency of 8%. Nine independent mutants were obtained and the Bcchs1 mutant phenotype compared to that of transformants in which the gene was not disrupted. These disruption mutants were dramatically reduced in their in vitro Mg2+, Mn2+, and Co2+-dependent Chitin Synthase activity. Chitin content was reduced by 30%, indicating that Bcchs1p contributes substantially to cell wall composition. Enzymatic degradation by a cocktail of glucanases revealed cell wall weakening in the mutant. Bcchs1 was transcribed at a constant level during vegetative exponential growth, suggesting that it was necessary throughout hyphal development. Bcchs1 mutant growth was identical to undisrupted control transformant growth, however, the mutant exhibited reduced pathogenicity on vine leaves. It can be assumed that disruption of Bcchs1 leads to cell wall weakening which might slow down in planta fungal progression.

Atsuhiko Hasegawa - One of the best experts on this subject based on the ideXlab platform.

  • direct detection of dermatophytes in skin samples based on sequences of the Chitin Synthase 1 chs1 gene
    Journal of Veterinary Medical Science, 2003
    Co-Authors: Rui Kano, Asuka Hirai, Toshihiro Watari, Masato Muramatsu, Atsuhiko Hasegawa
    Abstract:

    For the direct detection of dermatophytes in skin scrapings and hairs from animals, a primer pair specific to the Chitin Synthase 1 (CHS1) gene of dermatophytes was constructed. By PCR analysis with the primer pair, dermatophyte DNA could be diagnosed directly and rapidly in clinical skin samples.

  • Chitin Synthase 1 gene of arthroderma benhamiae isolates in japan
    Mycoses, 2002
    Co-Authors: Rui Kano, Asuka Hirai, Atsuhiko Hasegawa
    Abstract:

    Summary.  In the present study, the Chitin Synthase 1 (CHS1) gene of eight clinical isolates of Arthroderma benhamiae in Japan was investigated. Nucleotide sequence analysis of the CHS1 gene fragments from clinical isolates of A. benhamiae and from standard strains of Americano-European race and African race A. benhamiae indicated more than 90% similarities among these dermatophytes. An especially high degree of similarity was noted in nucleotide sequence CHS1 gene fragments, with more than 99% among eight clinical isolates and standard strains of the Americano-European race of A. benhamiae. The phylogenetic analysis of their sequences revealed that the eight clinical isolates and the standard strains of the Americano-European race of A. benhamiae were included in the same cluster, and that the African race of A. benhamiae formed a cluster that was distinct from the Americano-European race of A. benhamiae, A. simii and A. vanbreuseghemii. Zusammenfassung.  An acht klinischen Isolaten von Arthroderma benhamiae wurde das ChitinSynthase-1-Gen untersucht. Die Nukleotidsequenzanalyse der CHS1-Genfragmente der klinischen A. benhamiae–Isolate und der amerikano-europaischen Standardstamme sowie der afrikanischen Standardstamme wiesen mehr als 90%Ahnlichkeit auf. Ein besonders hoher Ahnlichkeitsgrad von uber 99% bestand zwischen den klinischen Isolaten und der amerikano-europaischen Stammfamilie, wahrend die afrikanischen A. benhamiae-Stamme einen Cluster bilden, der sich von der amerikano-europaischen Stammfamilie von A. benhamiae sowie von A. simii und A. vanbreuseghemii unterscheidet.

  • differences among Chitin Synthase 1 gene sequences in trichophyton rubrum and t violaceum
    Medical Mycology, 2000
    Co-Authors: R Kano, Yuka Nakamura, S. Watanabe, Ken Okabayashi, Shiho Ooka, Masato Kashima, Masako Mizoguchi, Atsuhiko Hasegawa
    Abstract:

    Nucleotide sequences of Chitin Synthase 1 (CHS1) gene were analysed for the phylogenetic relation between Trichophyton violaceum and T. rubrum, including two isolates of T. raubitschekii and one isolate of T. rubrum var. nigricans. About 620-bp genomic DNA fragments of the CHS1 gene were amplified from these dermatophytes by polymerase chain reaction (PCR) and sequenced. The CHS1 nucleotide sequences of these dermatophytes showed more than 95% similarity between the species. The phylogenetic analysis of their sequences revealed that T. rubrum was genetically distinct from T. violaceum. The specific restriction endonuclease site for HinfI was present in the CHS1 gene sequence of T. rubrum but not in that of T. violaceum. A molecular analysis of CHS1 genes will provide useful information for the identification of these Trichophyton species and the understanding of their evolution.

  • Chitin Synthase 2 gene sequence of malassezia species
    Microbiology and Immunology, 1999
    Co-Authors: Yuka Nakamura, Rui Kano, S. Watanabe, Tomohiro Aizawa, Atsuhiko Hasegawa
    Abstract:

    Nucleotide sequences of the Chitin Synthase 2 (CHS2) gene of seven species, Malassezia furfur, M. globosa, M. obtusa, M. pachydermatis, M. restricta, M. slooffiae and M. sympodialis, were analyzed for their phylogenetic relationship. About 620-bp genomic DNA fragments of the CHS2 gene were amplified from these Malassezia species by polymerase chain reaction (PCR) and sequenced. The CHS2 nucleotide sequences of these Malassezia species showed more than 95% similarity between the species. A phylogenetic analysis of the nucleotide sequences of CHS2 gene fragments of seven Malassezia species revealed that the species were genetically distinct from each other.

  • Molecular Analysis of Chitin Synthase 1 (CHS1) Gene Sequences of Trichophyton mentagrophytes Complex and T. rubrum
    Current Microbiology, 1998
    Co-Authors: R Kano, Hajime Tsujimoto, Yuka Nakamura, Toshihiro Watari, Hisashi Takahashi, S. Watanabe, Atsuhiko Hasegawa
    Abstract:

    Nucleotide sequences of Chitin Synthase 1 (CHS1) gene of dermatophytes, Arthroderma benhamiae, A. simii, A. vanbreuseghemii, Trichophyton mentagrophytes var. interdigitale (T. interdigitale), and T. rubrum were analyzed for their phylogenetic relationship. About 620-bp genomic DNA fragments of the CHS1 gene were amplified from these dermatophytes by polymerase chain reaction (PCR) and sequenced. The CHS1 nucleotide sequences of these five dermatophytes showed more than 90% similarity between the species. The phylogenetic analysis of their sequences revealed that A. benhamiae, A. simii, A. vanbreuseghemii, and T. rubrum were genetically distinct from one another, but T. interdigitale was genetically very close to A. vanbreuseghemii. On the other hand, a specific restriction endonuclease site of HinfI was present in the CHS1 gene fragment of T. rubrum but not in those of A. benhamiae, A. simii, A. vanbreuseghemii and T. interdigitale. The molecular analysis of CHS1 genes will provide useful information for the identification of these Trichophyton species and the understanding of their evolution.