22.二酸化窒素の呼吸器に及ぼす影響に関する形態学的研究(第614回千葉医学会例会・第14回肺癌研究施設例会)

<p>The first two columns are the protein IDs of the JGI <i>P</i>. <i>ostreatus</i> genome database.</p

Transcription Factor <em>Amr1</em> Induces Melanin Biosynthesis and Suppresses Virulence in <em>Alternaria brassicicola</em>

<div><p><em>Alternaria brassicicola</em> is a successful saprophyte and necrotrophic plant pathogen. Several <em>A. brassicicola</em> genes have been characterized as affecting pathogenesis of <em>Brassica</em> species. To study regulatory mechanisms of pathogenesis, we mined 421 genes <em>in silico</em> encoding putative transcription factors in a machine-annotated, draft genome sequence of <em>A. brassicicola</em>. In this study, targeted gene disruption mutants for 117 of the transcription factor genes were produced and screened. Three of these genes were associated with pathogenesis. Disruption mutants of one gene (<em>AbPacC</em>) were nonpathogenic and another gene (<em>AbVf8</em>) caused lesions less than half the diameter of wild-type lesions. Unexpectedly, mutants of the third gene, <em>Amr1</em>, caused lesions with a two-fold larger diameter than the wild type and complementation mutants. <em>Amr1</em> is a homolog of <em>Cmr1</em>, a transcription factor that regulates melanin biosynthesis in several fungi. We created gene deletion mutants of Δ<em>amr1</em> and characterized their phenotypes. The Δ<em>amr1</em> mutants used pectin as a carbon source more efficiently than the wild type, were melanin-deficient, and more sensitive to UV light and glucanase digestion. The AMR1 protein was localized in the nuclei of hyphae and in highly melanized conidia during the late stage of plant pathogenesis. RNA-seq analysis revealed that three genes in the melanin biosynthesis pathway, along with the deleted <em>Amr1</em> gene, were expressed at low levels in the mutants. In contrast, many hydrolytic enzyme-coding genes were expressed at higher levels in the mutants than in the wild type during pathogenesis. The results of this study suggested that a gene important for survival in nature negatively affected virulence, probably by a less efficient use of plant cell-wall materials. We speculate that the functions of the <em>Amr1</em> gene are important to the success of <em>A. brassicicola</em> as a competitive saprophyte and plant parasite.</p> </div

Expression of melanin biosynthesis-associated genes and four hydrolytic enzyme-coding genes.

<p>A–C. Relative transcription abundance of each gene was determined in comparison to actin gene transcripts in the same tissue. Y-axes show relative abundance of the transcripts compared to the actin gene. D. Expression ratio between the Δ<i>amr1</i> and wild type during late stage of infection. A total of three biological replicates (N = 3) were used for this study. Bars represent standard error. wt = wild type, Δ<i>a</i> = Δ<i>amr1:Amr1p-GFP</i>, GY = glucose yeast extract broth. <i>SCD1</i> = Scytalone dehydratase, <i>Brn1</i> = T3HN reductase, <i>Brn2</i> = T4HN reductase, <i>Cbh7</i> = cellobiohydrolase, <i>Amr1</i> = Alternaria melanin regulation, chymo = chymotrypsin.</p

Effect of pectin on the vegetative growth of four strains of Δ<i>amr1</i> mutants and wild-type <i>Alternaria brassicicola</i>.

<p>A–D. Data show mean dry weight in milligrams. Each graph is the result of an independent experiment. The poor correlation between the absolute amount of dry biomass and the length of the incubation period was partially due to differences in the number of conidia used in each experiment. Values above the bars indicate the percent increase in dry biomass of the mutant compared to the wild type. Hours under each chart show the length of the incubation period. ** indicates <i>p</i><0.01. Error bars represent standard deviation. wt = wild type.</p

Loss of pathogenicity of the <i>abpacc</i> mutant and decreased virulence of Δ<i>abvf8</i> compared to wild-type <i>Alternaria brassicicola</i>.

<p>d.f. = degrees of freedom, wt = wild type, p-value = probability, <i>AbVf8-10*</i> is an ectopic mutant.</p

Increased virulence of Δ<i>amr1</i> mutants on individual host plants (<i>Brassica oleracea</i>).

<p>A. Lesions on <i>B. oleracea</i> 5 days after inoculation with ∼2,000 conidia of the wild type, a Δ<i>amr1</i> mutant, and three complemented mutants with two types of constructs. B. Lesions produced by ∼440 and ∼400 conidia respectively of the wild type (left) and a Δ<i>amr1</i> mutant (right). C. Lesions produced by ∼330 and ∼300 conidia respectively of the wild-type (left) and a Δ<i>amr1</i> mutant (right). The three images in panels A–C are replicate experiments with the same pattern of inoculation. Note that the size of lesions caused by the mutant is similar on all leaves, while lesions caused by the wild type are smaller on young leaves than on old leaves. Δ<i>amr1-1</i> and Δ<i>amr1-4</i> produced similar results. D. Pathogenicity assay results for three mutants (Δ<i>amr1-1</i>, Δ<i>amr1-4</i>, and Δ<i>amr1-5</i>) compared to the wild type. E. Pathogenicity assay results with average lesion diameter (mm) and standard deviation. Abbreviations: wt = wild-type <i>A. brassicicola</i>; Δ<i>amr1 = Amr1</i> deletion mutant; compl = mutant complemented with a native allele of the <i>Amr1</i> gene (Δ<i>amr1:Amr1</i>); cons = mutant complemented with chimeric constructs of the <i>TrpC</i> promoter and a native allele of the <i>Amr1</i> coding region (Δ<i>amr1:TrpCp-Amr1</i>).</p

Melanin deficiency and its effects on fungal morphology and responses to stressors.

<p>A–D. Conidial chains produced by each strain. Insets: fungal growth of each strain showing colony color and pink pigment secreted by Δ<i>amr1</i> mutants during saprophytic growth on PDA. E. Pink exudate from the tips of conidial chains of the Δ<i>amr1</i> mutant. F. Germination and hyphal growth after UV irradiation. G. Growth comparisons between mutants and wild type either under high temperature (33°C), or in the presence of the indicated chemical. Abbreviations: Δ<i>amr1</i> = <i>Amr1</i> deletion mutant; Δ<i>amr1:Amr1</i> = Δ<i>amr1</i> mutant complemented with the <i>Amr1</i> allele; Δ<i>amr1:TrpCp-Amr1</i> = mutant that constitutively expresses the <i>Amr1</i> gene under control of the <i>TrpC</i> promoter.</p

Summary of <i>Alternaria brassicicola</i> transcription factor domains based on Pfam scans.

a<p>Genes of previously screened targeted deletion mutants <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002974#ppat.1002974-Cho2" target="_blank">[15]</a>.</p>b<p>Genes of mutants created and screened in this study.</p>c<p>Genes whose functions were previously characterized <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002974#ppat.1002974-Cho2" target="_blank">[15]</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002974#ppat.1002974-Srivastava1" target="_blank">[20]</a>.</p

Increased virulence of four strains of Δ<i>amr1</i> deletion mutants compared to wild-type <i>Alternaria brassicicola</i>.

<p>d.f. = degrees of freedom, P = probability, lesion size indicates the average lesion diameter. Results marked with an asterisk (*) were from pathogenicity assays with whole plants, others were from assays with detached leaves.</p

Comparison of differentially expressed genes in the Δ<i>amr1</i> and Δ<i>abvf19</i> mutants compared to wild-type <i>Alternaria brassicicola</i>.

<p>A. Comparison between up-regulated genes in the Δ<i>amr1</i> mutant and down-regulated genes in the Δ<i>abvf19</i> mutant during the late stage of infection. B. Number of differentially expressed glycoside hydrolase genes. C. Comparison of expression levels for the 24 genes differentially expressed in the Δ<i>amr1</i> and Δ<i>abvf19</i> mutants compared to the wild type. Y-axis shows Log₂ (mutant)_expression-Log₂ (wild type)_expression).</p