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1.
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
2.
Pestic Biochem Physiol ; 159: 51-58, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31400784

RESUMO

Isoquinoline alkaloids possess broad pharmacological activities. In this study, the antifungal activity of twelve isoquinoline alkaloids, including berberine (1), jatrorrhizine (2), coptisine (3), corydaline (4), tetrahydroberberine (5), chelidonine (6), dihydrosanguinarine (7), chelerythrine (8), sanguinarine (9), palmatine (10), tetrahydropalmatine (11) and columbamine (12) were evaluated against eight plant pathogenic fungi in vitro. All the tested compounds showed varying degrees of inhibition against the eight tested plant fungi. Among them, sanguinarine exhibited high antifungal activity (EC50 ranging from 6.96-59.36 µg/mL). It displayed the best inhibitory activity against Magnaporthe oryzae (EC50 = 6.96 µg/mL), compared with azoxystrobin (EC50 = 12.04 µg/mL), and significantly suppressed spore germination of M. oryzae with the inhibition rate reaching 100% (50 µg/mL). The optical microscopy and scanning electron microscopy observations revealed that after treating M. oryzae mycelia with sanguinarine at 10 µg/mL, the mycelia appeared curved, collapsed and the cell membrane integrity was eventually damaged. Furthermore, the reactive oxygen species production, mitochondrial membrane potential and nuclear morphometry of mycelia had been changed, and the membrane function and cell proliferation of mycelia were destroyed. These results will enrich our insights into action mechanisms of antifungal activity of sanguinarine against M. oryzae.


Assuntos
Alcaloides/farmacologia , Antifúngicos/farmacologia , Benzofenantridinas/farmacologia , Isoquinolinas/farmacologia , Berberina/análogos & derivados , Berberina/farmacologia , Alcaloides de Berberina/farmacologia , Magnaporthe/metabolismo , Magnaporthe/patogenicidade , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Testes de Sensibilidade Parasitária , Espécies Reativas de Oxigênio/metabolismo
3.
Appl Microbiol Biotechnol ; 103(16): 6617-6627, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31175429

RESUMO

The inhibitor of apoptosis protein (IAP) family has been identified in a variety of organisms. All IAPs contain one to three baculoviral IAP repeat (BIR) domains, which are required for anti-apoptotic activity. Here, we identified a type II BIR domain-containing protein, MoBir1, in the rice blast fungus Magnaporthe oryzae. Expression of the MoBIR1 gene in Saccharomyces cerevisiae suppressed hydrogen peroxide-induced cell death and delayed yeast cell chronological aging. Delayed aging was found to require the carboxyl terminus of MoBir1. M. oryzae transformants overexpressing the MoBIR1 gene demonstrated increased growth rate and biomass, delayed mycelial aging, and enhanced resistance to hydrogen peroxide but reduced reactive oxygen species generation and virulence. Moreover, MoBIR1-overexpressing transformants exhibited anti-apoptotic activity. However, MoBIR1 silencing resulted in no obvious phenotypic changes, compared with the wild-type M. oryzae strain Guy11. Our findings broaden the knowledge on fungal type II BIR domain-containing proteins.


Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Peróxido de Hidrogênio/toxicidade , Magnaporthe/enzimologia , Magnaporthe/patogenicidade , Oryza/microbiologia , Oxidantes/toxicidade , Doenças das Plantas/microbiologia , Apoptose , Proteínas Reguladoras de Apoptose/genética , Clonagem Molecular , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Deleção de Genes , Expressão Gênica , Magnaporthe/efeitos dos fármacos , Magnaporthe/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo
4.
Plant Mol Biol ; 100(6): 579-589, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31093900

RESUMO

KEY MESSAGE: CYP71Z18 exhibited plastic substrate specificity to catalyze oxidation of multiple rice diterpenes and elevated chemical defense against the blast fungus in transgenic rice. Diversified plant specialized metabolism relies on corresponding biosynthetic enzymes with differential substrate specificity. CYP71Z18 catalyzed formation of maize phytoalexins including zealexin A1, the sesquiterpenoid phytoalexin, and diterpenoid phytoalexin dolabralexin, indicating catalytic promiscuity on different terpene substrates. Here substrate specificity of CYP71Z18 was further explored through microbial metabolic engineering and it was identified to accept multiple rice diterpenes as substrates for oxidation. One CYP71Z18 enzymatic product derived from syn-pimaradiene was identified as 15,16-epoxy-syn-pimaradiene by NMR analysis, which was further elaborated by CYP99A3 to generate C19 hydroxylated product. 15,16-epoxy-syn-pimaradien-19-ol exhibited inhibitory effect on spore germination and appressorium formation of the blast pathogen Magnaporthe oryzae. Overexpression of CYP71Z18 in rice resulted in accumulation of several new diterpenoids, indicating promiscuous activity in planta. Transgenic rice also showed stronger resistance against M. oryzae infection, suggesting elevated chemical defense through changed diterpenoid metabolism by CYP71Z18 overexpression. This investigation sheds light on plant metabolic engineering using plastic substrate specificity of P450s to strengthen disease resistance and potentially provide abundant lead compounds.


Assuntos
Oryza/genética , Oxigênio/química , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Catálise , Resistência à Doença , Diterpenos/química , Cromatografia Gasosa-Espectrometria de Massas , Magnaporthe/metabolismo , Engenharia Metabólica , Doenças das Plantas/microbiologia , Folhas de Planta/metabolismo , Plantas Geneticamente Modificadas/genética , Proteínas Recombinantes/metabolismo , Sesquiterpenos , Especificidade por Substrato , Terpenos/metabolismo
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 Apr 02.
Artigo em Inglês | MEDLINE | ID: mdl-30987045

RESUMO

Secreted effectors from Magnaporthe oryzae play critical roles in the interaction with rice to facilitate fungal infection and disease development. M. oryzae-secreted protein MoHrip1 can improve plant defense as an elicitor in vitro, however, its biological function in fungal infection is not clear. In this study, we found that the expression of mohrip1 was significantly induced in the stages of fungal penetration and colonization. Although dispensable for the growth and conidiation, MoHrip1 was necessary for the full virulence of M. oryzae. Deletion of mohrip1 remarkably compromised fungal virulence on rice seedlings and even on rice leaves with wounds. Rice sheath inoculation assay further demonstrated the defects of mohrip1-deleted mutants on penetration and proliferation in rice cells. Additionally, compared with WT and complementation strain, the inoculation of mohrip1-deleted mutants induced a higher expression of specific defense related genes and a higher production of specific defensive compounds in rice leaves. These data collectively indicated that MoHrip1 is necessary for fungal penetration and invasive expansion, and further full virulence of rice blast fungus.


Assuntos
Proteínas Fúngicas/metabolismo , Magnaporthe/metabolismo , Magnaporthe/patogenicidade , Proteínas Fúngicas/genética , Regulação Fúngica da Expressão Gênica , Genes de Plantas , Magnaporthe/crescimento & desenvolvimento , Magnaporthe/fisiologia , Mutação/genética , Oryza/genética , Oryza/imunologia , Oryza/microbiologia , Imunidade Vegetal , Virulência
7.
Mar Drugs ; 17(4)2019 Mar 29.
Artigo em Inglês | MEDLINE | ID: mdl-30934847

RESUMO

This study was initiated to screen for marine bacterial agents to biocontrol Magnaporthe grisea, a serious fungal pathogen of cereal crops. A bacterial strain, isolated from the cold seep in deep sea, exhibited strong growth inhibition against M. grisea, and the strain was identified and designated as Bacillus sp. CS30. The corresponding antifungal agents were purified by acidic precipitation, sequential methanol extraction, Sephadex LH-20 chromatography, and reversed phase high-performance liquid chromatography (RP-HPLC), and two antifungal peaks were obtained at the final purification step. After analysis by mass spectrometry (MS) and tandem MS, two purified antifungal agents were deduced to belong to the surfactin family, and designated as surfactin CS30-1 and surfactin CS30-2. Further investigation showed that although the antifungal activity of surfactin CS30-1 is higher than that of surfactin CS30-2, both of them induced the increased generation of reactive oxygen species (ROS) and caused serious damage to the cell wall and cytoplasm, thus leading to the cell death of M. grisea. Our results also show the differences of the antifungal activity and antifungal mechanism of the different surfactin homologs surfactin CS30-1 and surfactin CS30-2, and highlight them as potential promising agents to biocontrol plant diseases caused by M. grisea.


Assuntos
Antifúngicos/farmacologia , Bacillus/metabolismo , Lipopeptídeos/farmacologia , Magnaporthe/efeitos dos fármacos , Tensoativos/farmacologia , Antifúngicos/isolamento & purificação , Antifúngicos/metabolismo , Bactérias/metabolismo , Lipopeptídeos/biossíntese , Lipopeptídeos/isolamento & purificação , Magnaporthe/metabolismo , Doenças das Plantas/microbiologia , Doenças das Plantas/terapia , Espécies Reativas de Oxigênio/metabolismo , Tensoativos/isolamento & purificação , Tensoativos/metabolismo
8.
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
9.
PLoS Pathog ; 15(2): e1007382, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30802274

RESUMO

Regulator of G-protein signaling (RGS) proteins primarily function as GTPase-accelerating proteins (GAPs) to promote GTP hydrolysis of Gα subunits, thereby regulating G-protein mediated signal transduction. RGS proteins could also contain additional domains such as GoLoco to inhibit GDP dissociation. The rice blast fungus Magnaporthe oryzae encodes eight RGS and RGS-like proteins (MoRgs1 to MoRgs8) that have shared and distinct functions in growth, appressorium formation and pathogenicity. Interestingly, MoRgs7 and MoRgs8 contain a C-terminal seven-transmembrane domain (7-TM) motif typical of G-protein coupled receptor (GPCR) proteins, in addition to the conserved RGS domain. We found that MoRgs7, but not MoRgs8, couples with Gα MoMagA to undergo endocytic transport from the plasma membrane to the endosome upon sensing of surface hydrophobicity. We also found that MoRgs7 can interact with hydrophobic surfaces via a hydrophobic interaction, leading to the perception of environmental hydrophobiccues. Moreover, we found that MoRgs7-MoMagA endocytosis is regulated by actin patch-associated protein MoCrn1, linking it to cAMP signaling. Our studies provided evidence suggesting that MoRgs7 could also function in a GPCR-like manner to sense environmental signals and it, together with additional proteins of diverse functions, promotes cAMP signaling required for developmental processes underlying appressorium function and pathogenicity.


Assuntos
Magnaporthe/metabolismo , Proteínas dos Microfilamentos/metabolismo , Oryza/microbiologia , Proteínas RGS/metabolismo , AMP Cíclico/metabolismo , Endocitose , Proteínas Fúngicas/metabolismo , Subunidades alfa de Proteínas de Ligação ao GTP/metabolismo , Doenças das Plantas/microbiologia , Domínios Proteicos , Transdução de Sinais
10.
Mol Plant Microbe Interact ; 32(4): 437-451, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30451565

RESUMO

The actin cytoskeleton and actin-coupled endocytosis are conserved cellular processes required for the normal growth and pathogenesis of the rice blast fungus Magnaporthe oryzae. We have previously shown that actin regulating kinase MoArk1 regulates actin dynamics and endocytosis to play a key role in virulence of the fungus. To understand the underlying mechanism, we have characterized the actin-binding protein MoAbp1 that interacts with MoArk1 from M. oryzae. The ΔMoabp1 mutant exhibited delayed endocytosis and defects in growth, host penetration, and invasive growth. Consistent with its putative function associated with actin-binding, MoAbp1 regulates the localization of actin patches and plays a role in MoArk1 phosphorylation. In addition, MoAbp1 interacts with MoCap (adenylyl cyclase-associated protein) affecting its normal patch localization pattern and the actin protein MoAct1 through its conserved domains. Taken together, our results support a notion that MoAbp1 functions as a protein scaffold linking MoArk1, MoCap1, and MoAct1 to regulate actin cytoskeleton dynamics critical in growth and pathogenicity of the blast fungus.


Assuntos
Citoesqueleto de Actina , Endocitose , Magnaporthe , Virulência , Citoesqueleto de Actina/metabolismo , Endocitose/genética , Proteínas Fúngicas/metabolismo , Magnaporthe/crescimento & desenvolvimento , Magnaporthe/metabolismo , Magnaporthe/patogenicidade , Proteínas dos Microfilamentos/metabolismo , Oryza/microbiologia , Virulência/genética
11.
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
12.
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
13.
Brief Bioinform ; 20(2): 448-456, 2019 03 22.
Artigo em Inglês | MEDLINE | ID: mdl-29040362

RESUMO

Rice blast disease caused by the fungus Magnaporthe grisea (M. grisea) is one of the most serious diseases for the cultivated rice Oryza sativa (O. sativa). A key factor causing rice blast disease and defense might be protein-protein interactions (PPIs) between rice and fungus. In this research, we have developed a computational pipeline to predict PPIs between blast fungus and rice. After cross-prediction by interolog-based and domain-based method, we achieved 532 potential PPIs between 27 fungus proteins and 236 rice proteins. Accuracy of jackknife test, 10-fold cross-validation test and independent test for these PPIs were 90.43, 93.85 and 84.67%, respectively, by using support vector machine classification method. Meanwhile, the pathogenic genes of blast fungus were enriched in the predicted PPIs network when compared with 1000 random interaction networks. The rice regulatory network was downloaded and divided into 228 subnetworks with over six nodes, and the top seven subnetworks affected by blast fungus through PPIs were investigated. The results indicated that 34 upregulated and 12 downregulated master regulators in rice interacting with the fungus proteins in response to the infection of blast fungus. The common master regulators in rice in response to the infection of M. grisea, Xanthomonas oryzae pv.oryzae and rice stripe virus were analyzed. The ubiquitin proteasome pathway was the common pathway in rice regulated by these three pathogens, while apoptosis signaling pathway was induced by fungus and bacteria. In summary, the results in this article provide insight into the process of blast fungus infection.


Assuntos
Biologia Computacional/métodos , Proteínas Fúngicas/metabolismo , Redes Reguladoras de Genes , Magnaporthe/metabolismo , Oryza/metabolismo , Proteínas de Plantas/metabolismo , Mapas de Interação de Proteínas , Proteínas Fúngicas/genética , Magnaporthe/patogenicidade , Oryza/microbiologia , Proteínas de Plantas/genética
14.
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
15.
Sci Rep ; 8(1): 14461, 2018 09 27.
Artigo em Inglês | MEDLINE | ID: mdl-30262874

RESUMO

Members of the Yippee-like (YPEL) gene family are highly conserved in eukaryotes and are homologous to the Drosophila yippee gene. In this study, we functionally characterized two YPEL-homologous genes, MoYPEL1 and MoYPEL2, in the rice blast pathogen Magnaporthe oryzae using the deletion mutants ΔMoypel1, ΔMoypel2, and ΔΔMoypel1,2. The MoYPEL1 deletion mutant was significantly defective in conidiation and unable to undergo appressorium development; however, deletion of MoYPEL2 resulted in a significant increase in conidiation and the abnormal development of two appressoria per conidium. These data demonstrate the opposite roles of each member of the YPEL gene family during the development of M. oryzae. The double mutant was phenotypically similar to the ΔMoypel1 mutant in conidiation, but similar to the ΔMoypel2 mutant in appressorium development. Subcellular localization of the MoYPEL1 protein was dynamic during appressorium development, while the MoYPEL2 protein consistently localized within the nuclei during developmental stages. Our studies indicate that the two YPEL gene family members play distinct roles in the developmental stages of M. oryzae, furthering our understanding of disease dissemination and development in fungi.


Assuntos
Proteínas Fúngicas , Magnaporthe , Proteínas Nucleares , Oryza/microbiologia , Doenças das Plantas/microbiologia , Fatores de Virulência , Núcleo Celular/genética , Núcleo Celular/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Deleção de Genes , Magnaporthe/genética , Magnaporthe/metabolismo , Magnaporthe/patogenicidade , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fatores de Virulência/genética , Fatores de Virulência/metabolismo
16.
Methods Mol Biol ; 1848: 81-91, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30182230

RESUMO

A number of challenges have to be overcome to identify a complete complement of phosphorylated proteins, the phosphoproteome, from cells and tissues. Phosphorylated proteins are typically of low abundance and moreover, the proportion of phosphorylated sites on a given protein is generally low. The challenge is further compounded when the tissue from which protein can be recovered is limited. Global phosphoproteomics primarily relies on efficient enrichment methods for phosphopeptides involving affinity binding coupled with analysis by fast high-resolution mass spectrometry (MS) and subsequent identification using various software packages. Here, we describe an effective protocol for phosphopeptide enrichment using an Iron-IMAC resin in combination with titanium dioxide (TiO2) beads from trypsin digested protein samples of the filamentous fungus Magnaporthe oryzae. Representative protocols for LC-MS/MS analysis and phosphopeptide identification are also described.


Assuntos
Magnaporthe/metabolismo , Fosfopeptídeos/metabolismo , Fosfoproteínas/metabolismo , Proteoma , Proteômica , Cromatografia de Afinidade , Cromatografia Líquida , Biologia Computacional/métodos , Análise de Dados , Proteínas Fúngicas , Humanos , Fosfopeptídeos/química , Fosfopeptídeos/isolamento & purificação , Fosfoproteínas/química , Fosfoproteínas/isolamento & purificação , Proteômica/métodos , Espectrometria de Massas em Tandem , Titânio/química
17.
Environ Microbiol ; 20(11): 3964-3979, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30246284

RESUMO

In the rice blast fungus Magnaporthe oryzae, the cell wall integrity (CWI) signalling pathway governs cell wall changes in response to external cues and normal CWI signalling is critical for appressorium function and pathogenicity. We previously characterized the mitogen-activated protein kinase (MAPK) kinase MoMkk1 as an integral component of the CWI pathway. Using the affinity purification approach, we have identified MoMkk1-interacting MoPpe1 as a homologue of Saccharomyces cerevisiae serine/threonine protein phosphatase Sit4/Ppe1. We found that MoPpe1 is required for vegetative growth, conidiation and full virulence. In addition, we found that MoPpe1 interacts with MoSap1, a protein with functions similar to MoPpe1. Intriguingly, we found that MoPpe1-MoSap1 interaction is related to CWI and target of rapamycin (TOR) pathways. We presented evidence suggesting that MoPpe1 and MoSap1 function as an adaptor complex linking CWI and TOR signalling and that the activation of the TOR pathway leads to suppression of CWI signalling, resulting in defects in appressorium function and pathogenicity. Taken together, our studies not only reveal important functions of MoMkk1-MoPpe1-MoSap1 interactions in growth and pathogenicity of the blast fungus, but also highlight the complexity of regulatory networks involving conserved yet novel regulatory mechanisms of CWI and TOR signalling.


Assuntos
Parede Celular/metabolismo , Proteínas Fúngicas/metabolismo , Sistema de Sinalização das MAP Quinases , Magnaporthe/metabolismo , Fosfoproteínas Fosfatases/metabolismo , Sirolimo/metabolismo , Magnaporthe/crescimento & desenvolvimento , Magnaporthe/patogenicidade , Oryza/microbiologia , Proteína Fosfatase 2 , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae , Virulência
18.
Sci Rep ; 8(1): 14355, 2018 09 25.
Artigo em Inglês | MEDLINE | ID: mdl-30254203

RESUMO

The rice blast fungus Magnaporthe oryzae is the most serious pathogen of cultivated rice and a significant threat to global food security. To accelerate targeted mutation and specific genome editing in this species, we have developed a rapid plasmid-free CRISPR-Cas9-based genome editing method. We show that stable expression of Cas9 is highly toxic to M. oryzae. However efficient gene editing can be achieved by transient introduction of purified Cas9 pre-complexed to RNA guides to form ribonucleoproteins (RNPs). When used in combination with oligonucleotide or PCR-generated donor DNAs, generation of strains with specific base pair edits, in-locus gene replacements, or multiple gene edits, is very rapid and straightforward. We demonstrate a co-editing strategy for the creation of single nucleotide changes at specific loci. Additionally, we report a novel counterselection strategy which allows creation of precisely edited fungal strains that contain no foreign DNA and are completely isogenic to the wild type. Together, these developments represent a scalable improvement in the precision and speed of genetic manipulation in M. oryzae and are likely to be broadly applicable to other fungal species.


Assuntos
Sistemas CRISPR-Cas/genética , Edição de Genes/métodos , Magnaporthe/genética , Magnaporthe/fisiologia , Oryza/microbiologia , Doenças das Plantas/microbiologia , Ribonucleoproteínas/metabolismo , Sequência de Bases , Magnaporthe/metabolismo , Melaninas/biossíntese , Mutação , Polimorfismo de Nucleotídeo Único
19.
Environ Microbiol ; 20(9): 3427-3441, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-30126031

RESUMO

Pyricularia oryzae is a plant pathogen causing rice blast, a serious disease spreading in cultivated rice globally. Transcription factors play important regulatory roles in fungal development and pathogenicity. Here, we characterized the biological functions of Crf1, a basic helix-loop-helix (bHLH) transcription factor, in the development and pathogenicity of P. oryzae with functional genetics, molecular and biochemical approaches. We found that CRF1 is necessary for virulence and plays an indispensable role in the regulation of carbohydrate and lipid metabolism in P. oryzae. Deletion of CRF1 led to defects in utilization of lipids, ethanol, glycerol and L-arabinose, and down-regulation of many important genes in lipolysis, ß-oxidation, gluconeogenesis, as well as glycerol and arabinose metabolism. CRF1 is also essential for peroxisome and vacuole function, and conidial cell death during appressorium formation. The appressorium turgor, penetration ability and virulence in Δcrf1 were restored by supplementation of exogenous glucose. The virulence of Crf1 mutant was also recovered by adding exogenous D-xylose, but not by addition of ethanol, pyruvate, leucine or L-arabinose. These data showed that Crf1 plays an important role in the complex regulatory network of carbohydrate and lipid metabolism that governs fungal development and pathogenicity.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Metabolismo dos Carboidratos , Proteínas Fúngicas/metabolismo , Metabolismo dos Lipídeos , Magnaporthe/metabolismo , Magnaporthe/patogenicidade , Oryza/microbiologia , Doenças das Plantas/microbiologia , Fatores de Transcrição/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Proteínas Fúngicas/genética , Regulação Fúngica da Expressão Gênica , Magnaporthe/genética , Magnaporthe/crescimento & desenvolvimento , Deleção de Sequência , Esporos Fúngicos/genética , Esporos Fúngicos/crescimento & desenvolvimento , Esporos Fúngicos/metabolismo , Esporos Fúngicos/patogenicidade , Fatores de Transcrição/genética , Virulência
20.
Nat Plants ; 4(8): 576-585, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-29988155

RESUMO

Accelerated adaptive evolution is a hallmark of plant-pathogen interactions. Plant intracellular immune receptors (NLRs) often occur as allelic series with differential pathogen specificities. The determinants of this specificity remain largely unknown. Here, we unravelled the biophysical and structural basis of expanded specificity in the allelic rice NLR Pik, which responds to the effector AVR-Pik from the rice blast pathogen Magnaporthe oryzae. Rice plants expressing the Pikm allele resist infection by blast strains expressing any of three AVR-Pik effector variants, whereas those expressing Pikp only respond to one. Unlike Pikp, the integrated heavy metal-associated (HMA) domain of Pikm binds with high affinity to each of the three recognized effector variants, and variation at binding interfaces between effectors and Pikp-HMA or Pikm-HMA domains encodes specificity. By understanding how co-evolution has shaped the response profile of an allelic NLR, we highlight how natural selection drove the emergence of new receptor specificities. This work has implications for the engineering of NLRs with improved utility in agriculture.


Assuntos
Imunidade Inata , Magnaporthe/imunologia , Proteínas NLR/fisiologia , Oryza/imunologia , Proteínas de Plantas/fisiologia , Polimorfismo Genético , Proteínas Fúngicas/imunologia , Proteínas Fúngicas/metabolismo , Interações Hospedeiro-Patógeno/imunologia , Magnaporthe/metabolismo , Magnaporthe/patogenicidade , Modelos Imunológicos , Modelos Moleculares , Proteínas NLR/metabolismo , Oryza/genética , Oryza/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo
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