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1.
New Phytol ; 219(3): 1031-1047, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-29663402

RESUMO

Protein post-translational modifications play critical roles in cellular processes, development and stress response. The small ubiquitin-like modifier (SUMO) to proteins is one of the essential modifications in eukaryotes, but its function remains largely unknown in plant pathogenic fungi. We present a comprehensive analysis combined with proteomic, molecular and cellular approaches to explore the roles of sumoylation in the model plant fungal pathogen, Magnaporthe oryzae. We found the SUMO pathway plays key roles in colony growth, conidia formation and virulence to the host, as well as cell-cycle-related phenotypes. Sumoylation is also involved in responding to different stresses. Affinity purification identified 940 putative SUMO substrates, many of which were reported to be involved in development, stress response and infection. Interestingly, four septins were also shown to be sumoylated. Mutation of consensus sumoylation sites in each septin all resulted in reduced virulence to the host and dislocation of septins in appressoria. Moreover, sumoylation is also involved in extracellular secretion of different effector proteins. Our study on the functions of sumoylation provides novel insight into development and infection of the rice blast fungus.


Assuntos
Magnaporthe/patogenicidade , Oryza/microbiologia , Doenças das Plantas/microbiologia , Sumoilação , Ciclo Celular , Proteínas Fúngicas/metabolismo , Deleção de Genes , Genes de Plantas , Interações Hospedeiro-Patógeno , Hifas/crescimento & desenvolvimento , Magnaporthe/citologia , Magnaporthe/genética , Oryza/fisiologia , Reprodutibilidade dos Testes , Septinas/metabolismo , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Estresse Fisiológico , Especificidade por Substrato , Sumoilação/genética , Virulência
2.
Autophagy ; 13(8): 1318-1330, 2017 Aug 03.
Artigo em Inglês | MEDLINE | ID: mdl-28594263

RESUMO

Magnaporthe oryzae, the ascomycete fungus that causes rice blast disease, initiates conidiation in response to light when grown on Prune-Agar medium containing both carbon and nitrogen sources. Macroautophagy/autophagy was shown to be essential for M. oryzae conidiation and induced specifically upon exposure to light but is undetectable in the dark. Therefore, it is inferred that autophagy is naturally induced by light, rather than by starvation during M. oryzae conidiation. However, the signaling pathway(s) involved in such phototropic induction of autophagy remains unknown. We identified an M. oryzae ortholog of GCN5 (MGG_03677), encoding a histone acetyltransferase (HAT) that negatively regulates light- and nitrogen-starvation-induced autophagy, by acetylating the autophagy protein Atg7. Furthermore, we unveiled novel regulatory mechanisms on Gcn5 at both transcriptional and post-translational levels, governing its function associated with the unique phototropic response of autophagy in this pathogenic fungus. Thus, our study depicts a signaling network and regulatory mechanism underlying the autophagy induction by important environmental clues such as light and nutrients.


Assuntos
Autofagia , Biocatálise , Proteínas Fúngicas/metabolismo , Magnaporthe/citologia , Magnaporthe/metabolismo , Processos Fototróficos , Acetilação , Autofagia/efeitos da radiação , Regulação Fúngica da Expressão Gênica/efeitos da radiação , Genes Fúngicos , Luz , Magnaporthe/genética , Magnaporthe/efeitos da radiação , Processos Fototróficos/efeitos da radiação , Ligação Proteica , Processamento de Proteína Pós-Traducional/efeitos da radiação , Esporos Fúngicos/metabolismo , Esporos Fúngicos/efeitos da radiação , Transcrição Genética/efeitos da radiação
3.
Sci Rep ; 7(1): 1259, 2017 04 28.
Artigo em Inglês | MEDLINE | ID: mdl-28455525

RESUMO

ER retention receptor is a seven trans-membrane protein that plays pivotal roles in function and integrity of endoplasmic reticulum (ER). Insertional mutagenesis of Magnaporthe oryzae identified MoERR1 as a pathogenicity gene encoding putative ER retention receptor orthologous to ERD2 in Saccharomyces cerevisiae. Search through the genome identified that M. oryzae possesses another ortholog of ERD2, which is designated as MoERR2. When MoERR1 and MoERR2 were tagged with GFP, both were localized to ER. Targeted disruption of MoERR1 showed pleiotropic effects on phenotypes, while deletion of MoERR2 had no effect on phenotypes we examined. The disruption mutant of MoERR1 showed growth retardation and produced significantly reduced number of conidia with aberrant morphology. Appressoria from the mutant were unable to penetrate into plant tissues presumably due to defect in cell wall integrity, thereby rendering the mutant non-pathogenic. The MoERR1 mutant also appeared to display abnormal ER structure and mis-regulation of genes involved in chaperone function and unfolded protein response under ER stress condition. Taken together, these results suggest that MoERR1 is a ER retention receptor required for function and integrity of ER, and that MoERR1-mediated ER functionalities are essential for fungal development and pathogenesis.


Assuntos
Proteínas Fúngicas/metabolismo , Magnaporthe/crescimento & desenvolvimento , Magnaporthe/patogenicidade , Doenças das Plantas/microbiologia , Receptores Estrogênicos/metabolismo , Proteínas Fúngicas/genética , Técnicas de Inativação de Genes , Magnaporthe/citologia , Adesão à Medicação , Oryza/microbiologia , Receptores Estrogênicos/genética , Saccharomyces cerevisiae , Esporos Fúngicos/citologia , Esporos Fúngicos/crescimento & desenvolvimento
4.
BMC Cell Biol ; 18(1): 11, 2017 01 26.
Artigo em Inglês | MEDLINE | ID: mdl-28125974

RESUMO

BACKGROUND: To cause an economically important blast disease on rice, the filamentous fungus Magnaporthe oryzae forms a specialized infection structure, called an appressorium, to penetrate host cells. Once inside host cells, the fungus produces a filamentous primary hypha that differentiates into multicellular bulbous invasive hyphae (IH), which are surrounded by a host-derived membrane. These hyphae secrete cytoplasmic effectors that enter host cells presumably via the biotrophic interfacial complex (BIC). The first IH cell, also known as the side BIC-associated cell, is a specialized effector-secreting cell essential for a successful infection. This study aims to determine cellular processes that lead to the development of this effector-secreting first IH cell inside susceptible rice cells. RESULTS: Using live-cell confocal imaging, we determined a series of cellular events by which the appressorium gives rise to the first IH cell in live rice cells. The filamentous primary hypha extended from the appressorium and underwent asymmetric swelling at its apex. The single nucleus in the appressorium divided, and then one nucleus migrated into the swollen apex. Septation occurred in the filamentous region of the primary hypha, establishing the first IH cell. The tip BIC that was initially associated with the primary hypha became the side BIC on the swollen apex prior to nuclear division in the appressorium. The average distance between the early side BIC and the nearest nucleus in the appressorium was estimated to be more than 32 µm. These results suggest an unknown mechanism by which effectors that are expressed in the appressorium are transported through the primary hypha for their secretion into the distantly located BIC. When M. oryzae was inoculated on heat-killed rice cells, penetration proceeded as normal, but there was no differentiation of a bulbous IH cell, suggesting its specialization for establishment of biotrophic infection. CONCLUSIONS: Our studies reveal cellular dynamics associated with the development of the effector-secreting first IH cell. Our data raise new mechanistic questions concerning hyphal differentiation in response to host environmental cues and effector trafficking from the appressorium to the BIC.


Assuntos
Núcleo Celular/metabolismo , Magnaporthe/citologia , Oryza/microbiologia , Células Vegetais/microbiologia , Morte Celular , Divisão do Núcleo Celular , Temperatura Alta , Hifas/citologia , Mitose , Modelos Biológicos
5.
PLoS Genet ; 13(1): e1006557, 2017 01.
Artigo em Inglês | MEDLINE | ID: mdl-28072818

RESUMO

The conserved target of rapamycin (TOR) pathway integrates growth and development with available nutrients, but how cellular glucose controls TOR function and signaling is poorly understood. Here, we provide functional evidence from the devastating rice blast fungus Magnaporthe oryzae that glucose can mediate TOR activity via the product of a novel carbon-responsive gene, ABL1, in order to tune cell cycle progression during infection-related development. Under nutrient-free conditions, wild type (WT) M. oryzae strains form terminal plant-infecting cells (appressoria) at the tips of germ tubes emerging from three-celled spores (conidia). WT appressorial development is accompanied by one round of mitosis followed by autophagic cell death of the conidium. In contrast, Δabl1 mutant strains undergo multiple rounds of accelerated mitosis in elongated germ tubes, produce few appressoria, and are abolished for autophagy. Treating WT spores with glucose or 2-deoxyglucose phenocopied Δabl1. Inactivating TOR in Δabl1 mutants or glucose-treated WT strains restored appressorium formation by promoting mitotic arrest at G1/G0 via an appressorium- and autophagy-inducing cell cycle delay at G2/M. Collectively, this work uncovers a novel glucose-ABL1-TOR signaling axis and shows it engages two metabolic checkpoints in order to modulate cell cycle tuning and mediate terminal appressorial cell differentiation. We thus provide new molecular insights into TOR regulation and cell development in response to glucose.


Assuntos
Ciclo Celular , Proteínas Fúngicas/metabolismo , Glucose/metabolismo , Transdução de Sinais , Serina-Treonina Quinases TOR/metabolismo , Autofagia , Morte Celular , Proteínas Fúngicas/genética , Magnaporthe/citologia , Magnaporthe/metabolismo , Esporos Fúngicos/crescimento & desenvolvimento , Esporos Fúngicos/metabolismo , Serina-Treonina Quinases TOR/genética
6.
Curr Opin Microbiol ; 34: 147-153, 2016 12.
Artigo em Inglês | MEDLINE | ID: mdl-27816794

RESUMO

Rice blast disease is a major constraint on worldwide rice production and understanding the biology of plant infection is a priority for development of new disease control strategies. Recent advances in live cell imaging, coupled with tractability of both host and pathogen to molecular genetics and genomics, has made the rice blast pathosystem an important model for understanding plant disease. Here we review recent advances in understanding the cell biology of plant infection and, in particular, the remarkable ability of the rice blast fungus to invade plant tissue and manipulate the host plant using a battery of secreted effector proteins. These fungal effectors suppress plant immunity, alter cellular organisation, and facilitate rapid fungal growth.


Assuntos
Magnaporthe/fisiologia , Oryza/microbiologia , Doenças das Plantas/microbiologia , Proteínas Fúngicas/metabolismo , Genômica , Interações Hospedeiro-Patógeno , Magnaporthe/citologia , Magnaporthe/crescimento & desenvolvimento , Magnaporthe/patogenicidade , Doenças das Plantas/prevenção & controle , Imunidade Vegetal
7.
Sci Rep ; 6: 30963, 2016 08 08.
Artigo em Inglês | MEDLINE | ID: mdl-27498554

RESUMO

Conidiation patterning is evolutionarily complex and mechanism concerning conidiogenous cell differentiation remains largely unknown. Magnaporthe oryzae conidiates in a sympodial way and uses its conidia to infect host and disseminate blast disease. Arrestins are multifunctional proteins that modulate receptor down-regulation and scaffold components of intracellular trafficking routes. We here report an alpha-arrestin that regulates patterns of conidiation and contributes to pathogenicity in M. oryzae. We show that disruption of ARRDC1 generates mutants which produce conidia in an acropetal array and ARRDC1 significantly affects expression profile of CCA1, a virulence-related transcription factor required for conidiogenous cell differentiation. Although germ tubes normally develop appressoria, penetration peg formation is dramatically impaired and Δarrdc1 mutants are mostly nonpathogenic. Fluorescent analysis indicates that EGFP-ARRDC1 puncta are well colocalized with DsRed2-Atg8, and this distribution profile could not be altered in Δatg9 mutants, suggesting ARRDC1 enters into autophagic flux before autophagosome maturation. We propose that M. oryzae employs ARRDC1 to regulate specific receptors in response to conidiation-related signals for conidiogenous cell differentiation and utilize autophagosomes for desensitization of conidiogenous receptor, which transmits extracellular signal to the downstream elements of transcription factors. Our investigation extends novel significance of autophagy-associated alpha-arrestin signaling to fungal parasites.


Assuntos
Arrestina/metabolismo , Autofagia , Magnaporthe/citologia , Magnaporthe/crescimento & desenvolvimento , Transdução de Sinais , Esporos Fúngicos/citologia , Esporos Fúngicos/crescimento & desenvolvimento , Arrestina/genética , Perfilação da Expressão Gênica , Técnicas de Inativação de Genes , Magnaporthe/genética , Magnaporthe/patogenicidade , Fatores de Transcrição/análise , Fatores de Transcrição/genética , Fatores de Virulência/genética , Fatores de Virulência/metabolismo
8.
Microbiologyopen ; 4(6): 952-66, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26472068

RESUMO

The rice blast fungus Magnaporthe oryzae grows inside living host cells. Cytological analyses by live-cell imaging have revealed characteristics of the biotrophic invasion, particularly the extrainvasive hyphal membrane (EIHM) originating from the host plasma membrane and a host membrane-rich structure, biotrophic interfacial complex (BIC). Here, we observed rice subcellular changes associated with invasive hyphal growth using various transformants expressing specifically localized fluorescent proteins. The invasive hyphae did not penetrate across but were surrounded by the host vacuolar membrane together with EIHM even after branching. High-resolution imaging of BICs revealed that the host cytosol was accumulated at BIC with aggregated EIHM and a symplastic effector, Pwl2, in a punctate form. The vacuolar membrane did not aggregate in but closely surrounded the BIC. A good correlation was observed between the early collapse of vacuoles and damage of invasive hyphae in the first-invaded cell. Furthermore, a newly developed, long-term imaging method has revealed that the central vacuole gradually shrank until collapse, which was caused by the hyphal invasion occurring earlier in the neighboring cells than in the first-invaded cells. These data suggest that M. oryzae may suppress host vacuole collapse during early infection stages for successful infection.


Assuntos
Magnaporthe/fisiologia , Oryza/microbiologia , Doenças das Plantas/microbiologia , Vacúolos/microbiologia , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Hifas/citologia , Hifas/crescimento & desenvolvimento , Hifas/fisiologia , Magnaporthe/citologia , Magnaporthe/crescimento & desenvolvimento
9.
Mol Plant Microbe Interact ; 28(2): 107-21, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-25390188

RESUMO

As part of a large-scale project whose goal was to identify candidate effector proteins in Magnaporthe oryzae, we developed a suite of vectors that facilitate high-throughput protein localization experiments in fungi. These vectors utilize Gateway recombinational cloning to place a gene's promoter and coding sequences upstream and in frame with enhanced cyan fluorescent protein, green fluorescent protein (GFP), monomeric red fluorescence protein (mRFP), and yellow fluorescent protein or a nucleus-targeted mCHERRY variant. The respective Gateway cassettes were incorporated into Agrobacterium-based plasmids to allow efficient fungal transformation using hygromycin or geneticin resistance selection. mRFP proved to be more sensitive than the GFP spectral variants for monitoring proteins secreted in planta; and extensive testing showed that Gateway-derived fusion proteins produced localization patterns identical to their "directly fused" counterparts. Use of plasmid for fungal protein localization (pFPL) vectors with two different selectable markers provided a convenient way to label fungal cells with different fluorescent proteins. We demonstrate the utility of the pFPL vectors for identifying candidate effector proteins and we highlight a number of important factors that must be taken into consideration when screening for proteins that are translocated across the host plasma membrane.


Assuntos
Citoplasma/metabolismo , Proteínas Fúngicas/metabolismo , Magnaporthe/metabolismo , Oryza/microbiologia , Transporte Proteico/fisiologia , Agrobacterium tumefaciens/genética , Agrobacterium tumefaciens/fisiologia , Clonagem Molecular , Citoplasma/química , Proteínas Fúngicas/genética , Regulação Fúngica da Expressão Gênica/fisiologia , Magnaporthe/citologia , Oryza/citologia , Oryza/metabolismo , Células Vegetais , Plasmídeos/genética , Transformação Genética
10.
Fungal Genet Biol ; 69: 43-51, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-24875422

RESUMO

To cause disease on host plants, many phytopathogenic fungi undergo morphological transitions including development of reproductive structures as well as specialized infection structures called appressoria. Such morphological transitions display distinct nuclear dynamics. Here we report the developmental requirement of MoAND1-mediated nuclear positioning for pathogenesis of the rice blast fungus, Magnaporthe oryzae. The MoAND1 gene encodes a protein that shows high similarity to Num1 in Saccharomyces cerevisiae and ApsA in Aspergillus nidulans, both of which are cell cortex proteins involved in nuclear migration and positioning. Targeted deletion of MoAND1 did not affect radial growth of the fungus but impaired nuclear distribution along the hyphae, which is reminiscent of ApsA mutant. In contrast to the wild-type, which produces three to five spores in a sympodial manner on the conidiophore, only a single spore was borne on the conidiophore of ΔMoand1, resulting in ∼65% decrease in conidia production, compared to the wild-type. The mutant conidia displayed abnormalities in septation pattern and nuclear distribution, which were correlated with their inability to germinate. Spores of the mutant that did germinate were capable of differentiating appressoria but were defective in the execution of programmed nuclear migration and positioning during development. Furthermore, mutant appressoria were not fully functional, leading to delay in penetration of host plants. However, the ability of ΔMoand1 to grow inside host tissues was comparable to that of the wild-type. All these defects greatly decreased the virulence of the mutant. Taken together, our data suggest that there is a stringent but incomplete developmental requirement for proper migration and positioning of fungal nuclei mediated by MoAND1 during asexual reproduction and pre-penetration phase of fungal pathogenesis.


Assuntos
Proteínas do Citoesqueleto/metabolismo , Proteínas Fúngicas/metabolismo , Magnaporthe/metabolismo , Magnaporthe/patogenicidade , Oryza/microbiologia , Doenças das Plantas/microbiologia , Aspergillus nidulans/genética , Núcleo Celular/metabolismo , Proteínas do Citoesqueleto/genética , Proteínas Fúngicas/genética , Deleção de Genes , Magnaporthe/citologia , Magnaporthe/crescimento & desenvolvimento , Saccharomyces cerevisiae/genética , Homologia de Sequência de Aminoácidos , Esporos Fúngicos/crescimento & desenvolvimento
11.
Mol Plant Microbe Interact ; 27(9): 996-1004, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-24835254

RESUMO

Magnaporthe oryzae forms a highly specialized infection structure called an appressorium for plant penetration. In M. oryzae and many other plant-pathogenic fungi, surface attachment and surface recognition are two essential requirements for appressorium formation. Development of appressoria in the air has not been reported. In this study, we found that expression of a dominant active MoRAS2(G18V) allele in M. oryzae resulted in the formation of morphologically abnormal appressoria on nonconducive surfaces, in liquid suspensions, and on aerial hyphae without attachment to hard surfaces. Both the Pmk1 mitogen-activated protein kinase cascade and cAMP signaling pathways that regulate surface recognition and appressorium morphogenesis in M. oryzae were overactivated in the MoRAS2(G18V) transformant. In mutants deleted of PMK1 or CPKA, expression of MoRAS2(G18V) had no significant effects on appressorium morphogenesis. Furthermore, expression of dominant MoRAS2 in Colletotrichum graminicola and C. gloeosporioides also caused the formation of appressorium-like structures in aerial hyphae. Overall, our data indicate that MoRas2 functions upstream from both the cAMP-PKA and Pmk1 pathways and overactive Ras signaling leads to improper activation of these two pathways and appressorium formation without surface attachment in appressorium-forming pathogens.


Assuntos
Proteínas Fúngicas/metabolismo , Magnaporthe/genética , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Oryza/microbiologia , Doenças das Plantas/microbiologia , Transdução de Sinais , Proteínas ras/metabolismo , Alelos , Substituição de Aminoácidos , Colletotrichum/citologia , Colletotrichum/genética , Colletotrichum/crescimento & desenvolvimento , Colletotrichum/fisiologia , Proteínas Fúngicas/genética , Deleção de Genes , Regulação Fúngica da Expressão Gênica , Genes Dominantes , Genes Reporter , Hifas , Magnaporthe/citologia , Magnaporthe/crescimento & desenvolvimento , Magnaporthe/fisiologia , Proteínas Quinases Ativadas por Mitógeno/genética , Mutação , Proteínas Recombinantes de Fusão , Esporos Fúngicos , Virulência , Proteínas ras/genética
12.
PLoS One ; 9(1): e85252, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24454828

RESUMO

Peroxisomes are present ubiquitously and make important contributions to cellular metabolism in eukaryotes. They play crucial roles in pathogenicity of plant fungal pathogens. The peroxisomal matrix proteins and peroxisomal membrane proteins (PMPs) are synthesized in the cytosol and imported post-translationally. Although the peroxisomal import machineries are generally conserved, some species-specific features were found in different types of organisms. In phytopathogenic fungi, the pathways of the matrix proteins have been elucidated, while the import machinery of PMPs remains obscure. Here, we report that MoPEX19, an ortholog of ScPEX19, was required for PMPs import and peroxisomal maintenance, and played crucial roles in metabolism and pathogenicity of the rice blast fungus Magnaporthe oryzae. MoPEX19 was expressed in a low level and Mopex19p was distributed in the cytoplasm and newly formed peroxisomes. MoPEX19 deletion led to mislocalization of peroxisomal membrane proteins (PMPs), as well peroxisomal matrix proteins. Peroxisomal structures were totally absent in Δmopex19 mutants and woronin bodies also vanished. Δmopex19 exhibited metabolic deficiency typical in peroxisomal disorders and also abnormality in glyoxylate cycle which was undetected in the known mopex mutants. The Δmopex19 mutants performed multiple disorders in fungal development and pathogenicity-related morphogenesis, and lost completely the pathogenicity on its hosts. These data demonstrate that MoPEX19 plays crucial roles in maintenance of peroxisomal and peroxisome-derived structures and makes more contributions to fungal development and pathogenicity than the known MoPEX genes in the rice blast fungus.


Assuntos
Proteínas Fúngicas/metabolismo , Magnaporthe/crescimento & desenvolvimento , Magnaporthe/metabolismo , Proteínas de Membrana/metabolismo , Oryza/microbiologia , Peroxissomos/metabolismo , Sequência de Aminoácidos , Parede Celular/metabolismo , Citoplasma/metabolismo , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Hifas/citologia , Hifas/crescimento & desenvolvimento , Hifas/metabolismo , Magnaporthe/citologia , Magnaporthe/patogenicidade , Proteínas de Membrana/química , Proteínas de Membrana/deficiência , Proteínas de Membrana/genética , Dados de Sequência Molecular , Deleção de Sequência , Homologia de Sequência do Ácido Nucleico , Esporos Fúngicos/citologia , Esporos Fúngicos/crescimento & desenvolvimento , Esporos Fúngicos/metabolismo
13.
Mol Plant Microbe Interact ; 27(5): 446-60, 2014 May.
Artigo em Inglês | MEDLINE | ID: mdl-24405033

RESUMO

The mitogen-activated protein kinase MoOsm1-mediated osmoregulation pathway plays crucial roles in stress responses, asexual and sexual development, and pathogenicity in Magnaporthe oryzae. Utilizing an affinity purification approach, we identified the putative transcriptional activator MoMsn2 as a protein that interacts with MoOsm1 in vivo. Disruption of the MoMSN2 gene resulted in defects in aerial hyphal growth, conidial production, and infection of host plants. Quantitative reverse transcription-polymerase chain reaction analysis showed that the expression of several genes involved in conidiophore formation was reduced in ΔMomsn2, suggesting that MoMsn2 might function as a transcriptional regulator of these genes. Subsequently, MoCos1 was identified as one of the MoMsn2 targets through yeast one-hybrid analysis in which MoMsn2 binds to the AGGGG and CCCCT motif of the MoCOS1 promoter region. Phenotypic characterization showed that MoMsn2 was required for appressorium formation and penetration and pathogenicity. Although the ΔMomsn2 mutant was tolerant to the cell-wall stressor Calcofluor white, it was sensitive to common osmotic stressors. Further analysis suggests that MoMsn2 is involved in the regulation of the cell-wall biosynthesis pathway. Finally, transcriptome data revealed that MoMsn2 modulates numerous genes participating in conidiation, infection, cell-wall integrity, and stress response. Collectively, our results led to a model in which MoMsn2 mediates a series of downstream genes that control aerial hyphal growth, conidiogenesis, appressorium formation, cell-wall biosynthesis, and infection and that also offer potential targets for the development of new disease management strategies.


Assuntos
Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica , Magnaporthe/genética , Oryza/microbiologia , Doenças das Plantas/microbiologia , Sequência de Aminoácidos , Parede Celular/metabolismo , Proteínas Fúngicas/genética , Hifas , Magnaporthe/citologia , Magnaporthe/crescimento & desenvolvimento , Magnaporthe/patogenicidade , Proteínas Quinases Ativadas por Mitógeno/genética , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Dados de Sequência Molecular , Motivos de Nucleotídeos , Pressão Osmótica , Fenótipo , Folhas de Planta/microbiologia , Mapeamento de Interação de Proteínas , Proteínas Recombinantes de Fusão , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Alinhamento de Sequência , Deleção de Sequência , Esporos Fúngicos , Transcriptoma , Dedos de Zinco
14.
Autophagy ; 9(11): 1818-27, 2013 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-23958498

RESUMO

Macroautophagy-mediated glycogen catabolism is required for asexual differentiation in the blast fungus, Magnaporthe oryzae. However, the function(s) of selective subtypes of autophagy has not been studied therein. Here, we report that mitophagy, selective autophagic delivery of mitochondria to the vacuoles for degradation, occurs during early stages of Magnaporthe conidiation. Specifically, mitophagy was evident in the foot cells while being undetectable in aerial hyphae and/or conidiophores. We show that loss of MoAtg24, a sorting nexin related to yeast Snx4, disrupts mitophagy and consequently leads to highly reduced conidiation, suggesting that mitophagy in the foot cells plays an important role during asexual development in Magnaporthe. Ectopic expression of yeast ScATG32 partially suppressed the conidiation initiation defects associated with MoATG24 deletion. MoAtg24 was neither required for pexophagy nor for macroautophagy, or for MoAtg8 localization per se, but directly associated with and likely recruited mitochondria to the autophagic structures during mitophagy. Lastly, MoAtg24 was also required for oxidative stress response in Magnaporthe.


Assuntos
Proteínas Fúngicas/metabolismo , Hifas/citologia , Hifas/metabolismo , Magnaporthe/citologia , Magnaporthe/metabolismo , Reprodução Assexuada , Proteínas Fúngicas/química , Proteínas de Fluorescência Verde/metabolismo , Mitocôndrias/metabolismo , Mutação/genética , Estresse Oxidativo , Estrutura Terciária de Proteína , Transporte Proteico , Proteínas Recombinantes de Fusão/metabolismo , Relação Estrutura-Atividade , Frações Subcelulares/metabolismo
15.
Biosci Biotechnol Biochem ; 77(7): 1539-47, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23832352

RESUMO

Roxithromycin (RXM), active against prokaryotes, has beneficial side effects such as anti-cancer activities on mammalian cells, but the mechanisms underlying these effects remain unclear. We found that RXM inhibited the cellular differentiation of the rice blast fungus Magnaporthe oryzae. Hence, we screened the targets of RXM by the T7 phage display method with fungal genomic DNA, and identified MoCDC27 (M. oryzae Cell Division Cycle 27) as a candidate. We generated mocdc27 knockdown mutants that the appressoria formation was less affected by RXM. A complemented mutant restored sensitivity against RXM to the level of the wild type. These results suggest that MoCDC27 was involved in the inhibition of appressorium formation by RXM, and that the complex of RXM-MoCDC27 affected another molecule involved in appressorium formation. The T7 phage display method with fungal genomic DNA can be a useful tool in the quest for drug target.


Assuntos
Magnaporthe/citologia , Magnaporthe/efeitos dos fármacos , Roxitromicina/farmacologia , Sequência de Aminoácidos , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Magnaporthe/genética , Dados de Sequência Molecular , Mutação , Biblioteca de Peptídeos , Fenótipo
16.
Nat Commun ; 4: 1996, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23774898

RESUMO

To cause plant diseases, pathogenic micro-organisms secrete effector proteins into host tissue to suppress immunity and support pathogen growth. Bacterial pathogens have evolved several distinct secretion systems to target effector proteins, but whether fungi, which cause the major diseases of most crop species, also require different secretory mechanisms is not known. Here we report that the rice blast fungus Magnaporthe oryzae possesses two distinct secretion systems to target effectors during plant infection. Cytoplasmic effectors, which are delivered into host cells, preferentially accumulate in the biotrophic interfacial complex, a novel plant membrane-rich structure associated with invasive hyphae. We show that the biotrophic interfacial complex is associated with a novel form of secretion involving exocyst components and the Sso1 t-SNARE. By contrast, effectors that are secreted from invasive hyphae into the extracellular compartment follow the conventional secretory pathway. We conclude that the blast fungus has evolved distinct secretion systems to facilitate tissue invasion.


Assuntos
Proteínas Fúngicas/metabolismo , Magnaporthe/patogenicidade , Oryza/microbiologia , Doenças das Plantas/microbiologia , Brefeldina A/farmacologia , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Citoplasma/efeitos dos fármacos , Citoplasma/metabolismo , Hifas/citologia , Hifas/efeitos dos fármacos , Hifas/metabolismo , Magnaporthe/citologia , Magnaporthe/efeitos dos fármacos , Modelos Biológicos , Mutação/genética , Oryza/efeitos dos fármacos , Proteínas SNARE/genética
17.
Fungal Biol ; 117(4): 239-49, 2013 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-23622718

RESUMO

The conidiophore stalk-less1 (COS1) gene encodes a novel transcription factor in Magnaporthe oryzae, and mutation of COS1 (M2942) resulted in developmental failure of conidiophores. COS1 putatively encodes a 491-amino-acid protein, which contains four multiple adjacent C2H2-type zinc-finger domains. The motifs are homologous to the zinc-finger protein Azf1 of Saccharomyces cerevisiae. Here, we report that the differences of expression profile between M2942 and the wild-type isolate Y34 by RNA-Sequence. DNA sequence analysis of promoter regions of those of COS1-dependent genes showed enrichment in the DNA sequence AAAAGAAA (A4GA3), the putative COS1-binding motif. Gel shift experiments showed that COS1 binds to DNA elements with A4GA3 motif. These suggest that many of the COS1-dependent transcripts may be regulated directly by COS1 binding.


Assuntos
Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica , Magnaporthe/genética , Fatores de Transcrição/metabolismo , Sítios de Ligação , DNA Fúngico/metabolismo , Ensaio de Desvio de Mobilidade Eletroforética , Proteínas Fúngicas/genética , Perfilação da Expressão Gênica , Técnicas de Inativação de Genes , Magnaporthe/citologia , Magnaporthe/crescimento & desenvolvimento , Regiões Promotoras Genéticas , Ligação Proteica , Esporos Fúngicos/citologia , Esporos Fúngicos/crescimento & desenvolvimento , Fatores de Transcrição/genética , Dedos de Zinco
18.
Fungal Genet Biol ; 56: 33-41, 2013 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-23591122

RESUMO

Melanized appressoria are highly specialized infection structures formed by germ tubes of the rice blast fungus Magnaporthe oryzae for plant infection. M. oryzae also forms appressorium-like structures on hyphal tips. Whereas appressorium formation by conidial germ tubes has been well characterized, formation of appressorium-like structures by hyphal tips is under-investigated. In a previous study, we found that the chs7 deletion mutant failed to form appressoria on germ tubes but were normal in the development of appressorium-like structures on artificial hydrophobic surfaces. In this study, we compared the differences between the formation of appressoria by germ tubes and appressorium-like structures by hyphal tips in M. oryzae. Structurally, both appressoria and appressorium-like structures had a melanin layer that was absent in the pore region. In general, the latters were 1.4-fold larger in size but had lower turgor pressure than appressoria, which is consistent with its lower efficiency in plant penetration. Treatments with cAMP, IBMX, or a cutin monomer efficiently induced appressorium formation but not the development of appressorium-like structures. In contrast, coating surfaces with waxes stimulated the formation of both infection structures. Studies with various signaling mutants indicate that Osm1 and Mps1 are dispensable but Pmk1 is essential for both appressorium formation and development of appressorium-like structures on hyphal tips. Interestingly, the cpkA mutant was reduced in the differentiation of appressorium-like structures but not appressorium formation. We also observed that the con7 mutant generated in our lab failed to form appressorium-like structures on hyphal tips but still produced appressoria by germ tubes on hydrophobic surfaces. Con7 is a transcription factor regulating the expression of CHS7. Overall, these results indicate that the development of appressorium-like structures by hyphal tips and formation of appressoria by germ tubes are not identical differentiation processes in M. oryzae and may involve different molecular mechanisms.


Assuntos
Hifas/citologia , Magnaporthe/citologia , Regulação Fúngica da Expressão Gênica , Genes Fúngicos , Hifas/química , Magnaporthe/química , Melaninas/análise , Microscopia , Mutação , Oryza/microbiologia , Doenças das Plantas/microbiologia , Transdução de Sinais
19.
Autophagy ; 8(7): 1058-70, 2012 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-22561104

RESUMO

The sorting nexins Atg20/Snx42 and Snx41 regulate membrane traffic and endosomal protein sorting and are essential for Cvt and/or pexophagy in yeast. Previously, we showed that macroautophagy is necessary for conidiation in the rice-blast fungus Magnaporthe oryzae. Here, we analyzed the physiological function(s) of selective autophagy in Magnaporthe through targeted deletion of MGG_12832, an ortholog of yeast SNX41 and ATG20/SNX42. Loss of MGG_12832 (hereafter SNX41) abolished conidia formation and pathogenesis in M. oryzae. Snx41-GFP localized as dynamic puncta or short tubules that are partially associated with autophagosomes and/or autophagic vacuoles. PX domain, but not macroautophagy per se, was required for such localization of Snx41-GFP in Magnaporthe. Although not required for nonselective autophagy, Snx41 was essential for pexophagy in Magnaporthe. We identified Oxp1, an ATP-dependent oxoprolinase in the gamma-glutamyl cycle, as a binding partner and potential retrieval target of Snx41-dependent protein sorting. The substrate of Oxp1, 5-oxoproline, could partially restore conidiation in the snx41Δ. Exogenous glutathione, a product of the gamma-glutamyl cycle, significantly restored pathogenicity in the snx41Δ mutant, likely through counteracting the oxidative stress imposed by the host. We propose that the gamma-glutamyl cycle and glutathione biosynthesis are subject to regulation by Snx41-dependent vesicular trafficking, and mediate antioxidant defense crucial for in planta growth and pathogenic differentiation of Magnaporthe at the onset of blast disease in rice.


Assuntos
Antioxidantes/metabolismo , Proteínas Fúngicas/metabolismo , Glutationa/metabolismo , Magnaporthe/crescimento & desenvolvimento , Magnaporthe/patogenicidade , Nexinas de Classificação/metabolismo , Esporos Fúngicos/metabolismo , Autofagia , Deleção de Genes , Proteínas de Fluorescência Verde/metabolismo , Hordeum/citologia , Hordeum/microbiologia , Magnaporthe/citologia , Magnaporthe/imunologia , Peroxissomos/metabolismo , Folhas de Planta/citologia , Folhas de Planta/microbiologia , Transporte Proteico , Proteínas Recombinantes de Fusão/metabolismo , Frações Subcelulares/metabolismo
20.
PLoS One ; 7(4): e36266, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22558413

RESUMO

The role of ß-oxidation and the glyoxylate cycle in fungal pathogenesis is well documented. However, an ambiguity still remains over their interaction in peroxisomes to facilitate fungal pathogenicity and virulence. In this report, we characterize a gene encoding an alanine, glyoxylate aminotransferase 1 (AGT1) in Magnaporthe oryzae, the causative agent of rice blast disease, and demonstrate that AGT1 is required for pathogenicity of M. oryzae. Targeted deletion of AGT1 resulted in the failure of penetration via appressoria; therefore, mutants lacking the gene were unable to induce blast symptoms on the hosts rice and barley. This penetration failure may be associated with a disruption in lipid mobilization during conidial germination as turgor generation in the appressorium requires mobilization of lipid reserves from the conidium. Analysis of enhanced green fluorescent protein expression using the transcriptional and translational fusion with the AGT1 promoter and open reading frame, respectively, revealed that AGT1 expressed constitutively in all in vitro grown cell types and during in planta colonization, and localized in peroxisomes. Peroxisomal localization was further confirmed by colocalization with red fluorescent protein fused with the peroxisomal targeting signal 1. Surprisingly, conidia produced by the Δagt1 mutant were unable to form appressoria on artificial inductive surfaces, even after prolonged incubation. When supplemented with nicotinamide adenine dinucleotide (NAD(+))+pyruvate, appressorium formation was restored on an artificial inductive surface. Taken together, our data indicate that AGT1-dependent pyruvate formation by transferring an amino group of alanine to glyoxylate, an intermediate of the glyoxylate cycle is required for lipid mobilization and utilization. This pyruvate can be converted to non-fermentable carbon sources, which may require reoxidation of NADH generated by the ß-oxidation of fatty acids to NAD(+) in peroxisomes. Therefore, it may provide a means to maintain redox homeostasis in appressoria.


Assuntos
Magnaporthe/citologia , Magnaporthe/fisiologia , Oryza/microbiologia , Peroxissomos/enzimologia , Doenças das Plantas/microbiologia , Transaminases/metabolismo , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Glioxilatos/metabolismo , Hordeum/microbiologia , Magnaporthe/efeitos dos fármacos , Magnaporthe/genética , NAD/farmacologia , Oxirredução , Peroxissomos/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Ácido Pirúvico/farmacologia , Deleção de Sequência , Propriedades de Superfície , Transaminases/deficiência , Transaminases/genética
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