Investigation of the pH signalling pathway in the phytopathogenic fungus Magnaporthe grisea

La perception de l’environnement extérieur est importante pour des interactions efficaces entre plantes et champignons. Chez les champignons filamenteux, l’information associée au pH extracellulaire est transmise via une voie de signalisation conservée et impliquant sept protéines. Parmi ces protéines, la protéine transmembranaire PalH est un récepteur présumé initier la réponse aux pH neutre ou alcalin. Le facteur de transcription PacC, présent sous forme inactive dans le cytoplasme de la cellule fongique, est clivé en réponse à l’activation de la voie, et migre dans le noyau où il active la transcription des « gènes alcalins » et réprime la transcription des « gènes acides ». Chez Magnaporthe grisea, un Ascomycète responsable de la principale maladie du riz, le rôle de cette voie dans la physiologie du champignon est encore inconnu. Les deux homologues des gènes PACC et PALH ont été identifiés. Dans le but d’analyser le rôle des deux protéines PacC et PalH chez M. grisea, la délétion de ces gènes a été réalisée. Plusieurs phénotypes ont été analysés chez les deux souches mutantes, notamment la croissance, la sporulation et le pouvoir infectieux. Ceci a permis d’étudier le rôle de la signalisation liée au pH extracellulaire dans le cycle de développement de M. grisea. De plus, une analyse du profil des gènes exprimés chez le mutant ΔpacC a été initiée. Les résultats obtenus indiquent que cette voie est importante pour l’adaptation du champignon à un environnement alcalin et qu’elle joue un rôle dans la pathogénicité du champignonPerception of external environment is important for successful infection of plants by fungi. In these organisms, the information about extracellular pH is provided to the cell by a conserved signalling pathway that involves seven proteins. Among these proteins, the transmembrane protein PalH is the putative receptor which would initiate the pH response. The transcription factor PacC, existing in an inactive form in the fungus cell cytoplasm, is activated through proteolysis in response to the pathway activation, and migrates into the nucleus where it activates the « alkaline » genes transcription and represses that of the « acidic » genes. In Magnaporthe grisea , an ascomycete responsible for the main rice disease, the role of this pathway is still unknown. In this work, the PACC and PALH genes have been identified. In order to analyse the role of the two corresponding proteins PacC et PalH, the deletion of these two genes has then been performed. Several phenotypes were studied in the two mutant strains, including growth rate, conidiation and ability to infect host plants. This enabled the investigation of the involvement of the pH signalling pathway in the M. grisea development cycle. Furthermore, a gene expression profiling analysis of the ΔpacC mutant has been undertaken and revealed the multiple cellular responses to pH changes. Taken all together, the results collected in this work indicate that the pH signalling pathway is important for M. grisea's adaptation to an alkaline environment and that it plays a significant role in the fungus pathogenicit

Adaptation to pH and role of PacC in the rice blast fungus Magnaporthe oryzae.

Fungi are known to adapt to pH partly via specific activation of the Pal signaling pathway and subsequent gene regulation through the transcription factor PacC. The role of PacC in pathogenic fungi has been explored in few species, and each time its partaking in virulence has been found. We studied the impact of pH and the role of PacC in the biology of the rice pathogen Magnaporthe oryzae. Conidia formation and germination were affected by pH whereas fungal growth and appressorium formation were not. Growth in vitro and in planta was characterized by alkalinization and ammonia accumulation in the surrounding medium. Expression of the MoPACC gene increased when the fungus was placed under alkaline conditions. Except for MoPALF, expression of the MoPAL genes encoding the pH-signaling components was not influenced by pH. Deletion of PACC caused a progressive loss in growth rate from pH 5 to pH 8, a loss in conidia production at pH 8 in vitro, a loss in regulation of the MoPALF gene, a decreased production of secreted lytic enzymes and a partial loss in virulence towards barley and rice. PacC therefore plays a significant role in M. oryzae's biology, and pH is revealed as one component at work during interaction between the fungus and its host plants

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