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1.
mSphere ; 4(5)2019 09 04.
Artigo em Inglês | MEDLINE | ID: mdl-31484736

RESUMO

Magnaporthe oryzae causes the rice blast disease, which is one of the most serious diseases of cultivated rice worldwide. Glycosylation is an important posttranslational modification of secretory and membrane proteins in all eukaryotes, catalyzed by glycosyltransferases (GTs). In this study, we identified and characterized a type 2 glycosyltransferase, MoGt2, in M. oryzae Targeted gene deletion mutants of MoGT2 (mogt2Δ strains) were nonpathogenic and were impaired in vegetative growth, conidiation, and appressorium formation at hyphal tips. Moreover, MoGT2 plays an important role in stress tolerance and hydrophobin function of M. oryzae Site-directed mutagenesis analysis showed that conserved glycosyltransferase domains (DxD and QxxRW) are critical for biological functions of MoGt2. MoGT2 deletion led to altered glycoproteins during M. oryzae conidiation. By liquid chromatography-tandem mass spectrometry (LC-MS/MS), we identified several candidate proteins as potential substrates of MoGt2, including several heat shock proteins, two coiled-coil domain-containing proteins, aminopeptidase 2, and nuclease domain-containing protein 1. On the other hand, we found that a conidiation-related gene, genes involved in various metabolism pathways, and genes involved in cell wall integrity and/or osmotic response were differentially regulated in the mogt2Δ mutant, which may potentially contribute to its condiation defects. Taken together, our results show that MoGt2 is important for infection-related morphogenesis and pathogenesis in M. oryzae IMPORTANCE The ascomycete fungus Magnapothe oryzae is the causal agent of rice blast disease, leading to severe loss in cultivated rice production worldwide. In this study, we identified a conserved type 2 glycosyltransferase named MoGt2 in M. oryzae The mogt2Δ targeted gene deletion mutants exhibited pleiotropic defects in vegetative growth, conidiation, stress response, hyphal appressorium-mediated penetration, and pathogenicity. Furthermore, conserved glycosyltransferase domains are critical for MoGt2 function. The comparative transcriptome analysis revealed potential target genes under MoGt2 regulation in M. oryzae conidiation. Identification of potential glycoproteins modified by MoGt2 provided information on its regulatory mechanism of gene expression and biological functions. Overall, our study represents the first report of type 2 glycosyltransferase function in M. oryzae infection-related morphogenesis and pathogenesis.

2.
Cell Microbiol ; : e13114, 2019 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-31487436

RESUMO

Nuclear import of proteins relies on nuclear import receptors called importins/karyopherins (Kaps), whose functions were reported in yeasts, fungi, plants, and animal cells, including cell cycle control, morphogenesis, stress sensing/response, and also fungal pathogenecity. However, limited is known about the physiological function and regulatory mechanism of protein import in the rice-blast fungus Magnaporthe oryzae. Here, we identified an ortholog of ß-importin in M. oryzae encoded by an ortholog of KAP119 gene. Functional characterisation of this gene via reverse genetics revealed that it is required for vegetative growth, conidiation, melanin pigmentation, and pathogenicity of M. oryzae. The mokap119Δ mutant was also defective in formation of appressorium-like structure from hyphal tips. By affinity assay and liquid chromatography-tandem mass spectrometry, we identified potential MoKap119-interacting proteins and further verified that MoKap119 interacts with the cyclin-dependent kinase subunit MoCks1 and mediates its nuclear import. Transcriptional profiling indicated that MoKap119 may regulate transcription of infection-related genes via MoCks1 regulation of MoSom1. Overall, our findings provide a novel insight into the regulatory mechanism of M. oryzae pathogenesis likely by MoKap119-mediated nuclear import of the cyclin-dependent kinase subunit MoCks1.

3.
Annu Rev Microbiol ; 73: 601-619, 2019 Sep 08.
Artigo em Inglês | MEDLINE | ID: mdl-31283431

RESUMO

The blast disease, caused by the ascomycete Magnaporthe oryzae, poses a great threat to rice production worldwide. Increasing use of fungicides and/or blast-resistant varieties of rice (Oryza sativa) has proved to be ineffective in long-term control of blast disease under field conditions. To develop effective and durable resistance to blast, it is important to understand the cellular mechanisms underlying pathogenic development in M. oryzae. In this review, we summarize the latest research in phototropism, autophagy, nutrient and redox signaling, and intrinsic phytohormone mimics in M. oryzae for cellular and metabolic adaptation(s) during its interactions with the host plants.

4.
Mol Plant Pathol ; 20(8): 1147-1162, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31218796

RESUMO

Magnaporthe oryzae causes blast disease, which is one of the most devastating infections in rice and several important cereal crops. Magnaporthe oryzae needs to coordinate gene regulation, morphological changes, nutrient acquisition and host evasion in order to invade and proliferate within the plant tissues. Thus far, the molecular mechanisms underlying the regulation of invasive growth in planta have remained largely unknown. We identified a precise filamentous-punctate-filamentous cycle in mitochondrial morphology during Magnaporthe-rice interaction. Interestingly, disruption of such mitochondrial dynamics by deletion of genes regulating either the mitochondrial fusion (MoFzo1) or fission (MoDnm1) machinery, or inhibition of mitochondrial fission using Mdivi-1 caused significant reduction in M. oryzae pathogenicity. Furthermore, exogenous carbon source(s) but not antioxidant treatment delayed such mitochondrial dynamics/transition during invasive growth. In contrast, carbon starvation induced the breakdown of the mitochondrial network and led to more punctate mitochondria in vitro. Such nutrient-based regulation of organellar dynamics preceded MoAtg24-mediated mitophagy, which was found to be essential for proper biotrophic development and invasive growth in planta. We propose that precise mitochondrial dynamics and mitophagy occur during the transition from biotrophy to necrotrophy and are required for proper induction and establishment of the blast disease in rice.

5.
mSphere ; 4(3)2019 05 29.
Artigo em Inglês | MEDLINE | ID: mdl-31142621

RESUMO

Sporisorium scitamineum is the fungal pathogen causing severe sugarcane smut disease that leads to massive economic losses globally. S. scitamineum invades host cane by dikaryotic hyphae, formed after sexual mating of two haploid sporidia of opposite mating type. Therefore, mating/filamentation is critical for S. scitamineum pathogenicity, while its molecular mechanisms remain largely unknown. The AGC (cyclic AMP [cAMP]-dependent protein kinase 1 [protein kinase A {PKA}], cGMP-dependent protein kinase [PKG], and protein kinase C [PKC]) kinase family is a group of serine/threonine (Ser/Thr) protein kinases conserved among eukaryotic genomes, serving a variety of physiological functions, including cell growth, metabolism, differentiation, and cell death. In this study, we identified an AGC kinase, named SsAgc1 (for S. scitamineum Agc1), and characterized its function by reverse genetics. Our results showed that SsAgc1 is critical for S. scitamineum mating/filamentation and pathogenicity, and oxidative stress tolerance under some circumstances. Transcriptional profiling revealed that the SsAgc1 signaling pathway may control expression of the genes governing fungal mating/filamentation and tryptophan metabolism, especially for tryptophol production. We showed that tryptophan and tryptophol could at least partially restore ssagc1Δ mating/filamentation. Overall, our work revealed a signaling pathway mediated by AGC protein kinases to regulate fungal mating/filamentation, possibly through sensing and responding to tryptophol as signal molecules.IMPORTANCE The AGC signaling pathway represents a conserved distinct signaling pathway in regulation of fungal differentiation and virulence, while it has not been identified or characterized in the sugarcane smut fungus Sporisorium scitamineum In this study, we identified a PAS domain-containing AGC kinase, SsAgc1, in S. scitamineum Functional analysis revealed that SsAgc1 plays a regulatory role on the fungal dimorphic switch.

6.
Environ Microbiol ; 21(3): 959-971, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30537399

RESUMO

The fungal pathogen Sporisorium scitamineum causes sugarcane smut disease. The formation and growth of dikaryotic hypha after sexual mating is critical for S. scitamineum pathogenicity, however regulation of S. scitimineum mating has not been studied in detail. We identified and characterized the core components of the conserved cAMP/PKA pathway in S. scitamineum by reverse genetics. Our results showed that cAMP/PKA signalling pathway is essential for proper mating and filamentation, and thus critical for S. scitamineum virulence. We further demonstrated that an elevated intracellular ROS (reactive oxygen species) level promotes S. scitamineum mating-filamentation, via transcriptional regulation of ROS catabolic enzymes, and is under regulation of the cAMP/PKA signalling pathway. Furthermore, we found that fungal cAMP/PKA signalling pathway is also involved in regulation of host ROS response. Overall, our work displayed a positive role of elevated intracellular ROS in fungal differentiation and virulence.

7.
mSystems ; 3(6)2018 Nov-Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30505942

RESUMO

The rice blast fungus Magnaporthe oryzae poses a great threat to global food security. During its conidiation (asexual spore formation) and appressorium (infecting structure) formation, autophagy is induced, serving glycogen breakdown or programmed cell death function, both essential for M. oryzae pathogenicity. Recently, we identified an M. oryzae histone acetyltransferase (HAT) Gcn5 as a key regulator in phototropic induction of autophagy and asexual spore formation while serving a cellular function other than autophagy induction during M. oryzae infection. To further understand the regulatory mechanism of Gcn5 on M. oryzae pathogenicity, we set out to identify more Gcn5 substrates by comparative acetylome between the wild-type (WT) and GCN5 overexpression (OX) mutant and between OX mutant and GCN5 deletion (knockout [KO]) mutant. Our results showed that Gcn5 regulates autophagy induction and other important aspects of fungal pathogenicity, including energy metabolism, stress response, cell toxicity and death, likely via both epigenetic regulation (histone acetylation) and posttranslational modification (nonhistone protein acetylation). IMPORTANCE Gcn5 is a histone acetyltransferase that was previously shown to regulate phototropic and starvation-induced autophagy in the rice blast fungus Magnaporthe oryzae, likely via modification on autophagy protein Atg7. In this study, we identified more potential substrates of Gcn5-mediated acetylation by quantitative and comparative acetylome analyses. By epifluorescence microscopy and biochemistry experiments, we verified that Gcn5 may regulate autophagy induction at both the epigenetic and posttranslational levels and regulate autophagic degradation of a critical metabolic enzyme pyruvate kinase (Pk) likely via acetylation. Overall, our findings reveal comprehensive posttranslational modification executed by Gcn5, in response to various external stimuli, to synergistically promote cellular differentiation in a fungal pathogen.

8.
Front Microbiol ; 9: 2555, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30416495

RESUMO

In the phytopathogenic fungus Sporisorium scitamineum, sexual mating between two compatible haploid cells and the subsequent formation of dikaryotic hyphae is essential for infection. This process was shown to be commonly regulated by a mitogen-activated protein kinase (MAPK) and a cAMP/PKA signaling pathway in the corn smut fungus Ustilago maydis but remains largely unknown in S. scitamineum. In this study, we identified a conserved putative MAP kinase Kpp2 in S. scitamineum and named it as SsKpp2. The sskpp2Δ mutant displayed significant reduction in mating/filamentation, which could be partially restored by addition of cAMP or tryptophol, a quorum-sensing molecule identified in budding yeast. Transcriptional profiling showed that genes governing S. scitamineum mating or tryptophol biosynthesis were significantly differentially regulated in the sskpp2Δ mutant compared to the WT, under mating condition. Our results demonstrate that the MAP kinase SsKpp2 is required for S. scitamineum mating/filamentation likely through regulating the conserved pheromone signal transduction pathway and tryptophol production.

9.
Mycology ; 9(3): 233-237, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30181929

RESUMO

Phytohormones (also named as plant hormones) are chemicals produced by plants in order to modulate various aspects of plant development, stress responses and defence. Recent studies revealed that fungi can also produce phytohormones or phytohormone-mimiking molecules, while it remains poorly understood about the details in the role and regulatory mechanism of such fungal produced phytohormonal molecules in plant-fungus interactions. The rice-blast fungus Magnaporthe oryzae imposes a great threat to global food security. Intensive investigation has been conducted to elucidate M. oryzae pathogenicity and rice (Oryza sativa L.) defense mechanism against blast disease, in order to provide theoretical basis and/or identify potential target(s) for developing novel disease control strategies, as well as for breeding of resistance varieties. Phytohormones have been demonstrated to play conserved and divergent roles in fine-tuning the balance of rice growth and immunity towards M. oryzae. Meanwhile, M. oryzae evolved elaborate strategy to manipulate the rice phytohormones metabolism, or even directly produce and secrete phytohormones, during their invasion process. In this review, we discuss the chemical communication in term of phytohormones in M. oryzae-rice pathosystem.

10.
3 Biotech ; 8(9): 380, 2018 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-30148030

RESUMO

To develop a new strategy that controls vascular pathogen infections in economic crops, we examined a possible enhancer of the vascular activity of XYLOGEN PROTEIN 1 promoter (Px). This protein is specifically expressed in the vascular tissues of Arabidopsis thaliana and plays an important role in xylem development. Although Px is predicted as vascular-specific, its activity is hard to detect and highly susceptible to plant and environmental conditions. The cauliflower mosaic virus 35S promoter (35S) is highly active in directing transgene expression. To test if 35S could enhance Px activity, while vascular specificity of the promoter is retained, we examined the expression of the uidA reporter gene, which encodes ß-glucuronidase (GUS), under the control of a chimeric promoter (35S-Px) or Px by generating 35S-Px-GUS and Px-GUS constructs, which were transformed into tobacco seedlings. Both 35S-Px and Px regulated gene expression in vascular tissues. However, GUS expression driven by 35S-Px was not detected in 30- and 60-day-old plants. Quantitative real-time PCR analysis showed that GUS gene expression regulated by 35S-Px was 6.2-14.9-fold higher in vascular tissues than in leaves. Histochemical GUS staining demonstrated that 35S-Px was strongly active in the xylem and phloem. Thus, fusion of 35S and Px might considerably enhance the strength of Px and increase its vascular specificity. In addition to confirming that 35S enhances the activity of a low-level tissue-specific promoter, these findings provide information for further improving the activity of such promoters, which might be useful for engineering new types of resistant genes against vascular infections.

11.
Front Microbiol ; 9: 426, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29568294

RESUMO

Mitogen-activated protein kinase (MAPK) pathways are ubiquitous and evolutionarily conserved signal transduction modules directing cellular respond to a diverse array of stimuli, in the eukaryotic organisms. In this study, PlMAPK10 was identified to encode a MAPK in Peronophythora litchii, the oomycete pathogen causing litchi downy blight disease. PlMAPK10, containing a specific and highly conserved dual phosphorylation lip sequence SEY (Serine-Glutamic-Tyrosine), represents a novel group of MAPKs as previously reported. Transcriptional profiling showed that PlMAPK10 expression was up-regulated in zoospore and cyst stages. To elucidate its function, the PlMAPK10 gene was silenced by stable transformation. PlMAPK10 silence did not impair oospore production, sporangium germination, zoospore encyst, or cyst germination but hindered hyphal growth, sporulation, pathogenicity, likely due to altering laccase activity. Over all, our results indicated that a MAPK encoded by PlMAPK10 gene in P. litchii is important for pathogenic development.

12.
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
13.
BMC Genomics ; 17: 354, 2016 05 16.
Artigo em Inglês | MEDLINE | ID: mdl-27185248

RESUMO

BACKGROUND: Sporisorium scitamineum causes the sugarcane smut disease, one of the most serious constraints to global sugarcane production. S. scitamineum possesses a sexual mating system composed of two mating-type loci, a and b locus. We previously identified and deleted the b locus in S. scitamineum, and found that the resultant SsΔMAT-1b mutant was defective in mating and pathogenicity. RESULTS: To further understand the function of b-mating locus, we carried out transcriptome analysis by comparing the transcripts of the mutant strain SsΔMAT-1b, from which the SsbE1 and SsbW1 homeodomain transcription factors have previously been deleted, with those from the wild-type MAT-1 strain. Also the transcripts from SsΔMAT-1b X MAT-2 were compared with those from wild-type MAT-1 X MAT-2 mating. A total of 209 genes were up-regulated (p < 0.05) in the SsΔMAT-1b mutant, compared to the wild-type MAT-1 strain, while 148 genes down-regulated (p < 0.05). In the mixture, 120 genes were up-regulated (p < 0.05) in SsΔMAT-1b X MAT-2, which failed to mate, compared to the wild-type MAT-1 X MAT-2 mating, and 271 genes down-regulated (p < 0.05). By comparing the up- and down-regulated genes in these two sets, it was found that 15 up-regulated and 37 down-regulated genes were common in non-mating haploid and mating mixture, which indeed could be genes regulated by b-locus. Furthermore, GO and KEGG enrichment analysis suggested that carbon metabolism pathway and stress response mediated by Hog1 MAPK signaling pathway were altered in the non-mating sets. CONCLUSIONS: Experimental validation results indicate that the bE/bW heterodimeric transcriptional factor, encoded by the b-locus, could regulate S. scitamineum sexual mating and/or filamentous growth via modulating glucose metabolism and Hog1-mediating oxidative response.


Assuntos
Basidiomycota/fisiologia , Meio Ambiente , Perfilação da Expressão Gênica , Reprodução Assexuada/genética , Transcriptoma , Metabolismo dos Carboidratos/genética , Biologia Computacional/métodos , Bases de Dados de Ácidos Nucleicos , Regulação Fúngica da Expressão Gênica , Genes Fúngicos Tipo Acasalamento , Sistema de Sinalização das MAP Quinases , Modelos Biológicos , Anotação de Sequência Molecular
14.
Fungal Genet Biol ; 86: 1-8, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26563415

RESUMO

Sporisorium scitamineum is the causal agent of sugarcane smut, which is one of the most serious constraints to global sugarcane production. S. scitamineum and Ustilago maydis are two closely related smut fungi, that are predicted to harbor similar sexual mating processes/system. To elucidate the molecular basis of sexual mating in S. scitamineum, we identified and deleted the ortholog of mating-specific U. maydis locus b, in S. scitamineum. The resultant b-deletion mutant was defective in mating and pathogenicity in S. scitamineum. Furthermore, a functional b locus heterodimer could trigger filamentous growth without mating in S. scitamineum, and functionally replace the b locus in U. maydis in terms of triggering aerial filament production and forming solopathogenic strains, which do not require sexual mating prior to pathogenicity on the host plants.


Assuntos
Genes Fúngicos Tipo Acasalamento , Saccharum/microbiologia , Ustilaginales/genética , Ustilaginales/patogenicidade , Sequência de Aminoácidos , Dados de Sequência Molecular , Doenças das Plantas/microbiologia , Genética Reversa , Ustilaginales/crescimento & desenvolvimento , Ustilago/genética , Ustilago/patogenicidade , Virulência
15.
PLoS Pathog ; 11(6): e1004972, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-26102503

RESUMO

Phototropic regulation of circadian clock is important for environmental adaptation, organismal growth and differentiation. Light plays a critical role in fungal development and virulence. However, it is unclear what governs the intracellular metabolic response to such dark-light rhythms in fungi. Here, we describe a novel circadian-regulated Twilight (TWL) function essential for phototropic induction of asexual development and pathogenesis in the rice-blast fungus Magnaporthe oryzae. The TWL transcript oscillates during circadian cycles and peaks at subjective twilight. GFP-Twl remains acetylated and cytosolic in the dark, whereas light-induced phosphorylation (by the carbon sensor Snf1 kinase) drives it into the nucleus. The mRNA level of the transcription/repair factor TFB5, was significantly down regulated in the twl∆ mutant. Overexpression of TFB5 significantly suppressed the conidiation defects in the twl∆ mutant. Furthermore, Tfb5-GFP translocates to the nucleus during the phototropic response and under redox stress, while it failed to do so in the twl∆ mutant. Thus, we provide mechanistic insight into Twl-based regulation of nutrient and redox homeostasis in response to light during pathogen adaptation to the host milieu in the rice blast pathosystem.


Assuntos
Regulação Fúngica da Expressão Gênica , Homeostase/fisiologia , Luz , Magnaporthe/metabolismo , Fototropismo/genética , Doenças das Plantas/microbiologia , Ritmo Circadiano , Alimentos , Proteínas Fúngicas/genética , Regulação Fúngica da Expressão Gênica/genética , Hifas/genética , Magnaporthe/efeitos dos fármacos , Magnaporthe/genética , Magnaporthe/crescimento & desenvolvimento , Oryza/microbiologia , Oxirredução , Fototropismo/fisiologia , Esporos Fúngicos/crescimento & desenvolvimento
16.
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 , Degradação Mitocondrial , 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
17.
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
18.
Autophagy ; 6(4): 455-61, 2010 May.
Artigo em Inglês | MEDLINE | ID: mdl-20383057

RESUMO

Nutrient limitation acts as a trigger for the synthesis of glycogen, which serves as a carbon and energy reserve during starvation. Recently, we reported that an autophagy-deficient mutant (atg8Δ) shows severe reduction in aerial hyphal growth and conidiation in the rice-blast fungus Magnaporthe oryzae, and proposed that autophagy plays an important role in facilitating glycogen homeostasis to ensure proper asexual differentiation in Magnaporthe. Here, we identify and characterize a vacuolar glucoamylase function (Sga1) that hydrolyses glycogen to meet the energy requirements during asexual development in Magnaporthe. Loss of SGA1 resulted in significant reduction in conidiation compared to the wild-type Magnaporthe strain. More importantly, an sga1Δ atg8Δ double deletion mutant showed further reduction in conidiation compared to the atg8Δ mutant in Magnaporthe. Forced localization of GFP-Sga1 to the cytoplasm (through removal of the predicted signal peptide) led to increased conidiation in wild type and the sga1Δ, but more interestingly, significantly restored conidiation in the atg8Δ mutant. Our results indicate that autophagy and Sga1 act cooperatively in vacuolar glycogen breakdown, which is essential for conidia formation but dispensable for pathogenicity in Magnaporthe.


Assuntos
Autofagia , Proteínas Fúngicas/metabolismo , Glucana 1,4-alfa-Glucosidase/metabolismo , Glicogênio/metabolismo , Magnaporthe/citologia , Magnaporthe/enzimologia , Reprodução Assexuada/fisiologia , Biocatálise , Hidrólise , Magnaporthe/fisiologia , Transporte Proteico , Esporos Fúngicos/crescimento & desenvolvimento , Frações Subcelulares/enzimologia , Vacúolos/enzimologia
19.
Autophagy ; 5(1): 33-43, 2009 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-19115483

RESUMO

Autophagy, a conserved pathway for bulk cellular degradation and recycling in eukaryotes, regulates proper turnover of organelles, membranes and certain proteins. Such regulated degradation is important for cell growth and development particularly during environmental stress conditions, which act as key inducers of autophagy. We found that autophagy and MoATG8 were significantly induced during asexual development in Magnaporthe oryzae. An RFP-tagged MoAtg8 showed specific localization and enrichment in aerial hyphae, conidiophores and conidia. We confirmed that loss of MoATG8 results in dramatically reduced ability to form conidia, the asexual spores that propagate rice-blast disease. Exogenous supply of glucose or sucrose significantly suppressed the conidiation defects in a MoATG8-deletion mutant. Comparative proteomics based identification and characterization of Gph1, a glycogen phosphorylase that catalyzes glycogen breakdown, indicated that autophagy-assisted glycogen homeostasis is likely important for proper aerial growth and conidiation in Magnaporthe. Loss of Gph1, or addition of G6P significantly restored conidiation in the Moatg8Delta mutant. Overproduction of Gph1 led to reduced conidiation in wild-type Magnaporthe strain. We propose that glycogen autophagy actively responds to and regulates carbon utilization required for cell growth and differentiation during asexual development in Magnaporthe.


Assuntos
Autofagia , Glicogênio/metabolismo , Magnaporthe/citologia , Magnaporthe/metabolismo , Reprodução Assexuada/fisiologia , Sequência de Aminoácidos , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Homeostase , Hordeum/microbiologia , Hifas/crescimento & desenvolvimento , Hifas/metabolismo , Hifas/ultraestrutura , Magnaporthe/crescimento & desenvolvimento , Magnaporthe/ultraestrutura , Dados de Sequência Molecular , Mutação/genética , Fagossomos/metabolismo , Fagossomos/microbiologia , Processamento de Proteína Pós-Traducional , Transporte Proteico , Proteínas Recombinantes de Fusão/metabolismo , Esporos Fúngicos/citologia , Esporos Fúngicos/metabolismo , Esporos Fúngicos/ultraestrutura , Frações Subcelulares/metabolismo
20.
Methods Enzymol ; 451: 295-310, 2008.
Artigo em Inglês | MEDLINE | ID: mdl-19185728

RESUMO

Autophagy is a bulk degradative process responsible for the turnover of membranes, organelles, and proteins in eukaryotic cells. Genetic and molecular regulation of autophagy has been independently elucidated in budding yeast and mammalian cells. In filamentous fungi, autophagy is required for several important physiological functions, such as asexual and sexual differentiation, pathogenic development, starvation stress and programmed cell death during heteroincompatibility. Here, we detail biochemical and microscopy methods useful for measuring the rate of induction of autophagy in filamentous fungi, and we summarize the methods that have been routinely used for monitoring macroautophagy in both yeast and filamentous fungi. The role of autophagy in carbohydrate catabolism and cell survival is discussed along with the specific functions of macroautophagy in fungal development and pathogenesis.


Assuntos
Autofagia/fisiologia , Bioensaio/métodos , Fungos/metabolismo , Animais , Cadaverina/análogos & derivados , Cadaverina/metabolismo , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Corantes Fluorescentes/metabolismo , Fungos/citologia , Fungos/genética , Deleção de Genes , Glicogênio/metabolismo , Microscopia/métodos , Fagossomos/metabolismo , Fagossomos/ultraestrutura , Proteômica/métodos
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