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PubMed2026

Evolutionary constraints and regulatory plasticity shape host specialization in the Magnaporthe oryzae species complex.

Rice blast caused by Magnaporthe oryzae threatens global rice production, and wheat blast emergence highlights the pathogen's capacity for host shifts. Although numerous studies have described M. oryzae genome organization and infection mechanisms, critical questions remain regarding the evolutionary drivers of stable host specialization. Importantly, the blast pathogen comprises a species complex of genetically differentiated, host-adapted lineages rather than single homogeneous species. To address this knowledge gap, we integrate evidence from evolutionary genomics, transcriptomics, and metabolomics to develop the "Constrained Plasticity" framework. We argue that host adaptation arises from three interacting layers: genomic scaffolding (including epigenetic and noncoding RNA regulation), regulatory networks (enabling transcriptional plasticity), and metabolic compatibility (determining physiological success). This systems-level perspective explains the long-term stability of host-adapted lineages and the rare breakdowns resulting in host shifts, such as wheat blast. This framework generates testable predictions for pathogen emergence and provides a roadmap for developing lineage-aware resistance strategies.

Oryza↗
PubMed2004

Proteome analysis of rice blast fungus (Magnaporthe grisea) proteome during appressorium formation.

We used two-dimensional gel electrophoresis (2-DE) to identify the proteins that are induced in the rice blast fungus Magnaporthe grisea during appressorium formation. Proteins were extracted from conidia that had germinated on hydrophilic glass plates or from germinated and appressoria-forming conidia on leaf wax-coated hydrophobic glass plates after 4, 8, and 12 h of incubation. Differentially expressed protein spots during appressorium formation were confirmed from gels after 2-DE analysis where proteins had been labeled with (35)S methionine and stained with silver. Internal amino acid sequencing identified five proteins among several proteins induced during appressorium formation. Two denoted as M. grisea proteasome homolgues (MgP1 and MgP5) were 20S proteasome alpha subunits. The remaining three were scytalone dehydratase (SCD), and serine carboxypeptidase Y (CPY). None of the five have been reported previously in the rice blast fungus apart from SCD. We further investigated the role the alpha subunit of 20S proteasome plays in appressorium formation. We confirmed by Western blot analysis that MgP5 is highly expressed during appressorium formation and found that it is also markedly induced by nitrogen- and carbon-starvation, in particular by the former. These observations suggest that the 20S proteasome may be involved in remobilizing storage proteins, which then help to build the appressorium. Thus, fungal proteome analysis may provide important clues about developmental changes such as the generation of the appressorium.

Amino Acid Sequence↗
PubMed2004

Proteomic analysis of pathogen-responsive proteins from rice leaves induced by rice blast fungus, Magnaporthe grisea.

Proteomic approaches using two-dimensional gel electrophoresis (2-DE) were adopted to identify proteins from rice leaf that are differentially expressed in response to the rice blast fungus, Magnaporthe grisea. Microscopic observation of inoculated leaf with M. grisea revealed that callose deposition and hypersensitive response was clearly visible in incompatible interactions but excessive invading hypha with branches were evident in compatible interactions. Proteins were extracted from leaves 24, 48, and 72 hours after rice blast fungus inoculation. Eight proteins resolved on the 2-DE gels were induced or increased in the inoculated leaf. Matrix-assisted laser desorption/ionization-time of flight analysis of these differentially displayed proteins showed them to be two receptor-like protein kinases (RLK), two beta-1.3-glucanases (Glu1, Glu2), thaumatin-like protein (TLP), peroxidase (POX 22.3), probenazole-inducible protein (PBZ1), and rice pathogenesis-related 10 (OsPR-10). Of these proteins, RLK, TLP, PBZ, and OsPR-10 proteins were induced more in the incompatible interactions than in compatible ones. A phytohormone, jasmonic acid also induced all eight proteins in leaves. To confirm whether the expression profile is equal to the 2-DE data, seven cDNA clones were used as probes in Northern hybridization experiments using total RNA from leaf tissues inoculated with incompatible and compatible rice blast fungal races. The genes encoding POX22.3, Glu1, Glu2, TLP, OsRLK, PBZ1, and OsPR-10 were activated in inoculated leaves, with TLP, OsRLK, PBZ1, and OsPR-10 being expressed earlier and more in incompatible than in compatible interactions. These results suggest that early and high induction of these genes may provide host plants with leading edges to defend themselves. The localization of two rice PR-10 proteins, PBZ1 and OsPR-10, was further examined by immunohistochemical analysis. PBZ1 accumulated highly in mesophyll cells under the attachment site of the appressorium. In contrast, OsPR-10 expression was mainly localized to vascular tissue.

Blotting, Northern↗
PubMed2003

Impact of alternative respiration and target-site mutations on responses of germinating conidia of Magnaporthe grisea to Qo-inhibiting fungicides.

Qo-inhibiting fungicides act as respiration inhibitors by binding to the Qo center of cytochrome b. Sensitivities of fungi to Qo inhibitors can be influenced by the induction of alternative respiration or by mutational changes of the cytochrome b target site. Previous studies on both mechanisms in Magnaporthe grisea (Hebert) Barr were focused on the mycelial stage of the pathogen. The present study describes the expression and impact of both resistance mechanisms during the stage of conidia germination. In the absence of a host, alternative respiration provided a >500-fold rescue from azoxystrobin during the germination of conidia derived from four wild-type isolates of M. grisea. This rescue potential during conidia gemination was substantially more pronounced than for mycelial growth. However, the pronounced effectiveness of alternative respiration during conidia germination was not apparent when barley leaves were protected with azoxystrobin prior to inoculation with conidia. In a comparison of a wild-type strain and an alternative respiration-deficient mutant, azoxystrobin efficacies in suppressing symptom development differed by a factor of two, with full disease control achieved for both genotypes at 1 microg ml(-1) azoxystrobin. In contrast, conidia derived from two QoI-resistant target site mutants were highly resistant to azoxystrobin and trifloxystrobin and fully capable of infecting leaf surfaces protected with 10 microg ml(-1) of azoxystrobin. Both target-site mutants had emerged spontaneously in the presence of high azoxystrobin doses when residual mycelial growth was supported by alternative respiration. The effective silencing of alternative respiration in protective applications of Qo-inhibiting fungicides might constitute a strategy of slowing the emergence of highly resistant target site mutants.

Acetates↗
PubMed2004

Mechanism of resistance to carpropamid in Magnaporthe grisea.

The inhibitory activity of carpropamid on scytalone dehydratase (SDH) extracted from a carpropamid-resistant strain of Magnaporthe grisea (Hebert) Barr was dramatically reduced in comparison with that on SDH extracted from the sensitive strain. A single-point mutation (G to A) located at the upstream region (233 bp downstream from the ATG codon) resulting in a one-amino-acid substitution (valine [GTG] 75 to methionine [ATG]: V75M) was found in the resistant strain. To examine whether the V75M mutation is the primary reason for decreasing the sensitivity of SDH to carpropamid, the SDH cDNAs of both the sensitive and the resistant strain were cloned into a GST-fused protein expression vector-system. The recombinant SDHs of both strains exhibited the same sensitivities to carpropamid as those extracted from the mycelia of the respective strains. These data clearly revealed that the V75M mutation causes the low sensitivities of the SDHs of the carpropamid-resistant strains, and strongly suggests that the V75M mutation confers resistance of these strains to carpropamid.

Amides↗
PubMedDate not supplied

Isolation of pheromone precursor genes of Magnaporthe grisea.

In heterothallic ascomycetes one mating partner serves as the source of female tissue and is fertilized with spermatia from a partner of the opposite mating type. The role of pheromone signaling in mating is thought to involve recognition of cells of the opposite mating type. We have isolated two putative pheromone precursor genes of Magnaporthe grisea. The genes are present in both mating types of the fungus but they are expressed in a mating type-specific manner. The MF1-1 gene, expressed in Mat1-1 strains, is predicted to encode a 26-amino-acid polypeptide that is processed to produce a lipopeptide pheromone. The MF2-1 gene, expressed in Mat1-2 strains, is predicted to encode a precursor polypeptide that is processed by a Kex2-like protease to yield a pheromone with striking similarity to the predicted pheromone sequence of a close relative, Cryphonectria parasitica. Expression of the M. grisea putative pheromone precursor genes was observed under defined nutritional conditions and in field isolates. This suggests that the requirement for complex media for mating and the poor fertility of field isolates may not be due to limitation of pheromone precursor gene expression. Detection of putative pheromone precursor gene mRNA in conidia suggests that pheromones may be important for the fertility of conidia acting as spermatia.

Amino Acid Sequence↗
PubMed2001

Regions of microsynteny in Magnaporthe grisea and Neurospora crassa.

A bacterial artificial chromosome (BAC) clone containing 110,467 bp of genomic DNA from Magnaporthe grisea was sequenced, annotated, and compared to the genomes of Neurospora crassa, Candida albicans, and Saccharomyces cerevisiae. Twenty-six open reading frames (ORFs), involved in multiple biochemical pathways, were identified in the BAC sequence. A region of 53 kb, containing 18 of the 26 ORFs, was found to be syntenic to a portion of the N. crassa genome. Subregions of complete colinearity as well as interrupted colinearity were present. No synteny was evident with either C. albicans or S. cerevisiae. The identification of syntenic regions containing highly conserved genes across two genera that have been evolutionarily separated for approximately 200 million years elicits many biological questions as to the function and identity of these genes.

Candida albicans↗
PubMed2001

The vacuole as central element of the lytic system and sink for lipid droplets in maturing appressoria of Magnaporthe grisea.

Histochemical and ultrastructural studies were carried out on a wild-type strain (Guy11) and a melanin-deficient mutant (buf1) of the rice-blast pathogen, Magnaporthe grisea (= Pyricularia oryzae), in order to investigate the destination of lipid storage reserves during appressorium development. Lipid droplets were abundant in conidia and were mobilised upon germination, accumulating in the appressorial hook which developed at the tip of each germ tube. Following the formation of a septum at the base of the nascent appressorium, one or a few closely appressed central vacuoles became established and were observed to enlarge in the course of appressorium maturation. On unyielding artificial surfaces such as glass or plastic, appressoria matured to completion within 36-48 h, by which time the enlarged vacuole filled most of the inside volume of the appressorium. Light and transmission electron microscopical observations revealed that the lipid droplets entered the vacuole by autophagocytosis and were degraded therein. Histochemical approaches confirmed the vacuole as the key lytic element in maturing appressoria. Endocytosis of a vital dye, Neutral Red, progressed via endosomes which migrated into the vacuole and lysed there, releasing their dye content into the vacuolar lumen. Furthermore, activity of the lysosomal marker enzyme, acid phosphomonoesterase, was strongly localised in the vacuole at all stages of appressorium maturation. It is therefore envisaged that vacuoles are involved in the degradation of lipid storage reserves which may act as sources of energy and/or osmotically active metabolites such as glycerol, which generate the very high turgor pressure known to be crucial for penetration of hard surfaces. On softer surfaces such as onion epidermis, appressoria of M. grisea were able to penetrate before degradation of lipid droplets had been completed.

Acid Phosphatase↗
PubMed2003

Identification and fine mapping of Pi33, the rice resistance gene corresponding to the Magnaporthe grisea avirulence gene ACE1.

Rice blast disease is a major constraint for rice breeding. Nevertheless, the genetic basis of resistance remains poorly understood for most rice varieties, and new resistance genes remain to be identified. We identified the resistance gene corresponding to the cloned avirulence gene ACE1 using pairs of isogenic strains of Magnaporthe grisea differing only by their ACE1 allele. This resistance gene was mapped on the short arm of rice chromosome 8 using progenies from the crosses IR64 (resistant) x Azucena (susceptible) and Azucena x Bala (resistant). The isogenic strains also permitted the detection of this resistance gene in several rice varieties, including the differential isogenic line C101LAC. Allelism tests permitted us to distinguish this gene from two other resistance genes [ Pi11 and Pi-29(t)] that are present on the short arm of chromosome 8. Segregation analysis in F(2) populations was in agreement with the existence of a single dominant gene, designated as Pi33. Finally, Pi33 was finely mapped between two molecular markers of the rice genetic map that are separated by a distance of 1.6 cM. Detection of Pi33 in different semi-dwarf indica varieties indicated that this gene could originate from either one or a few varieties.

Chromosome Mapping↗
PubMed2004

Use of Pi5(t) markers in marker-assisted selection to screen for cultivars with resistance to Magnaporthe grisea.

Identification of the PCR markers tightly linked to genes that encode important agronomic traits is useful for marker-assisted selection (MAS). The rice Pi5(t) locus confers broad-spectrum resistance to Magnaporthe grisea, the causal agent of rice blast disease. It has been hypothesized that the Pi5(t) locus carries the same gene as that encoded by the Pi3(t) and Pii(t) loci. We developed three PCR-based dominant markers (JJ80-T3, JJ81-T3, and JJ113-T3) from three previously identified BIBAC clones-JJ80, JJ81, and JJ113-that are linked to the Pi5(t) locus. PCR analysis of 24 monogenic lines revealed that these markers are present only in lines that carry Pi5(t), Pi3(t), and Pii(t). PCR and DNA gel-blot analysis of candidate resistance lines using JJ80-T3, JJ81-T3, and JJ113-T3 indicated that Tetep is the likely donor of Pi5(t). Of the 184 rice varieties tested, 34 carried the JJ80-T3-, JJ81-T3-, and JJ113-T3-specific bands. Disease evaluation of those 34 varieties revealed that all conferred resistance to PO6-6. The genomic structure of three of these resistant varieties (i.e., IR72, Taebaeg, Jahyangdo) is most similar to that of Pi5(t). Our results demonstrate the usefulness of the JJ80-T3, JJ81-T3, and JJ113-T3 markers for MAS for M. grisea resistance.

Breeding↗
PubMed2006

Identification and fine mapping of AvrPi15, a novel avirulence gene of Magnaporthe grisea.

Avirulence of Magnaporthe grisea isolate CHL346 on rice cultivar GA25 was studied with 242 ascospore progenies derived from the cross CHL346 x CHL42. Segregation analysis of the avirulence in the progeny population was in agreement with the existence of a single avirulence (Avr) gene, designated as AvrPi15. For mapping the Avr gene, we developed a total of 121 microsatellite DNA markers [simple sequence repeat (SSR)], which evenly distributed in the whole-genome of M. grisea through bioinformatics analysis (BIA) using the publicly available sequence. Linkage analysis of the AvrPi15 gene with these SSR markers showed that six markers on chromosome 6, MS6-1, MS6-2, MS6-3, MS6-7, MS6-8 and MS6-10, were linked to the AvrPi15 locus. To further define the chromosomal location of the AvrPi15 locus, two additional markers, MS6-17 and STS6-6, which were developed based on the sequences of telomeric region 11 (TEL11), were subjected to linkage analysis. The results showed that MS6-17 and STS6-6 were associated with the locus by 3.3 and 0.8 cM, respectively. To finely map the Avr gene, two additional candidate avirulence gene (CAG) markers, CAG6-1 and CAG6-2, were developed based on the gene annotation of the sequence of TEL 11. Linkage analysis of the Avr gene with these two markers revealed that both of them completely cosegregated with the AvrPi15 locus. Finally, this locus was physically mapped into approximately 7.2-kb interval of the TEL11 by BIA using these sequence-ready markers. This is the key step toward positional cloning of the AvrPi15 gene.

Chromosome Segregation↗
PubMed2006

Biotechnologically relevant enzymes and proteins. Antifungal mechanism of the Aspergillus giganteus AFP against the rice blast fungus Magnaporthe grisea.

The mold Aspergillus giganteus produces a basic, low molecular weight protein showing antifungal properties against economically important plant pathogens, the AFP (Antifungal Protein). In this study, we investigated the mechanisms by which AFP exerts its antifungal activity against Magnaporthe grisea. M. grisea is the causal agent of rice blast, one of the most devastating diseases of cultivated rice worldwide. AFP was purified from the extracellular medium of A. giganteus cultures. The AFP protein was found to induce membrane permeabilization in M. grisea cells. Electron microscopy studies revealed severe cellular degradation and damage of plasma membranes in AFP-treated fungal cells. AFP however failed to induce membrane permeabilization on rice or human HeLa cells. Furthermore, AFP enters the fungal cell and targets to the nucleus, as revealed by co-localization experiments of Alexa-labeled AFP with the SYTOX Green dye. Finally, AFP binds to nucleic acids, including M. grisea DNA. Our results suggest that the combination of fungal cell permeabilization, cell-penetrating ability and nucleic acid-binding activity of AFP determines its potent antifungal activity against M. grisea. These results are discussed in relation to the potential of the AFP protein to enhance crop protection against fungal diseases.

Antifungal Agents↗
PubMed2003

Characterization of spontaneous mutants of Magnaporthe grisea expressing stable resistance to the Qo-inhibiting fungicide azoxystrobin.

The class of Qo-inhibiting fungicides (QoIs) act as respiration inhibitors by binding to the Qo center of cytochrome b. The longevity of these fungicides has been challenged by the selection of fungal sub-populations resisting high doses of QoI fungicides, with a G143A amino acid exchange in the cytochrome b target site identified as the most common cause of resistance. In contrast, the mechanism of alternative respiration, as another mechanism of fungal QoI resistance, has thus far not been affiliated with practical resistance. In the present study, azoxystrobin-resistant mutants of Magnaporthe grisea were generated and characterized. Emergence of these spontaneous mutants was facilitated when resting melanized mycelia were allowed to escape full inhibition by azoxystrobin. This escape was related to the intactness of alternative respiration, indicating that residual expression of this rescue mechanism was involved in the spontaneous emergence of target-site mutants. The two mutants characterized resisted high doses of the QoI, azoxystrobin, with resistance factors exceeding 1,000. Two different mutations of the cytochrome b gene were identified as exchanges of guanine, leading to a G143A or a G143S amino acid exchange. Resistance of both target-site mutants remained stable during four consecutive disease cycles in the absence of azoxystrobin. Several parameters tested to measure fitness penalties inherent to the mutational changes revealed that the G143A mutant was not compromised. In contrast, the conidia production of the G143S mutant was significantly lower under both saprophytic and pathogenic conditions of reproduction.

Acrylates↗
PubMed2003

Occan, a novel transposon in the Fot1 family, is ubiquitously found in several Magnaporthe grisea isolates.

We investigated a DNA fragment and its flanking region deleted in the spontaneous Pi-a virulent mutant of Magnaporthe grisea Ina168. A new transposon-like sequence was identified from a region adjacent to the deleted fragment and was named Occan. Occan contained a 2,259-bp ORF interrupted by one 63-bp intron and had both a TA dinucleotide and 77 bp of perfect inverted repeats at both termini, without direct repeats. These features indicated that Occan is a member of the Fot1 family. RT-PCR analysis confirmed the expression of the putative transposase and the presence of an intron. Southern analysis of pulse-field gel electrophoresis-separated chromosomes indicated that Occan was dispersed in all chromosomes of the rice pathogen, Ina168. Copy numbers of Occan were also preserved in a host-specific manner amongst M. grisea isolates. In particular, rice pathogens contained a large number of the element inserted into their genome. Phylogenetic analysis with other known members of the Fot1 family revealed that Occan was dissimilar to any other known elements and it is thus proposed that Occan be separated to a new subfamily.

Amino Acid Sequence↗
PubMed2003

Meiotic behavior of a supernumerary chromosome in Magnaporthe oryzae.

A 1.2-Mb DNA band from an isolate of Magnaporthe oryzae was detected in a pulsed-field gel. A chromosomal entity corresponding to this band was observed at the mitotic metaphase stage. This minichromosome, carrying many transposable elements and two telomeres, was transmitted to ascosporic F(1) cultures in a non-Mendelian manner with frequent changes in its size and number. Segregation analysis with RFLP markers indicated that the minichromosome underwent structural rearrangements, such as deletion and duplication, not only during meiosis but also after meiosis. An ectopic sister chromatid recombination may cause the size variation of the minichromosomes.

Blotting, Southern↗
PubMed2005

Enhanced resistance to the rice blast fungus Magnaporthe grisea conferred by expression of a cecropin A gene in transgenic rice.

Cecropins are a family of antimicrobial peptides, which constitute an important key component of the immune response in insects. Here, we demonstrate that transgenic rice (Oryza sativa L.) plants expressing the cecropin A gene from the giant silk moth Hyalophora cecropia show enhanced resistance to Magnaporthe grisea, the causal agent of the rice blast disease. Two plant codon-optimized synthetic cecropin A genes, which were designed either to retain the cecropin A peptide in the endoplasmic reticulum, the ER-CecA gene, or to secrete cecropin A to the extracellular space, the Ap-CecA gene, were prepared. Both cecropin A genes were efficiently expressed in transgenic rice. The inhibitory activity of protein extracts prepared from leaves of cecropin A-expressing plants on the in vitro growth of M. grisea indicated that the cecropin A protein produced by the transgenic rice plants was biologically active. Whereas no effect on plant phenotype was observed in ER-CecA plants, most of the rice lines expressing the Ap-CecA gene were non-fertile. Cecropin A rice plants exhibited resistance to rice blast at various levels. Transgene expression of cecropin A genes was not accompanied by an induction of pathogenesis-related (PR) gene expression supporting that the transgene product itself is directly active against the pathogen. Taken together, the results presented in this study suggest that the cecropin A gene, when designed for retention of cecropin A into the endoplasmic reticulum, could be a useful candidate for protection of rice plants against the rice blast fungus M. grisea.

Animals↗
PubMed2002

Genetic and physical mapping of a rice blast resistance locus, Pi-CO39(t), that corresponds to the avirulence gene AVR1-CO39 of Magnaporthe grisea.

We have identified, genetically mapped and physically delineated the chromosomal location of a new rice blast resistance locus, designated Pi-CO39(t). This locus confers resistance to Magnaporthe grisea isolates carrying the AVR1-CO39 avirulence locus. The AVR1-CO39 locus is conserved in non-rice (cereals and grasses)-infecting isolates of M. grisea, making Pi-CO39(t) useful for engineering M. grisea resistance in rice and other cereals. The resistance in the rice line CO39 was inherited as a single dominant locus in segregating populations derived from F(2) and F(3) crosses between disease-resistant (CO39) and susceptible (51583) rice genotypes. Microsatellite, RFLP and resistance gene analog (RGA) markers were used to map the Pi-CO39(t) locus to a 1.2-cM interval between the probenazole-responsive ( RPR1) gene (0.2 cM) and RFLP marker S2712 (1.0 cM) on the short arm of rice chromosome 11. RFLP markers G320 and F5003, and resistance gene analogs RGA8, RGA38 and RGACO39 were tightly linked to the Pi-CO39(t) locus (no recombination detected in a sample of ~2400 gametes). A large-insert genomic library of CO39 was constructed in the binary plant transformation vector pCLD04541. A library screen using RGA8, RGA38 and probes derived from the ends of CO39 clones, as well as BAC end probes from the corresponding locus in the rice cv. Nipponbare, resulted in the assembly of three CO39 contigs of 180 kb, 110 kb and 145 kb linked to the Pi-CO39(t) locus. A 650-kb contig was also constructed representing the susceptible locus, pi-CO39(t), in the Nipponbare genome. The two genomes are highly divergent with respect to additions, deletions and translocations at the Pi-CO39(t) locus, as revealed by the presence or absence of mapping markers.

Blotting, Southern↗
PubMed2003

Serial Analysis of Gene Expression (SAGE) of Magnaporthe grisea: genes involved in appressorium formation.

Treatment with cyclic AMP (cAMP) induces appressorium formation in the phytopathogenic fungus Magnaporthe grisea, the causative agent of rice blast disease. In a search for the M. grisea genes responsible for appressorium formation and host invasion, SAGE (Serial Analysis of Gene Expression) was carried out using mRNA isolated from fungal conidia germinating in the presence and absence of cAMP. From cAMP-treated conidia 5087 tags including 2889 unique tags were isolated, whereas untreated conidia yielded 2342 unique tags out of total of 3938. cAMP treatment resulted in up- and down-regulation of genes corresponding to 57 and 53 unique tags, respectively. Upon consultation of EST/cDNA databases, 22 tags with higher representation in cAMP-treated conidia were annotated with putative gene names. Furthermore, 28 tags corresponding to cAMP-induced genes could be annotated with the help of the recently published genome sequence of M. grisea. cAMP-induced genes identified by SAGE included many genes that have not been described so far, as well as a number of genes known to be involved in pathogenicity, e.g. MPG1, MAS1 and MAC1. RT-PCR of 13 randomly selected genes confirmed the SAGE results, verifying the fidelity of the SAGE data.

Base Sequence↗