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

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

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

Confocal microscopic images showing <i>Amr1</i> gene expression and its protein localization in fungal nuclei during conidiation and pathogenesis.

<p>A–C. Green fluorescence indirectly shows localization of Amr1 protein in fungal nuclei: GFP is expressed as the Amr1-GFP fusion protein. D–F. Green fluorescence in fungal tissues indicates an abundance of GFP, whose gene is under the regulation of the <i>Amr1</i> promoter in the Δ<i>amr1:Amr1p-GFP</i> mutant. G. Fluorescence shows co-localization of Amr1-GFP proteins, and 4′,6-diamidino-2-phenylindole (DAPI) stains that bind to nucleotides. Pink color represents auto-fluorescence of plant tissues. Arrowheads indicate conidia. Arrows point to fungal hyphae with localized green fluorescence in the nuclei during late plant infection. hpi =  hours post-inoculation.</p

Verification of <i>Δamr1</i> deletion mutants.

<p>A. Replacement of the <i>Amr1</i> coding region with the selectable marker, Hygromycin B transferase (HygB) resistance cassette. On the HygB-probed blot, the expected 4.5 Kb band indicates a single copy insertion of the Hyg B resistance cassette in lane d4. Additional bands in the other lanes suggest multiple insertions of the cassette. B. Replacement of the <i>Amr1</i> coding region with a green fluorescent protein (GFP) coding region and a HygB cassette. The upper panel of both A and B is a schematic diagram of wild-type (wt) and mutant loci and the lower panel consists of five Southern blots showing loss of the <i>Amr1</i> gene in selected mutants. Dots (•) indicate lanes of mutants used in this study. Probe regions are marked by P_h and P_a. Abbreviations: E = <i>Eco</i>RI enzyme digestion site. d1 = Δ<i>amr1-1</i>, d4<i> = </i>Δ<i>amr1-4</i>, d5<i> = </i> Δ<i>amr1-5</i>, and D3 = Δ<i>amr1:Amr1p-GFP.</i> e1-e5, E1, E3, and E4 are ectopic insertion mutants.</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

Schematic diagram of the PCR strategy used to make each construct.

<p>A. Construct for replacement of the <i>Amr1</i> gene with a Hygromycin B resistance cassette. B. Construct for replacement of the <i>Amr1</i> gene with a GFP coding region and Hygromycin B resistance cassette. C. Amplification of the wild-type allele of the <i>Amr1</i> gene. D. Construct for the constitutive <i>Amr1</i> expression cassette. E. Construct for the Amr1-GFP fusion protein expression.</p

Effect of the <i>Amr1</i> gene on lesion diameters (mm) following inoculation with wild-type <i>Alternaria brassicicola</i>, Δ<i>amr1:Amr1p-GFP</i>, Δ<i>amr1:Amr1</i>,and Δ<i>amr1:TrpCp-Amr1</i>.

<p>Mean lesion diameters (mm) and their standard deviations in each column followed by the same letter were not significantly different based on the Waller-Duncan <i>k</i>-ratio (<i>k</i> = 100) t-test. * and ** indicate a significant effect of leaf age based on analysis of variance at <i>p</i><0.05 and <i>p</i><0.01, respectively. Increased virulence was calculated by the sum of lesion size differences between the wild type and mutant divided by the sum of wild-type lesion sizes multiplied by 100. combined = all data with lesions caused by wild type, Δ<i>amr1</i>, Comp, and Cons mutants. Δ<i>amr1 = Δamr1:Amr1p-GFP</i>, Compl = Δ<i>amr1:Amr1</i>, Cons = Δ<i>amr1:TrpCp-Amr1.</i></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