NIP1, a small phytotoxic protein secreted by the barley pathogen Rhynchosporium secalis, is a race-specific elicitor of defense responses in barley cultivars carrying the resistance gene, Rrs1. Co-inoculation employing spores from a virulent fungal race together with the NIP1 protein converted the phenotype of the interaction from compatible to incompatible only on Rrs1-containing plants. In addition, transformation of a virulent fungal race with the nip1 gene yielded avirulent transformants. This demonstrated that the protein is the product of a fungal avirulence gene. The fungal genome was found to contain a single copy of the nip1 gene. Sequence analysis of nip1 cDNA and genomic clones revealed that the gene consists of two exons and one intron. The derived amino acid sequence comprised a secretory signal peptide of 22 amino acids and a cysteine-rich mature protein of 60 amino acids. All fungal races that were avirulent on barley cultivars of the Rrs1 resistance genotype carry and express the nip1 gene and secrete an elicitor-active NIP1 polypeptide. In contrast, races lacking this gene were virulent. In addition, single nucleotide exchanges were detected in the coding region of the nip1 alleles in one virulent fungal race and in a race whose interaction with barley is not controlled by the Rrs1 gene. The resulting exchanges of single amino acids render the gene products elicitor-inactive. Thus, the R.secalis-barley interaction provides the first example of a pathosystem conforming to the gene-for-gene hypothesis in which a plant with a particular resistance gene recognizes a pathogen by a virulence factor, i.e. one of its offensive weapons. On the fungal side, in turn, recognition by the host plant is eluded by either deletion of the encoding gene or alteration of the primary structure of the gene product.