Genetic analysis of developmental mutants and rapid chromosome mapping of APP1, a gene required for appressorium formation in Magnaporthe grisea

Infection of rice by Magnaporthe grisea, the rice blast pathogen, requires formation of a specialized, darkly pigmented, dome-shaped appressorium. To elucidate the mechanism(s) involved in formation of this structure, several developmental mutants were isolated by UV mutagenesis. Two mutants (243-7 and 370-7) with greatly reduced appressorium formation on the hydrophobic surface of GelBond were obtained. An additional mutant, 174/3, which was able to form appressoria on the hydrophilic surface of GelBond, was also isolated. A fourth mutant, 138-1, was obtained that produced conidia on complete medium. Genetic analysis of the mutants indicated that the phenotypes are under single gene control. Pairwise crosses between mutants demonstrated that the mutations are at separate loci. The genetic loci in mutants 243-7, 3707, 17-4/3, and 138-1 were termed APP1, APP2, APP3, and CCN1, respectively. From a cross between mutants containing either app1^- or app3^-, two recombinant progeny were obtained from a total of 62, indicating that the two loci are closely linked. The other loci are independent. The addition of cyclic AMP or 1,16-hexadecanediol to the mutant containing app2^- restored the ability to form appressoria, but was ineffective on the app1^- mutant. DNA bulked segregation analysis from a cross with mapping strain 2539 and restriction fragment length polymorphism markers from genetic maps of M. grisea were used to localize the APP1 locus to a central region of chromosome 2. Fine mapping resulted in the identification of flanking markers cos94 and A14B10 located 0.5 cM on either side of APP1, as well as several co- segregating cosmid markers, 4-10, cos91, cos167, and A14D8. The results indicate the utility of linkage maps for efficiently positioning genes of interest in the M. grisea genome and should expedite gene isolation by positional cloning.