Alignment of the repeated DPR domains of AbDhn1, AbDhn2 and AbDhn3.

<p>Conserved amino acids are boxed in black (identical) or grey (similar). DHN1.1–DHN1.2 designate the two domains from AbDhn1, DHN2.1–DHN2.5 the five domains of AbDhn2, and DHN3.1–DHN3.3 the three domains from AbDhn3. Numbers indicate the amino acid positions. The conserved DPR motif is boxed in red.</p

Effects of <i>AbDhn</i> knockouts on pathogenicity.

<p><i>B. oleracea</i> leaves were inoculated with 5 µL drops of conidia suspension (10⁵, 10⁴ or 10³ conidia mL⁻¹ in water). Mutants were inoculated on the right part of the central vein and compared on the same leaf with the parental strain (inoculated on the left part of the central vein). A: Representative result at 5 dpi. B: Percentage of successful infection at 5 dpi. The experiment was repeated twice and for each experiment each genotype was inoculated on 30 leaves at the three inocula concentrations. Error bars indicate standard deviations and asterisks indicate a significant difference with respect to the wild-type aggressiveness using the Student test (<i>P</i><0.01).</p

Expression of <i>AbDhn</i> genes in different genetic backgrounds estimated by real-time PCR.

<p>A: <i>A. brassicicola</i> wild-type (WT) and Δ<i>abhog1</i> strains were exposed to 350 mM NaCl for 30 min prior RNA extraction. For the three <i>AbDhn</i> genes, expression induction is represented as a log₂ ratio (fold induction) of their relative expression under stress condition to their relative expression in the control without stress. B: Basal transcript levels of <i>AbDhn</i> genes in the Δ<i>abhacA</i> mutant relative to their expression levels in the reference wild-type strain <i>Abra 43</i> (Log₂ values). Each value is the mean of two independent experiments, each with three replicates. Asterisks indicate values that are significantly (<i>P</i><0.01) different than that of the wild-type.</p

Alternative splicing of the <i>AbDhn2</i> transcript.

<p>A: Electrophoresis gel of PCR products obtained after amplification of the <i>AbDhn2</i> coding sequence using genomic DNA (lane g) or first-strand cDNA (lane RT) as template; Lane L: DNA ladder. B: Schematic representation of the splicing events leading to α and β forms of mature transcripts. Exons are indicated as black or hatched boxes.</p

Characteristics of the dehydrin-like proteins found in <i>A. brassicicola.</i>

*<p>values indicate percentage of total amino acids.</p

Expression of AbDhn-GFP proteins under salt stress.

<p>Germinated conidia (24 h-old) from strains expressing AbDhn-GFP fusions under the control of their own promoters or from a strain constitutively expressing GFP (cGFP) were either exposed to 350 mM NaCl for 2 h or left without stress. Proteins extracts were immunoblotted with HRP-coupled antibodies directed against GFP. Numbers correspond to molecular weights in kDa. The two AbDhn2 isoforms are indicated.</p

Expression levels of <i>AbDhn1</i>, <i>AbDhn2</i> and <i>AdDhn3</i> sequences in <i>A. brassicicola</i> exposed to various stresses.

<p>First-strand cDNAs were prepared from RNA samples extracted from germinated conidia either exposed to 125 µM camalexin (CAM), 125 µM brassinin (BRA), 2.5 mM allyl-isothiocyanate (Al-ITC), 5 mM menadione (MEN), 5 mM hydrogen peroxide (H2O2), 1 M sorbitol (SORB), 350 mM sodium chloride (NACL), 20 mM dithiothreitol (DTT) or incubated at 4°C (COLD) for the indicated times and used as template for real-time PCR. For each gene, expression induction is represented as a ratio (fold induction) of its relative expression (studied gene transcript abundance/β-tubulin transcript abundance) in each inductive condition to its relative expression in the corresponding control. Each value is the mean of two independent experiments, each with three replicates. For easier visualization of the results, numerical data were transformed into colour-grid representations using JColorGrid software <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075143#pone.0075143-Joachimiak1" target="_blank">[45]</a> in which the fold gene expression induction (Log₂ values) is represented by a grey scale (on the right).</p

Susceptibility of AbDhn-deficient mutants to temperature stress.

<p>Calibrated water suspensions of conidia from the wild-type (WT) strain <i>Abra43</i> and AbDhn-deficient mutants were left for 10 h at various temperatures (−20°C, +4°C, +20°C, +40°C). Conidia were then used to inoculate microplate wells and nephelometric growth curves were established over a 33 h period. ΔLag time was calculated as the difference between the lag time at the tested temperature and the lag time at 20°C and was used as a parameter to estimate the effect of the treatment on spore viability. Error bars indicate standard deviations and asterisks indicate values that are significantly (<i>P</i><0.01) higher than that of the wild-type. Each genotype was analysed in triplicate and the experiments were repeated twice times per growth condition.</p

Subcellular localization of the AbDhn1-GFP fusion protein.

<p>Double-labelled strains expressing AbDhn1<i>-</i>GFP and DsRed-SKL were exposed to 350 mM NaCl for 2 h. Co-localization analyses in conidia (A) and hyphae (B) were examined using confocal microscopy. Bars = 10 µm.</p

Susceptibility of AbDhn-deficient mutants to oxidative stress.

<p>Nephelometric monitoring of growth of wild-type strain (black symbols) and AbDhn-deficient mutants (open symbols; Δ<i>abdhn1</i>: triangles, Δ<i>abdhn2</i>: circles, Δ<i>abdh3</i>: diamonds) was automatically recorded for 33 h at 24°C. The unit of the Y-axis corresponds to the Relative Nephelometric Unit (RNU). Conidia were used to inoculate microplate wells containing standard PDB medium that was supplemented with either 10 mM menadione, 5 mM H₂O₂, 5 mM Al-ITC or 100 µM brassinin. Error bars indicate standard deviations. Each genotype was analysed in triplicate and the experiments were repeated twice times per growth condition. Lag time and maximal growth rate variables were calculated from the growth curves using a calculation method described by Joubert et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075143#pone.0075143-Joubert3" target="_blank">[19]</a>. For each parameter, Student’s T-test was used to assess significant difference between the treated and untreated samples or between mutants and parental isolate.</p