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The rice blast fungus Magnaporthe oryzae differentiates specialized cells called appressoria that are required for fungal penetration into host leaves. In this study, we identified the novel basic leucine zipper (bZIP) transcription factor BIP1 (B-ZIP Involved in Pathogenesis-1) that is essential for pathogenicity. BIP1 is required for the infection of plant leaves, even if they are wounded, but not for appressorium-mediated penetration of artificial cellophane membranes. This phenotype suggests that BIP1 is not implicated in the differentiation of the penetration peg but is necessary for the initial establishment of the fungus within plant cells. BIP1 expression was restricted to the appressorium by both transcriptional and post-transcriptional control. Genome-wide transcriptome analysis showed that 40 genes were down regulated in a BIP1 deletion mutant. Most of these genes were specifically expressed in the appressorium. They encode proteins with pathogenesis-related functions such as enzymes involved in secondary metabolism including those encoded by the ACE1 gene cluster, small secreted proteins such as SLP2, BAS2, BAS3, and AVR-Pi9 effectors, as well as plant cuticle and cell wall degrading enzymes. Interestingly, this BIP1 network is different from other known infection-related regulatory networks, highlighting the complexity of gene expression control during plant-fungal interactions. Promoters of BIP1-regulated genes shared a GCN4/bZIP-binding DNA motif (TGACTC) binding in vitro to BIP1. Mutation of this motif in the promoter of MGG_08381.7 from the ACE1 gene cluster abolished its appressorium-specific expression, showing that BIP1 behaves as a transcriptional activator. In summary, our findings demonstrate that BIP1 is critical for the expression of early invasion-related genes in appressoria. These genes are likely needed for biotrophic invasion of the first infected host cell, but not for the penetration process itself. Through these mechanisms, the blast fungus strategically anticipates the host plant environment and responses during appressorium-mediated penetration., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Lambou et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

The rice blast fungus Pyricularia oryzae (syn. Magnaporthe oryzae, Magnaporthe grisea), a member of the order Magnaporthales in the class Sordariomycetes, is an important plant pathogen and a model species for studying pathogen infection and plant-fungal interaction. In this study, we generated genome sequence data from five additional Magnaporthales fungi including non-pathogenic species, and performed comparative genome analysis of a total of 13 fungal species in the class Sordariomycetes to understand the evolutionary history of the Magnaporthales and of fungal pathogenesis. Our results suggest that the Magnaporthales diverged ca. 31 millon years ago from other Sordariomycetes, with the phytopathogenic blast clade diverging ca. 21 million years ago. Little evidence of inter-phylum horizontal gene transfer (HGT) was detected in Magnaporthales. In contrast, many genes underwent positive selection in this order and the majority of these sequences are clade-specific. The blast clade genomes contain more secretome and avirulence effector genes, which likely play key roles in the interaction between Pyricularia species and their plant hosts. Finally, analysis of transposable elements (TE) showed differing proportions of TE classes among Magnaporthales genomes, suggesting that species-specific patterns may hold clues to the history of host/environmental adaptation in these fungi.

Delineating species and epidemic lineages in fungal plant pathogens is critical to our understanding of disease emergence and the structure of fungal biodiversity and also informs international regulatory decisions. Pyricularia oryzae (syn. Magnaporthe oryzae ) is a multihost pathogen that infects multiple grasses and cereals, is responsible for the most damaging rice disease (rice blast), and is of growing concern due to the recent introduction of wheat blast to Bangladesh from South America. However, the genetic structure and evolutionary history of M. oryzae , including the possible existence of cryptic phylogenetic species, remain poorly defined. Here, we use whole-genome sequence information for 76 M. oryzae isolates sampled from 12 grass and cereal genera to infer the population structure of M. oryzae and to reassess the species status of wheat-infecting populations of the fungus. Species recognition based on genealogical concordance, using published data or extracting previously used loci from genome assemblies, failed to confirm a prior assignment of wheat blast isolates to a new species ( Pyricularia graminis-tritici ). Inference of population subdivisions revealed multiple divergent lineages within M. oryzae , each preferentially associated with one host genus, suggesting incipient speciation following host shift or host range expansion. Analyses of gene flow, taking into account the possibility of incomplete lineage sorting, revealed that genetic exchanges have contributed to the makeup of multiple lineages within M. oryzae These findings provide greater understanding of the ecoevolutionary factors that underlie the diversification of M. oryzae and highlight the practicality of genomic data for epidemiological surveillance in this important multihost pathogen. IMPORTANCE Infection of novel hosts is a major route for disease emergence by pathogenic microorganisms. Understanding the evolutionary history of multihost pathogens is therefore important to better predict the likely spread and emergence of new diseases. Magnaporthe oryzae is a multihost fungus that causes serious cereal diseases, including the devastating rice blast disease and wheat blast, a cause of growing concern due to its recent spread from South America to Asia. Using whole-genome analysis of 76 fungal strains from different hosts, we have documented the divergence of M. oryzae into numerous lineages, each infecting a limited number of host species. Our analyses provide evidence that interlineage gene flow has contributed to the genetic makeup of multiple M. oryzae lineages within the same species. Plant health surveillance is therefore warranted to safeguard against disease emergence in regions where multiple lineages of the fungus are in contact with one another.

Background: In February 2016, a new fungal disease was spotted in wheat fields across eight districts in Bangladesh. The epidemic spread to an estimated 15,000 hectares, about 16 % of the cultivated wheat area in Bangladesh, with yield losses reaching up to 100 %. Within weeks of the onset of the epidemic, we performed transcriptome sequencing of symptomatic leaf samples collected directly from Bangladeshi fields., Results: Reinoculation of seedlings with strains isolated from infected wheat grains showed wheat blast symptoms on leaves of wheat but not rice. Our phylogenomic and population genomic analyses revealed that the wheat blast outbreak in Bangladesh was most likely caused by a wheat-infecting South American lineage of the blast fungus Magnaporthe oryzae., Conclusion: Our findings suggest that genomic surveillance can be rapidly applied to monitor plant disease outbreaks and provide valuable information regarding the identity and origin of the infectious agent.

In this study we identified the L-arabinose-responsive regulator of Pyricularia oryzae that regulates L-arabinose release and catabolism. Previously we identified the Zn2Cys6 transcription factor (TF), AraR, that has this role in the Trichocomaceae family (Eurotiales), but is absent in other fungi. Candidate Zn2Cys6 TF genes were selected according to their transcript profiles on L-arabinose. Deletion mutants of these genes were screened for their growth phenotype on L-arabinose. One mutant, named Δara1, was further analyzed. Our analysis demonstrated that Ara1 from P. oryzae is the functional analog of AraR from A. niger, while there is no significant sequence similarity between them., (© 2016 Federation of European Biochemical Societies.)

Deciphering the genetic bases of pathogen adaptation to its host is a key question in ecology and evolution. To understand how the fungus Magnaporthe oryzae adapts to different plants, we sequenced eight M. oryzae isolates differing in host specificity (rice, foxtail millet, wheat, and goosegrass), and one Magnaporthe grisea isolate specific of crabgrass. Analysis of Magnaporthe genomes revealed small variation in genome sizes (39-43 Mb) and gene content (12,283-14,781 genes) between isolates. The whole set of Magnaporthe genes comprised 14,966 shared families, 63% of which included genes present in all the nine M. oryzae genomes. The evolutionary relationships among Magnaporthe isolates were inferred using 6,878 single-copy orthologs. The resulting genealogy was mostly bifurcating among the different host-specific lineages, but was reticulate inside the rice lineage. We detected traces of introgression from a nonrice genome in the rice reference 70-15 genome. Among M. oryzae isolates and host-specific lineages, the genome composition in terms of frequencies of genes putatively involved in pathogenicity (effectors, secondary metabolism, cazome) was conserved. However, 529 shared families were found only in nonrice lineages, whereas the rice lineage possessed 86 specific families absent from the nonrice genomes. Our results confirmed that the host specificity of M. oryzae isolates was associated with a divergence between lineages without major gene flow and that, despite the strong conservation of gene families between lineages, adaptation to different hosts, especially to rice, was associated with the presence of a small number of specific gene families. All information was gathered in a public database (http://genome.jouy.inra.fr/gemo)., (© The Author(s) 2015. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

Methionine is a sulfur amino acid standing at the crossroads of several biosynthetic pathways. In fungi, the last step of methionine biosynthesis is catalyzed by a cobalamine-independent methionine synthase (Met6, EC 2.1.1.14). In the present work, we studied the role of Met6 in the infection process of the rice blast fungus, Magnaporthe oryzae. To this end MET6 null mutants were obtained by targeted gene replacement. On minimum medium, MET6 null mutants were auxotrophic for methionine. Even when grown in presence of excess methionine, these mutants displayed developmental defects, such as reduced mycelium pigmentation, aerial hypha formation and sporulation. They also displayed characteristic metabolic signatures such as increased levels of cysteine, cystathionine, homocysteine, S-adenosylmethionine, S-adenosylhomocysteine while methionine and glutathione levels remained unchanged. These metabolic perturbations were associated with the over-expression of MgCBS1 involved in the reversed transsulfuration pathway that metabolizes homocysteine into cysteine and MgSAM1 and MgSAHH1 involved in the methyl cycle. This suggests a physiological adaptation of M. oryzae to metabolic defects induced by the loss of Met6, in particular an increase in homocysteine levels. Pathogenicity assays showed that MET6 null mutants were non-pathogenic on both barley and rice leaves. These mutants were defective in appressorium-mediated penetration and invasive infectious growth. These pathogenicity defects were rescued by addition of exogenous methionine and S-methylmethionine. These results show that M. oryzae cannot assimilate sufficient methionine from plant tissues and must synthesize this amino acid de novo to fulfill its sulfur amino acid requirement during infection.

Magnaporthe oryzae is a fungal plant pathogen of many grasses including rice. Since arabinoxylan is one of the major components of the plant cell wall of grasses, M. oryzae is likely to degrade this polysaccharide for supporting its growth in infected leaves. D-Xylose is released from arabinoxylan by fungal depolymerising enzymes and catabolized through the pentose pathway. The expression of genes involved in these pathways is under control of the transcriptional activator XlnR/Xlr1, conserved among filamentous ascomycetes. In this study, we identified M. oryzae genes involved in the pentose catabolic pathway (PCP) and their function during infection, including the XlnR homolog, XLR1, through the phenotypic analysis of targeted null mutants. Growth of the Δxlr1 strain was reduced on D-xylose and xylan, but unaffected on L-arabinose and arabinan. A strong reduction of PCP gene expression was observed in the Δxlr1 strain on D-xylose and L-arabinose. However, there was no significant difference in xylanolytic and cellulolytic enzyme activities between the Δxlr1 mutant and the reference strain. These data demonstrate that XLR1 encodes the transcriptional activator of the PCP in M. oryzae, but does not appear to play a role in the regulation of the (hemi-) cellulolytic system in this fungus. This indicates only partial similarity in function between Xlr1 and A. niger XlnR. The deletion mutant of D-xylulose kinase encoding gene (XKI1) is clearly unable to grow on either D-xylose or L-arabinose and showed reduced growth on xylitol, L-arabitol and xylan. Δxki1 displayed an interesting molecular phenotype as it over-expressed other PCP genes as well as genes encoding (hemi-) cellulolytic enzymes. However, neither Δxlr1 nor Δxki1 showed significant differences in their pathogeny on rice and barley compared to the wild type, suggesting that D-xylose catabolism is not required for fungal growth in infected leaves., (Copyright © 2013 Elsevier Inc. All rights reserved.)

A gene (MoPRD1), related to xylose reductases, was identified in Magnaporthe oryzae. Recombinant MoPRD1 displays its highest specific reductase activity toward L-arabinose and D-xylose. Km and Vmax values using L-arabinose and D-xylose are similar. MoPRD1 was highly overexpressed 2-8h after transfer of mycelium to D-xylose or L-arabinose, compared to D-glucose. Therefore, we conclude that MoPDR1 is a novel pentose reductase, which combines the activities and expression patterns of fungal L-arabinose and D-xylose reductases. Phylogenetic analysis shows that PRD1 defines a novel family of pentose reductases related to fungal D-xylose reductases, but distinct from fungal L-arabinose reductases. The presence of PRD1, L-arabinose and D-xylose reductases encoding genes in a given species is variable and likely related to their life style., (Copyright © 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

Effector proteins are key elements in plant-fungal interactions. The rice blast fungus Magnaporthe oryzae secretes numerous effectors that are suspected to be translocated inside plant cells. However, their cellular targets and the mechanisms of translocation are still unknown. Here, we have identified the open reading frame (ORF3) corresponding to the M. oryzae avirulence gene AVR1-CO39 that interacts with the rice resistance gene Pi-CO39 and encodes a small secreted protein without homology to other proteins. We demonstrate that AVR1-CO39 is specifically expressed and secreted at the plant-fungal interface during the biotrophic phase of infection. Live-cell imaging with M. oryzae transformants expressing a translational fusion between AVR1-CO39 and the monomeric red fluorescent protein (mRFP) indicated that AVR1-CO39 is translocated into the cytoplasm of infected rice cells. Transient expression of an AVR1-CO39 isoform without a signal peptide in rice protoplasts triggers a Pi-CO39-specific hypersensitive response, suggesting that recognition of AVR1-CO39 by the Pi-CO39 gene product occurs in the cytoplasm of rice cells. The native AVR1-CO39 protein enters the secretory pathway of rice protoplasts as demonstrated by the ER localization of AVR1-CO39:mRFP:HDEL translational fusions, and is correctly processed as shown by Western blotting. However, this secreted AVR1-CO39 isoform triggers a Pi-CO39-specific hypersensitive response and accumulates inside rice protoplasts as shown by Western blotting and localization of AVR1-CO39:mRFP translational fusions. This indicates that AVR1-CO39 is secreted by rice protoplasts and re-enters into the cytoplasm by unknown mechanisms, suggesting that translocation of AVR1-CO39 into rice cells occurs independently of fungal factors., (© 2012 The Authors The Plant Journal © 2012 Blackwell Publishing Ltd.)

Magnaporthe oryzae is the most damaging fungal pathogen of rice (Oryza sativa). In this study, we characterized the TIG1 transducin beta-like gene required for infectious growth and its interacting genes that are required for plant infection in this model phytopathogenic fungus. Tig1 homologs in yeast and mammalian cells are part of a conserved histone deacetylase (HDAC) transcriptional corepressor complex. The tig1 deletion mutant was nonpathogenic and defective in conidiogenesis. It had an increased sensitivity to oxidative stress and failed to develop invasive hyphae in plant cells. Using affinity purification and coimmunoprecipitation assays, we identified several Tig1-associated proteins, including two HDACs that are homologous to components of the yeast Set3 complex. Functional analyses revealed that TIG1, SET3, SNT1, and HOS2 were core components of the Tig1 complex in M. oryzae. The set3, snt1, and hos2 deletion mutants displayed similar defects as those observed in the tig1 mutant, but deletion of HST1 or HOS4 had no detectable phenotypes. Deletion of any of these core components of the Tig1 complex resulted in a significant reduction in HDAC activities. Our results showed that TIG1, like its putative yeast and mammalian orthologs, is one component of a conserved HDAC complex that is required for infectious growth and conidiogenesis in M. oryzae and highlighted that chromatin modification is an essential regulatory mechanism during plant infection.

Comparative analyses of genome sequences from several plant-infecting fungi have shown conservation and expansion of protein families with plant disease-related functions. Here, we show that this hypothesis can be extended to mutualistic symbiotic fungi. We have identified a gene encoding an Era (Escherichia coli Ras)-like GTPase in the rice blast fungus Magnaporthe oryzae and found that it is orthologous to the mature amino terminal part of the Gin1 protein from the arbuscular mycorrhizal (AM) fungus Glomus intraradices. M. oryzae Erl1 is required for full root virulence. Appressoria formation was not severely affected in Deltaerl1 strains, but invasive hyphae grew slower than in the wild type. Root browning defect of Deltaerl1 strains could be complemented by the AM gene under the control of the ERL1 promoter. Erl1 and Gin-N localized to the nucleus when carboxy-terminally labeled with green fluorescent protein (GFP). However, amino-terminal GFP-tagged versions of the proteins expressed in Aspergillus nidulans were shown to localize in the cytoplasm and to cause polarity defects. These data suggest that Erl1 and Gin-N are orthologs and might be involved in the control of hyphal growth in planta. This is the first characterization of an Era-like GTPase in filamentous fungi.

Fungal secondary metabolites are an important source of bioactive compounds for agrochemistry and pharmacology. Over the past decade, many studies have been undertaken to characterize the biosynthetic pathways of fungal secondary metabolites. This effort has led to the discovery of new compounds, gene clusters, and key enzymes, and has been greatly supported by the recent releases of fungal genome sequences. In this review, we present results from a search for genes involved in secondary metabolism and their clusters in the genome of the rice pathogen, Magnaporthe grisea, as well as in other fungal genomes. We have also performed a phylogenetic analysis of recently discovered genes encoding hybrids between a polyketide synthase and a single non-ribosomal peptide synthetase module (PKS-NRPS), as M. grisea seems rich in these enzymes compared with other fungi. Using results from expression and functional studies, we discuss the role of these PKS-NRPS in the avirulence and pathogenicity of M. grisea.

The ascomycete Magnaporthe grisea is a model species for the study of plant fungal interactions. As in many filamentous fungi, targeted gene replacement occurs at low frequency in M. grisea (average 7%). mus52/KU80 is a gene essential for non-homologous end joining (NHEJ) of DNA double-strand breaks. Its deletion increases the frequency of targeted gene replacement in fungi [Ninomiya, Y., Suzuki, K., Ishii, C., Inoue, H., 2004. Highly efficient gene replacements in Neurospora strains deficient for non-homologous end joining. Proc. Natl. Acad. Sci. USA 101(33), 12248-53]. M. grisea KU80 deletion mutants were constructed and displayed wild-type phenotypes regarding pathogenicity, growth, sporulation and mating. MgADE4 targeted gene replacement frequency was increased in Deltaku80 mutants (80% vs 5%) and high frequencies (>80%) were observed at seven other loci. However, the deletion of MgKU80 did not increase the frequency of ACE1 replacement indicating that this locus has an intrinsic reduced ability for gene replacement. These results open the way to large-scale reverse genetics experiments in M. grisea facilitating the study of the infection process.

The interaction between rice and the blast fungus Magnaporthe grisea is the focus of extensive studies on rice disease resistance and fungal infection mechanisms. Here, we review the characteristics of susceptible rice blast infections in terms of physiology, cytology and both host and pathogen transcriptional responses. The success of the infection and the type of disease symptoms strongly depend on environmental and developmental cues. After its penetration into a host cell, the fungus differentiates invasive hyphae that fill up the plant cell lumen and are in direct contact with the membrane of the infected cell. The infected plant cell is alive, displaying considerable vesicle accumulation near the fungus, which is consistent with the establishment of a biotrophic phase at this stage of the infection. Colonization of host tissues by the fungus occurs through the perforation of cell walls from adjacent cells, likely using plasmodesmata as breaking points, or through hyphal growth in the apoplasm. After a few days of biotrophic growth within rice tissues, the fungus switches to a necrotrophic-like phase associated with the onset of sporulation, leading to visible lesions. Genome-wide transcriptomic studies have shown that classical plant defence responses are triggered during a susceptible infection, although the kinetics and amplitude of these responses are slower and lower than in resistant interactions. Infected rice cells are submitted to an intense transcriptional reprogramming, where responses to hormones such as auxins, abscissic acid and jasmonates are likely involved. Consistent with the extensive plant-fungal exchanges during the biotrophic phase, many rice genes expressed during infection encode plasma membrane proteins. At the onset of lesion formation (5 days after the start of infection), M. grisea is actively reprogramming its transcription towards active DNA, RNA and protein syntheses to sustain its rapid growth in infected tissues. A striking characteristic of M. grisea genes expressed at this stage of the infection is the over-representation of genes encoding secreted proteins, mainly of unknown function. However, some of these secreted proteins are enzymes involved in cell wall, protein and lipid degradation, suggesting that the fungus is starting to degrade host polymers and cell walls or is remodelling its own cell wall. The next challenge will be to decipher the role of these induced plant and fungal genes in the susceptible interaction.

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.

The best characterized form of resistance is gene-for-gene resistance. Less well characterized is nonhost resistance in which an entire plant species is resistant to an entire pathogen species. Here, different rice genotypes were inoculated with host and nonhost strains of Magnaporthe isolated from rice, wheat and crabgrass. The different types of interactions were characterized at a cytological level using a 3,3'-diaminobenzidine (DAB) stain to investigate the occurrence of reactive oxygen intermediates or by observing the occurrence of cellular autofluorescence. Gene expression of a set of selected PR-genes was analysed using quantitative real-time polymerase chain reaction. Inoculation with the isolate from crabgrass resulted in a lack of penetration. The wheat isolate induced a hypersensitive response with varying degrees of pathogen growth inside the invaded cell according to the rice genotype. Expression analysis of our PR-gene set revealed clear differences between the different types of interactions in both kinetic and magnitude of gene induction. Our integrated study opens the way to the dissection of molecular components leading to nonhost reactions to Magnaporthe grisea in rice and points to novel sources of durable resistance to fungal plant pathogens in other cereal crops.

Background: A systematic survey of loci carrying retrotransposons in the genome of the rice blast fungus Magnaporthe grisea allowed the identification of novel non-canonical retropseudogenes. These elements are chimeric retrogenes composed of DNA copies from different cellular transcripts directly fused to each other. Their components are copies of a non protein-coding highly expressed RNA of unknown function termed WEIRD and of two fungal retrotransposons: MGL and Mg-SINE. Many of these chimeras are transcribed in various M. grisea tissues and during plant infection. Chimeric retroelements with a similar structure were recently found in three mammalian genomes. All these chimeras are likely formed by RNA template switches during the reverse transcription of diverse LINE elements., Results: We have shown that in M. grisea template switching occurs at specific sites within the initial template RNA which contains a characteristic consensus sequence. We also provide evidence that both single and double template switches may occur during LINE retrotransposition, resulting in the fusion of three different transcript copies. In addition to the 33 bipartite elements, one tripartite chimera corresponding to the fusion of three retrotranscripts (WEIRD, Mg-SINE, MGL-LINE) was identified in the M. grisea genome. Unlike the previously reported two human tripartite elements, this fungal retroelement is flanked by identical 14 bp-long direct repeats. The presence of these short terminal direct repeats demonstrates that the LINE enzymatic machinery was involved in the formation of this chimera and its integration in the M. grisea genome., Conclusion: A survey of mammalian genomic databases also revealed two novel tripartite chimeric retroelements, suggesting that double template switches occur during reverse transcription of LINE retrotransposons in different eukaryotic organisms.

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 delta cpkA and delta mac1 sum1-99 and tetraspanin mutant delta 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.

The ACE1 avirulence gene allele from the rice blast fungus Magnaporthe grisea was characterized in virulent isolate 2/0/3, revealing the insertion of a 1.9 kb MINE retrotransposon in the last ACE1 exon. MINE is a novel chimeric element composed of a transcribed non-coding sequence of 1.1 kb (WEIRD) fused to a 5'-truncated MGL retrotransposon. MINEs were found in high copy number in M. grisea isolates from rice (68 copies) and as a single copy in isolate CD156 from Eleusine. MINEs vary in size (1.3-6.7 kb) with conserved 5' WEIRD sequences and variable 3' MGL sequences. MGLs fused to WEIRDs correspond to different 5'-truncated MGLs with conserved 3' ends. The organization and diversity of MINEs suggest that these retrotransposons result from independent fusions between WEIRD and 5'-truncated MGLs. Such chimera could be formed during MGL reverse transcription as proposed for human U6-LINE1 chimeric retrotransposons and integrated into M. grisea genome using MGL machinery.

Rice, as a widely and intensively cultivated crop, should be a target for parasite host shifts and a source for shifts to co-occurring weeds. Magnaporthe oryzae, of the M. grisea species complex, is the most important fungal pathogen of rice, with a high degree of host specificity. On the basis of 10 loci from six of its seven linkage groups, 37 multilocus haplotypes among 497 isolates of M. oryzae from rice and other grasses were identified. Phylogenetic relationships among isolates from rice (Oryza sativa), millet (Setaria spp.), cutgrass (Leersia hexandra), and torpedo grass (Panicum repens) were predominantly tree like, consistent with a lack of recombination, but from other hosts were reticulate, consistent with recombination. The single origin of rice-infecting M. oryzae followed a host shift from a Setaria millet and was closely followed by additional shifts to weeds of rice, cutgrass, and torpedo grass. Two independent estimators of divergence time indicate that these host shifts predate the Green Revolution and could be associated with rice domestication. The rice-infecting lineage is characterized by high copy number of the transposable element MGR586 (Pot3) and, except in two haplotypes, by a loss of AVR-Co39. Both mating types have been retained in ancestral, well-distributed rice-infecting haplotypes 10 (mainly temperate) and 14 (mainly tropical), but only one mating type was recovered from several derived, geographically restricted haplotypes. There is evidence of a common origin of both ACE1 virulence genotypes in haplotype 14. Host-haplotype association is evidenced by low pathogenicity on hosts associated with other haplotypes.

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.

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 beta-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.

Although microsatellite or simple sequence repeat (SSR) markers have several advantages, few have been developed in fungi. The goal of this study was to identify and characterize SSR-containing loci in the filamentous ascomycete Magnaporthe grisea, the causal agent of rice blast disease, and to add these markers to an integrated genetic map of this species [Theor. Appl. Genet. 95 (1997) 20]. We have constructed and screened a microsatellite-enriched small-insert genomic library as well as exploited both publicly available and one proprietary databases for identification of M. grisea SSR containing sequences. Twenty-four out of 49 primer pairs designed to amplify SSR, produced unambiguous polymorphic products in our test population of six isolates. The number of alleles at each locus ranged from two to six when assayed on 3% agarose gels. Twenty-three of the primer pairs amplified polymorphic products between Guy11 and 2539, the parents of a cross from which a genetic map for M. grisea has been established. Genetic analysis showed that all the markers segregated in the expected 1:1 ratio and map positions were determined for all 23 loci.