Mevalonate pathway, which uses acetyl-CoA as substrate to produce mevalonate, is the main route of ergosterol production in fungi.Ergosterol does not only participate in the synthesis of cell membrane but also contribute to the prenylation of membrane targeting CAAX motif of small GTPases.In mevalonate pathway, two acetyl-CoA condensed into acetoacetyl-CoA by acetoacetyl-CoA acetyltransferase or acetoacetyl-CoA thiolase, this acetoacetyl-CoA is further transformed into mevalonate under the catalysis of HMG-CoA reductase, and followed by series of reactions to yield ergosterol as a final product.Acetoacetyl-CoA acetyltransferase, the first catalytic enzyme of mevalonate pathway, has two homologus genes in Magnaporthe oryzae, while S.cerevisiae has only one homologue of this gene which plays multiple functions.To characterize the functions acetoacetyl-CoA acetyltransferase in M.oryzae, we generated the deletion mutants of acetoacetyl-CoA acetyltransferase (MoAcat) ΔMoAcatl and ΔMoAcat2.Phenotypic analysis found MoAcat2 plays important role in mevalonate pathway, its deletion lead to auxotrophic growth on minimal medium with glucose, acetate sodium, glycerol, olive oil, this deficiency can however be rescued with mevalonate.Further study also showed mevalonate is essential for hyphae morphological structure and pathogenicity.By contrast, MoAcat1 deletion does not affect mevalonate pathway, but its null mutant fail to infect rice.Subcelluar localization showed MoAcat2 is localized in cytoplasm, while MoAcat1 is localized in mitochondrial.Our results suggested the two acetoacetyl-CoA acetyltransferase genes may play distinct roles in M.oryzae.