TmpL, a Transmembrane Protein Required for Intracellular Redox Homeostasis and Virulence in a Plant and an Animal Fungal Pathogen
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{"title"=>"TmpL, a transmembrane protein required for intracellular redox homeostasis and virulence in a plant and an animal fungal pathogen", "type"=>"journal", "authors"=>[{"first_name"=>"Kwang Hyung", "last_name"=>"Kim", "scopus_author_id"=>"10339111800"}, {"first_name"=>"Sven D.", "last_name"=>"Willger", "scopus_author_id"=>"25724786800"}, {"first_name"=>"Sang Wook", "last_name"=>"Park", "scopus_author_id"=>"57157561800"}, {"first_name"=>"Srisombat", "last_name"=>"Puttikamonkul", "scopus_author_id"=>"25724607900"}, {"first_name"=>"Nora", "last_name"=>"Grahl", "scopus_author_id"=>"25724317300"}, {"first_name"=>"Yangrae", "last_name"=>"Cho", "scopus_author_id"=>"7404469218"}, {"first_name"=>"Biswarup", "last_name"=>"Mukhopadhyay", "scopus_author_id"=>"7101857372"}, {"first_name"=>"Robert A.", "last_name"=>"Cramer", "scopus_author_id"=>"7102501409"}, {"first_name"=>"Christopher B.", "last_name"=>"Lawrence", "scopus_author_id"=>"7402265726"}], "year"=>2009, "source"=>"PLoS Pathogens", "identifiers"=>{"sgr"=>"73549085891", "pmid"=>"19893627", "isbn"=>"1553-7374 (Electronic)\\r1553-7366 (Linking)", "pui"=>"358060463", "issn"=>"15537366", "scopus"=>"2-s2.0-73549085891", "doi"=>"10.1371/journal.ppat.1000653"}, "id"=>"c91b6eb8-8ef3-3493-80c3-5fdd87494285", "abstract"=>"The regulation of intracellular levels of reactive oxygen species (ROS) is critical for developmental differentiation and virulence of many pathogenic fungi. In this report we demonstrate that a novel transmembrane protein, TmpL, is necessary for regulation of intracellular ROS levels and tolerance to external ROS, and is required for infection of plants by the necrotroph Alternaria brassicicola and for infection of mammals by the human pathogen Aspergillus fumigatus. In both fungi, tmpL encodes a predicted hybrid membrane protein containing an AMP-binding domain, six putative transmembrane domains, and an experimentally-validated FAD/NAD(P)-binding domain. Localization and gene expression analyses in A. brassicicola indicated that TmpL is associated with the Woronin body, a specialized peroxisome, and strongly expressed during conidiation and initial invasive growth in planta. A. brassicicola and A. fumigatus DeltatmpL strains exhibited abnormal conidiogenesis, accelerated aging, enhanced oxidative burst during conidiation, and hypersensitivity to oxidative stress when compared to wild-type or reconstituted strains. Moreover, A. brassicicola DeltatmpL strains, although capable of initial penetration, exhibited dramatically reduced invasive growth on Brassicas and Arabidopsis. Similarly, an A. fumigatus DeltatmpL mutant was dramatically less virulent than the wild-type and reconstituted strains in a murine model of invasive aspergillosis. Constitutive expression of the A. brassicicola yap1 ortholog in an A. brassicicola DeltatmpL strain resulted in high expression levels of genes associated with oxidative stress tolerance. Overexpression of yap1 in the DeltatmpL background complemented the majority of observed developmental phenotypic changes and partially restored virulence on plants. Yap1-GFP fusion strains utilizing the native yap1 promoter exhibited constitutive nuclear localization in the A. brassicicola DeltatmpL background. Collectively, we have discovered a novel protein involved in the virulence of both plant and animal fungal pathogens. Our results strongly suggest that dysregulation of oxidative stress homeostasis in the absence of TmpL is the underpinning cause of the developmental and virulence defects observed in these studies.", "link"=>"http://www.mendeley.com/research/tmpl-transmembrane-protein-required-intracellular-redox-homeostasis-virulence-plant-animal-fungal-pa-8", "reader_count"=>5, "reader_count_by_academic_status"=>{"Student > Doctoral Student"=>2, "Student > Ph. D. Student"=>1, "Professor"=>2}, "reader_count_by_user_role"=>{"Student > Doctoral Student"=>2, "Student > Ph. D. Student"=>1, "Professor"=>2}, "reader_count_by_subject_area"=>{"Agricultural and Biological Sciences"=>4, "Biochemistry, Genetics and Molecular Biology"=>1}, "reader_count_by_subdiscipline"=>{"Agricultural and Biological Sciences"=>{"Agricultural and Biological Sciences"=>4}, "Biochemistry, Genetics and Molecular Biology"=>{"Biochemistry, Genetics and Molecular Biology"=>1}}, "group_count"=>0}

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  • {"files"=>["https://ndownloader.figshare.com/files/877860"], "description"=>"<p>(<b>A</b>) Accumulation of excess superoxide in the conidia of the <i>A. brassicicola ΔtmpL</i> mutants. 7-day-old conidia of <i>A. brassicicola</i> wild-type and <i>ΔtmpL</i> mutant grown on a nutrient-rich medium were strained with nitroblue tetrazolium (NBT) to detect superoxide. Each micrograph shows the picture of conidium before (−) and after (+) NBT staining. (<b>B</b>) Accumulation of excess superoxide in the mature appressoria and emerging infection hyphae of the <i>A. brassicicola ΔtmpL</i> mutants. Conidia of <i>A. brassicicola</i> wild-type and <i>ΔtmpL</i> mutant were inoculated on onion epidermis, incubated at room temperature for 8 and 12 hr, and stained with NBT. Insets show another example of appressorium stained with NBT from each strain. Bars = 10 µm. Abbreviations: a, appressorium; ih, infection hypha. (<b>C</b>) Accumulation of excess ROS in the conidia of the <i>A. brassicicola ΔtmpL</i> mutants. Conidia released from 7-day-old colonies were stained with H<sub>2</sub>DCFDA and viewed by epifluorescence microscopy. Insets show a magnified view of conidia stained with H<sub>2</sub>DCFDA from each strain. Bars = 50 µm. (<b>D</b>) Accumulation of excess H<sub>2</sub>O<sub>2</sub> in mature appressoria of the <i>A. brassicicola ΔtmpL</i> mutants. Conidia of the <i>A. brassicicola</i> wild-type and <i>ΔtmpL</i> mutant were inoculated on green cabbage cotyledons and incubated at room temperature for 12 hr before being stained with 3,3′-diaminobenzidine tetrahydrochloride (DAB). Insets are the magnified view of each appressorium. Right panels are the pictures of two more appressoria (arrows) stained with DAB from each strain, showing a typical range of staining intensity. Bars = 10 µm. Abbreviations: a, appressorium; ih, infection hypha. (<b>E</b>) Accumulation of excess ROS in the conidia of the <i>A. fumigatus ΔtmpL</i> mutants. Conidia and conidiophores of the <i>A. fumigatus</i> wild-type (CEA10) and <i>ΔtmpL</i> mutant were released from 3-day-old colonies and subsequently stained with H<sub>2</sub>DCFDA for 30 min and 1 hr, respectively, and viewed by epifluorescence microscopy. Bars = 20 µm.</p>", "links"=>[], "tags"=>["ros", "conidiation"], "article_id"=>548314, "categories"=>["Virology", "Cell Biology", "Infectious Diseases", "Biochemistry", "Genetics"], "users"=>["Kwang-Hyung Kim", "Sven D. Willger", "Sang-Wook Park", "Srisombat Puttikamonkul", "Nora Grahl", "Yangrae Cho", "Biswarup Mukhopadhyay", "Robert A. Cramer Jr.", "Christopher B. Lawrence"], "doi"=>"https://dx.doi.org/10.1371/journal.ppat.1000653.g007", "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Excess_ROS_production_during_conidiation_and_infection_in_916_tmpL_mutants_/548314", "title"=>"Excess ROS production during conidiation and infection in <i>ΔtmpL</i> mutants.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2009-11-06 02:18:34"}
  • {"files"=>["https://ndownloader.figshare.com/files/877098"], "description"=>"<p>(<b>A</b>) Domain organization of TmpL. The predicted protein encoded by the <i>tmpL</i> gene is comprised of 1025 amino acid residues. This protein contains an AMP-binding domain, six putative transmembrane domains, and a FAD/NAD(P)-binding domain. The bottom picture shows predicted topological map of the TmpL protein. Regions of TmpL proposed to be hydrophobic membrane-spanning domains or hydrophilic domains facing the cytosol or subcompartmental matrix were identified using the TMHMM (<a href=\"http://www.cbs.dtu.dk/services/TMHMM-2.0/\" target=\"_blank\">http://www.cbs.dtu.dk/services/TMHMM-2.0/</a>) and PRED-TMR (<a href=\"http://athina.biol.uoa.gr/PRED-TMR2/\" target=\"_blank\">http://athina.biol.uoa.gr/PRED-TMR2/</a>). (<b>B</b>) UV-visible spectra of TmpL partial recombinant protein containing FAD/NAD(P)-binding region. The absorbance spectrum shown indicates that the protein contains bound flavin (black line), demonstrating that TmpL is a FAD/NAD(P)-binding flavoprotein. Bovine serum albumin (BSA) was used as a non-flavin binding protein control (blue line). A solution of the FAD-incubated protein (2.0 mg ml<sup>−1</sup>) in 50 mM sodium phosphate buffer, pH 7.5, was analyzed.</p>", "links"=>[], "tags"=>["putative", "membrane"], "article_id"=>547549, "categories"=>["Virology", "Cell Biology", "Infectious Diseases", "Biochemistry", "Genetics"], "users"=>["Kwang-Hyung Kim", "Sven D. Willger", "Sang-Wook Park", "Srisombat Puttikamonkul", "Nora Grahl", "Yangrae Cho", "Biswarup Mukhopadhyay", "Robert A. Cramer Jr.", "Christopher B. Lawrence"], "doi"=>"https://dx.doi.org/10.1371/journal.ppat.1000653.g001", "stats"=>{"downloads"=>1, "page_views"=>10, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_TmpL_is_a_putative_membrane_flavoprotein_/547549", "title"=>"TmpL is a putative membrane flavoprotein.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2009-11-06 02:05:49"}
  • {"files"=>["https://ndownloader.figshare.com/files/878003"], "description"=>"<p>(<b>A</b>) Transcript levels of antioxidant-related genes in vegetative mycelia and 36 hr air-exposed mycelia of <i>A. brassicicola</i> wild-type and <i>ΔtmpL</i> mutant. Relative transcript abundance was determined by comparing each gene transcript level with the transcript level of the same gene in vegetative mycelia of wild-type (set to transcript level = 1). Data are mean±SD of three independent experiments. (<b>B</b>) Constitutive nuclear localization of GFP-Yap1 in <i>A. brassicicola ΔtmpL</i> mutant conidia. Distribution of GFP-Yap1 in the wild-type (WT:pYap1GFP-Yap1) and the Ab<i>ΔtmpL</i> mutant (Ab<i>ΔtmpL</i>:pYap1GFP-Yap1) during normal conidiation on CM (0 mM H<sub>2</sub>O<sub>2</sub>) and following treatment of wild-type and Ab<i>ΔtmpL</i> mutant with H<sub>2</sub>O<sub>2</sub> for 1 hr (1 mM H<sub>2</sub>O<sub>2</sub>).</p>", "links"=>[], "tags"=>["antioxidant-related", "genes", "localization", "gfp-yap1", "brassicicola"], "article_id"=>548451, "categories"=>["Virology", "Cell Biology", "Infectious Diseases", "Biochemistry", "Genetics"], "users"=>["Kwang-Hyung Kim", "Sven D. Willger", "Sang-Wook Park", "Srisombat Puttikamonkul", "Nora Grahl", "Yangrae Cho", "Biswarup Mukhopadhyay", "Robert A. Cramer Jr.", "Christopher B. Lawrence"], "doi"=>"https://dx.doi.org/10.1371/journal.ppat.1000653.g008", "stats"=>{"downloads"=>1, "page_views"=>3, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Expression_of_antioxidant_related_genes_and_nuclear_localization_of_GFP_Yap1_in_A_brassicicola_916_tmpL_mutants_/548451", "title"=>"Expression of antioxidant-related genes and nuclear localization of GFP-Yap1 in <i>A. brassicicola ΔtmpL</i> mutants.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2009-11-06 02:20:51"}
  • {"files"=>["https://ndownloader.figshare.com/files/878309"], "description"=>"<p>(<b>A</b>) Light micrographs of onion epidermis inoculated with <i>A. brassiciola</i> wild-type and <i>ΔtmpL</i> mutants at 12 and 24 hpi. Note that penetration hyphae (inset) were frequently visible under the mutant appressoria, but no further development was observed in the Ab<i>ΔtmpL</i> mutant infection. Bars = 20 µm, except for the inset where it denotes 5 µm. Abbreviations: a, appressorium; gt, germ tube; ih, infection hypha; ph, penetration hypha. (<b>B</b>) Frequency of infection hyphae formation beneath appressoria of the <i>A. brassicicola</i> wild-type and <i>ΔtmpL</i> mutants on onion epidermis. Columns and error bars represent average and SD, respectively, of four independent experiments. (<b>C</b>) Transmission electron micrographs showing transverse sections of green cabbage leaves inoculated with <i>A. brassicicola</i> wild-type and <i>ΔtmpL</i> mutants. Leaves were collected at 24 hpi and prepared for transmission electron microscopy. Arrow indicates papilla formation (callose deposition) around a fungal penetration hypha. Bars = 2 µm. Abbreviations: a, appressorium; cu, cuticle layer; ih, secondary infection hypha; ph, penetration hypha. (<b>D</b>) Callose deposition on green cabbage cotyledons inoculated with <i>A. brassicicola</i> wild-type and <i>ΔtmpL</i> mutants. White spots indicate callose deposition of the inoculation sites stained by aniline blue and viewed by epifluorescence microscopy. The tiny black spots dispersed on plant surface are fungal conidia (cd). Bars = 50 µm.</p>", "links"=>[], "tags"=>["hyphae", "callose", "detection", "assays", "brassicicola", "mutant"], "article_id"=>548753, "categories"=>["Virology", "Cell Biology", "Infectious Diseases", "Biochemistry", "Genetics"], "users"=>["Kwang-Hyung Kim", "Sven D. Willger", "Sang-Wook Park", "Srisombat Puttikamonkul", "Nora Grahl", "Yangrae Cho", "Biswarup Mukhopadhyay", "Robert A. Cramer Jr.", "Christopher B. Lawrence"], "doi"=>"https://dx.doi.org/10.1371/journal.ppat.1000653.g010", "stats"=>{"downloads"=>1, "page_views"=>4, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Appressoria_and_infection_hyphae_formation_ultrastructure_and_callose_detection_assays_of_A_brassicicola_916_tmpL_mutant_infection_/548753", "title"=>"Appressoria and infection hyphae formation, ultrastructure, and callose detection assays of <i>A. brassicicola ΔtmpL</i> mutant infection.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2009-11-06 02:25:53"}
  • {"files"=>["https://ndownloader.figshare.com/files/878486"], "description"=>"<p>Mock = 0.01% Tween inoculated, CEA10 = WT, <i>ΔtmpL</i> = <i>A. fumigatus ΔtmpL</i> mutant. Mice were inoculated with 1×10<sup>5</sup> conidia intranasally, euthanized on days +2 and +4 after inoculation, lungs removed, fixed in formaldehyde, and stained with hematoxylin and eosin (H&E) or Gommori's methenamine silver (GMS) stain. On day +2, Af<i>ΔtmpL</i> mice generally displayed less necrotic lesions and less fungal burden as the wild-type. Significant inflammation, necrosis, and an influx of immune effector cells (primarily neutrophils) were observed on day +4 in all infected animals but not the mock control. However, lesions are more localized and not as extensive and we clearly observed more open alveoli in mice infected with the Af<i>ΔtmpL</i> strain. Interestingly, GMS staining revealed that fungal growth is less extensive in the Af<i>ΔtmpL</i> strain as well. Bar = 500 µm at 40× magnification.</p>", "links"=>[], "tags"=>["histopathology", "cd-1", "infected", "wild-type"], "article_id"=>548930, "categories"=>["Virology", "Cell Biology", "Infectious Diseases", "Biochemistry", "Genetics"], "users"=>["Kwang-Hyung Kim", "Sven D. Willger", "Sang-Wook Park", "Srisombat Puttikamonkul", "Nora Grahl", "Yangrae Cho", "Biswarup Mukhopadhyay", "Robert A. Cramer Jr.", "Christopher B. Lawrence"], "doi"=>"https://dx.doi.org/10.1371/journal.ppat.1000653.g011", "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Representative_histopathology_of_CD_1_mouse_model_infected_with_A_fumigatus_wild_type_and_916_tmpL_mutants_/548930", "title"=>"Representative histopathology of CD-1 mouse model infected with <i>A. fumigatus</i> wild-type and <i>ΔtmpL</i> mutants.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2009-11-06 02:28:50"}
  • {"files"=>["https://ndownloader.figshare.com/files/877212"], "description"=>"<p>(<b>A</b>) Co-localization analyses with a TmpL-GFP and DsRed-AbHex1 double-labeled strain in an <i>A. brassicicola</i> conidium (upper panel) and vegetative hypha (lower panel) were examined using confocal microscopy. TmpL-GFP localizes over cell cortex-associated or cytoplasmic DsRed-AbHex1 signals (arrowheads), not the septal pore-associated signals. Note that no TmpL-GFP signal is observed in the growing vegetative hyphae. Bars = 10 µm. (<b>B</b>) Co-localization analyses with a TmpL-GFP and DsRed-PTS1 double-labeled strain in <i>A. brassicicola</i> conidia. DsRed-PTS1 fluorescence reveals peroxisomes. A large peroxisome is completely associated with TmpL-GFP signal (a green dotted circle, top panel). The other circles denote a partial association between TmpL-GFP and DsRed-PTS1 signals (white dotted circles, top and middle panels) and a complete dissociation of TmpL-GFP with DsRed-PTS1 signals (red dotted circles, middle and bottom panels). Note that different conidial age determined by collected sites, from the center to the edge of fungal colony, shows different types of association between two fluorescence signals. Insets indicate a magnified view of each white dotted circle representing a partial association between TmpL-GFP and DsRed-PTS1 signals. Bars = 10 µm. (<b>C</b>) Localization analyses with <i>Δpex14</i> mutants on a background of either a TmpL-GFP or DsRed-AbHex1 strain. Note that the deletion of <i>pex14</i> resulted in cytoplasmic redistribution of TmpL-GFP and DsRed-AbHex1 fluorescence signals. Bars = 10 µm.</p>", "links"=>[], "tags"=>["tmpl-gfp", "fusion"], "article_id"=>547663, "categories"=>["Virology", "Cell Biology", "Infectious Diseases", "Biochemistry", "Genetics"], "users"=>["Kwang-Hyung Kim", "Sven D. Willger", "Sang-Wook Park", "Srisombat Puttikamonkul", "Nora Grahl", "Yangrae Cho", "Biswarup Mukhopadhyay", "Robert A. Cramer Jr.", "Christopher B. Lawrence"], "doi"=>"https://dx.doi.org/10.1371/journal.ppat.1000653.g002", "stats"=>{"downloads"=>0, "page_views"=>5, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Subcellular_distribution_of_TmpL_GFP_fusion_protein_/547663", "title"=>"Subcellular distribution of TmpL-GFP fusion protein.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2009-11-06 02:07:43"}
  • {"files"=>["https://ndownloader.figshare.com/files/435021", "https://ndownloader.figshare.com/files/435083", "https://ndownloader.figshare.com/files/435218", "https://ndownloader.figshare.com/files/435383", "https://ndownloader.figshare.com/files/435415", "https://ndownloader.figshare.com/files/435449", "https://ndownloader.figshare.com/files/435623", "https://ndownloader.figshare.com/files/435669"], "description"=>"<div><p>The regulation of intracellular levels of reactive oxygen species (ROS) is critical for developmental differentiation and virulence of many pathogenic fungi. In this report we demonstrate that a novel transmembrane protein, TmpL, is necessary for regulation of intracellular ROS levels and tolerance to external ROS, and is required for infection of plants by the necrotroph <em>Alternaria brassicicola</em> and for infection of mammals by the human pathogen <em>Aspergillus fumigatus</em>. In both fungi, <em>tmpL</em> encodes a predicted hybrid membrane protein containing an AMP-binding domain, six putative transmembrane domains, and an experimentally-validated FAD/NAD(P)-binding domain. Localization and gene expression analyses in <em>A. brassicicola</em> indicated that TmpL is associated with the Woronin body, a specialized peroxisome, and strongly expressed during conidiation and initial invasive growth <em>in planta</em>. <em>A. brassicicola</em> and <em>A. fumigatus ΔtmpL</em> strains exhibited abnormal conidiogenesis, accelerated aging, enhanced oxidative burst during conidiation, and hypersensitivity to oxidative stress when compared to wild-type or reconstituted strains. Moreover, <em>A. brassicicola ΔtmpL</em> strains, although capable of initial penetration, exhibited dramatically reduced invasive growth on Brassicas and Arabidopsis. Similarly, an <em>A. fumigatus ΔtmpL</em> mutant was dramatically less virulent than the wild-type and reconstituted strains in a murine model of invasive aspergillosis. Constitutive expression of the <em>A. brassicicola yap1</em> ortholog in an <em>A. brassicicola ΔtmpL</em> strain resulted in high expression levels of genes associated with oxidative stress tolerance. Overexpression of <em>yap1</em> in the <em>ΔtmpL</em> background complemented the majority of observed developmental phenotypic changes and partially restored virulence on plants. Yap1-GFP fusion strains utilizing the native <em>yap1</em> promoter exhibited constitutive nuclear localization in the <em>A. brassicicola ΔtmpL</em> background. Collectively, we have discovered a novel protein involved in the virulence of both plant and animal fungal pathogens. Our results strongly suggest that dysregulation of oxidative stress homeostasis in the absence of TmpL is the underpinning cause of the developmental and virulence defects observed in these studies.</p></div>", "links"=>[], "tags"=>["transmembrane", "intracellular", "redox", "homeostasis", "virulence", "fungal", "pathogen"], "article_id"=>145808, "categories"=>["Cancer", "Cell Biology", "Biochemistry", "Genetics"], "users"=>["Kwang-Hyung Kim", "Sven D. Willger", "Sang-Wook Park", "Srisombat Puttikamonkul", "Nora Grahl", "Yangrae Cho", "Biswarup Mukhopadhyay", "Robert A. Cramer Jr.", "Christopher B. Lawrence"], "doi"=>["https://dx.doi.org/10.1371/journal.ppat.1000653.s001", "https://dx.doi.org/10.1371/journal.ppat.1000653.s002", "https://dx.doi.org/10.1371/journal.ppat.1000653.s003", "https://dx.doi.org/10.1371/journal.ppat.1000653.s004", "https://dx.doi.org/10.1371/journal.ppat.1000653.s005", "https://dx.doi.org/10.1371/journal.ppat.1000653.s006", "https://dx.doi.org/10.1371/journal.ppat.1000653.s007", "https://dx.doi.org/10.1371/journal.ppat.1000653.s008"], "stats"=>{"downloads"=>17, "page_views"=>31, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/TmpL_a_Transmembrane_Protein_Required_for_Intracellular_Redox_Homeostasis_and_Virulence_in_a_Plant_and_an_Animal_Fungal_Pathogen/145808", "title"=>"TmpL, a Transmembrane Protein Required for Intracellular Redox Homeostasis and Virulence in a Plant and an Animal Fungal Pathogen", "pos_in_sequence"=>0, "defined_type"=>4, "published_date"=>"2009-11-06 01:36:48"}
  • {"files"=>["https://ndownloader.figshare.com/files/877708"], "description"=>"<p>(<b>A</b>) Increased sensitivity of <i>A. brassicicola ΔtmpL</i> mutants to oxidative stress generated by KO<sub>2</sub> or H<sub>2</sub>O<sub>2</sub> compared with wild-type. Conidial suspensions of <i>A. brassicicola</i> wild-type (WT), <i>ΔtmpL</i> mutant (Ab<i>ΔtmpL</i>), and reconstituted strain (AbtmpL rec) were cultured on minimal agar medium containing different concentrations of KO<sub>2</sub> or H<sub>2</sub>O<sub>2</sub> and evaluated 5 days after inoculation. (<b>B</b>) Increased sensitivity of <i>A. fumigatus ΔtmpL</i> mutant germlings to oxidative stress generated by H<sub>2</sub>O<sub>2</sub> compared with wild-type. Plates with the germlings of <i>A. fumigatus</i> wild-type strain (CEA10), <i>ΔtmpL</i> mutant (Af<i>ΔtmpL</i>), and reconstituted strain (AftmpL rec) were overlaid with 1.25mM H<sub>2</sub>O<sub>2</sub> solution, incubated at 37°C for 10 minutes. After washing the plate with sterile distilled water the plates were incubated until colonies were large enough to count. Samples were prepared in triplicate, and error bars on the graph represent SD of two independent experiments.</p>", "links"=>[], "tags"=>["mutants", "hypersensitive", "extracellular", "oxidative"], "article_id"=>548157, "categories"=>["Virology", "Cell Biology", "Infectious Diseases", "Biochemistry", "Genetics"], "users"=>["Kwang-Hyung Kim", "Sven D. Willger", "Sang-Wook Park", "Srisombat Puttikamonkul", "Nora Grahl", "Yangrae Cho", "Biswarup Mukhopadhyay", "Robert A. Cramer Jr.", "Christopher B. Lawrence"], "doi"=>"https://dx.doi.org/10.1371/journal.ppat.1000653.g006", "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_916_tmpL_mutants_are_hypersensitive_to_extracellular_oxidative_stress_/548157", "title"=>"<i>ΔtmpL</i> mutants are hypersensitive to extracellular oxidative stress.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2009-11-06 02:15:57"}
  • {"files"=>["https://ndownloader.figshare.com/files/878136"], "description"=>"<p>(<b>A</b>) Virulence assay on green cabbage leaves using two conidial concentrations, 20×10<sup>4</sup> and 5×10<sup>4</sup> conidia ml<sup>−1</sup> of <i>A. brassicicola</i> wild-type (WT), ectopic (A1E1), two <i>ΔtmpL</i> mutants (A1–3 and A1–4), and a reconstituted strain (A1C2). Five days after inoculation (graph), disease severity was calculated based on the lesion diameter. Columns and error bars represent average and SD, respectively, of five independent experiments. (<b>B</b>) Virulence assay on mice mock inoculated or inoculated intranasally with 10<sup>6</sup> conidia/ 25 µl of <i>A. fumigatus</i> wild-type (CEA10), <i>ΔtmpL</i> mutant (Af<i>ΔtmpL</i>), and reconstituted strain (AftmpL rec). P value for comparison between Af<i>ΔtmpL</i> mutant and wild-type CEA10, <i>P</i> = 0.0006. Af<i>ΔtmpL</i> is significantly less virulent than the wild-type CEA10 and the reconstituted strain AftmpL rec.</p>", "links"=>[], "tags"=>["virulence"], "article_id"=>548590, "categories"=>["Virology", "Cell Biology", "Infectious Diseases", "Biochemistry", "Genetics"], "users"=>["Kwang-Hyung Kim", "Sven D. Willger", "Sang-Wook Park", "Srisombat Puttikamonkul", "Nora Grahl", "Yangrae Cho", "Biswarup Mukhopadhyay", "Robert A. Cramer Jr.", "Christopher B. Lawrence"], "doi"=>"https://dx.doi.org/10.1371/journal.ppat.1000653.g009", "stats"=>{"downloads"=>1, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Reduced_virulence_of_916_tmpL_mutants_/548590", "title"=>"Reduced virulence of <i>ΔtmpL</i> mutants.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2009-11-06 02:23:10"}
  • {"files"=>["https://ndownloader.figshare.com/files/877331"], "description"=>"<p>Organelle targeting of partial or complete TmpL-GFP fusion proteins. At left schematic representations: gray boxes represent the AMP-binding (A) domain of TmpL protein; six tandem black boxes represent the transmembrane (T) domain; and a blue box represents the FAD/NAD(P)-binding (FN) domain. Right micrographs show GFP signal localization patterns of each fusion protein in <i>A. brassicicola</i> conidia. Bars = 10 µm.</p>", "links"=>[], "tags"=>["transmembrane", "tmpl", "carries", "organelle", "targeting"], "article_id"=>547778, "categories"=>["Virology", "Cell Biology", "Infectious Diseases", "Biochemistry", "Genetics"], "users"=>["Kwang-Hyung Kim", "Sven D. Willger", "Sang-Wook Park", "Srisombat Puttikamonkul", "Nora Grahl", "Yangrae Cho", "Biswarup Mukhopadhyay", "Robert A. Cramer Jr.", "Christopher B. Lawrence"], "doi"=>"https://dx.doi.org/10.1371/journal.ppat.1000653.g003", "stats"=>{"downloads"=>1, "page_views"=>11, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_The_transmembrane_domain_of_the_TmpL_protein_carries_an_organelle_targeting_signal_/547778", "title"=>"The transmembrane domain of the TmpL protein carries an organelle targeting signal.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2009-11-06 02:09:38"}
  • {"files"=>["https://ndownloader.figshare.com/files/877429"], "description"=>"<p>(<b>A</b>) The phase specific expression of <i>tmpL</i> was quantified by quantitative real-time (QRT)-PCR after synthesis of cDNA of each developmental RNA including infectious growth, vegetative growth, and conidiation. Relative abundance of <i>tmpL</i> transcripts during infectious growth (from ungerminated conidia to <i>in planta</i> fungal cells 120 hpi) and conidial development (from 12 hr air-exposed mycelia to 24 hr air-exposed mycelia) was normalized by comparing with vegetative growth in liquid CM (set to transcript level = 1). (<b>B</b>) Epifluorescence microscopy of <i>in planta</i> GFP expression for the TmpL-GFP strain. The germ tubes and appressoria did not exhibit any GFP signal on the plant surface at 6 hpi. However, a GFP signal was detectable at 12 hpi in a punctate pattern in the appressoria and infection hyphae growing within the plant tissue, consistent with the QRT-PCR results. The wild-type strain was used as a control. Abbreviations: a, appressorium; ih, infection hypha.</p>", "links"=>[], "tags"=>["brassicicola"], "article_id"=>547882, "categories"=>["Virology", "Cell Biology", "Infectious Diseases", "Biochemistry", "Genetics"], "users"=>["Kwang-Hyung Kim", "Sven D. Willger", "Sang-Wook Park", "Srisombat Puttikamonkul", "Nora Grahl", "Yangrae Cho", "Biswarup Mukhopadhyay", "Robert A. Cramer Jr.", "Christopher B. Lawrence"], "doi"=>"https://dx.doi.org/10.1371/journal.ppat.1000653.g004", "stats"=>{"downloads"=>0, "page_views"=>1, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Phase_specific_expression_of_A_brassicicola_tmpL_/547882", "title"=>"Phase specific expression of <i>A. brassicicola tmpL</i>.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2009-11-06 02:11:22"}
  • {"files"=>["https://ndownloader.figshare.com/files/878695"], "description"=>"<p>(<b>A</b>) Transcript levels of <i>yap1</i>, <i>sod1</i>, and <i>ctt1</i> in vegetative mycelia and 36 hr air-exposed mycelia of <i>A. brassicicola</i> wild-type (WT), <i>yap1</i> overexpression mutant on wild-type background (WT:pToxAYap1), <i>ΔtmpL</i> mutant (Ab<i>ΔtmpL</i>), and <i>yap1</i> overexpression mutant on <i>ΔtmpL</i> mutant background (Ab<i>ΔtmpL</i>:pToxAYap1). Relative transcript abundance was determined by comparing each gene transcript level with the transcript level of the same gene in vegetative mycelia of wild-type (set to transcript level = 1). Data are mean±SD of two independent experiments. (<b>B</b>) Hypersensitivity to extracellular oxidative stress generated by H<sub>2</sub>O<sub>2</sub> was recovered by <i>yap1</i> overexpression in Ab<i>ΔtmpL</i> mutants. Conidial suspensions of WT, Ab<i>ΔtmpL</i>, and Ab<i>ΔtmpL</i>:pToxAYap1 strain were cultured on minimal agar medium containing different concentrations of H<sub>2</sub>O<sub>2</sub> and evaluated 5 days after inoculation. (<b>C</b>) Light micrographs showing restoration of the abnormal conidiogenesis of Ab<i>ΔtmpL</i> mutants in Ab<i>ΔtmpL</i>:pToxAYap1 overexpression strain. Conidia released from 7-day-old colonies of WT, WT:pToxAYap1, Ab<i>ΔtmpL</i>, and Ab<i>ΔtmpL</i>:pToxAYap1 strain were observed with a light microscope. Note that <i>yap1</i> overexpression in the wild-type has no significant effect on the conidiogenesis, resulting in similar conidia production. Bars = 20 µm. (<b>D</b>) Partial restoration of the reduced virulence of Ab<i>ΔtmpL</i> mutants in Ab<i>ΔtmpL</i>:pToxAYap1 overexpression strain. Note that <i>yap1</i> overexpression in the wild-type negatively affected <i>A. brassicicola</i> pathogenicity, resulting in smaller lesion development compared to the wild-type. Pictures were taken 5 days after inoculation.</p>", "links"=>[], "tags"=>["abnormal", "phenotypes", "reduced", "virulence", "brassicicola", "mutants", "redox"], "article_id"=>549139, "categories"=>["Virology", "Cell Biology", "Infectious Diseases", "Biochemistry", "Genetics"], "users"=>["Kwang-Hyung Kim", "Sven D. Willger", "Sang-Wook Park", "Srisombat Puttikamonkul", "Nora Grahl", "Yangrae Cho", "Biswarup Mukhopadhyay", "Robert A. Cramer Jr.", "Christopher B. Lawrence"], "doi"=>"https://dx.doi.org/10.1371/journal.ppat.1000653.g012", "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Restoration_of_the_abnormal_phenotypes_and_reduced_virulence_of_A_brassicicola_916_tmpL_mutants_by_redox_regulator_yap1_overexpression_/549139", "title"=>"Restoration of the abnormal phenotypes and reduced virulence of <i>A. brassicicola ΔtmpL</i> mutants by redox regulator <i>yap1</i> overexpression.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2009-11-06 02:32:19"}
  • {"files"=>["https://ndownloader.figshare.com/files/877567"], "description"=>"<p>(<b>A</b>) Fungal colony grown on solid CM plates (colony). Note that the <i>A. brassicicola ΔtmpL</i> mutant colony is a light brown compared with the dark brown color of the wild-type colony. Light micrographs (LM) of less-pigmented conidia and abnormal branching of the conidial chain of the Ab<i>ΔtmpL</i> mutants compared to the normal conidiogenesis of the wild-type. Transmission electron micrographs (TEM) depicting the less electron-dense and thinner cell wall of an Ab<i>ΔtmpL</i> mutant conidium compared to a wild-type conidium. Bars = 500 nm. Abbreviations: cw, conidial cell wall; cy, conidial cytoplasm; v, vacuole. (<b>B</b>) Quantification of protein concentration from conidial suspensions of <i>A. brassicicola</i> wild-type and <i>ΔtmpL</i> mutant. Note color difference of conidial suspensions between the wild-type and mutants (inset). Values indicate the total quantity of protein released by different-aged fungal cultures from each strain. Average values and SD of three independent quantitations are shown. (<b>C</b>) Light and transmission electron micrographs of 21-day-old conidia of <i>A. brassicicola</i> wild-type and <i>ΔtmpL</i> mutant. Arrows indicate cytoplasmic bleeding due to cell burst of the Ab<i>ΔtmpL</i> mutant conidia. Bars = 2 µm. (<b>D</b>) Transmission electron micrographs showing sections of 10-day-old conidia of <i>A. fumigatus</i> wild-type CEA10 and <i>ΔtmpL</i> mutants. Compared to the normal nucleus and subcellular structures of the wild-type conidia, more than half of the Af<i>ΔtmpL</i> mutant conidia showed at least one of the apoptotic histological markers: (a) discontinuous or missing mitochondrial outer membrane, (b) chromatin condensation and margination (arrowheads; a and b), (c) accumulation of huge electron dense materials (arrows) in cytoplasm, and (d) conidia with features of necrotic cell death. Bars = 500 nm. Abbreviations: er, endoplasmic reticulum; m, mitochondria; N, nuclei.</p>", "links"=>[], "tags"=>["conidiogenesis", "aged", "conidia"], "article_id"=>548018, "categories"=>["Virology", "Cell Biology", "Infectious Diseases", "Biochemistry", "Genetics"], "users"=>["Kwang-Hyung Kim", "Sven D. Willger", "Sang-Wook Park", "Srisombat Puttikamonkul", "Nora Grahl", "Yangrae Cho", "Biswarup Mukhopadhyay", "Robert A. Cramer Jr.", "Christopher B. Lawrence"], "doi"=>"https://dx.doi.org/10.1371/journal.ppat.1000653.g005", "stats"=>{"downloads"=>5, "page_views"=>3, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Abnormal_conidiogenesis_and_rapid_loss_of_cell_integrity_in_aged_conidia_of_the_916_tmpL_mutants_/548018", "title"=>"Abnormal conidiogenesis and rapid loss of cell integrity in aged conidia of the <i>ΔtmpL</i> mutants.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2009-11-06 02:13:38"}

PMC Usage Stats | Further Information

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