clk1, a serine/threonine protein kinase-encoding gene, is involved in pathogenicity of Colletotrichum lindemuthianum on common bean.

A random insertional mutagenesis in Colletotrichum lindemuthianum, the causal agent of common bean anthracnose, generated four mutants that showed altered pathogenicity when tested on intact seedlings, excised leaves, and/or excised hypocotyls. One of these mutants, H290, produced very few lesions on bean leaves and appeared affected in its ability to penetrate the leaf cuticle. Molecular analyses showed that the border sequences of the unique integration site of the disrupting pAN7-1 plasmid in the mutant exhibited homology with conserved domains of serine/threonine protein kinases. The corresponding wild-type sequences were cloned and a gene replacement vector with a mutated copy harboring a selection marker constructed. Transformation of the wild-type pathogen produced a strain with a phenotype identical to the original mutant. Genomic and cDNA sequences indicated that the disrupted gene is a member of the serine/threonine protein kinase family. The gene, called clk1 (Colletotrichum lindemuthianum kinase 1), was weakly expressed in the mycelium of the wild-type strain grown on rich and minimal synthetic media but was undetectable during the infection even when a sensitive reverse transcriptase-polymerase chain reaction methodology was used. This study represents the first characterization of altered pathogenicity mutants in C. lindemuthianum produced by random mutagenesis and demonstrates the involvement of a member of the serine/threonine kinase gene family in the early steps of the infection process.

[1]  T. Hunter,et al.  The protein kinase family: conserved features and deduced phylogeny of the catalytic domains. , 1988, Science.

[2]  I. Herskowitz,et al.  Identification of fuz7, a Ustilago maydis MEK/MAPKK homolog required for a-locus-dependent and -independent steps in the fungal life cycle. , 1994, Genes & development.

[3]  F. Sanger,et al.  DNA sequencing with chain-terminating inhibitors. , 1977, Proceedings of the National Academy of Sciences of the United States of America.

[4]  R. O’Connell,et al.  Cytology and physiology of infection of Phaseolus vulgaris by Colletotrichum lindemuthianum , 1985 .

[5]  R. O’Connell,et al.  Identification of glycoproteins specific to biotrophic intracellular hyphae formed in the Colletotrichum lindemuthianum-bean interaction. , 1994, The New phytologist.

[6]  O. Yoder,et al.  Selectable genes for transformation of the fungal plant pathogen Glomerella cingulata f. sp. phaseoli (Colletotrichum lindemuthianum). , 1987, Gene.

[7]  D. Ballance.  Sequences important for gene expression in filamentous fungi , 1986, Yeast.

[8]  J. Xu,et al.  MAP kinase and cAMP signaling regulate infection structure formation and pathogenic growth in the rice blast fungus Magnaporthe grisea. , 1996, Genes & development.

[9]  R. Dean,et al.  The cAMP-dependent protein kinase catalytic subunit is required for appressorium formation and pathogenesis by the rice blast pathogen Magnaporthe grisea. , 1995, The Plant cell.

[10]  N. Talbot,et al.  Identification and characterization of MPG1, a gene involved in pathogenicity from the rice blast fungus Magnaporthe grisea. , 1993, The Plant cell.

[11]  D. Parisot,et al.  Analysis of diverse isolates of Colletotrichum lindemuthianum infecting common bean using molecular markers , 1995 .

[12]  M. Hensel,et al.  Molecular genetic approaches for the study of virulence in both pathogenic bacteria and fungi. , 1996, Microbiology.

[13]  R. Dean,et al.  cAMP Regulates Infection Structure Formation in the Plant Pathogenic Fungus Magnaporthe grisea. , 1993, The Plant cell.

[14]  R. Oliver,et al.  Transformation of Aspergillus based on the hygromycin B resistance marker from Escherichia coli. , 1987, Gene.

[15]  P. Lefebvre,et al.  Cloning of flagellar genes in Chlamydomonas reinhardtii by DNA insertional mutagenesis. , 1993, Genetics.

[16]  A. Kuspa,et al.  Tagging developmental genes in Dictyostelium by restriction enzyme-mediated integration of plasmid DNA. , 1992, Proceedings of the National Academy of Sciences of the United States of America.

[17]  A. Osbourn,et al.  Molecular dissection of fungal phytopathogenicity. , 1995, Microbiology.

[18]  P. Pouwels,et al.  Functional elements in the promoter region of the Aspergillus nidulans gpdA gene encoding glyceraldehyde-3-phosphate dehydrogenase. , 1990, Gene.

[19]  J. Hamer,et al.  The CPKA gene of Magnaporthe grisea is essential for appressorial penetration , 1997 .

[20]  F. Chumley,et al.  Mutations at the smo genetic locus affect the shape of diverse cell types in the rice blast fungus. , 1989, Genetics.

[21]  W. Schafer.  MOLECULAR MECHANISMS OF FUNGAL PATHOGENICITY TO PLANTS , 1994 .

[22]  R. Metzenberg,et al.  Insertional mutagenesis in Neurospora crassa: cloning and molecular analysis of the preg+ gene controlling the activity of the transcriptional activator NUC-1. , 1993, Genetics.

[23]  T. Hunter,et al.  Protein kinases and phosphatases: The Yin and Yang of protein phosphorylation and signaling , 1995, Cell.

[24]  J. A. Bailey,et al.  Cell wall degrading enzymes of Colletotrichum lindemuthianum: their role in the development of bean anthracnose , 1989 .

[25]  I. Herskowitz.  MAP kinase pathways in yeast: For mating and more , 1995, Cell.

[26]  W. Schäfer,et al.  The role of cutinase in fungal pathogenicity. , 1993, Trends in microbiology.

[27]  M. Flaishman,et al.  Cloning of a gene expressed during appressorium formation by Colletotrichum gloeosporioides and a marked decrease in virulence by disruption of this gene. , 1995, The Plant cell.

[28]  C. Pieterse,et al.  Isolation of putative pathogenicity genes of the potato late blight fungus Phytophthora infestans by differential hybridization of a genomic library , 1993 .

[29]  M. Dron,et al.  Fungal-and plant-specific gene markers to follow the bean anthracnose infection process and normalize a bean chitinase mRNA induction , 1992 .

[30]  John Carbon,et al.  A colony bank containing synthetic CoI EI hybrid plasmids representative of the entire E. coli genome , 1976, Cell.