Sequence conservation and model structure of Bos1 HAMP domains.

<p>The amino-acid sequences of the six HAMP domains of the Bos1 protein were aligned with Clustal W. Amino acids identical over 80% are in bold. In the bottom panel “*” denotes hydrophobic core residues at critical heptad positions and hydrophobic residues of the connector. “g” corresponds to the conserved glycine residues of the connector motif (according to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042520#pone.0042520-Airola1" target="_blank">[35]</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042520#pone.0042520-Parkinson1" target="_blank">[32]</a>). Interacting residues derived from <i>in silico</i> structures are highlighted in yellow. Mutated residues are shaded with the following color code according to the phenotypes indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042520#pone-0042520-t003" target="_blank">Table 3</a>: red = HR to iprodione and phenylpyrroles, osmosensitivity; green = LR to iprodione; purple = MR to iprodione and osmosensitivity. The structure of the HAMP domain 3 was predicted on the SWISS-MODEL server using the alignment mode (for details see text). Portions of the HAMP sequences involved in the typical HAMP structures AS1, AS2 and the connector are indicated above the sequence.</p

Homology based models of HAMP domains with focus on interactions between AS1 and AS2.

<p>A/ HAMP3 wt and mutant peptides. In the last panel I365S (light grey) and I365S M427T (dark grey) isoforms are shown in an overlay. B/ HAMP4 wt and mutant peptides. The orientation of the peptides is from up (N-terminus) to down (C-terminus) or from right to left (A). Solely, the side chains from amino-acids of the connector proximal helices are displayed. Residues potentially involved in interactions are labelled; modified residues are written in italics. In overlaid models, the wild-type peptide is shaded in light grey, the mutant peptide in dark grey.</p

Homology based models of HAMP domains with focus on interactions between AS1 and AS2.

<p>A/ HAMP1 wild-type (wt) and mutant peptides. B/ HAMP2 wt and mutant peptides. The orientation of the peptides is from up (N-terminus) to down (C-terminus). Solely, the side chains from amino-acids of the connector proximal helices are displayed. Residues potentially involved in interactions are labelled; modified residues are written in italics.</p

Modifications in Bos1 peptide sequence and associated phenotypic and structural changes.

a<p>S: sensitive, LR: low resistance, MR: moderate resistance, HR: high resistance, according to the resistance level classification explained in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042520#s2" target="_blank">Materials & Methods</a>. nd: not determined.</p>b<p>modified HAMP; numbering according to the peptide modifications cited in the first column.</p>c<p>AS: amphiphilic helix in HAMP.</p>d<p>Characteristics of amino acids: PU = polar uncharged, NH = non polar hydrophobic, + = positively charged, − = negatively charged, ali = aliphatic, aro = aromatic; na: not applicable.</p>e<p>Structural changes as presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042520#pone-0042520-g002" target="_blank">Figure 2</a> and explained in the text.</p>f<p>Flu = Fludioxonil; Fen = Fenpiclonil.</p

Comparison of fitness parameters between the iprodione-induced mutants and the parental strains.

a<p>G0 is the wild-type parent isolate, G20C is the 20^th transfer generation produced on PDA medium without iprodione (control) and G20I is the 20^th transfer generation produced on PDA supplemented with iprodione.</p>b<p>Daily radial growth rate between the 3^rd and 4^th day after inoculation.</p>c<p>Spore produced on PDA medium 14 days after inoculation.</p>d<p>Spore produced on tomato stem 7 days after inoculation.</p>e<p>Data are means of two to three independent repetitions. For each isolate, means within a column followed by the same letter were not significantly different (ANOVA, α = 0.05; Newman–Keuls test).</p

Phenotypes and changes in Bos1 peptide sequence in pyrrolnitrine- and iprodione-induced mutants.

a<p>G0 is the wild-type parent isolate, G20C is the 20^th transfer generation produced on PDA medium (control), G20P is the the 20^th transfer generation produced on PDA supplemented with pyrrolnitrin and G20I is the 20^th transfer generation produced on PDA supplemented with iprodione.</p>b<p>S: sensitive, LR: low resistance, MR: moderate resistance, HR: high resistance, according to the resistance level classification explained in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042520#s2" target="_blank">Materials & Methods</a> section and EC₅₀ values indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042520#pone.0042520.s002" target="_blank">Table S1</a>.</p><p>nd: not determined.</p

<i>Botrytis cinerea</i> strains used in this study.

<p>Fitness characteristics were measured as indicated in Material in Methods.</p

Schematic view of the bikaverin gene clusters in <i>F. fujikuroi</i>[<b>14</b>], and in gray (B05.10) and pink (1750) <i>B. cinerea</i> strains.

<p>Complete clusters include a PKS-encoding gene (<i>bik1</i>), a putative FAD-dependent monooxygenase (<i>bik2</i>), an O-methyltransferase (<i>bik</i>3), a putative NmrA-like transcriptional regulator (<i>bik4</i>), a Zn(II)₂Cys₆ fungal type transcription factor (<i>bik5</i>) and an MFS-type transporter (<i>bik6</i>).</p

Comparative phylogenies of the conserved elongation factor EF1 α and the BIK1.

<p><i>Maximum likelihood</i> phylogenies from alignments of the protein sequences were estimated using the PhyML program with the substitution model WAG and a number of bootstraps of 100 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053729#pone.0053729-Dereeper1" target="_blank">[52]</a> (<a href="http://www.phylogeny.fr" target="_blank">http://www.phylogeny.fr</a>). A. Phylogeny of EF1α represents the evolution of fungal species. Selected proteins are from <i>Botrytis cinerea</i> (BROAD: BC1G_09492.1), <i>Sclerotinia sclerotiorum</i> (GenBank: EDO03042.1), <i>Fusarium verticillioides</i> (BROAD: FVEG_02381.3), <i>Fusarium oxysporum</i> (GenBank: EGU83230.1), <i>Fusarium fujikuroi</i> (B. Tudzynski, unpublished data), <i>Fusarium graminearum</i> (BROAD: FGSG_08811.3), <i>Podospora anserina</i> (NCBI: XP_001907437.1), <i>Aspergillus oryzae</i> (GenBank: BAA76296.1), <i>Aspergillus niger</i> (NCBI: XP_001398942.1), <i>Ajellomyces capsulatus</i> (GenBank: AAB17119.1), <i>Coccidioides immitis</i> (GenBank: AAK54650.1), <i>Schizosaccharomyces pombe</i> (NCBI: NP_594440.1), <i>Saccharomyces cerevisiae</i> (GenBank: AAA34585.1). B. Phylogeny of BIK1 indicates a non-vertical inheritance. Selected proteins are from the pink <i>B. cinerea</i> strain 1750 (EMBL: HE802550), <i>F. fujikuroi</i> (GenBank: CAB92399.1), <i>F. oxysporum</i> (BROAD: FOXG_04757), and <i>F. verticillioides</i> (BROAD: FVEG_03379). The PKS sequence from <i>A. niger</i> (JGI: ANG51499) was used to root the tree.</p

Heterologous expression of <i>bcbik4</i> and <i>bcbik5</i> of the gray strain B05.10 in the respective <i>Fusarium fujikuroi</i> (IMI58289) deletion mutants.

<p>A. Expression of <i>ffbik2</i> and <i>ffbik4/bcbik4.</i> Wild type (IMI58289) and mutants were grown for three days in 10% liquid ICI (0.6 mM glutamine) medium at 28°C and 180 rpm. The northern blots were hybridized with probes for <i>ffbik2</i> or simultaneously hybridized with probes for <i>ffbik4</i> and <i>bcbik4</i>. rRNA is shown as loading control. B. Identification of bikaverin and norbikaverin production. Wild type (IMI58289) and mutants were grown in liquid ICI medium containing 6 mM glutamine for three days. Samples were taken and used for HPLC-DAD analyses (detected at 450 nm) as described in Material and Methods. Peaks for norbikaverin (1) and bikaverin (2) are labeled in the chromatograms.</p