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1.
Methods Mol Biol ; 2085: 189-198, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31734926

RESUMO

Liquid chromatography-mass spectrometry (LC-MS) is one of the most important analytical chemistry techniques for the detection and characterization of biologically active compounds of low abundance-for example, hormones. Gas chromatography (GC) coupled with mass spectrometry has been a method of choice to detect jasmonic acid, the well-known defense hormone in plants. Recently, we identified structural and functional analogs of phytohormone jasmonic acid (JA) and its derivatives, in the rice-blast fungus Magnaporthe oryzae. Here, we describe protocols of LC-MS/MS-based identification and quantification of fungal jasmonates, especially during pathogenic development in the rice blast fungus.

2.
mBio ; 10(4)2019 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-31431550

RESUMO

The blast fungus initiates infection using a heavily melanized, dome-shaped infection structure known as the appressorium, which forcibly ruptures the cuticle to enter the rice leaf tissue. How this process takes place remains not fully understood. Here, we used untargeted metabolomics analyses to profile the metabolome of developing appressoria and identified significant changes in six key metabolic pathways, including early sphingolipid biosynthesis. Analyses employing small molecule inhibitors, gene disruption, or genetic and chemical complementation demonstrated that ceramide compounds of the sphingolipid biosynthesis pathway are essential for normal appressorial development controlled by mitosis. In addition, ceramide was found to act upstream from the protein kinase C-mediated cell wall integrity pathway during appressorium repolarization and pathogenicity in rice blast. Further discovery of the sphingolipid biosynthesis pathway revealed that glucosylceramide (GlcCer) synthesized by ceramide is the key substance affecting the pathogenicity of Magnaporthe oryzae Our results provide new insights into the chemical moieties involved in the infection-related signaling networks, thereby revealing a potential target for the development of novel control agents against the major disease of rice and other cereals.IMPORTANCE Our untargeted analysis of metabolomics throughout the course of pathogenic development gave us an unprecedented high-resolution view of major shifts in metabolism that occur in the topmost fungal pathogen that infects rice, wheat, barley, and millet. Guided by these metabolic insights, we demonstrated their practical application by using two different small-molecule inhibitors of sphingolipid biosynthesis enzymes to successfully block the pathogenicity of M. oryzae Our study thus defines the sphingolipid biosynthesis pathway as a key step and potential target that can be exploited for the development of antifungal agents. Furthermore, future investigations that exploit such important metabolic intermediates will further deepen our basic understanding of the molecular mechanisms underlying the establishment of fungal blast disease in important cereal crops.

3.
mBio ; 10(4)2019 07 30.
Artigo em Inglês | MEDLINE | ID: mdl-31363034

RESUMO

Precise kinetochore-microtubule interactions ensure faithful chromosome segregation in eukaryotes. Centromeres, identified as scaffolding sites for kinetochore assembly, are among the most rapidly evolving chromosomal loci in terms of the DNA sequence and length and organization of intrinsic elements. Neither the centromere structure nor the kinetochore dynamics is well studied in plant-pathogenic fungi. Here, we sought to understand the process of chromosome segregation in the rice blast fungus Magnaporthe oryzae High-resolution imaging of green fluorescent protein (GFP)-tagged inner kinetochore proteins CenpA and CenpC revealed unusual albeit transient declustering of centromeres just before anaphase separation of chromosomes in M. oryzae Strikingly, the declustered centromeres positioned randomly at the spindle midzone without an apparent metaphase plate per se Using CenpA chromatin immunoprecipitation followed by deep sequencing, all seven centromeres in M. oryzae were found to be regional, spanning 57-kb to 109-kb transcriptionally poor regions. Highly AT-rich and heavily methylated DNA sequences were the only common defining features of all the centromeres in rice blast. Lack of centromere-specific DNA sequence motifs or repetitive elements suggests an epigenetic specification of centromere function in M. oryzae PacBio genome assemblies and synteny analyses facilitated comparison of the centromeric/pericentromeric regions in distinct isolates of rice blast and wheat blast and in Magnaporthiopsis poae Overall, this study revealed unusual centromere dynamics and precisely identified the centromere loci in the top model fungal pathogens that belong to Magnaporthales and cause severe losses in the global production of food crops and turf grasses.IMPORTANCE Magnaporthe oryzae is an important fungal pathogen that causes a loss of 10% to 30% of the annual rice crop due to the devastating blast disease. In most organisms, kinetochores are clustered together or arranged at the metaphase plate to facilitate synchronized anaphase separation of sister chromatids in mitosis. In this study, we showed that the initially clustered kinetochores separate and position randomly prior to anaphase in M. oryzae Centromeres in M. oryzae occupy large genomic regions and form on AT-rich DNA without any common sequence motifs. Overall, this study identified atypical kinetochore dynamics and mapped functional centromeres in M. oryzae to define the roles of centromeric and pericentric boundaries in kinetochore assembly on epigenetically specified centromere loci. This study should pave the way for further understanding of the contribution of heterochromatin in genome stability and virulence of the blast fungus and its related species of high economic importance.

4.
BMC Plant Biol ; 19(1): 326, 2019 Jul 19.
Artigo em Inglês | MEDLINE | ID: mdl-31324141

RESUMO

BACKGROUND: Autophagy is a conserved, highly-regulated catabolic process that plays important roles in growth, development and innate immunity in plants. In this study, we compared the rate of autophagy induction in Nicotiana benthamiana plants infected with Tobacco mosaic virus or the TMV 24A + UPD mutant variant, which replicates at a faster rate and induces more severe symptoms. Using a BirA* tag and proximity-dependent biotin identification (BioID) analysis, we identified host proteins that interact with the core autophagy protein, ATG8 in TMV 24A + UPD infected plants. By combining the use of a fast replicating TMV mutant and an in vivo protein-protein screening technique, we were able to gain functional insight into the role of autophagy in a compatible virus-host interaction. RESULTS: Our study revealed an increased autophagic flux induced by TMV 24A + UPD, as compared to TMV in N. benthamiana. Analysis of the functional proteome associated with ATG8 revealed a total of 67 proteins, 16 of which are known to interact with ATG8 or its orthologs in mammalian and yeast systems. The interacting proteins were categorized into four functional groups: immune system process, response to ROS, sulphur amino acid metabolism and calcium signalling. Due to the presence of an ubiquitin-associated (UBA) domain, which is demonstrated to interact with ATG8, the Huntingtin-interacting protein K-like (HYPK) was selected for validation of the physical interaction and function. We used yeast two hybrid (Y2H), bimolecular fluorescence complementation (BiFC) and subcellular localization to validate the ATG8-HYPK interaction. Subsequent down-regulation of ATG8 by virus-induced gene silencing (VIGS) showed enhanced TMV symptoms, suggesting a protective role for autophagy during TMV 24A + UPD infection. CONCLUSION: This study presents the use of BioID as a suitable method for screening ATG8 interacting proteins in planta. We have identified many putative binding partners of ATG8 during TMV 24A + UPD infection in N. benthamiana plants. In addition, we have verified that NbHYPK is an interacting partner of ATG8. We infer that autophagy plays a protective role in TMV 24A + UPD infected plants.


Assuntos
Família da Proteína 8 Relacionada à Autofagia/metabolismo , Doenças das Plantas/virologia , Proteínas de Plantas/metabolismo , Tabaco/virologia , Autofagossomos/metabolismo , Autofagia/genética , Autofagia/fisiologia , Biotinilação , Imunidade Vegetal , Tabaco/metabolismo , Vírus do Mosaico do Tabaco
5.
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.

6.
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.

7.
Curr Opin Microbiol ; 46: 1-6, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-29452844

RESUMO

Phytohormone networks are crucial for maintaining the delicate balance between growth and biotic stress responses in plants. Jasmonic acid, salicylic acid, ethylene, and the associated signaling crosstalk are important for pathogen defense; whereas gibberellin and cytokinin function in growth and development in plants. Plant pathogenic fungi have evolved remarkable strategies to manipulate and/or hijack such phytohormone signaling cascades for their own benefit, thus leading to susceptibility and disease in host plants. Interestingly, these hormones are also targeted by fungal endosymbionts and mutualists during beneficial interactions with plants. We highlight current advances in our understanding of the role of fungal effectors in such antagonistic manipulation of phytohormones during pathogenic as well as symbiotic association with plant hosts. In addition to the aforementioned effector-based control, certain phytohormone mimics have recently emerged as a powerful molecular language in fungal manipulation of defense responses and innate immunity in plants.


Assuntos
Proteínas Fúngicas/metabolismo , Fungos/metabolismo , Doenças das Plantas/microbiologia , Reguladores de Crescimento de Planta/metabolismo , Plantas/microbiologia , Proteínas Fúngicas/genética , Fungos/genética , Interações Hospedeiro-Patógeno , Plantas/metabolismo , Transdução de Sinais
8.
Front Microbiol ; 8: 2289, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29209297

RESUMO

The cAMP-Protein Kinase A signaling, anchored on CpkA, is necessary for appressorium development and host penetration, but indispensable for infectious growth in Magnaporthe oryzae. In this study, we identified and characterized the gene encoding the second catalytic subunit, CPK2, whose expression was found to be lower compared to CPKA at various stages of pathogenic growth in M. oryzae. Deletion of CPK2 caused no alterations in vegetative growth, conidiation, appressorium formation, or pathogenicity. Surprisingly, the cpkAΔcpk2Δ double deletion strain displayed significant reduction in growth rate and conidiation compared to the single deletion mutants. Interestingly, loss of CPKA and CPK2 resulted in morphogenetic defects in germ tubes (with curled/wavy and serpentine growth pattern) on hydrophobic surfaces, and a complete failure to produce appressoria therein, thus suggesting an important role for CPK2-mediated cAMP-PKA in surface sensing and response pathway. CPKA promoter-driven expression of CPK2 partially suppressed the defects in host penetration and pathogenicity in the cpkAΔ. Such ectopic CPK2 expressing strain successfully penetrated the rice leaves, but was unable to produce proper secondary invasive hyphae, thus underscoring the importance of CpkA in growth and differentiation in planta. The Cpk2-GFP localized to the nuclei and cytoplasmic vesicles in conidia and germ tubes. The Cpk2-GFP colocalized with CpkA-mCherry on vesicles in the cytosol, but such overlap was not evident in the nuclei. Our studies indicate that CpkA and Cpk2 share overlapping functions, but also play distinct roles during pathogenesis-associated signaling and morphogenesis in the rice blast fungus.

10.
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
11.
Mol Microbiol ; 105(3): 484-504, 2017 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-28544028

RESUMO

The cAMP-dependent PKA signalling plays a central role in growth, asexual development and pathogenesis in fungal pathogens. Here, we functionally characterised RPKA, the regulatory subunit of cAMP/PKA and studied the dynamics and organisation of the PKA subunits in the rice blast pathogen Magnaporthe oryzae. The RPKA subunit was essential for proper vegetative growth, asexual sporulation and surface hydrophobicity in M. oryzae. A spontaneous suppressor mutation, SMR19, that restored growth and conidiation in the RPKA deletion mutant was isolated and characterised. SMR19 enhanced conidiation and appressorium formation but failed to suppress the pathogenesis defects in rpkAΔ. The PKA activity was undetectable in the mycelial extracts of SMR19, which showed a single mutation (val242leu) in the highly conserved active site of the catalytic subunit (CPKA) of cAMP/PKA. The two subunits of cAMP/PKA showed different subcellular localisation patterns with RpkA being predominantly nucleocytoplasmic in conidia, while CpkA was largely cytosolic and/or vesicular. The CpkA anchored RpkA in cytoplasmic vesicles, and localisation of PKA in the cytoplasm was governed by CpkA in a cAMP-dependant or independent manner. We show that there exists a tight regulation of PKA subunits at the level of transcription, and the cAMP signalling is differentially compartmentalised in a stage-specific manner in rice blast.


Assuntos
Subunidades Catalíticas da Proteína Quinase Dependente de AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Magnaporthe/genética , Sequência de Aminoácidos , AMP Cíclico/metabolismo , Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica/genética , Magnaporthe/metabolismo , Mutação , Oryza/microbiologia , Doenças das Plantas/microbiologia , Transdução de Sinais , Esporos Fúngicos/genética , Supressão Genética/genética , Virulência/genética
12.
New Phytol ; 214(1): 330-342, 2017 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-27898176

RESUMO

The interaction of Magnaporthe oryzae, the rice blast fungus, and rice begins when M. oryzae establishes contact with the host plant surface. On perception of appropriate surface signals, M. oryzae forms appressoria and initiates host invasion. Pth11, an important G-protein-coupled receptor necessary for appressorium formation in M. oryzae, contains seven transmembrane regions and a CFEM (common in several fungal extracellular membrane proteins) domain with the characteristic eight cysteine residues. We focused on gaining further insight into the role of the CFEM domain in the putative surface sensing/response function of Pth11. Increased/constitutive expression of CFEM resulted in precocious, albeit defective, appressoria formation in wild-type M. oryzae. The Pth11C63A/C65A mutant, probably with disrupted disulfide bonds in the CFEM, showed delayed appressorium formation and reduced virulence. Furthermore, the accumulation of reactive oxygen species (ROS) was found to be altered in the pth11Δ strain. Strikingly, antioxidant treatment induced appressorium formation in pth11Δ. The Gα subunit MagB and the mitogen-activated protein (MAP) kinase Pmk1 were required for the formation of antioxidant-induced appressoria. We conclude that the CFEM domain of Pth11 is required for proper development of the appressoria, appressoria-like structures and pathogenicity. Highly regulated ROS homeostasis is important for Pth11-mediated appressorium formation in M. oryzae.


Assuntos
Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Magnaporthe/metabolismo , Oryza/microbiologia , Doenças das Plantas/microbiologia , Motivos de Aminoácidos , Sequência de Aminoácidos , Antioxidantes/farmacologia , Sequência Conservada , Cisteína , Subunidades alfa de Proteínas de Ligação ao GTP/metabolismo , Interações Hidrofóbicas e Hidrofílicas , Magnaporthe/patogenicidade , Oxirredução , Domínios Proteicos , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacos , Esporos Fúngicos/efeitos dos fármacos , Esporos Fúngicos/crescimento & desenvolvimento , Relação Estrutura-Atividade , Frações Subcelulares/metabolismo
13.
PLoS Genet ; 12(10): e1006383, 2016 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-27749909

RESUMO

Cytokinesis in many organisms requires a plasma membrane anchored actomyosin ring, whose contraction facilitates cell division. In yeast and fungi, actomyosin ring constriction is also coordinated with division septum assembly. How the actomyosin ring interacts with the plasma membrane and the plasma membrane-localized septum synthesizing machinery remains poorly understood. In Schizosaccharomyces pombe, an attractive model organism to study cytokinesis, the ß-1,3-glucan synthase Cps1p / Bgs1p, an integral membrane protein, localizes to the plasma membrane overlying the actomyosin ring and is required for primary septum synthesis. Through a high-dosage suppressor screen we identified an essential gene, sbg1+ (suppressor of beta glucan synthase 1), which suppressed the colony formation defect of Bgs1-defective cps1-191 mutant at higher temperatures. Sbg1p, an integral membrane protein, localizes to the cell ends and to the division site. Sbg1p and Bgs1p physically interact and are dependent on each other to localize to the division site. Loss of Sbg1p results in an unstable actomyosin ring that unravels and slides, leading to an inability to deposit a single contiguous division septum and an important reduction of the ß-1,3-glucan proportion in the cell wall, coincident with that observed in the cps1-191 mutant. Sbg1p shows genetic and / or physical interaction with Rga7p, Imp2p, Cdc15p, and Pxl1p, proteins known to be required for actomyosin ring integrity and efficient septum synthesis. This study establishes Sbg1p as a key member of a group of proteins that link the plasma membrane, the actomyosin ring, and the division septum assembly machinery in fission yeast.


Assuntos
Actomiosina/genética , Citocinese/genética , Glucosiltransferases/genética , Proteínas de Membrana/genética , Proteínas de Schizosaccharomyces pombe/genética , Schizosaccharomyces/genética , Citoesqueleto de Actina/genética , Actomiosina/metabolismo , Proteínas de Ciclo Celular/genética , Divisão Celular/genética , Membrana Celular , Parede Celular/genética , Parede Celular/metabolismo , Glucosiltransferases/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , beta-Glucanas/metabolismo
14.
Semin Cell Dev Biol ; 57: 84-92, 2016 09.
Artigo em Inglês | MEDLINE | ID: mdl-27133541

RESUMO

Pathogenic fungi have evolved highly varied and remarkable strategies to invade and infect their plant hosts. Typically, such fungal pathogens utilize highly specialized infection structures, morphologies or cell types produced from conidia or ascospores on the cognate host surfaces to gain entry therein. Such diverse infection strategies require intricate coordination in cell signaling and differentiation in phytopathogenic fungi. Here, we present an overview of our current understanding of cell signaling and infection-associated development that primes host penetration in the top ten plant pathogenic fungi, which utilize specific receptors to sense and respond to different surface cues, such as topographic features, hydrophobicity, hardness, plant lipids, phytohormones, and/or secreted enzymes. Subsequently, diverse signaling components such as G proteins, cyclic AMP/Protein Kinase A and MAP kinases are activated to enable the differentiation of infection structures. Recent studies have also provided fascinating insights into the spatio-temporal dynamics and specialized sequestration and trafficking of signaling moieties required for proper development of infection structures in phytopathogenic fungi. Molecular insight in such infection-related morphogenesis and cell signaling holds promise for identifying novel strategies for intervention of fungal diseases in plants.


Assuntos
Fungos/metabolismo , Plantas/microbiologia , Transdução de Sinais , Modelos Biológicos , Doenças das Plantas/microbiologia , Receptores de Superfície Celular/metabolismo
15.
PLoS Genet ; 11(12): e1005704, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26658729

RESUMO

The retromer mediates protein trafficking through recycling cargo from endosomes to the trans-Golgi network in eukaryotes. However, the role of such trafficking events during pathogen-host interaction remains unclear. Here, we report that the cargo-recognition complex (MoVps35, MoVps26 and MoVps29) of the retromer is essential for appressorium-mediated host penetration by Magnaporthe oryzae, the causal pathogen of the blast disease in rice. Loss of retromer function blocked glycogen distribution and turnover of lipid bodies, delayed nuclear degeneration and reduced turgor during appressorial development. Cytological observation revealed dynamic MoVps35-GFP foci co-localized with autophagy-related protein RFP-MoAtg8 at the periphery of autolysosomes. Furthermore, RFP-MoAtg8 interacted with MoVps35-GFP in vivo, RFP-MoAtg8 was mislocalized to the vacuole and failed to recycle from the autolysosome in the absence of the retromer function, leading to impaired biogenesis of autophagosomes. We therefore conclude that retromer is essential for autophagy-dependent plant infection by the rice blast fungus.


Assuntos
Magnaporthe/genética , Oryza/genética , Doenças das Plantas/genética , Transporte Proteico/genética , Sequência de Aminoácidos , Autofagia/genética , Glicogênio/metabolismo , Interações Hospedeiro-Patógeno/genética , Gotículas Lipídicas/metabolismo , Magnaporthe/patogenicidade , Oryza/microbiologia , Doenças das Plantas/microbiologia , Folhas de Planta/genética , Folhas de Planta/metabolismo , Folhas de Planta/microbiologia , Vacúolos/genética , Vacúolos/microbiologia , Rede trans-Golgi/genética
16.
Nat Chem Biol ; 11(9): 733-40, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-26258762

RESUMO

Distinct modifications fine-tune the activity of jasmonic acid (JA) in regulating plant growth and immunity. Hydroxylated JA (12OH-JA) promotes flower and tuber development but prevents induction of JA signaling, plant defense or both. However, biosynthesis of 12OH-JA has remained elusive. We report here an antibiotic biosynthesis monooxygenase (Abm) that converts endogenous free JA into 12OH-JA in the model rice blast fungus Magnaporthe oryzae. Such fungal 12OH-JA is secreted during host penetration and helps evade the defense response. Loss of Abm in M. oryzae led to accumulation of methyl JA (MeJA), which induces host defense and blocks invasive growth. Exogenously added 12OH-JA markedly attenuated abmΔ-induced immunity in rice. Notably, Abm itself is secreted after invasion and most likely converts plant JA into 12OH-JA to facilitate host colonization. This study sheds light on the chemical arms race during plant-pathogen interaction, reveals Abm as an antifungal target and outlines a synthetic strategy for transformation of a versatile small-molecule phytohormone.


Assuntos
Ciclopentanos/metabolismo , Proteínas Fúngicas/imunologia , Regulação Fúngica da Expressão Gênica , Magnaporthe/genética , Oxigenases de Função Mista/imunologia , Oryza/imunologia , Oxilipinas/metabolismo , Reguladores de Crescimento de Planta/metabolismo , Ciclopentanos/química , Ciclopentanos/imunologia , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Interações Hospedeiro-Patógeno/imunologia , Hidroxilação , Magnaporthe/imunologia , Magnaporthe/patogenicidade , Metilação , Oxigenases de Função Mista/química , Oxigenases de Função Mista/genética , Modelos Moleculares , Oryza/microbiologia , Oxilipinas/química , Oxilipinas/imunologia , Doenças das Plantas/imunologia , Doenças das Plantas/microbiologia , Reguladores de Crescimento de Planta/química , Reguladores de Crescimento de Planta/imunologia , Imunidade Vegetal , Folhas de Planta/imunologia , Folhas de Planta/microbiologia , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Transdução de Sinais
17.
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
18.
New Phytol ; 206(4): 1463-75, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-25659573

RESUMO

We identified the Magnaporthe oryzae avirulence effector AvrPi9 cognate to rice blast resistance gene Pi9 by comparative genomics of requisite strains derived from a sequential planting method. AvrPi9 encodes a small secreted protein that appears to localize in the biotrophic interfacial complex and is translocated to the host cell during rice infection. AvrPi9 forms a tandem gene array with its paralogue proximal to centromeric region of chromosome 7. AvrPi9 is expressed highly at early stages during initiation of blast disease. Virulent isolate strains contain Mg-SINE within the AvrPi9 coding sequence. Loss of AvrPi9 did not lead to any discernible defects during growth or pathogenesis in M. oryzae. This study reiterates the role of diverse transposable elements as off-switch agents in acquisition of gain-of-virulence in the rice blast fungus. The prevalence of AvrPi9 correlates well with the avirulence pathotype in diverse blast isolates from the Philippines and China, thus supporting the broad-spectrum resistance conferred by Pi9 in different rice growing areas. Our results revealed that Pi9 and Piz-t at the Pi2/9 locus activate race specific resistance by recognizing sequence-unrelated AvrPi9 and AvrPiz-t genes, respectively.


Assuntos
Resistência à Doença/genética , Proteínas Fúngicas/metabolismo , Genes de Plantas , Genômica/métodos , Magnaporthe/patogenicidade , Oryza/imunologia , Oryza/microbiologia , Doenças das Plantas/microbiologia , Alelos , Cromossomos de Plantas/genética , Regulação Fúngica da Expressão Gênica , Teste de Complementação Genética , Genoma Fúngico , Interações Hospedeiro-Patógeno/genética , Magnaporthe/genética , Dados de Sequência Molecular , Mutagênese Insercional/genética , Oryza/citologia , Oryza/crescimento & desenvolvimento , Doenças das Plantas/imunologia , Análise de Sequência de DNA , Elementos Nucleotídeos Curtos e Dispersos/genética , Virulência/genética
19.
Fungal Genet Biol ; 68: 71-6, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-24769367

RESUMO

A sulfonylurea-resistant allele of the ILV2 gene encoding an acetolactate synthase from the rice-blast fungus Magnaporthe oryzae has been extensively used in fungal transformation as a dominant selectable marker that confers resistance to chlorimuron ethyl. We devised a novel strategy for site-specific integration of foreign DNA via sulfonylurea resistance reconstitution (SRR) by replacing the native ILV2 with the sulfonylurea-resistant ILV2(SUR) variant. In contrast to random ectopic integration, SRR-based targeted incorporation at a defined locus eliminates position/orientation effects, unnecessary mutations and/or variation in gene expression. Independent transformants derived from the same SRR construct showed consistent and reproducible fluorescent signal in M. oryzae. Furthermore, the high frequency (>95%) of ILV2-specific targeted integration via SRR circumvents the need for a deficiency in non-homologous end joining (NHEJ) pathway in the recipient strain. Unlike the split-marker technique, which is particularly suitable for targeted gene replacement, the SRR strategy should prove useful for promoter analyses, gene tagging and/or complementation analyses in filamentous fungi.


Assuntos
Acetolactato Sintase/genética , Magnaporthe/genética , Compostos de Sulfonilureia/farmacologia , Genes Fúngicos , Vetores Genéticos , Magnaporthe/efeitos dos fármacos , Transformação Genética
20.
PLoS One ; 8(11): e79128, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24302988

RESUMO

Pexophagy, the degradation of peroxisomes via selective autophagy, depends on Atg20/Snx42 function in Saccharomyces cerevisiae. Besides its role in selective autophagy, Atg20/Snx42 is also involved in an autophagy-independent endosomal retrieval trafficking, in cooperation with two other sorting nexins, Snx41 and Snx4. Recently, we reported that the sorting nexin MoSnx41, which showed high sequence similarity to yeast Snx41 and Snx42/Atg20 proteins, regulates the gamma-glutamyl cycle and GSH production and is essential for conidiation and pathogenicity in Magnaporthe oryzae. Pexophagy was also found to be defective in Mosnx41Δ mutant. These findings indicate that MoSnx41 likely serves combined functions of Snx42/Atg20 and Snx41 in M. oryzae.. In this study, we performed complementation analyses and demonstrate that MoSnx41 alone serves the dual function of protein sorting (ScSnx41) and pexophagy (ScSnx42/Atg20). To study the potential biological function of pexophagy in fungal pathogenic life cycle, we created deletion mutants of potential pexophagy-specific genes, and characterized them in terms of pexophagy, conidiation and pathogenesis. We identified Pex14 as an essential protein for pexophagy in M. oryzae. Overall, our results show that pexophagy per se is not essential for asexual development or virulence in M. oryzae.


Assuntos
Proteínas Fúngicas/metabolismo , Magnaporthe/fisiologia , Peroxissomos/metabolismo , Nexinas de Classificação/metabolismo , Autofagia/fisiologia , Proteínas Fúngicas/genética , Magnaporthe/patogenicidade , Mutação , Nexinas de Classificação/genética
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