RESUMEN

Blast fungus (Magnaporthe oryzae B.C. Couch) is an imminent threat to global food security because it causes serious yield losses in rice (Oryza sativa L.) and wheat (Triticum aestivum L.). The investigation of infection processes in resistant and susceptible varieties, as well as the cellular responses involved in resistance, can help us to better understand the process of interaction of the M. oryzae-Poaceae pathosystems. Thus, the objectives of this study were to evaluate the processes of pre- and post-infection of M. oryzae in leaves of wheat varieties with different levels of resistance. The percentage of germinated conidia, appressorium formed, tissue penetration and colonization, and the reaction of leaf tissue to infection were evaluated. A decrease was observed in the percentage of germinated conidia, appressorium formation, tissue penetration and colonization, especially in the tissues of resistant varieties, in addition to an increase in the plant's response to infection, with cell wall reinforcement, cell death, and autofluorescent cytoplasm aggregation. Nevertheless, our data produced a different temporal perspective regarding the expression of the known types of resistance. We found that, for a single genotype, recognition can start as early as 6 h after inoculation and continue to evolve until very late during the infection cycle, culminating in cell death. The combined and overlapping pre- and post-haustorial resistance mechanisms were sufficient to prevent disease symptoms, with a few punctual lesions observed in one of the resistant varieties (BR 18) and no visible symptoms in the other two (Ônix or BRS229) as opposed to susceptible variety.

Asunto(s)

Magnaporthe , Oryza , Ascomicetos , Magnaporthe/genética , Oryza/microbiología , Enfermedades de las Plantas/microbiología , Hojas de la Planta/microbiología , Esporas Fúngicas , Triticum/microbiología

RESUMEN

Rapid detection is key to managing emerging diseases because it allows their spread around the world to be monitored and limited. The first major wheat blast epidemics were reported in 1985 in the Brazilian state of Paraná. Following this outbreak, the disease quickly spread to neighboring regions and countries and, in 2016, the first report of wheat blast disease outside South America was released. This Asian outbreak was due to the trade of infected South American seed, demonstrating the importance of detection tests in order to avoid importing contaminated biological material into regions free from the pathogen. Genomic analysis has revealed that one particular lineage within the fungal species Pyricularia oryzae is associated with this disease: the Triticum lineage. A comparison of 81 Pyricularia genomes highlighted polymorphisms specific to the Triticum lineage, and this study developed a real-time PCR test targeting one of these polymorphisms. The test's performance was then evaluated in order to measure its analytical specificity, analytical sensitivity, and robustness. The C17 quantitative PCR test detected isolates belonging to the Triticum lineage with high sensitivity, down to 13 plasmid copies or 1 pg of genomic DNA per reaction tube. The blast-based approach developed here to study P. oryzae can be transposed to other emerging diseases.

Asunto(s)

Agricultura , Genoma Fúngico , Magnaporthe , Reacción en Cadena en Tiempo Real de la Polimerasa , Triticum , Agricultura/métodos , Genes Fúngicos/genética , Genómica , Magnaporthe/genética , Enfermedades de las Plantas/microbiología , América del Sur , Triticum/microbiología

RESUMEN

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.

Asunto(s)

Flujo Génico , Magnaporthe/genética , Bangladesh , Biota , Grano Comestible/microbiología , Transferencia de Gen Horizontal , Variación Genética , Magnaporthe/clasificación , Magnaporthe/aislamiento & purificación , Poaceae/microbiología , Análisis de Secuencia de ADN , América del Sur , Secuenciación Completa del Genoma

RESUMEN

Magnaporthe oryzae is an important model system in studies of plant pathogenic fungi, and nitrogen is a key nutrient source affecting microbial growth and development. In order to understand how nitrogen stress causes changes in mycelial proteins, we analyzed differentially expressed mycelial proteins from the M. oryzae virulent strain CH-63 using two-dimensional electrophoresis and mass spectrometry in complete medium or under nitrogen starvation conditions. A total of 975 ± 70 and 1169 ± 90 protein spots were detected in complete medium and under nitrogen starvation conditions, respectively. Forty-nine protein spots exhibited at least 2-fold up-regulation or down-regulation at the protein level according to PDQuest7.4. Moreover, 43 protein spots were successfully identified by matrix-assisted laser desorption/ionization-time-of-flight/time-of-flight mass spectrometry. Among these spots, 6 proteins were functionally unknown and 37 proteins were categorized into 5 groups according to their functions, including development, metabolism, biosynthesis, and biological process. These 37 proteins were further analyzed for their enriched metabolic pathways by KOBAS2.0, and 14 proteins were found to be involved in glycolysis, tricarboxylic acid cycle, and nitrogen metabolism. Taken together, the regulation of M. oryzae growth under the nitrogen starvation conditions appears to be complex because of the various proteins and enzymes involved.

Asunto(s)

Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Magnaporthe/genética , Magnaporthe/metabolismo , Regulación Fúngica de la Expresión Génica , Nitrógeno/metabolismo , Oryza , Enfermedades de las Plantas/microbiología , Proteoma/genética , Proteómica/métodos , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción

RESUMEN

Previous studies have shown that the blast fungus, Magnaporthe oryzae, may experience nitrogen starvation during infection of its plant host (rice,Oryza sativa). Here, we studied the expression of seven genes encoding cysteine-rich proteins with N-terminal signal peptides during nitrogen limitation and throughout the infection process. Some genes were upregulated to a greater extent in weak pathogenic strains than in strong pathogenic strains when they were cultured in complete media, and the expression of some genes was higher in both weak and strong pathogenic strains cultured in 1/10-N and nitrogen starvation media. Furthermore, the expression of these genes was upregulated to different extents in the early stages of M. oryzae infection. These data demonstrate that the genes of interest are highly expressed in weak and strong pathogenic strains cultured under nitrogen limitation and at the early stage of the infection process. This indicates that cysteine-rich secreted proteins in the blast fungus might be involved in establishing disease in the host and that they are sensitive to nitrogen levels. Thus, their role in sensing nitrogen availability within the host is implied, which provides a basis for further functional identification of these genes and their products during plant infection.

Asunto(s)

Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Interacciones Huésped-Patógeno , Magnaporthe/genética , Magnaporthe/metabolismo , Nitrógeno/metabolismo , Oryza/microbiología , Regulación Fúngica de la Expresión Génica , Enfermedades de las Plantas/microbiología

RESUMEN

The aim of the current study was to investigate the prokaryotic expression of the Magnaporthe oryzae effector genes BAS1 and BAS4 fused to the fluorescent protein mCherry. Based on previous polymorphic analysis of BAS1 and BAS4 in rice blast strains using PCR, blast strains containing the PCR products of BAS1 and BAS4 were selected for liquid culture for total RNA extraction. For PCR analysis, cDNA was selected as a template to amplify the coding region of BAS1 and BAS4, the plasmid pXY201 was selected as template to amplify the mCherry sequence, and the three sequences were cloned into pMD®19-T vectors. Positive recombinant plasmids were digested using two restriction enzymes and the cleaved fragments of BAS1 and mCherry and BAS4 and mCherry were ligated to pGEX-4T-1 vectors and expression was induced using IPTG. The PCR results showed that the sequence sizes of BAS1, BAS4, and mCherry were 348, 309, and 711 bp, respectively, and these were cloned into pMD®19-T vectors. After digestion and gel purification, the fragments of BAS1 and mCherry, BAS4 and mCherry were ligated into pGEX-4T-1 vectors and expressed in Escherichia coli BL21 competent cells. The expressed proteins were approximately 60 kDa, corresponding to their theoretical size. Prokaryotic expression products of BAS1 and BAS4 fused to mCherry were presented in this study, providing a base for constructing prokaryotic expression vectors of pathogen effector genes fused to mCherry, which will contribute to further study of the subcellular localization, function, and protein interactions of these effectors.

Asunto(s)

Proteínas Fúngicas/genética , Proteínas Luminiscentes/genética , Magnaporthe/genética , Proteínas Recombinantes de Fusión/genética , Fusión Artificial Génica/métodos , Clonación Molecular/métodos , ADN Complementario/genética , Proteínas Fúngicas/biosíntesis , Proteínas Fúngicas/química , Amplificación de Genes , Expresión Génica , Genes Fúngicos , Vectores Genéticos/química , Vectores Genéticos/genética , Proteínas Luminiscentes/biosíntesis , Proteínas Luminiscentes/química , Oryza/microbiología , Plásmidos/genética , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/química , Proteína Fluorescente Roja

RESUMEN

The aim of this study was to construct overexpression vectors and selecting strains of the Magnaporthe oryzae effectors BAS1 and BAS4. Primer pairs of BAS1, BAS4, and mCherry were designed based on their known nucleotide sequences. The coding sequences of BAS1 and BAS4 were amplified, and the pXY201 plasmid was selected as a template to amplify the mCherry sequence. Fragments of BAS1 and mCherry, and BAS4 and mCherry were ligated into the pCAMBIA1302 vector. The recombinant pCAMBIA-BAS1-mCherry and pCAMBIA-BAS4-mCherry plasmids were transformed into E. coli DH5α competent cells. Transformants were screened by PCR, and plasmids from the positive transformants were extracted by enzymatic digestion to obtain pCAMBIA-BAS1-mCherry and pCAMBIA-BAS4-mCherry. The pCAMBIA-BAS1-mCherry and pCAMBIA-BAS4-mCherry plasmids were transformed into protoplasts of rice blast strains and the transformed strains were screened by PCR using primer pairs against the hygromycin gene. The result showed that the PCR products corresponded with the theoretical sizes. RT-PCR was used to analyze the expression of BAS1 and BAS4 in five transformed strains of BAS1 and BAS4, and the result showed that the higher expression level of the two genes was occurred in five transformant strains comparing to wild-type strain A3467-40 (the strain containing BAS1 and BAS4), but there was no difference among the five overexpression strains. The sporulation and spore germination of transformed strains was higher than in wild type strain, and there was no difference in the germination time. Construction of overexpression vectors and strains of M. oryzae effectors BAS1 and BAS4 provide reference material for other new effectors.

Asunto(s)

Proteínas Fúngicas/genética , Vectores Genéticos/metabolismo , Proteínas Luminiscentes/genética , Magnaporthe/genética , Plásmidos/metabolismo , Transactivadores/genética , Clonación Molecular , Cartilla de ADN/síntesis química , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas Fúngicas/metabolismo , Expresión Génica , Vectores Genéticos/química , Proteínas Luminiscentes/metabolismo , Magnaporthe/metabolismo , Oryza/microbiología , Enfermedades de las Plantas/microbiología , Plásmidos/química , Ingeniería de Proteínas , Protoplastos/microbiología , Protoplastos/patología , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Transactivadores/metabolismo , Transformación Bacteriana , Proteína Fluorescente Roja

RESUMEN

Wheat blast, caused by Magnaporthe oryzae, is an important disease across central and southern Brazil. Control has relied mainly on strobilurin fungicides (quinone-outside inhibitors [QoIs]). Here, we report the widespread distribution of QoI resistance in M. oryzae populations sampled from wheat fields and poaceous hosts across central and southern Brazil and the evolution of the cytochrome b (cyt b) gene. Sequence analysis of the cyt b gene distinguished nine haplotypes, with four haplotypes carrying the G143A mutation associated with QoI resistance and two haplotypes shared between isolates sampled from wheat and other poaceous hosts. The frequency of the G143A mutation in the wheat-infecting population increased from 36% in 2005 to 90% in 2012. The G143A mutation was found in many different nuclear genetic backgrounds of M. oryzae. Our findings indicate an urgent need to reexamine the use of strobilurins to manage fungal wheat diseases in Brazil.

Asunto(s)

Citocromos b/genética , Farmacorresistencia Fúngica/genética , Magnaporthe/genética , Metacrilatos , Pirimidinas , Secuencia de Bases , Haplotipos , Datos de Secuencia Molecular , Estrobilurinas , Triticum/microbiología

RESUMEN

The in vitro sensitivity of AvrPik allele isolates of Magnaporthe oryzae to isoprothiolane was examined and the virulence fitness costs of AvrPik allele isolates to isoprothiolane were assessed. Isoprothiolane was found to suppress the radial growth of AvrPik allele isolates at all concentrations (1, 5, 10, 15, and 20 µg/mL). Generally, a higher isoprothiolane concentration has a stronger inhibitory effect on mycelial growth in AvrPik allele isolates at 6 and 10 days after inoculation. The inhibitory effect of isoprothiolane also increased with treatment time. To determine whether a correlation existed between the in vitro sensitivity of AvrPik allele isolates and virulence, the half-maximal inhibitor concentration and 75% of the maximum inhibitor concentration were calculated for each mutation isolate and wild-type isolate. Based on these values and virulence, no significant correlation between the susceptibility of AvrPik allele isolates and virulence was detected. In summary, no fitness costs were associated with sensitivity of blast isolates carrying specific AvrPik alleles to different virulence.

Asunto(s)

Aptitud Genética/efectos de los fármacos , Magnaporthe/efectos de los fármacos , Mutación , Micelio/efectos de los fármacos , Tiofenos/farmacología , Alelos , Relación Dosis-Respuesta a Droga , Genotipo , Magnaporthe/genética , Magnaporthe/patogenicidad , Micelio/genética , Micelio/patogenicidad , Oryza/microbiología , Enfermedades de las Plantas/microbiología , Factores de Tiempo , Virulencia

RESUMEN

Magnaporthe oryzae, the causal agent of wheat blast, was characterized on a molecular level with 38 newly isolated genomic SSR loci. Among the 31 wheat isolates analyzed, 15 polymorphic loci were detected, with an average of 1.7 alleles per locus, 28.9% of them being highly or reasonably informative. The number of polymorphic loci was higher in isolates from Londrina in the Brazilian state of Paraná and Coromandel in Minas Gerais compared with Goiânia in Goiás and São Borja in Rio Grande do Sul. The rice isolate was clearly different from the wheat isolates, and the size difference in polymorphic SSR loci between one isolate from wheat and one isolate from rice was associated with the number of repeats. Some isolates collected from different states and in different years demonstrated similarities of 100%. The markers developed here are useful for the genetic analysis of M. oryzae isolated from wheat, and isolates representing the variability detected in the field can be used to search for better wheat blast resistance.

Asunto(s)

Magnaporthe/genética , Triticum/microbiología , Brasil , Marcadores Genéticos , Genotipo , Oryza/microbiología , Enfermedades de las Plantas/microbiología , Polimorfismo Genético

RESUMEN

Since its first report in Brazil in 1985, wheat blast, caused by Magnaporthe oryzae (anamorph: Pyricularia oryzae), has become increasingly important in South America, where the disease is still spreading. We used 11 microsatellite loci to elucidate the population structure of the wheat blast pathogen in wheat fields in central-western, southeastern, and southern Brazil. No subdivision was found among the wheat-infecting populations, consistent with high levels of gene flow across a large spatial scale. Although the clonal fraction was relatively high and the two mating type idiomorphs (MAT1-1 and MAT1-2) were not at similar frequencies, the clone-corrected populations from Distrito Federal and Goiás, Minas Triangle, and São Paulo were in gametic equilibrium. Based on these findings, we propose that populations of the wheat blast pathogen exhibit a mixed reproductive system in which sexual reproduction is followed by the local dispersal of clones. Seedling virulence assays with local wheat cultivars differentiated 14 pathotypes in the current population. Detached head virulence assays differentiated eight virulence groups on the same wheat cultivars. There was no correlation between seedling and head reactions.

Asunto(s)

Variación Genética , Genética de Población , Magnaporthe/genética , Oryza/microbiología , Enfermedades de las Plantas/microbiología , Triticum/microbiología , Brasil , Flujo Génico , Genes Fúngicos/genética , Genes del Tipo Sexual de los Hongos/genética , Inflorescencia/microbiología , Magnaporthe/patogenicidad , Repeticiones de Microsatélite/genética , Plantones/microbiología , Virulencia

RESUMEN

The rice blast disease caused by the ascomycete Magnaporthe grisea continues to cause a tremendous impact in rice (Oryza sativa) cultures around the world. Elucidating the molecular basis of the fungus interactions with its host might help increase the general understanding of the pathogen-host relationship. At the moment of invasion, the fungus secretes effectors that modify host defenses and cellular processes as they successively invade living rice cells. PWL2, an effector protein, is a known AVR (avirulence) gene product. The PWL2 gene prevents the fungus from infecting weeping lovegrass (Eragrostis curvula). In this study, we identified a PWL2 allele gene (which we termed PWL2D) in a strain of M. grisea. The sequence of PWL2D has only two bases different from that of PWL2, producing alterations in residue 90 and residue 142. However, the alteration of residue 90 (from D(90) to N(90)) is critical to gene function. Here, we cloned the gene PWL2D in a pET System vector, expressed the gene product in Escherichia coli and evaluated by spectroscopic techniques some aspects of the PWL2D structure. While TRX-tagged PWL2D is prone to aggregation, the solubility of PWL2D is improved when it is overexpressed without its original signal peptide. Expression and purification procedures for these constructs are described. Finally, we found out that the protein seems to be an intrinsically disordered protein. Results from these studies will facilitate structural analysis of PWL2D and might contribute to understanding the gene's function and of fungal/plant interactions.

Asunto(s)

Proteínas Fúngicas/genética , Proteínas Fúngicas/aislamiento & purificación , Magnaporthe/genética , Mutación , Alelos , Secuencia de Aminoácidos , Dicroismo Circular , Clonación Molecular , Escherichia coli/genética , Proteínas Fúngicas/química , Genes Fúngicos , Vectores Genéticos/genética , Datos de Secuencia Molecular , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/aislamiento & purificación , Resonancia Magnética Nuclear Biomolecular , Conformación Proteica , Alineación de Secuencia , Tiorredoxinas/química , Regulación hacia Arriba

RESUMEN

Isolates of Pyricularia grisea from wheat (Triticum aestivum Lam.) and triticale (x Triticosecale Wittmack) spikes with blast symptoms were analyzed by classical (VCG) and molecular (RAPD) techniques. P. grisea mutants, unable to use sodium nitrate (nit) as nitrogen source, were obtained with potassium chlorate. For vegetative compatibility (VCG) tests, genetically complementary nit mutant pairs were inoculated in a medium with sodium nitrate as a single nitrogen source. P. grisea isolates were divided into two vegetative compatibility groups and two RAPD groups. Since vegetative compatible strains may mutually exchange genetic and cytoplasmatic material, the contribution of the parasexual cycle in the genetic variability of Brazilian P. grisea isolates is discussed.

Asunto(s)

ADN de Hongos/genética , Grano Comestible/microbiología , Magnaporthe/aislamiento & purificación , Triticum/microbiología , Brasil , Proteínas Fúngicas/genética , Proteínas Fúngicas/fisiología , Genes Fúngicos , Prueba de Complementación Genética , Variación Genética , Magnaporthe/genética , Magnaporthe/metabolismo , Magnaporthe/fisiología , Nitratos/metabolismo , Nitrógeno/metabolismo , Enfermedades de las Plantas/microbiología , Técnica del ADN Polimorfo Amplificado Aleatorio , Recombinación Genética

RESUMEN

Isolates of Magnaporthe grisea causing gray leaf spot on rice were collected in Argentina and analyzed for mating distribution and fertility. One hundred and twenty-five isolates of M. grisea were collected from rice plants between 2000 and 2003. Each isolate was tested for mating type through a polymerase chain reaction based assay. All M. grisea isolates from Argentina belonged to a single mating type, MAT1.1. The fertility status of isolates was determined using controlled crosses in vitro, pairing each isolate with GUY11 and KA9 (MAT1.2 standard hermaphroditic testers). Production of perithecia was scarce among isolates of the blast pathogen since a low percentage of them (7.2%) developed perithecia with only one of the fertile tester (KA9); all crosses failed with the other tester strain. Asci and ascospores were not observed. The presence of only one mating type and the absence of female fertile isolates indicate that sexual reproduction is rare or absent in M. grisea populations associated with rice in Argentina.

Asunto(s)

Genes del Tipo Sexual de los Hongos , Magnaporthe/fisiología , Oryza , Enfermedades de las Plantas/microbiología , Argentina , Cruzamientos Genéticos , ADN de Hongos/química , ADN de Hongos/genética , Magnaporthe/genética , Magnaporthe/crecimiento & desarrollo , Reacción en Cadena de la Polimerasa

Asunto(s)

Magnaporthe/clasificación , Oryza/microbiología , Enfermedades de las Plantas/microbiología , Inmunidad Innata , Magnaporthe/genética , Magnaporthe/crecimiento & desarrollo , Magnaporthe/aislamiento & purificación , Filogenia , Plantones/microbiología , Uruguay

RESUMEN

Microorganisms with large genomes are commonly the subjects of single-round partial sequencing of cDNA, generating expressed sequence tags (ESTs). Usually there is a great distance between gene discovery by EST projects and submission of amino acid sequences to public databases. We analyzed the relationship between available ESTs and protein sequences and used the sequences available in the secondary database, clusters of orthologous groups (COG), to investigate ESTs from eight microorganisms of medical and/or economic relevance, selecting for candidate ESTs that may be further pursued for protein characterization. The organisms chosen were Paracoccidioides brasiliensis, Dictyostelium discoideum, Fusarium graminearum, Plasmodium yoelii, Magnaporthe grisea, Emericella nidulans, Chlamydomonas reinhardtii and Eimeria tenella, which have more than 10,000 ESTs available in dbEST. A total of 77,114 protein sequences from COG were used, corresponding to 3,201 distinct genes. At least 212 of these were capable of identifying candidate ESTs for further studies (E. tenella). This number was extended to over 700 candidate ESTs (C. reinhardtii, F. graminearum). Remarkably, even the organism that presents the highest number of ESTs corresponding to known proteins, P. yoelii, showed a considerable number of candidate ESTs for protein characterization (477). For some organisms, such as P. brasiliensis, M. grisea and F. graminearum, bioinformatics has allowed for automatic annotation of up to about 20% of the ESTs that did not correspond to proteins already characterized in the organism. In conclusion, 4093 ESTs from these eight organisms that are homologous to COG genes were selected as candidates for protein characterization.

Asunto(s)

Bases de Datos de Proteínas , Etiquetas de Secuencia Expresada , Análisis de Secuencia de Proteína , Animales , Chlamydomonas reinhardtii/genética , Dictyostelium/genética , Eimeria tenella/genética , Emericella/genética , Fusarium/genética , Genoma , Magnaporthe/genética , Paracoccidioides/genética , Plasmodium yoelii/genética , Proteínas/genética , Homología de Secuencia de Aminoácido

RESUMEN

Microorganisms with large genomes are commonly the subjects of single-round partial sequencing of cDNA, generating expressed sequence tags (ESTs). Usually there is a great distance between gene discovery by EST projects and submission of amino acid sequences to public databases. We analyzed the relationship between available ESTs and protein sequences and used the sequences available in the secondary database, clusters of orthologous groups (COG), to investigate ESTs from eight microorganisms of medical and/or economic relevance, selecting for candidate ESTs that may be further pursued for protein characterization. The organisms chosen were Paracoccidioides brasiliensis, Dictyostelium discoideum, Fusarium graminearum, Plasmodium yoelii, Magnaporthe grisea, Emericella nidulans, Chlamydomonas reinhardtii and Eimeria tenella, which have more than 10,000 ESTs available in dbEST. A total of 77,114 protein sequences from COG were used, corresponding to 3,201 distinct genes. At least 212 of these were capable of identifying candidate ESTs for further studies (E. tenella). This number was extended to over 700 candidate ESTs (C. reinhardtii, F. graminearum). Remarkably, even the organism that presents the highest number of ESTs corresponding to known proteins, P. yoelii, showed a considerable number of candidate ESTs for protein characterization (477). For some organisms, such as P. brasiliensis, M. grisea and F. graminearum, bioinformatics has allowed for automatic annotation of up to about 20 of the ESTs that did not correspond to proteins already characterized in the organism. In conclusion, 4093 ESTs from these eight organisms that are homologous to COG genes were selected as candidates for protein characterization

Asunto(s)

Animales , Bases de Datos de Proteínas , Etiquetas de Secuencia Expresada , Análisis de Secuencia de Proteína , Chlamydomonas reinhardtii/genética , Dictyostelium/genética , Eimeria tenella/genética , Emericella/genética , Fusarium/genética , Genoma , Magnaporthe/genética , Paracoccidioides/genética , Plasmodium yoelii/genética , Proteínas/genética , Homología de Secuencia de Aminoácido

RESUMEN

The AVR1-CO39 gene that came from a Magnaporthe grisea isolate from weeping lovegrass controls avirulence on the rice cultivar CO39. AVR1-CO39 was not present in the genome of the rice-infecting M. grisea isolate Guyll from French Guyana, suggesting that the gene had been deleted. Molecular analysis of the deletion breakpoints in the AVR1-CO39 locus revealed the presence of a truncated copy of a previously unknown retrotransposon at the left-hand border. At the right-hand border was a truncated copy of another repetitive element that is present at multiple locations in the genome of Guyll. The structures of avr1-CO39 loci were further examined in 45 rice-infecting isolates collected in Brazil, China, Japan, India, Indonesia, Mali, and the Philippines. Most isolates showed no hybridization signal with the AVR1-CO39 probe and had the same locus structure as Guyll. Some isolates from Japan showed a signal with the AVR1-CO39 probe, but the region specifying avirulence activity was rearranged. These findings suggest that widespread virulence to 'CO39' among rice-infecting M. grisea isolates is due to ancestral rearrangements at the AVR1-CO39 locus that may have occurred early in the evolution of pathogenicity to rice.

Asunto(s)

Mapeo Cromosómico , Magnaporthe/genética , Magnaporthe/patogenicidad , Oryza/microbiología , Virulencia/genética , Secuencia de Bases , Brasil , China , Genes Fúngicos , Geografía , Japón , Magnaporthe/aislamiento & purificación , Datos de Secuencia Molecular