In order to clone and analyse the avirulence gene AVR-Pia from Japanese field isolates of Magnaporthe oryzae, a mutant of the M. oryzae strain Ina168 was isolated. This mutant, which was named Ina168m95-1, gained virulence towards the rice cultivar Aichi-asahi, which contains the resistance gene Pia. A DNA fragment (named PM01) that was deleted in the mutant and that co-segregated with avirulence towards Aichi-asahi was isolated. Three cosmid clones that included the regions that flanked

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... e isolated from a genomic DNA library. One of these clones (46F3) could complement the mutant phenotype, which indicated clearly that this clone contained the avirulence gene AVR-Pia. Clone 46F3 contained insertions of transposable elements. The 46F3 insert was divided into fragments I-VI, and these were cloned individually into a hygromycin-resistant vector for the transformation of the mutant Ina168m95-1. An inoculation assay of the transformants revealed that fragment V (3.5 kb) contained AVR-Pia. By deletion analysis of fragment V, AVR-Pia was localised to an 1199-bp DNA fragment, which included a 255-bp open reading frame with weak homology to a bacterial cytochrome-c-like protein. Restriction fragment length polymorphism analysis of this region revealed that this DNA sequence co-segregated with the AVR-Pia locus in a genetic map that was constructed using Chinese isolates. extra domain between the nucleocapsid and protease domains. Nucleic Acids Res. 29, 4106-4113. Orbach, M. J. (1994) A cosmid with a Hy R marker for fungal library construction and screening. Gene 150, 159-162. A telomeric avirulence gene determines efficacy for the rice blast resistance gene Pi-ta. Plant Cell 12, 2019-2032. Peyyala R., and Farman, M. L. (2006) Magnaporthe oryzae isolates causing gray leaf spot of perennial ryegrass possess a functional copy of the AVR1-CO39 avirulence gene. Mol. Plant Pathol. 7, 157-165. Soanes, D. M., Richards, T. A. and Talbot, N. J. (2007) Insights from sequencing fungal and oomycete genomes: What can we learn about plant disease and the evolution of pathogenicity? Comparative genome analysis of filamentous fungi reveals gene family expansions associated with fungal pathogenesis. PLoS ONE 3, e2300.