Localization of XopN_KXO85, OsVOZ2, and OsXNP in plant cells.

<p>Subcellular localization of the XopN_KXO85-GFP, OsVOZ2-GFP, and OsXNP-GFP fusion proteins in maize mesophyll cells. OsABF1-RFP was used as a nuclear marker. GFP (green) fluorescence was merged with RFP (red) fluorescence. Bars = 10 µm.</p

Pathogenicity test for <i>xop</i> mutants of <i>Xoo</i> KXO85 in rice.

<p><b>A</b>. Disease severity of each <i>xop</i> mutant in 3-month-old rice leaves. W, water; 85, wild-type KXO85; Q, KXO85 <i>xopQ</i>_<i>KXO85</i>::EZ-Tn<i>5</i>; X, KXO85 <i>xopX</i>_<i>KXO85</i>::EZ-Tn<i>5</i>; P1, KXO85 <i>xopP1</i>_<i>KXO85</i>::EZ-Tn<i>5</i>; P2, KXO85 <i>xopP2</i>_<i>KXO85</i>::EZ-Tn<i>5</i>; N, KXO85 <i>xopN</i>_<i>KXO85</i>::EZ-Tn<i>5</i>. <b>B</b>. Disease severity of the <i>xopN</i>_<i>KXO85</i> mutants in the flag leaves of rice grown in a paddy field. W, water; 85, KXO85; N, KXO85 <i>xopN</i>_<i>KXO85</i>::EZ-Tn<i>5</i>; and N^C, KXO85 <i>xopN</i>_<i>KXO85</i>::EZ-Tn<i>5</i> (pML122G2). Photographs were taken and lesion lengths were determined 21 days after inoculation. Vertical error bars indicate the standard deviations (SD). The data are the averages of 12–15 replicates for each treatment. Columns and lines not connected by the same letter are significantly different (P<0.05) as determined by a one-way ANOVA (P<0.001) followed by post hoc Tukey HSD analysis. <b>C</b>. Bacterial growth patterns of the KXO85, <i>xopN</i>_<i>KXO85</i> mutant, and complemented <i>xopN</i>_<i>KXO85</i> mutant strains in flag leaves of wild-type Dongjin. The data are shown as the average values for three replicates; vertical bars indicate the error ranges (±SD). The bacterial populations were assessed every 3 days after inoculation. Different letters at day 21 indicate significant differences (P<0.05) as determined by a one-way ANOVA (P<0.001) followed by post hoc Tukey HSD analysis.</p

Interactions between XopN_KXO85 and OsVOZ2 and OsXNP.

<p><b>A</b>. Screening for interactors of XopN_KXO85 in rice using a yeast two-hybrid system. S (strong: pEXP ^TM32/Krev1 + pEXP ^TM22/RalGDS-wt), W (weak: pEXP ^TM32/Krev1 + pEXP ^TM22/RalGDS-m1), and A (absent: pEXP ^TM32/Krev1 + pEXP ^TM22/RalGDS-m2) indicate the strength of each interaction. Three independent and representative colonies are shown for each bait–prey combination. <b>B</b>. <i>In vivo</i> pull-down analysis of XopN_KXO85 and OsVOZ2 (left panel) and XopN_KXO85 and OsXNP (right panel). Total proteins from <i>N</i><i>. benthamiana</i> leaves co-expressing XopN_KXO85-6× His and Flag-OsVOZ2 or XopN_KXO85-6× His and OsXNP-Flag protein were purified by Ni⁺ affinity chromatography followed by Western blotting using anti-His and anti-Flag antibodies. The expected molecular weights were as follows: XopN_KXO85-6× His = 78.7 kDa; Flag-OsVOZ2 = 74.6 kDa; OsXNP-Flag = 40.1 kDa; +, protein expressed; and -, vector control. <b>C</b>. BiFC analysis of XopN_KXO85 -OsVOZ2, XopN_KXO85 -OsXNP, and XopN_KXO85 -OsVOZ1 interactions in <i>N</i><i>. benthamiana</i> leaves. Negative, pDEST-SCYNE(R)^GW + pDEST-SCYCE(R)^GW; positive, pEXP-SCYNE(R)-Cnx7 + pEXP-SCYCE(R)-Cnx6. Bars = 50 µm.</p

Virulence assay in wild-type Dongjin rice and the OsVOZ2 mutant line PFG_3A-07565.

<p><b>A</b>. Schematic representation of the T-DNA insertion in OsVOZ2 T₇ transgenic rice. <i>OsVOZ2</i> consists of four exons (orange boxes) and three introns (line between the orange boxes). The T-DNA was located in the second intron from the translational start site. F and R are the primers used for RT-PCR analysis, which showed the expected size of <i>OsVOZ2</i> in wild-type Dongjin but not in the <i>OsVOZ2</i> mutant rice PFG_3A-07565. Actin1 was used for normalization of the cDNA quantity. <b>B</b>. Virulence assay of the <i>xopN</i>_<i>KXO85</i> mutant in wild-type Dongjin rice and OsVOZ2 mutant rice. W, water; 85, KXO85; N, KXO85 <i>xopN</i>_<i>KXO85</i>::EZ-Tn<i>5</i>; and N^C, KXO85 <i>xopN</i>_<i>KXO85</i>::EZ-Tn<i>5</i> (pML122G2). Photographs were taken 21 days after inoculation. <b>C</b>. Measurement of disease severity in flag leaves of wild-type Dongjin rice (□) and OsVOZ2 mutant rice (▨). W, water; 85, KXO85; N, KXO85 <i>xopN</i>_<i>KXO85</i>::EZ-Tn<i>5</i>; and N^C, KXO85 <i>xopN</i>_<i>KXO85</i>::EZ-Tn<i>5</i> (pML122G2). Lesion lengths were determined 21 days after inoculation. Vertical error bars indicate the standard deviation (SD). The statistical significance was determined using a two-way ANOVA as compared to wild-type Dongjin rice with the post hoc Tukey HSD test (***, P<0.001). <b>D</b>. Growth patterns of the KXO85, <i>xopN</i>_<i>KXO85</i> mutant, and complemented <i>xopN</i>_<i>KXO85</i> mutant in the flag leaves of OsVOZ2 mutant rice (PFG_3A-07565). The data are the average values of three replicates; vertical bars indicate the error ranges (±SD). The bacterial populations were assessed every 3 days after inoculation. Different letters at day 21 indicate significant differences (P<0.05) as determined by a one-way ANOVA (P<0.001) followed by post hoc Tukey HSD analysis.</p

Heat map of expression of hydrophobin, <i>FVFD16</i>, <i>FVFD30</i>, and <i>FDS</i> genes at 3 developmental stages and in different tissues of <i>F</i>. <i>velutipes</i>.

<p>The bar at the top of the panel represents the expression values (log2 based normalization value). Detailed information is given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0093560#pone.0093560.s017" target="_blank">Table S15</a>.</p

Comparative analysis of gene expression at 3 different developmental stages and in different tissues of <i>F. velutipes</i>.

<p>(a) The 3 developmental stages of <i>F. velutipes</i>. (b) The Venn diagram shows unique and common genes expressed across the different developmental stages and the different tissues. (c) Heat map of expression of AA genes at 3 developmental stages and in different tissues of <i>F. velutipes</i>. The bar at the top of the panel represents the expression values (log2 based normalization value). AA1, multicopper oxidase (laccases, ferroxidase, laccase-like multicopper oxidase); AA2, classII peroxidase (manganase peroxidase, lignin peroxidase, versatile peroxidase); AA3, GMC oxidoreductase (cellobiose dehydrogenase, aryl-alcohol oxidase/glucose oxidase, alcohol oxidase, pyranose oxidase); AA5, radical-copper oxidase (glyoxal oxidase, galatose oxidase); AA7, glucooligosaccharide oxidase. ◂ indicates a 2-fold increase in gene compared to the next highest expression level for a particular stage or tissue and exhibiting peaked expression (≥5.5 log2 values) during the mycelial stage. † indicates that the AA gene showed a higher expression level than the overall average expression level of all other AA genes during all stages and in all tissues. Detailed information is given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0093560#pone.0093560.s012" target="_blank">Table S10</a>.</p

Scaffold assignments by Southern hybridization analysis with CHEF-separated chromosomes.

<p>A DNA probe was generated for each of the 11 scaffolds representing chromosome regions. Arrows indicate the positive signals. 1, <i>F. velutipes</i> KACC42780 electrophoretic karyotype; 2, ctg26 probe (chromosome 1); 3, ctg02 probe (chromosome 2); 4, ctg01 probe (chromosome 3); 5, ctg06 probe (chromosome 4); 6, ctg29 probe (chromosome 5); 7, ctg13 probe (chromosome 6); 8, ctg33 probe (chromosome 7); 9, ctg03 probe (chromosome 8); 10, ctg11_1 probe (chromosome 9); 11, ctg11_2 probe (chromosome 10); 12, ctg05 probe (chromosome 11).</p

General features of the <i>F</i>. <i>velutipes</i> genome.

<p>General features of the <i>F</i>. <i>velutipes</i> genome.</p

Figure 1

<p>The <i>F. velutipes</i> genome map (a). A, GC content was calculated as the percentage of G+C in 100-kb non-overlapping windows. B, Gene density is represented as the number of genes in 100-kb non-overlapping windows. C, Transcription factors. D, Distribution of alcohol dehydrogenase genes. E, Distribution of CAZyme genes. F, Distribution of AA genes. G, Pseudochromosomes, represented clockwise starting from center above. Blocks represent 11 <i>F. velutipes</i> pseudochromosomes. Numbers indicate the chromosome number. H, Genome duplication: regions sharing more than 90% sequence similarity (e-value ≤1e-100) and more than 80% query coverage are connected by grey lines. (b) Presence of orthologs of the predicted <i>F. velutipes</i> genes in fungal taxons (right). Detailed information is given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0093560#pone.0093560.s003" target="_blank">Tables S1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0093560#pone.0093560.s008" target="_blank">S6</a>.</p

Comparison of genome characteristics between <i>F</i>. <i>velutipes</i> and other basidiomycetes.

<p>Comparison of genome characteristics between <i>F</i>. <i>velutipes</i> and other basidiomycetes.</p