Identification and characterization of a class III chitin synthase gene of Moniliophthora perniciosa, the fungus that causes witches’ broom disease of cacao

Chitin synthase (CHS) is a glucosyltransferase that converts UDP-N-acetylglucosamine into chitin, one of the main components of fungal cell wall. Class III chitin synthases act directly in the formation of the cell wall. They catalyze the conversion of the immediate precursor of chitin and are responsible for the majority of chitin synthesis in fungi. As such, they are highly specific molecular targets for drugs that can inhibit the growth and development of fungal pathogens. In this work, we have identified and characterized a chitin synthase gene of Moniliophthora perniciosa (Mopchs) by primer walking. The complete gene sequence is 3,443 bp, interrupted by 13 small introns, and comprises a cDNA with an ORF with 2,739 bp, whose terminal region was experimentally determined, encoding a protein with 913 aa that harbors all the motifs and domains typically found in class III chitin synthases. This is the first report on the characterization of a chitin synthase gene, its mature transcription product, and its putative protein in basidioma and secondary mycelium stages of M. perniciosa, a basidiomycotan fungus that causes witches’ broom disease of cacao.

[1]  B. Moerschbacher,et al.  Survey and expression analysis of five new chitin synthase genes in the biotrophic rust fungus Puccinia graminis , 2006, Current Genetics.

[2]  H. Merzendorfer,et al.  Chitin metabolism in insects: structure, function and regulation of chitin synthases and chitinases , 2003, Journal of Experimental Biology.

[3]  D. Higgins,et al.  T-Coffee: A novel method for fast and accurate multiple sequence alignment. , 2000, Journal of molecular biology.

[4]  D. H. Brown,et al.  The synthesis of chitin in cell-free extracts of Neurospora crassa. , 1957, The Journal of biological chemistry.

[5]  J. Ruiz-Herrera,et al.  Evolution and phylogenetic relationships of chitin synthases from yeasts and fungi. , 2002, FEMS yeast research.

[6]  J. Latgé The cell wall: a carbohydrate armour for the fungal cell , 2007, Molecular microbiology.

[7]  D. Lewis Fungi and sugars — a suite of interactions , 1991 .

[8]  Thomas L. Madden,et al.  Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. , 1997, Nucleic acids research.

[9]  Y. Asada,et al.  Isolation, characterization, and expression analysis of a class IV chitin synthase gene from the edible basidiomycetous mushroom Pleurotus ostreatus , 2007, Mycoscience.

[10]  E. Thines,et al.  Polar Localizing Class V Myosin Chitin Synthases Are Essential during Early Plant Infection in the Plant Pathogenic Fungus Ustilago maydis[W] , 2005, The Plant Cell Online.

[11]  Kenji Matsuura,et al.  Reconstructing the early evolution of Fungi using a six-gene phylogeny , 2006, Nature.

[12]  Roderic D. M. Page,et al.  TreeView: an application to display phylogenetic trees on personal computers , 1996, Comput. Appl. Biosci..

[13]  J. Doyle,et al.  A rapid DNA isolation procedure for small amounts of fresh leaf tissue , 1987 .

[14]  T G Burland,et al.  DNASTAR's Lasergene sequence analysis software. , 2000, Methods in molecular biology.

[15]  Sarah Calvo,et al.  Insights from the genome of the biotrophic fungal plant pathogen Ustilago maydis , 2006, Nature.

[16]  M. C. Aime,et al.  The causal agents of witches' broom and frosty pod rot of cacao (chocolate, Theobroma cacao) form a new lineage of Marasmiaceae. , 2005, Mycologia.

[17]  John P. Huelsenbeck,et al.  MrBayes 3: Bayesian phylogenetic inference under mixed models , 2003, Bioinform..

[18]  M. Choquer,et al.  Survey of the Botrytis cinerea chitin synthase multigenic family through the analysis of six euascomycetes genomes. , 2004, European journal of biochemistry.

[19]  Mario Stanke,et al.  Gene prediction with a hidden Markov model and a new intron submodel , 2003, ECCB.

[20]  P. Robbins,et al.  The S. cerevisiae structural gene for chitin synthase is not required for chitin synthesis in vivo , 1986, Cell.

[21]  Luciano Digiampietri,et al.  The mitochondrial genome of the phytopathogenic basidiomycete Moniliophthora perniciosa is 109 kb in size and contains a stable integrated plasmid. , 2008, Mycological research.

[22]  Carlos Priminho Pirovani,et al.  Knowledge Discovery in Genome Database: The Chitin Metabolic Pathway in Crinipellis Perniciosa (Stahel) Singer , 2005 .

[23]  K. Burton,et al.  Cloning and characterisation of a chitin synthase gene cDNA from the cultivated mushroom Agaricus bisporus and its expression during morphogenesis. , 2000, FEMS microbiology letters.

[24]  G. Griffith,et al.  Genetic variability and chromosome-length polymorphisms of the witches' broom pathogen Crinipellis perniciosa from various plant hosts in South America. , 2006, Mycological research.

[25]  B. Bago,et al.  Effect of Nikkomycin Z, a chitin-synthase inhibitor, on hyphal growth and cell wall structure of two arbuscular-mycorrhizal fungi , 1996, Protoplasma.

[26]  C. Pirovani,et al.  Use of response surface methodology to examine chitinase regulation in the basidiomycete Moniliophthora perniciosa. , 2008, Mycological research.

[27]  P. Rabinovitch,et al.  Targeted gene walking polymerase chain reaction. , 1991, Nucleic acids research.

[28]  W. Pearson Rapid and sensitive sequence comparison with FASTP and FASTA. , 1990, Methods in enzymology.

[29]  Jonathan E. Allen,et al.  The Genome of the Basidiomycetous Yeast and Human Pathogen Cryptococcus neoformans , 2005, Science.

[30]  S. Nagahashi,et al.  Characterization of Chitin Synthase 2 of Saccharomyces cerevisiae. , 1995, The Journal of Biological Chemistry.

[31]  Helaine Carrer,et al.  A genome survey of Moniliophthora perniciosa gives new insights into Witches' Broom Disease of cacao , 2008, BMC Genomics.

[32]  H. Merzendorfer,et al.  Insect chitin synthases: a review , 2005, Journal of Comparative Physiology B.

[33]  A. Krogh,et al.  Predicting transmembrane protein topology with a hidden Markov model: application to complete genomes. , 2001, Journal of molecular biology.

[34]  S. Free,et al.  The structure and synthesis of the fungal cell wall , 2006, BioEssays : news and reviews in molecular, cellular and developmental biology.

[35]  E. Cabib,et al.  Chitin synthetase zymogen is attached to the yeast plasma membrane. , 1975, Proceedings of the National Academy of Sciences of the United States of America.

[36]  Rolf Apweiler,et al.  InterPro and InterProScan , 2007 .

[37]  Søren Brunak,et al.  Analysis and prediction of gene splice sites in four Aspergillus genomes. , 2009, Fungal genetics and biology : FG & B.

[38]  R. Durbin,et al.  GeneWise and Genomewise. , 2004, Genome research.

[39]  J. Behr Chitin Synthase As an Antifungal Target: Recent Advances , 2003 .

[40]  C. Roncero The genetic complexity of chitin synthesis in fungi , 2002, Current Genetics.

[41]  J. T. Kadonaga,et al.  The RNA polymerase II core promoter. , 2003, Annual review of biochemistry.

[42]  G. Griffith,et al.  Witches’ brooms and frosty pods: Two major pathogens of cacao , 2003 .

[43]  D. Swofford PAUP*: Phylogenetic analysis using parsimony (*and other methods), Version 4.0b10 , 2002 .