Influence of pH on the biology of <i>M. oryzae</i>.

<p>(a) The wild type strain Guy11 was grown for 14 and 21 days on solid rice medium buffered to pH 5 (grey) or to pH 8 (black) and conidia from one 9 mm plate were collected and counted under a microscope; Student test analysis of the data revealed a Pvalue of 0,004 at 14 days and of 0.07 at 21 days, due to higher variance. (b) One hundred conidia collected from cultures on non-buffered solid rice medium were incubated for 8 hours in water adjusted to pH 3 to 8, and their germination was monitored by microscopic observation of the presence of a germ tube at least 3 µm in length. (c) Conidia collected from cultures on solid rice medium buffered to pH 5 (grey) or pH 8 (black) were sprayed onto rice or barley plants, and the lesions on 10 separate leaves were counted after 5 days of incubation; Student test analysis of the data revealed a Pvalue of 0.078 for the barley experiment and of 0.079 for the rice experiment. All experiments were run in triplicates and standard deviations are shown.</p

Impact of <i>MoPACC</i> deletion on the fungus biology.

<p>(a) Conidia production in the parental (P), control (C2 (ectopic)) and mutant (T2) strains grown for 14 days on solid rice medium buffered to pH 5 (grey) or to pH 8 (black). (b) Pathogenicity tests on barley leaves infected by conidia from the parent, mutant and complemented strains. Lesions on 10 separate leaves were counted after 5 days of incubation. All experiments were run in triplicates and standard deviations are shown.</p

Impact of <i>MoPACC</i> deletion on the fungal growth and alkalinization.

<p>(a) Growth of the parental (square) and deletion (triangle) strains on solid medium buffered to pH 5 (grey) or pH 8 (black). (b) Growth rate as a function of pH in the parental (square), control (circle) and mutant (triangle) strains grown on 10 solid media buffered to 10 pH values ranging from 5.5 to 7.9. (c) pH of the culture (big triangles) and ammonia accumulation (small triangles) during growth of the deletion strain in liquid non-buffered TNK-YE medium. (d) Expression of the <i>MoPAL</i> genes in the <i>MoPACC</i> mutant strains. Following 15 minutes transfer of the deletion strain from non-buffered liquid medium to fresh medium buffered to pH 5 (grey) or pH 8 (black), total mRNA was extracted and quantitative PCR analysis was carried out using primers specific of the <i>MoPACC</i> and all the six <i>MoPAL</i> genes (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0069236#pone.0069236.s002" target="_blank">Table S1</a>). Three independent biological replicates were analyzed and quantification was based on the 2^ΔCT method using the <i>MoEF1</i>α gene as reference. The experiments were performed in parallel to those reported for the wild type strain in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0069236#pone-0069236-g003" target="_blank">figure 3</a>. Left panel : Gene expression after transfer to pH 5 or to pH 8. Right panel : Change in expression between pH 5 and pH 8 (ratios are plotted using a logarithmic scale).</p

Impact of <i>MoPACC</i> deletion on the production of secreted lytic enzymes.

<p>The parental (circle) and mutant (triangle) strains were grown in liquid TNK-YE and the cultures were filtrated at 4, 24, 72 and 96 hours post-inoculation (X axis). Proteins present in the filtrates (top left panel) and seven different enzymatic activities were measured (inside dotted frame). For each assay, specific activities are shown as the average results of 4 experiments. Standard deviations are indicated.</p

Deletion of the <i>MoPACC</i> gene.

<p>(a) Schematic representation of the <i>MoPACC</i> gene replacement by the hygromycin resistance gene flanked by 0.9 kb of downstream and 1.1 Kb of upstream sequences from the <i>MoPACC</i> locus. Primers (short arrows; <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0069236#pone.0069236.s002" target="_blank">Table S1</a>) used for PCR analysis of the transformants are indicated together with the probe (black bar) used for Southern analysis and distances between the EcoRV restriction sites (dotted double arrow). (b) Southern analysis of the parental (P) strain, three control transformants (C1–C3; transformed with plasmid pFV8 carrying a <i>HPH</i>-only DNA cassette) and eight transformants of interest (T1–T11) using the DNA probe shown in (a). (c) PCR amplification of <i>MoPACC</i> using primers pacC1 and pacC2. (d) PCR amplification using pacC3 and hygro1. (e) PCR amplification using pacC4 and hygro2.</p

<i>M. oryzae</i> modulates its environmental pH.

<p>(a) pH of the medium (white circles) and ammonia accumulation (small black circles) during growth of the wild type strain Guy11 in liquid non-buffered TNK-YE medium. (b) pH of the infection zone during plant infection by the wild type Guy11 strain; detached barley (black) or rice (grey) leaves were infected with drops of conidia and the pH was recorded over time using a surface electrode. Results are the average of three independent experiments and standard deviations are shown.</p

Expression of the <i>M. oryzae</i> pH-signaling related genes.

<p>Following 15 minutes (grey) or 2 hours (black) transfer of the wild type Guy11 strain from non-buffered liquid medium to fresh medium buffered to pH 5 or pH 8, total mRNA was extracted and quantitative PCR analysis was carried out using primers specific of the <i>MoPACC</i> and all the six <i>MoPAL</i> genes (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0069236#pone.0069236.s002" target="_blank">Table S1</a>). Quantification was based on the 2^ΔCT method using the <i>MoEF1</i>α gene as reference. Three independent biological replicates were analyzed for each studied gene. a) Gene expression after transfer to pH 5. b) Gene expression after transfer to pH 8. c) Change in expression between pH 5 and pH 8 (ratios are plotted using a logarithmic scale). d) <i>MoPACC</i> expression following 2 hours transfer of the fungus to media buffered to different pH values.</p

Results of the complementation studies.

<p>Growth and conidiation <i>in vitro</i> were measured for the complemented strain Δ<i>PACC+PACC</i> under the same conditions used for the study of the Δ<i>PACC</i> mutant strain (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0069236#pone-0069236-g005" target="_blank">Fig. 5</a>).</p

<i>MoPACC</i> and <i>MoPAL</i> genes.

<p>The <i>M. oryzae PAL</i> and <i>PACC</i> genes are presented. Introns were searched using the Softberry software and both their number (bold) and positions (start-end) are given. The length of the corresponding predicted proteins and the identity of these proteins to their <i>A. nidulans</i> counterparts is provided.</p