Genetic Analysis of Sporulation inMagnaporthe griseaby Chemical and Insertional Mutagenesis

Chemical and plasmid insertional mutagenesis were used to genetically define critical steps in the sporulation pathway of the rice blast fungus. Six mutants with altered conidiogenesis and spore morphology were genetically and phenotypically characterized. Two mutations, designated con5- and con6-, completely abolish conidial production. A series of mutations (con1-, con2-, con4-, and con7-) downstream from con5- and con6- affect the development of conidia and reduce sporulation. The con1- and con2- mutations block early steps in conidiogenesis, resulting in >90% reduction in sporulation and the production of abnormally shaped conidia. The con2- mutant is completely aconidial in the dark but produces mostly nonseptate or two-celled conidia under continuous illumination. An independent mutation (con3-) that regulates light response for sporulation was isolated from the con2- mutant. The con4- and con7- mutants produce conidia of abnormal cell shape and reduce sporulation by approximately 35%. Formation of appressoria, the infection structure required for penetration of plant cells, is blocked in the con1- and con7- mutants, and reduced by 70 and 22% in the con2- and con4- mutants, respectively. Pathogenicity on rice is lost in the con1- and con7- mutants and significantly reduced in the con2- and con4- mutants. Five mutations (con1-B-, con4-, con5-, con6-, and con7-) derived from plasmid transformation showed perfect cosegregation with hygromycin B resistance, indicating that the mutations are caused by insertional inactivation. Pairwise crosses between mutants suggested linkages between CON2 and CON1 (19 cM apart), and between CON5 and CON6. A schematic sporulation pathway is deduced based on mutant phenotypes and the epistatic relationships among different mutations.