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1.
Plant Dis ; 104(1): 60-70, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31647693

RESUMO

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.


Assuntos
Agricultura , Genoma Fúngico , Magnaporthe , Reação em Cadeia da Polimerase em Tempo Real , Triticum , Agricultura/métodos , Genes Fúngicos/genética , Genômica , Magnaporthe/genética , Doenças das Plantas/microbiologia , América do Sul , Triticum/microbiologia
2.
J Microbiol ; 57(12): 1115-1125, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31758396

RESUMO

Histone acetylation/deacetylation represent a general and efficient epigenetic mechanism through which fungal cells control gene expression. Here we report developmental requirement of MoHOS2-mediated histone deacetylation (HDAC) for the rice blast fungus, Magnaporthe oryzae. Structural similarity and nuclear localization indicated that MoHOS2 is an ortholog of Saccharomyces cerevisiae Hos2, which is a member of class I histone deacetylases and subunit of Set3 complex. Deletion of MoHOS2 led to 25% reduction in HDAC activity, compared to the wild-type, confirming that it is a bona-fide HDAC. Lack of MoHOS2 caused decrease in radial growth and impinged dramatically on asexual sporulation. Such reduction in HDAC activity and phenotypic defects of ΔMohos2 were recapitulated by a single amino acid change in conserved motif that is known to be important for HDAC activity. Expression analysis revealed up-regulation of MoHOS2 and concomitant down-regulation of some of the key genes involved in asexual reproduction under sporulation-promoting condition. In addition, the deletion mutant exhibited defect in appressorium formation from both germ tube tip and hyphae. As a result, ΔMohos2 was not able to cause disease symptoms. Wound-inoculation showed that the mutant is compromised in its ability to grow inside host plants as well. We found that some of ROS detoxifying genes and known effector genes are de-regulated in the mutant. Taken together, our data suggest that MoHOS2-dependent histone deacetylation is pivotal for proper timing and induction of transcription of the genes that coordinate developmental changes and host infection in M. oryzae.


Assuntos
Histona Desacetilases/genética , Histona Desacetilases/metabolismo , Magnaporthe/enzimologia , Magnaporthe/crescimento & desenvolvimento , Magnaporthe/metabolismo , Reprodução Assexuada/fisiologia , Parede Celular/metabolismo , Epigênese Genética , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Deleção de Genes , Regulação Fúngica da Expressão Gênica , Histona Desacetilases/química , Magnaporthe/genética , Modelos Moleculares , Mutagênese Sítio-Dirigida , Oryza/microbiologia , Fenótipo , Doenças das Plantas/microbiologia , Conformação Proteica , Processamento de Proteína Pós-Traducional , Reprodução Assexuada/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Virulência/genética , Virulência/fisiologia
3.
Plant Dis ; 103(11): 2759-2763, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31509496

RESUMO

Rice blast caused by the fungus Magnaporthe oryzae is one of the most destructive diseases of rice. Its control through the deployment of host resistance genes would be facilitated by understanding the pathogen's race structure. Here, dynamics of race structures in this decade in Heilongjiang province were characterized by Chinese differential cultivars. Two patterns of dynamics of the race structures emerged: both race diversity and population-specific races increased gradually between 2006 and 2011, but they increased much more sharply between 2011 and 2015, with concomitant falls in both the population-common races and dominant races. Four races (ZD1, ZD3, ZD5, and ZE1) were among the top three dominant races over the whole period, indicating that the core of the race structure remained stable through this decade. On the host side, the composition of resistance in the cultivar differential set could be divided in two: the three indica-type entries of the differential set expressed a higher level of resistance to the population of M. oryzae isolates tested than did the four japonica-type entries. The cultivars Tetep and Zhenlong 13 as well as two additional resistance genes α and ε were confirmed as the most promising donors of blast resistance for the local rice improvement programs.[Formula: see text]Copyright © 2019 The Author(s). This is an open-access article distributed under the CC BY-NC-ND 4.0 International license.


Assuntos
Magnaporthe , Oryza , China , Resistência à Doença/genética , Variação Genética , Magnaporthe/classificação , Magnaporthe/genética , Oryza/genética , Oryza/microbiologia
4.
Plant Dis ; 103(8): 1967-1973, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31188736

RESUMO

Rice blast is one of the most serious diseases for rice, and controlling the filamentous fungus Magnaporthe oryzae that causes rice blast is crucial for global food security. Typically, early infected rice does not show symptoms. Therefore, the early diagnosis of rice blast is particularly important to avoid uncontrollable propagation of rice blast fungus. In the present work, a rapid and efficient loop-mediated isothermal amplification (LAMP) method was developed to detect the pathogen at the early infected stage of rice. The Alb1 superfamily hypothetical protein MGG_04322, a nuclear shuttling factor involved in ribosome and melanin biogenesis, was chosen as the target for designing the LAMP primers. The LAMP assay enabled rapid detection of as little as 10 pg of pure genomic DNA of M. oryzae. In addition, we established the quantitative LAMP (q-LAMP) detection system to quantify the conidia of rice blast fungus. The q-LAMP assay enabled rapid detection (within 35 min) of the fungal spores at a sensitivity of 3.2 spores/ml. In addition, the assay sets up the linearization formula of the standard curve as y = 0.3066 + 15.33x (where x = amplification of time), inferring that spore number = 100.60y. In addition, the q-LAMP assay was successfully used to detect the presence of the virulence strains of M. oryzae (wild type) in comparison with that of the two mutant strains by quantifying the biomass within host tissue. These results provide a useful and convenient tool for detecting M. oryzae that could be applied in the incubation period of rice blast before symptoms appear.


Assuntos
Magnaporthe , Técnicas de Amplificação de Ácido Nucleico , Oryza , Primers do DNA , DNA Fúngico/genética , Limite de Detecção , Magnaporthe/genética , Oryza/microbiologia , Doenças das Plantas/microbiologia
5.
BMC Plant Biol ; 19(1): 204, 2019 May 16.
Artigo em Inglês | MEDLINE | ID: mdl-31096914

RESUMO

BACKGROUND: Rice blast disease is one of the most destructive fungal disease of rice worldwide. The avirulence (AVR) genes of Magnaporthe oryzae are recognized by the cognate resistance (R) genes of rice and trigger race-specific resistance. The variation in AVR is one of the major drivers of new races. Detecting the variation in the AVR gene in isolates from a population of Magnaporthe oryzae collected from rice production fields will aid in evaluating the effectiveness of R genes in rice production areas. The Pik gene contains 5 R alleles (Pik, Pikh, Pikp, Pikm and Piks) corresponding to the AVR alleles (AVR-Pik/kh/kp/km/ks) of M. oryzae. The Pik gene specifically recognizes and prevents infections by isolates of M. oryzae that contain AVR-Pik. The molecular variation in AVR-Pik alleles of M. oryzae and Pik alleles of rice remains unclear. RESULTS: We studied the possible evolutionary pathways of AVR-Pik alleles by analyzing their DNA sequence variation and assaying their avirulence to the cognate Pik alleles of resistance genes under field conditions in China. The results of PCR products from genomic DNA showed that 278 of the 366 isolates of M. oryzae collected from Yunnan Province, China, carried AVR-Pik alleles. Among the isolates from six regions of Yunnan, 66.7-90.3% carried AVR-Pik alleles. Moreover, 10 AVR-Pik haplotypes encoding five novel AVR-Pik variants were identified among 201 isolates. The AVR-Pik alleles evolved to virulent from avirulent forms via stepwise base substitution. These findings demonstrate that AVR-Pik alleles are under positive selection and that mutations are responsible for defeating race-specific resistant Pik alleles in nature. CONCLUSIONS: We demonstrated the polymorphism and distribution of AVR-Pik alleles in Yunnan Province, China. By pathogenicity assays used to detect the function of the different haplotypes of AVR-Pik, for the first time, we showed the avoidance and stepwise evolution of AVR-Pik alleles in rice production areas of Yunnan. The functional AVR-Pik possesses diversified sequence structures and is under positive selection in nature.


Assuntos
Genes Fúngicos/genética , Magnaporthe/genética , Oryza/microbiologia , Doenças das Plantas/microbiologia , Alelos , Evolução Biológica , Frequência do Gene , Variação Genética , Haplótipos , Magnaporthe/metabolismo , Magnaporthe/patogenicidade
6.
Int J Mol Sci ; 20(7)2019 Mar 29.
Artigo em Inglês | MEDLINE | ID: mdl-30934953

RESUMO

Manganese, as an essential trace element, participates in many physiological reactions by regulating Mn associated enzymes. Magnaporthe oryzae is a serious pathogen and causes destructive losses for rice production. We identified a cytochrome P450 gene, MoMCP1, involving the alleviation of manganese toxin and pathogenicity. To identify the underlying mechanisms, transcriptomics were performed. The results indicated that many pathogenicity related genes were regulated, especially hydrophobin related genes in ∆Momcp1. Furthermore, the Mn2+ toxicity decreased the expressions of genes involved in the oxidative phosphorylation and energy production, and increased the reactive oxygen species (ROS) levels, which might impair the functions of mitochondrion and vacuole, compromising the pathogenicity and development in ∆Momcp1. Additionally, our results provided further information about Mn associated the gene network for Mn metabolism in cells.


Assuntos
Sistema Enzimático do Citocromo P-450/genética , Proteínas Fúngicas/genética , Perfilação da Expressão Gênica , Genes Fúngicos , Magnaporthe/genética , Manganês/toxicidade , Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica , Proteínas de Fluorescência Verde/metabolismo , Peróxido de Hidrogênio/metabolismo , Hifas/metabolismo , Espaço Intracelular/metabolismo , Magnaporthe/patogenicidade , Mutação/genética , Oryza/microbiologia , Superóxido Dismutase/metabolismo , Transcriptoma/genética
7.
Arch Microbiol ; 201(6): 807-816, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-30874825

RESUMO

Rice blast, caused by Magnaporthe oryzae, is a serious threat to global rice production. In recent years, many pathogenicity genes of M. oryzae have been identified, although most of their functions remain unknown. In this study, we report the synergistic deletion of RGS1 and COS1 that may reduce the pathogenicity of M. oryzae. The investigation involved comparing ΔMorgs1, ΔMocos1, and ΔMorgs1/ΔMocos1 mutants. The ΔMorgs1/ΔMocos1 mutant showed a weak reduction in vegetative growth, and the colonies displayed fewer and smoother aerial hyphae. The ΔMorgs1/ΔMocos1 mutant exhibited delayed appressorium-like structure formation and 'low pathogenicity' on detached rice seedling leaves when compared with ΔMocos1. Moreover, the melanin content of the single and double mutants was remarkably lower than that of the WT type. Thus, our results indicate that the synergy between RGS1 and COS1 may be crucial in the pathogenicity of M. oryzae.


Assuntos
Proteínas Fúngicas/genética , Deleção de Genes , Magnaporthe/genética , Magnaporthe/patogenicidade , Oryza/microbiologia , Doenças das Plantas/microbiologia , Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica , Hifas/genética , Hifas/crescimento & desenvolvimento , Hifas/metabolismo , Hifas/patogenicidade , Magnaporthe/crescimento & desenvolvimento , Magnaporthe/metabolismo , Plântula/microbiologia , Esporos Fúngicos/genética , Esporos Fúngicos/crescimento & desenvolvimento , Esporos Fúngicos/metabolismo , Esporos Fúngicos/patogenicidade , Virulência
8.
Fungal Genet Biol ; 127: 23-34, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30822500

RESUMO

Short-chain acyl-CoA dehydrogenase (Scad) mediated ß-oxidation serves as the fastest route for generating essential energies required to support the survival of organisms under stress or starvation. In this study, we identified three putative SCAD genes in the genome of the globally destructive rice blast pathogen Magnaporthe oryzae, named as MoSCAD1, MoSCAD2, and MoSCAD3. To elucidate their function, we deployed targeted gene deletion strategy to investigate individual and the combined influence of MoSCAD genes on growth, stress tolerance, conidiation and pathogenicity of the rice blast fungus. First, localization and co-localization results obtained from this study showed that MoScad1 localizes to the endoplasmic reticulum (ER), MoScad2 localizes exclusively to the mitochondria while MoScad3 partially localizes to the mitochondria and peroxisome at all developmental stages of M. oryzae. Results obtained from this investigation showed that the deletion of MoSCAD1 and MoSCAD2 caused a minimal but significant reduction in the growth of ΔMoscad1 and ΔMoscad2 strains, while, growth characteristics exhibited by the ΔMoscad3 strain was similar to the wild-type strain. Furthermore, we observed that deletion of MoSCAD2 resulted in drastic reduction in conidiation, delayed conidia germination, triggered the development of abnormal appressorium and suppressed host penetration and colonization efficiencies of the ΔMoscad1 strain. This study provides first material evidence confirming the possible existence of ER ß-oxidation pathway in M. oryzae. We also infer that mitochondria ß-oxidation rather than peroxisomal and ER ß-oxidation play an essential role in the vegetative growth, conidiation, appressorial morphogenesis and progression of pathogenesis in M. oryzae.


Assuntos
Butiril-CoA Desidrogenase/genética , Proteínas Fúngicas/genética , Magnaporthe/genética , Magnaporthe/patogenicidade , Esporos Fúngicos/crescimento & desenvolvimento , Retículo Endoplasmático , Radicais Livres/metabolismo , Deleção de Genes , Regulação Fúngica da Expressão Gênica , Magnaporthe/enzimologia , Mitocôndrias/metabolismo , Oryza/microbiologia , Oxirredução , Peroxissomos/metabolismo , Doenças das Plantas/microbiologia , Esporos Fúngicos/genética
9.
Mol Plant Microbe Interact ; 32(6): 740-749, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30601714

RESUMO

Higher yield potential and greater yield stability are common targets for crop breeding programs, including those in rice. Despite these efforts, biotic and abiotic stresses continue to impact rice production. Rice blast disease, caused by Magnaporthe oryzae, is the most devastating disease affecting rice worldwide. In the field, resistant varieties are unstable and can become susceptible to disease within a few years of release due to the adaptive potential of the blast fungus, specifically in the effector (avirulence [AVR]) gene pool. Here, we analyzed genetic variation of the effector gene AVR-Pik in 58 rice blast isolates from Thailand and examined the interaction between AVR-Pik and the cognate rice resistance gene Pik. Our results reveal that Thai rice blast isolates are very diverse. We observe four AVR-Pik variants in the population, including three previously identified variants, AVR-PikA, AVR-PikD, and AVR-PikE, and one novel variant, which we named AVR-PikF. Interestingly, 28 of the isolates contained two copies of AVR-Pik, always in the combination of AVR-PikD and AVR-PikF. Blast isolates expressing only AVR-PikF show high virulence to rice cultivars encoding allelic Pik resistance genes, and the AVR-PikF protein does not interact with the integrated heavy metal-associated domain of the Pik resistance protein in vitro, suggesting a mechanism for immune evasion.


Assuntos
Alelos , Duplicação Gênica , Magnaporthe , Mutação , Oryza , Animais , Variação Genética , Magnaporthe/genética , Magnaporthe/patogenicidade , Oryza/microbiologia , Doenças das Plantas/microbiologia , Virulência/genética
10.
Phytopathology ; 109(5): 870-877, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30501464

RESUMO

The Magnaporthe oryzae avirulence gene AvrPib is required for the resistance mediated by its cognate resistance gene Pib, which has been intensively used in indica rice breeding programs in many Asian countries. However, the sequence diversity of AvrPib among geographically distinct M. oryzae populations was recently shown to be increasing. Here, we selected a field population consisting of 248 rice blast isolates collected from a disease hotspot in Philippine for the analysis of AvrPib haplotypes and their pathogenicity against Pib. We found that all of the isolates were virulent to Pib and each of them contained an insertion of Pot3 transposon in AvrPib. Moreover, Pot3 insertion was detected in different genomic positions, resulting in three different AvrPib haplotypes, designated avrPib-H1 to H3. We further conducted a genome-wide Pot2 fingerprinting analysis by repetitive element palindromic polymerase chain reaction (PCR) and identified seven different lineages out of 47 representative isolates. The isolates belonging to the same lineage often had the same AvrPib haplotype. In contrast, the isolates having the same AvrPib haplotypes did not always belong to the same lineages. Both mating types MAT1-1 and MAT1-2 were identified in the population in Bohol and the latter appeared dominant. On the host side, we found that 32 of 52 released rice varieties in the Philippines contained Pib diagnosed by PCR gene-specific primers and DNA sequencing of gene amplicons, suggesting that it was widely incorporated in different rice varieties. Our study highlights the genetic dynamics of rice blast population at both the AvrPib locus and the genome-wide levels, providing insight into the mechanisms of the mutations in AvrPib leading to the breakdown of Pib-mediated resistance in rice.


Assuntos
Magnaporthe/genética , Oryza/microbiologia , Doenças das Plantas/microbiologia , Elementos de DNA Transponíveis , Resistência à Doença/genética , Variação Genética , Magnaporthe/patogenicidade , Mutagênese Insercional , Oryza/genética , Filipinas , Doenças das Plantas/genética , Virulência
11.
Genomics ; 111(4): 661-668, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-29775784

RESUMO

Magnaporthe oryzae is a fungal pathogen causing blast disease in many plant species. In this study, seventy three isolates of M. oryzae collected from rice (Oryza sativa) in 1996-2014 were genotyped using a genotyping-by-sequencing approach to detect genetic variation. An association study was performed to identify single nucleotide polymorphisms (SNPs) associated with virulence genes using 831 selected SNP and infection phenotypes on local and improved rice varieties. Population structure analysis revealed eight subpopulations. The division into eight groups was not related to the degree of virulence. Association mapping showed five SNPs associated with fungal virulence on chromosome 1, 2, 3, 4 and 7. The SNP on chromosome 1 was associated with virulence against RD6-Pi7 and IRBL7-M which might be linked to the previously reported AvrPi7.


Assuntos
Proteínas Fúngicas/genética , Genoma Fúngico , Magnaporthe/genética , Fatores de Virulência/genética , Proteínas Fúngicas/metabolismo , Magnaporthe/patogenicidade , Oryza/microbiologia , Polimorfismo de Nucleotídeo Único , Fatores de Virulência/metabolismo
12.
FEMS Microbiol Lett ; 366(1)2019 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-30535195

RESUMO

N6-methyladenosine (m6A) RNA methylation is a conserved modification of RNA in eukaryotes. Pyricularia oryzae, a filamentous phytopathogenic fungus, is the cause of a destructive rice blast disease that can lead to significant declines in rice production. Here, we characterized the function of m6A RNA methylation in the development and virulence of P. oryzae by studying four genes with functional genomics. We found that PoIme4 is an N6-adenosine-methyltransferase, and deletion of PoIME4 led to decreased levels of m6A RNA methylation. PoYth1 and PoYth2 are two m6A-binding proteins, and deletion of PoYTH2 led to reduced conidiation. Co-localization experiments showed that PoAlkb1 (an mRNA:m6A demethylase) and PoYth1 were co-localized with PoDcp1 in the processing bodies involved in mRNA decay. Virulence tests showed that PoIME4, PoALKB1, PoYTH1 and PoYTH2 were involved in virulence on rice in P. oryzae. Therefore, these experimental evidences provide new and important information about the roles of m6A RNA methylation in fungal asexual reproduction and pathogenicity.


Assuntos
Adenosina/análogos & derivados , Magnaporthe/genética , Magnaporthe/patogenicidade , RNA Fúngico/metabolismo , Adenosina/metabolismo , Genes Fúngicos/genética , Metilação , Oryza/microbiologia , Virulência/genética
13.
Chembiochem ; 20(5): 693-700, 2019 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-30443971

RESUMO

Most fungal secondary metabolism genes are poorly expressed under laboratory conditions. Nectriapyrones are known as secondary metabolites produced mainly by symbiotic fungi, including endophytes and plant pathogens. Herein, we show the induction of nectriapyrone production in the rice blast fungus Pyricularia oryzae. The two-component signal transduction system was disturbed by disrupting OSM1 and PoYPD1, which encoded a HOG MAP kinase and a His-containing phosphotransfer (HPt) protein, respectively. This induced the production of two polyketide compounds: nectriapyrone and its hydroxylated analogue. The nectriapyrone biosynthetic gene cluster consists of a polyketide synthase gene (NEC1) and an O-methyltransferase gene (NEC2). Overexpression of the two genes induced overproduction of nectriapyrone and five nectriapyrone analogues, including a new derivative. Nectriapyrone production was not required for the infection of rice. The structure of nectriapyrone is similar to that of the germicidins produced by Streptomyces spp., and nectriapyrone inhibited the growth of Streptomyces griseus.


Assuntos
Magnaporthe , Monoterpenos/metabolismo , Metabolismo Secundário/genética , Genes Fúngicos , Magnaporthe/genética , Magnaporthe/metabolismo , Família Multigênica , Transdução de Sinais/genética
14.
Genetics ; 211(1): 151-167, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30446520

RESUMO

The pathogenic life cycle of the rice blast fungus Magnaporthe oryzae involves a series of morphogenetic changes, essential for its ability to cause disease. The smo mutation was identified > 25 years ago, and affects the shape and development of diverse cell types in M. oryzae, including conidia, appressoria, and asci. All attempts to clone the SMO1 gene by map-based cloning or complementation have failed over many years. Here, we report the identification of SMO1 by a combination of bulk segregant analysis and comparative genome analysis. SMO1 encodes a GTPase-activating protein, which regulates Ras signaling during infection-related development. Targeted deletion of SMO1 results in abnormal, nonadherent conidia, impaired in their production of spore tip mucilage. Smo1 mutants also develop smaller appressoria, with a severely reduced capacity to infect rice plants. SMO1 is necessary for the organization of microtubules and for septin-dependent remodeling of the F-actin cytoskeleton at the appressorium pore. Smo1 physically interacts with components of the Ras2 signaling complex, and a range of other signaling and cytoskeletal components, including the four core septins. SMO1 is therefore necessary for the regulation of RAS activation required for conidial morphogenesis and septin-mediated plant infection.


Assuntos
Proteínas Fúngicas/genética , Magnaporthe/genética , Receptor Smoothened/genética , Esporos Fúngicos/crescimento & desenvolvimento , Citoesqueleto de Actina/metabolismo , Proteínas Fúngicas/metabolismo , Magnaporthe/crescimento & desenvolvimento , Magnaporthe/patogenicidade , Microtúbulos/metabolismo , Morfogênese , Oryza/microbiologia , Septinas/metabolismo , Transdução de Sinais , Receptor Smoothened/metabolismo , Esporos Fúngicos/genética , Virulência/genética
15.
Appl Microbiol Biotechnol ; 103(1): 327-337, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30357439

RESUMO

The biosynthesis of branched-chain amino acids (BCAAs) is conserved in fungi and plants, but not in animals. The Leu1 gene encodes isopropylmalate isomerase that catalyzes the conversion of α-isopropylmalate into ß-isopropylmalate in the second step of leucine biosynthesis in yeast. Here, we identified and characterized the functions of MoLeu1, an ortholog of yeast Leu1 in the rice blast fungus Magnaporthe oryzae. The transcriptional level of MoLEU1 was increased during conidiation and in infectious stages. Cellular localization analysis indicated that MoLeu1 localizes to the cytoplasm at all stages of fungal development. Targeted gene deletion of MoLEU1 led to leucine auxotrophy, and phenotypic analysis of the generated ∆Moleu1 strain revealed that MoLeu1-mediated leucine biosynthesis was required for vegetative growth, asexual development, and pathogenesis of M. oryzae. We further observed that invasive hyphae produced by the ∆Moleu1 strain were mainly limited to the primary infected host cells. The application of exogenous leucine fully restored vegetative growth and partially restored conidiation as well as pathogenicity defects in the ∆Moleu1 strain. In summary, our results suggested that MoLeu1-mediated leucine biosynthesis crucially promotes vegetative growth, conidiogenesis, and pathogenicity of M. oryzae. This study helps unveil the regulatory mechanisms that are essential for infection-related morphogenesis and pathogenicity of the rice blast fungus.


Assuntos
Proteínas Fúngicas/metabolismo , Isomerases/metabolismo , Leucina/biossíntese , Magnaporthe/metabolismo , Magnaporthe/patogenicidade , Citoplasma/metabolismo , Proteínas Fúngicas/genética , Deleção de Genes , Regulação Fúngica da Expressão Gênica , Teste de Complementação Genética , Hifas/patogenicidade , Isomerases/genética , Magnaporthe/genética , Magnaporthe/crescimento & desenvolvimento , Oryza/microbiologia , Doenças das Plantas/microbiologia , Reprodução Assexuada/genética , Esporos Fúngicos/crescimento & desenvolvimento
16.
Phytopathology ; 109(4): 504-508, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30253117

RESUMO

The blast fungus Magnaporthe oryzae is comprised of lineages that exhibit varying degrees of specificity on about 50 grass hosts, including rice, wheat, and barley. Reliable diagnostic tools are essential given that the pathogen has a propensity to jump to new hosts and spread to new geographic regions. Of particular concern is wheat blast, which has suddenly appeared in Bangladesh in 2016 before spreading to neighboring India. In these Asian countries, wheat blast strains are now co-occurring with the destructive rice blast pathogen raising the possibility of genetic exchange between these destructive pathogens. We assessed the recently described MoT3 diagnostic assay and found that it did not distinguish between wheat and rice blast isolates from Bangladesh. The assay is based on primers matching the WB12 sequence corresponding to a fragment of the M. oryzae MGG_02337 gene annotated as a short chain dehydrogenase. These primers could not reliably distinguish between wheat and rice blast isolates from Bangladesh based on DNA amplification experiments performed in separate laboratories in Bangladesh and in the United Kingdom. Specifically, all eight rice blast isolates tested in this study produced the WB12 amplicon. In addition, comparative genomics of the WB12 nucleotide sequence revealed a complex underlying genetic structure with related sequences across M. oryzae strains and in both rice and wheat blast isolates. We, therefore, caution against the indiscriminate use of this assay to identify wheat blast and encourage further development of the assay to ensure its value in diagnosis.


Assuntos
Magnaporthe , Técnicas de Diagnóstico Molecular , Oryza , Doenças das Plantas , Ásia , Bangladesh , Genótipo , Índia , Magnaporthe/classificação , Magnaporthe/genética , Doenças das Plantas/genética , Doenças das Plantas/microbiologia , Poaceae , Triticum , Reino Unido
17.
Mol Microbiol ; 111(3): 662-677, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30537256

RESUMO

The fungicide fludioxonil causes hyperactivation of the Hog1p MAPK within the high-osmolarity glycerol signaling pathway essential for osmoregulation in pathogenic fungi. The molecular regulation of MoHog1p phosphorylation is not completely understood in pathogenic fungi. Thus, we identified and characterized the putative MoHog1p-interacting phosphatase gene MoPTP2 in the filamentous rice pathogen Magnaporthe oryzae. We found overexpression of MoPTP2 conferred fludioxonil resistance in M. oryzae, whereas the 'loss of function' mutant ΔMoptp2 was more susceptible toward the fungicide. Additionally, quantitative phosphoproteome profiling of MoHog1p phosphorylation revealed lower phosphorylation levels of MoHog1p in the MoPtp2p overexpression mutant compared to the wild-type strain, whereas MoHog1p phosphorylation increased in the ΔMoptp2 mutant. Furthermore, we identified a set of MoHog1p-dependent genes regulated by the MoPtp2p expression level. Our results indicate that the phosphatase MoPtp2p is involved in the regulation of MoHog1p phosphorylation and that overexpression of the gene MoPTP2 is a novel molecular mechanism of fungicide resistance.


Assuntos
Dioxóis/farmacologia , Farmacorresistência Fúngica , Fungicidas Industriais/farmacologia , Magnaporthe/efeitos dos fármacos , Magnaporthe/genética , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Proteínas Tirosina Fosfatases/biossíntese , Pirróis/farmacologia , Proteínas Fúngicas/análise , Deleção de Genes , Expressão Gênica , Oryza/microbiologia , Fosfoproteínas/análise , Fosforilação , Doenças das Plantas/microbiologia , Processamento de Proteína Pós-Traducional , Proteoma/análise
18.
Curr Genet ; 65(1): 223-239, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-29946987

RESUMO

In fungi, O-mannosylation is one type of conserved protein modifications that add the carbohydrate residues to specific residues of target proteins by protein O-mannosyltransferases. Previously, three members of O-mannosyltransferases were identified in Magnaporthe oryzae, with MoPmt2 playing important roles in fungal growth and pathogenicity. However, the biological roles of the rest Pmt proteins remain unclear. In this study, to understand if O-mannosyltransferases are crucial for fungal pathogenicity of M. oryzae, the Pmt-coding genes MoPmt1 and MoPmt4 were separately disrupted and their roles in pathogenesis were analyzed. Of the two genes, only MoPmt4 is specifically required for full virulence of M. oryzae. Deletion of MoPmt4 resulted in defects on radial growth, with more branching hyphae and septa as compared to Guy11. The MoPmt4 mutant was severely impaired not only in conidiation, but also in both penetration and biotrophic invasion in susceptible rice plants. This mutant also had defects in suppression of host-derived ROS-mediated plant defense responses that might be ascribed from the reduced activities of extracellular enzymes. Furthermore, like their fungi counterparts, MoPmt4 localized in the ER and had O-mannosyltransferase activity. Domain disruption analysis indicated that mannosyltransferase activity regulated by PMT domain of MoPmt4 is crucial for fungal development and pathogenicity of M. oryzae. Taken together, these data suggest that MoPmt4 is a protein O-mannosyltransferase essential for fungal development and full virulence of M. oryzae.


Assuntos
Proteínas Fúngicas/genética , Pleiotropia Genética/genética , Magnaporthe/genética , Manosiltransferases/genética , Proteínas Fúngicas/metabolismo , Deleção de Genes , Regulação da Expressão Gênica no Desenvolvimento , Regulação Fúngica da Expressão Gênica , Hifas/genética , Hifas/crescimento & desenvolvimento , Hifas/metabolismo , Magnaporthe/crescimento & desenvolvimento , Magnaporthe/patogenicidade , Manosiltransferases/metabolismo , Oryza/metabolismo , Oryza/microbiologia , Espécies Reativas de Oxigênio/metabolismo , Esporos Fúngicos/genética , Esporos Fúngicos/crescimento & desenvolvimento , Esporos Fúngicos/metabolismo , Virulência/genética
19.
New Phytol ; 221(1): 399-414, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30169888

RESUMO

Generation of mRNA isoforms by alternative polyadenylation (APA) and their involvement in regulation of fungal cellular processes, including virulence, remains elusive. Here, we investigated genome-wide polyadenylation site (PAS) selection in the rice blast fungus to understand how APA regulates pathogenicity. More than half of Magnaporthe oryzae transcripts undergo APA and show novel motifs in their PAS region. Transcripts with shorter 3'UTRs are more stable and abundant in polysomal fractions, suggesting they are being translated more efficiently. Importantly, rice colonization increases the use of distal PASs of pathogenicity genes, especially those participating in signalling pathways like 14-3-3B, whose long 3'UTR is required for infection. Cleavage factor I (CFI) Rbp35 regulates expression and distal PAS selection of virulence and signalling-associated genes, tRNAs and transposable elements, pointing its potential to drive genomic rearrangements and pathogen evolution. We propose a noncanonical PAS selection mechanism for Rbp35 that recognizes UGUAH, unlike humans, without CFI25. Our results showed that APA controls turnover and translation of transcripts involved in fungal growth and environmental adaptation. Furthermore, these data provide useful information for enhancing genome annotations and for cross-species comparisons of PASs and PAS usage within the fungal kingdom and the tree of life.


Assuntos
Regiões 3' não Traduzidas , Proteínas Fúngicas/genética , Magnaporthe/genética , Magnaporthe/patogenicidade , Oryza/microbiologia , Carbono/metabolismo , Elementos de DNA Transponíveis , Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica , Interações Hospedeiro-Patógeno/fisiologia , Magnaporthe/metabolismo , Mutação , Doenças das Plantas/microbiologia , Poli A/genética , Poli A/metabolismo , Poliadenilação , RNA não Traduzido , Transdução de Sinais/genética , Estresse Fisiológico/genética , Virulência/genética
20.
Pest Manag Sci ; 75(3): 772-778, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30123985

RESUMO

BACKGROUND: The mitogen-activated protein kinase MoHog1p was fused with a green fluorescent protein (GFP) in the filamentous fungus Magnaporthe oryzae. The MoHOG1::GFP mutant was found to be an excellent tool visualizing in vivo fungicide-dependent translocation of MoHog1p into the nucleus. Validation of pathway specificity was achieved by generating fluorescence-labelled MoHog1p in the ΔMohik1 'loss of function' mutant strain. RESULTS: GFP-labelled MoHog1p expressed in the wildtype and in ΔMohik1 demonstrates that fludioxonil is acting on the HOG pathway and even more precisely that fungicide action is dependent on the group III histidine kinase MoHik1p. GFP-tagged MoHog1p translocated into the nucleus upon fungicide treatment in the MoHOG1::GFP mutant within seconds, but did not do so in the ΔMohik1/HOG1::GFP mutant. CONCLUSION: Here, we developed a rapid in vivo tool for fluorescent-based validation of fungicides targeting the HOG-signaling pathway. Furthermore, using the fluorescent mutants generated in this study, we are able to visualize that fungicide action is dependent on the histidine kinase MoHik1p but operates in a different mechanism of pathway activation compared to osmotic stress. © 2018 Society of Chemical Industry.


Assuntos
Dioxóis/farmacologia , Fungicidas Industriais/farmacologia , Histidina Quinase/antagonistas & inibidores , Magnaporthe/efeitos dos fármacos , Pirróis/farmacologia , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Proteínas de Fluorescência Verde/genética , Magnaporthe/enzimologia , Magnaporthe/genética , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Transdução de Sinais
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