Cyclophilins are peptidyl prolyl cis-trans isomerases that are highly conserved throughout eukaryotes and that are best known for being the cellular target of the immunosuppressive drug cyclosporin A (CsA). The activity of CsA is caused by the drug forming a complex with cyclophilin A and inhibiting the calmodulin-dependent phosphoprotein phosphatase calcineurin. We have investigated the role of CYP1 , a cyclophilin-encoding gene in the phytopathogenic fungus Magnaporthe grisea , which is the causal agent of rice blast disease. CYP1 putatively encodes a mitochondrial and cytosolic form of cyclophilin, and targeted gene replacement has shown that CYP1 acts as a virulence determinant in rice blast. Cyp1 mutants show reduced virulence and are impaired in associated functions, such as penetration peg formation and appressorium turgor generation. CYP1 cyclophilin also is the cellular target for CsA in Magnaporthe, and CsA was found to inhibit appressorium development and hyphal growth in a CYP1 -dependent manner. These data implicate cyclophilins as virulence factors in phytopathogenic fungi and also provide evidence that calcineurin signaling is required for infection structure formation by Magnaporthe. INTRODUCTIONRice blast is the most serious disease of cultivated rice and causes serious recurrent epidemics throughout the ricegrowing regions of the world (Baker et al., 1997). The disease is caused by the ascomycete fungus Magnaporthe grisea , which produces specialized infection structures called appressoria to penetrate the leaves and stems of rice plants, allowing the fungus entry to the underlying tissue (for reviews, see Howard and Valent, 1996; Hamer and Talbot, 1998). Plant infection by appressoria is accomplished by the generation of enormous turgor pressure, which is translated into mechanical force to breach the plant cuticle (Howard et al., 1991). Once in the plant, the fungus develops bulbous secondary hyphae and quickly colonizes the host tissue (Bourett and Howard, 1990; Heath et al., 1990; Talbot et al., 1993). Damage to the rice crop results from either leaf blast, which kills or debilitates seedlings, or neck and panicle blast, which destroy the rice grain during the seed-setting stage (Ou, 1985). The economic cost of rice blast is significant, both in direct losses to the rice harvest and in the costs of control measures such as rice breeding programs, fungicide applications, and the development of new antifungal chemicals (Baker et al., 1997).Developing novel mechanisms to control rice blast will require detailed understanding of both the disease and the developmental biology of Magnaporthe. Considerable progress has been made in identifying the genes required for elaboration of appressoria because the fungus is tractable to molecular genetics (Talbot, 1995; Howard and Valent, 1996), but far less is known about the later stages of plant infection and how the growth of the fungus within rice tissue is regulated. Appressorium formation on the rice leaf surface requires a cAMP signaling pathway...

Restriction enzyme-mediated DNA integration (REMI) mutagenesis was used to identify mutants of Magnaporthe grisea impaired in pathogenicity. Three REMI protocols were evaluated and the frequency of REMIs determined. An REMI library of 3,527 M. grisea transformants was generated in three genetic backgrounds, and 1,150 transformants were screened for defects in pathogenicity with a barley cut leaf assay. Five mutants were identified and characterized. Two mutants (2029 and 2050) were impaired in appressorium function. Two other mutants, 125 and 130, were altered in conidial morphology, conidiogenesis, and appressorium function. Mutant 130 was also a methionine auxotroph and methionine auxotrophy co-segregated with the reduction in pathogenicity. An additional mutant, 80, showed reduced pathogenicity on blast-susceptible rice cultivars but was fully pathogenic on barley. The reduction of pathogenicity in mutant 80 was associated with a delay in conidial germination and appressorium development. Genetic analysis suggested single-gene segregation for each mutant, but only two of the mutations co-segregated with the hygromycin resistance marker. The genetic loci in mutants 2029, 2050, 125, 130, and 80 were termed PDE1, PDE2, IGD1, MET1, and GDE1, respectively. pde1 and pde2 were non-allelic to cpkA, a mutation in the catalytic subunit of cyclic AMP (cAMP)-dependent protein kinase A with a very similar phenotype. The results indicate the utility of REMI for studying fungal pathogenicity, but also highlight the requirement for rigorous genetic and phenotypic analysis.

Plant infection by the rice blast fungus Magnaporthe grisea is brought about by the action of specialized infection cells called appressoria. These infection cells generate enormous turgor pressure, which is translated into an invasive force that allows a narrow penetration hypha to breach the plant cuticle. The Magnaporthe pde1 mutant was identified previously by restriction enzyme-mediated DNA integration mutagenesis and is impaired in its ability to elaborate penetration hyphae. Here we report that the pde1 mutation is the result of an insertion into the promoter of a P-type ATPase-encoding gene. Targeted gene disruption confirmed the role of PDE1 in penetration hypha development and pathogenicity but highlighted potential differences in PDE1 regulation in different Magnaporthe strains. The predicted PDE1 gene product was most similar to members of the aminophospholipid translocase group of P-type ATPases and was shown to be a functional homolog of the yeast ATPase gene ATC8 . Spatial expression studies showed that PDE1 is expressed in germinating conidia and developing appressoria. These findings implicate the action of aminophospholipid translocases in the development of penetration hyphae and the proliferation of the fungus beyond colonization of the first epidermal cell. INTRODUCTIONOne of the principal reasons why pathogenic fungi are so successful at causing plant disease is their ability to penetrate the plant cuticle directly, often using specialized infection cells called appressoria (Mendgen et al., 1996). A wide variety of fungal pathogens form appressoria, and the development of these cells involves a complex morphogenetic program that results in rapid differentiation of a highly specialized structure (Dean, 1997). The rice blast fungus Magnaporthe grisea is an important pathogen of cultivated rice and has emerged as an experimental model for the study of plant infection processes (for reviews, see Howard and Valent, 1996;Hamer and Talbot, 1998). Magnaporthe develops dome-shaped appressoria, which form at the ends of germ tubes soon after spore germination on the leaf surface. Appressoria attach strongly to the leaf and then generate enormous turgor pressure (up to 8 MPa), which is used to rupture the plant cuticle (Howard et al., 1991). The turgor inside appressoria is generated by a rapid increase in intracellular glycerol levels, which is maintained by a specialized cell wall layer containing melanin (Howard and Ferrari, 1989;de Jong et al., 1997). If melanin biosynthesis is blocked, either by chemical intervention or mutation, then Magnaporthe is unable to penetrate the plant surface and cannot cause disease (Chida and Sisler, 1987;Chumley and Valent, 1990).How such enormous cellular turgor is translated into the substantial invasive force necessary to breach the plant cell wall is not clear, but it appears to involve polarization of the cytoskeleton to the point of infection and localized cell wall modification Howard, 1990, 1992;Bechinger et al., 1999). A narrow penetration hypha forms at t...