The rice blast fungus Magnaporthe oryzae is the most serious pathogen of cultivated rice and a significant threat to global food security. To accelerate targeted mutation and specific genome editing in this species, we have developed a rapid plasmid-free CRISPR-Cas9-based genome editing method. We show that stable expression of Cas9 is highly toxic to M. oryzae. However efficient gene editing can be achieved by transient introduction of purified Cas9 pre-complexed to RNA guides to form ribonucleoproteins (RNPs). When used in combination with oligonucleotide or PCR-generated donor DNAs, generation of strains with specific base pair edits, in-locus gene replacements, or multiple gene edits, is very rapid and straightforward. We demonstrate a co-editing strategy for the creation of single nucleotide changes at specific loci. Additionally, we report a novel counterselection strategy which allows creation of precisely edited fungal strains that contain no foreign DNA and are completely isogenic to the wild type. Together, these developments represent a scalable improvement in the precision and speed of genetic manipulation in M. oryzae and are likely to be broadly applicable to other fungal species.

Exocytosis involves the fusion of intracellular secretory vesicles with the plasma membrane, thereby delivering integral membrane proteins to the cell surface and releasing material into the extracellular space. Importantly, exocytosis also provides a source of lipid moieties for membrane extension. The tethering of the secretory vesicle before docking and fusion with the plasma membrane is mediated by the exocyst complex, an evolutionary conserved octameric complex of proteins. Recent findings indicate that the exocyst complex also takes part in other intra-cellular processes besides secretion. These various functions seem to converge toward defining a direction of membrane growth in a range of systems from fungi to plants and from neurons to cilia. In this review we summarize the current knowledge of exocyst function in cell polarity, signaling and cell-cell communication and discuss implications for plant and animal health and disease.

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

To cause rice blast disease, the fungal pathogen Magnaporthe oryzae develops a specialized infection structure called an appressorium. This dome-shaped, melanin-pigmented cell generates enormous turgor and applies physical force to rupture the rice leaf cuticle using a rigid penetration peg. Appressorium-mediated infection requires septin-dependent reorientation of the F-actin cytoskeleton at the base of the infection cell, which organizes polarity determinants necessary for plant cell invasion. Here, we show that plant infection by M. oryzae requires two independent S-phase cell-cycle checkpoints. Initial formation of appressoria on the rice leaf surface requires an S-phase checkpoint that acts through the DNA damage response (DDR) pathway, involving the Cds1 kinase. By contrast, appressorium repolarization involves a novel, DDR-independent S-phase checkpoint, triggered by appressorium turgor generation and melanization. This second checkpoint specifically regulates septin-dependent, NADPH oxidase-regulated F-actin dynamics to organize the appressorium pore and facilitate entry of the fungus into host tissue.fungi | pathogen | Pyricularia | appressorium | cell cycle

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