Dehydrin-like proteins in the necrotrophic fungus Alternaria brassicicola have a role in plant pathogenesis and stress response.

In this study, the roles of fungal dehydrin-like proteins in pathogenicity and protection against environmental stresses were investigated in the necrotrophic seed-borne fungus Alternaria brassicicola. Three proteins (called AbDhn1, AbDhn2 and AbDhn3), harbouring the asparagine-proline-arginine (DPR) signature pattern and sharing the characteristic features of fungal dehydrin-like proteins, were identified in the A. brassicicola genome. The expression of these genes was induced in response to various stresses and found to be regulated by the AbHog1 mitogen-activated protein kinase (MAPK) pathway. A knock-out approach showed that dehydrin-like proteins have an impact mainly on oxidative stress tolerance and on conidial survival upon exposure to high and freezing temperatures. The subcellular localization revealed that AbDhn1 and AbDhn2 were associated with peroxisomes, which is consistent with a possible perturbation of protective mechanisms to counteract oxidative stress and maintain the redox balance in AbDhn mutants. Finally, we show that the double deletion mutant ΔΔabdhn1-abdhn2 was highly compromised in its pathogenicity. By comparison to the wild-type, this mutant exhibited lower aggressiveness on B. oleracea leaves and a reduced capacity to be transmitted to Arabidopsis seeds via siliques. The double mutant was also affected with respect to conidiation, another crucial step in the epidemiology of the disease

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

Transmission capacity of <i>A. brassicicola</i> wild-type (WT) and AbDhn-deficient genotypes to <i>Arabidopsis thaliana</i> seeds (L<i>er</i> ecotype).

<p>The seed transmission capacity according to the silique stage and global seed transmission capacity (strain model) were measured as described by Pochon et al <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075143#pone.0075143-Pochon1" target="_blank">[26]</a>. The five youngest siliques of at least five plants were inoculated with each fungal genotype and the experiment was repeated twice. Contaminated siliques were harvested 10 dpi. After dissection, seeds were incubated separately on PDA medium for 2 days. A seed was considered contaminated when incubation resulted in typical <i>A. brassicicola</i> colony development. For each inoculated fungal genotype, the seed infection probability was evaluated from at least 1000 seeds. Values represent infection probabilities with 95% confidence interval.</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

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 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

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

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

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