Magnaporthe grisea is the most destructive pathogen of rice worldwide and the principal model organism for elucidating the molecular basis of fungal disease of plants. Here, we report the draft sequence of the M. grisea genome. Analysis of the gene set provides an insight into the adaptations required by a fungus to cause disease. The genome encodes a large and diverse set of secreted proteins, including those defined by unusual carbohydrate-binding domains. This fungus also possesses an expanded family of G-protein-coupled receptors, several new virulence-associated genes and large suites of enzymes involved in secondary metabolism. Consistent with a role in fungal pathogenesis, the expression of several of these genes is upregulated during the early stages of infection-related development. The M. grisea genome has been subject to invasion and proliferation of active transposable elements, reflecting the clonal nature of this fungus imposed by widespread rice cultivation.Outbreaks of rice blast disease are a serious and recurrent problem in all rice-growing regions of the world, and the disease is extremely difficult to control 1,2 . Rice blast, caused by the fungus Magnaporthe grisea, is therefore a significant economic and humanitarian problem. It is estimated that each year enough rice is destroyed by rice blast disease to feed 60 million people 3 . The life cycle of the rice blast fungus is shown in Fig. 1. Infections occur when fungal spores land and attach themselves to leaves using a special adhesive released from the tip of each spore 4 . The germinating spore develops an appressorium-a specialized infection cell-which generates enormous turgor pressure (up to 8 MPa) that ruptures the leaf cuticle, allowing invasion of the underlying leaf tissue 5,6 . Subsequent colonization of the leaf produces disease lesions from which the fungus sporulates and spreads to new plants. When rice blast infects young rice seedlings, whole plants often die, whereas spread of the disease to the stems, nodes or panicle of older plants results in nearly total loss of the rice grain 2 . Different host-limited forms of M. grisea also infect a broad range of grass species including wheat, barley and millet. Recent reports have shown that the fungus has the capacity to infect plant roots 7 .Here we present our preliminary analysis of the draft genome sequence of M. grisea, which has emerged as a model system for understanding plant-microbe interactions because of both its economic significance and genetic tractability 1,2 . Acquisition of the M. grisea genome sequenceThe genome of a rice pathogenic strain of M. grisea, 70-15, was sequenced through a whole-genome shotgun approach. In all, greater than sevenfold sequence coverage was produced, and a summary of the principal genome sequence data is provided in Table 1 and Supplementary Table S1. The draft genome sequence consists of 2,273 sequence contigs longer than 2 kilobases (kb), ordered and orientated within 159 scaffolds. The total length of all sequence contigs is 38.8 mega...

Isolates of the rice blast fungus Magnaporthe grisea that carry the gene encoding Avirulence Conferring Enzyme1 (ACE1) are specifically recognized by rice (Oryza sativa) cultivars carrying the resistance gene Pi33. This recognition enables resistant plants to activate a defense response. ACE1 was isolated by map-based cloning and encodes a putative hybrid between a polyketide synthase and a nonribosomal peptide synthetase, enzymes involved in microbial secondary metabolism. ACE1 is expressed exclusively during fungal penetration of host leaves, the time point at which plant defense reactions are triggered. Ace1 appears to be localized in the cytoplasm of the appressorium. Mutation of the putative catalytic site of the b-ketoacyl synthase domain of Ace1 abolishes recognition of the fungus by resistant rice. This suggests that Ace1 biosynthetic activity is required for avirulence. Our results are consistent with the hypothesis that the fungal signal recognized by resistant rice plants is the secondary metabolite whose synthesis depends on Ace1.

In the maize pathogenic fungus Ustilago maydis integration of transforming DNA at homologous or heterologous sites is often accompanied by duplications of the DNA. We show that it is possible to generate single-copy integration events with high efficiency by restriction enzyme-mediated integration (REMI). In about 50% of cases, a plasmid that contains a single BamHI site is integrated at chromosomal BamH1 sites, if BamHI is added to the transformation mixtures. In the other cases it appears that integration events have also occurred preferentially at BamHI sites, but without restoration of the recognition sites. Using REMI we have generated approximately 1000 insertion mutants. Pathogenicity tests demonstrated that about 1-2% of these mutants were unable to induce symptoms when tested in planta. For two of the mutants we have shown that the phenotype is linked to the insertion event.

Summary• The avirulence gene ACE1 from the rice blast fungus Magnaporthe grisea encodes a polyketide synthase (PKS) fused to a nonribosomal peptide synthetase (NRPS) probably involved in the biosynthesis of a secondary metabolite recognized by Pi33 resistant rice (Oryza sativa) cultivars.• Analysis of the M. grisea genome revealed that ACE1 is located in a cluster of 15 genes, of which 14 are potentially involved in secondary metabolism as they encode enzymes such as a second PKS-NRPS (SYN2), two enoyl reductases (RAP1 and RAP2) and a putative Zn(II) 2 Cys 6 transcription factor (BC2).• These 15 genes are specifically expressed during penetration into the host plant, defining an infection-specific gene cluster. A pORF3-GFP transcriptional fusion showed that the highly expressed ORF3 gene from the ACE1 cluster is only expressed in appressoria, as is ACE1. Phenotypic analysis of deletion or disruption mutants of SYN2 and RAP2 showed that they are not required for avirulence in Pi33 rice cultivars, unlike ACE1. Inactivation of other genes was unsuccessful because targeted gene replacement and disruption were inefficient at this locus.• Overall, the ACE1 gene cluster displays an infection-specific expression pattern restricted to the penetration stage which is probably controlled at the transcriptional level and reflects regulatory networks specific to early stages of infection.

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We identified a nonpathogenic strain of Ustilago maydis by tagging mutagenesis. The affected gene, glucosidase1 (gas1), displays similarity to catalytic a-subunits of endoplasmic reticulum (ER) glucosidase II. We have shown that Gas1 localizes to the ER and complements the temperature-sensitive phenotype of a Saccharomyces cerevisiae mutant lacking ER glucosidase II. gas1 deletion mutants were normal in growth and mating but were more sensitive to calcofluor and tunicamycin. Mutant infection hyphae displayed significant alterations in the distribution of cell wall material and were able to form appressoria and penetrate the plant surface but arrested growth in the epidermal cell layer. Electron microscopy analysis revealed that the plant-fungal interface between mutant hyphae and the plant plasma membrane was altered compared with the interface of penetrating wild-type hyphae. This may indicate that gas1 mutants provoke a plant response.

Magnaporthe grisea is responsible for a devastating fungal disease of rice called blast. Current control of this disease relies on resistant rice cultivars that recognize M. grisea signals corresponding to specific secreted proteins encoded by avirulence genes. The M. grisea ACE1 avirulence gene differs from others, since it controls the biosynthesis of a secondary metabolite likely recognized by rice cultivars carrying the Pi33 resistance gene. Using a transcriptional fusion between ACE1 promoter and eGFP, we showed that ACE1 is only expressed in appressoria during fungal penetration into rice and barley leaves, onion skin, and cellophane membranes. ACE1 is almost not expressed in appressoria differentiated on Teflon and Mylar artificial membranes. ACE1 expression is not induced by cellophane and plant cell wall components, demonstrating that it does not require typical host plant compounds. Cyclic AMP (cAMP) signaling mutants ⌬cpkA and ⌬mac1 sum1-99 and tetraspanin mutant ⌬pls1::hph differentiate melanized appressoria with normal turgor but are unable to penetrate host plant leaves. ACE1 is normally expressed in these mutants, suggesting that it does not require cAMP signaling or a successful penetration event. ACE1 is not expressed in appressoria of the buf1::hph mutant defective for melanin biosynthesis and appressorial turgor. The addition of hyperosmotic solutes to buf1::hph appressoria restores appressorial development and ACE1 expression. Treatments of young wild-type appressoria with actin and tubulin inhibitors reduce both fungal penetration and ACE1 expression. These experiments suggest that ACE1 appressorium-specific expression does not depend on host plant signals but is connected to the onset of appressorium-mediated penetration.Magnaporthe grisea species complex attacks a wide range of grasses, including wheat, barley, and rice (10,26), and is a model organism for the study of plant fungal interactions (11, 42). The M. grisea infection cycle is characteristic of grass leaf spot diseases. After spore attachment and germination, the fungus differentiates an appressorium through the perception of physical and chemical surface parameters (hydrophobicity, hardness, and cuticle monomers) (21, 42). This differentiation is the result of a complex morphogenetic process that involves cyclic AMP (cAMP), mitogen-activated protein kinases, and calcium signaling pathways (7,45,50). Early stages of appressorium development are associated with the deposition of a melanin layer between the cell wall and plasma membrane (21), migration of lipid bodies from spore to appressorium, mobilization of glycogen, and the formation of a septum sealing the appressorium (5, 43). Maturation of the appressorium is characterized by the degradation of lipid bodies and glycogen (43) and the generation of a high turgor (22). Finally, a reorganization of the cytoskeleton is induced at the point of emergence of the penetration peg that penetrates the host cuticle and cell wall (5,35). Inside the plant, M. grisea differentiates bulbous...

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