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Mitogen-activated protein kinases (MAPKs) are a group of protein kinases that execute a wide variety of roles in cellular signal transduction pathways such as osmoregulation, cell wall biosynthesis, growth, and differentiation. A polymerase chain reaction (PCR) with degenerate primers based on conserved regions of known MAPKs was used to clone the MAPK gene PTK1 from the leaf pathogen Pyrenophora teres (anamorph Drechslera teres), the causal agent of net blotch of barley (Hordeum vulgare L.). The predicted amino acid sequence shows high homology with MAPKs from other phytopathogenic fungi. The gene is present in the genome as a single copy. PTK1 is expressed during in vitro growth on complete medium, under conidiation-inducing conditions and during infection of barley leaves, as shown by reverse transcription-PCR studies. In order to assess the role of PTK1 in the life cycle of P. teres, targeted gene disruption was conducted. Mutants carrying an interrupted copy of the gene were deficient in conidiation, did not form appressoria on glass surfaces or on barley leaves, lost their ability to infect barley leaves, and could not colonize host tissues following artificial wounding.