The rice blast fungus Magnaporthe oryzae causes a devastating disease that threatens global rice (Oryza sativa) production. Despite intense study, the biology of plant tissue invasion during blast disease remains poorly understood. Here we report a high resolution, transcriptional profiling study of the entire plant-associated development of the blast fungus. Our analysis revealed major temporal changes in fungal gene expression during plant infection. Pathogen gene expression could be classified into 10 modules of temporally co-expressed genes, providing evidence for the induction of pronounced shifts in primary and secondary metabolism, cell signalling and transcriptional regulation. A set of 863 genes encoding secreted proteins are differentially expressed at specific stages of infection, and 546 genes named MEP (Magnaporthe  effector protein) genes were predicted to encode effectors. Computational prediction of structurally-related MEPs, including the MAX effector family, revealed their temporal co-regulation in the same co-expression modules. We characterised 32 MEP genes and demonstrate that Mep effectors are predominantly targeted to the cytoplasm of rice cells via the biotrophic interfacial complex (BIC) and use a common unconventional secretory pathway. Taken together, our study reveals major changes in gene expression associated with blast disease and identifies a diverse repertoire of effectors critical for successful infection.

ice blast disease is an important threat to global food security 1 . The disease starts when asexual spores of Magnaporthe oryzae, called conidia, land on the hydrophobic surface of a rice leaf inducing differentiation of an infection cell called an appressorium 1-3 . The appressorium develops turgor of up to 8.0 MPa due to glycerol accumulation, which generates osmotic pressure 4 . Glycerol is maintained in the appressorium by melanin in the cell wall, which reduces its porosity 4,5 . Development of the appressorium is tightly linked to the cell cycle, autophagy [6][7][8] and metabolic checkpoint control mediated by TOR kinase and the cAMP-dependent protein kinase A (PKA) pathway [9][10][11] . Appressorium turgor is monitored by a sensor kinase, Sln1, and once a threshold is reached 12 , septin GTPases in the appressorium pore form a hetero-oligomeric complex that scaffolds cortical F-actin at the base of the appressorium 13,14 . This leads to force generation to pierce the cuticle with a rigid penetration hypha. Once inside the leaf, invasive hyphae colonize the first epidermal cell before seeking out plasmodesmata-rich pit fields through which the fungus invades neighbouring cells 15 . M. oryzae actively suppresses plant immunity using fungal effector proteins delivered into plant cells 16 . After five days, disease lesions appear from which the fungus sporulates to colonize neighbouring plants.Formation of an appressorium by M. oryzae requires a conserved pathogenicity mitogen-activated protein kinase (MAPK), called Pmk1 (ref. 17 ). Pmk1 mutants cannot form appressoria or cause plant infection, even when plants are wounded 17 . Instead, Δpmk1 mutants produce undifferentiated germlings that undergo several rounds of mitosis and septation 17,18 . Pmk1 is also responsible for lipid and glycogen mobilization to the appressorium, autophagy in the conidium 4,8,19,20 and invasive cell-to-cell movement 15 . A set of pl surface sensors 21 that trigger cAMP-PKA signalling are required for Pmk1 activation 17 , and a TOR-dependent nutrient sensing pathway is necessary for appressorium formation, acting upstream, or perhaps independently, of Pmk1 (refs. [9][10][11] ). The mechanism by which Pmk1 exerts such an important role in plant infection has remained largely unknown and only one transcriptional regulator, Mst12, which may act downstream of Pmk1, has been characterized in detail. Mst12 mutants form appressoria normally but are non-functional and cannot cause disease 22 .In this study we set out to identify the mechanism by which major transcriptional changes are regulated during appressorium development by M. oryzae. We identified major temporal changes in gene expression in response to an appressorium-inductive hydrophobic