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

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Genetic studies have shown essential functions of N-glycosylation during infection of the plant pathogenic fungi, however, systematic roles of N-glycosylation in fungi is still largely unknown. Biological analysis demonstrated N-glycosylated proteins were widely present at different development stages of Magnaporthe oryzae and especially increased in the appressorium and invasive hyphae. A large-scale quantitative proteomics analysis was then performed to explore the roles of N-glycosylation in M. oryzae. A total of 559 N-glycosites from 355 proteins were identified and quantified at different developmental stages. Functional classification to the N-glycosylated proteins revealed N-glycosylation can coordinate different cellular processes for mycelial growth, conidium formation, and appressorium formation. N-glycosylation can also modify key components in N-glycosylation, O-glycosylation and GPI anchor pathways, indicating intimate crosstalk between these pathways. Interestingly, we found nearly all key components of the endoplasmic reticulum quality control (ERQC) system were highly N-glycosylated in conidium and appressorium. Phenotypic analyses to the gene deletion mutants revealed four ERQC components, Gls1, Gls2, GTB1 and Cnx1, are important for mycelial growth, conidiation, and invasive hyphal growth in host cells. Subsequently, we identified the Gls1 N-glycosite N497 was important for invasive hyphal growth and partially required for conidiation, but didn't affect colony growth. Mutation of N497 resulted in reduction of Gls1 in protein level, and localization from ER into the vacuole, suggesting N497 is important for protein stability of Gls1. Our study showed a snapshot of the N-glycosylation landscape in plant pathogenic fungi, indicating functions of this modification in cellular processes, developments and pathogenesis.

The rice blast fungus Magnaporthe oryzae causes a devastating disease which threatens global rice production. In spite of 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 of 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 were predicted to be effectors and named MEP (Magnaportheeffector protein) genes. Computational prediction of structurally-related MEPs, including the MAX effector family, revealed their temporal co-regulation in the same co- expression modules. We functionally 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 to successful infection.

In eukaryotes, N 6 -methyladenosine (m 6 A) is abundant on mRNA, and plays key roles in the regulation of RNA function. However, the roles and regulatory mechanisms of m 6 A in phytopathogenic fungi are still largely unknown.Combined with biochemical analysis, MeRIP-seq and RNA-seq methods, as well as biological analysis, we showed that Magnaporthe oryzae MTA1 gene is an orthologue of human METTL4, which is involved in m 6 A modification and plays a critical role in autophagy for fungal infection.The Dmta1 mutant showed reduced virulence due to blockage of appressorial penetration and invasive growth. Moreover, the autophagy process was severely disordered in the mutant. MeRIP-seq identified 659 hypomethylated m 6 A peaks covering 595 mRNAs in Dmta1 appressoria, 114 m 6 A peaks was negatively related to mRNA abundance, including several ATG gene transcripts. Typically, the mRNA abundance of MoATG8 was also increased in the single m 6 A site mutant Δatg8/MoATG8 A982C , leading to an autophagy disorder.Our findings reveal the functional importance of the m 6 A methylation in infection of M. oryzae and provide novel insight into the regulatory mechanisms of plant pathogenic fungi.

Upon fungal and bacterial pathogen attack, plants launch pattern-triggered immunity (PTI) by recognizing pathogen-associated molecular patterns (PAMPs) to defend against pathogens. Although PTI-mediated response has been widely studied, a systematic understanding of the reprogrammed cellular processes during PTI by multi-omics analysis is lacking. In this study, we generated metabolome, transcriptome, proteome, ubiquitome and acetylome data to investigate rice (Oryza sativa) PTI responses to two PAMPs, the fungi-derived chitin and the bacteriaderived flg22. Integrative multi-omics analysis uncovered convergence and divergence of rice responses to these PAMPs at multiple regulatory layers. Rice responded to chitin and flg22 in a similar manner at the transcriptome and proteome levels, but distinct at the metabolome level. We found that this was probably due to post-translational regulation including ubiquitination and acetylation, which reshaped gene expression by modulating enzymatic activities, and possibly led to distinct metabolite profiles. We constructed regulatory atlas of metabolic pathways, including the defence-related phenylpropanoid and flavonoid biosynthesis and linoleic acid derivative metabolism. The multi-level regulatory network generated in this study sets the foundation for in-depth mechanistic dissection of PTI in rice and potentially in other related poaceous crop species.

Plant infection by microbial pathogens is a dynamic process. Here, we investigated the heterogeneity of plant responses in the context of pathogen location. A single-cell atlas of Arabidopsis thaliana leaves challenged by the fungus Colletotrichum higginsianum revealed cell type-specific gene expression that highlights an enrichment of intracellular immune receptors in vasculature cells. Using trajectory inference, we assigned cells that directly interacted with the invasive hyphae. Further analysis of cells at these infection sites revealed transcriptional plasticity based on cell type. A reprogramming of abscisic acid signalling was specifically activated in guard cells. Consistently, a contact-dependent stomatal closure was observed, possibly representing a defense response that anticipates pathogen invasive growth. We defined cell type-specific deployments of genes activating indole glucosinolate biosynthesis at the infection sites, and determined their contribution to resistance. This research highlights the spatial dynamics of plant response during infection and reveals cell type-specific processes and gene functions.