Regulation of Cellular Diacylglycerol through Lipid Phosphate Phosphatases Is Required for Pathogenesis of the Rice Blast Fungus, <i>Magnaporthe oryzae</i>

<div><p>Considering implication of diacylglycerol in both metabolism and signaling pathways, maintaining proper levels of diacylglycerol (DAG) is critical to cellular homeostasis and development. Except the PIP₂-PLC mediated pathway, metabolic pathways leading to generation of DAG converge on dephosphorylation of phosphatidic acid catalyzed by lipid phosphate phosphatases. Here we report the role of such enzymes in a model plant pathogenic fungus, <i>Magnaporthe oryzae</i>. We identified five genes encoding putative lipid phosphate phosphatases (<i>MoLPP1</i> to <i>MoLPP5</i>). Targeted disruption of four genes (except <i>MoLPP4</i>) showed that <i>MoLPP3</i> and <i>MoLPP5</i> are required for normal progression of infection-specific development and proliferation within host plants, whereas <i>MoLPP1</i> and <i>MoLPP2</i> are indispensable for fungal pathogenicity. Reintroduction of <i>MoLPP3</i> and <i>MoLPP5</i> into individual deletion mutants restored all the defects. Furthermore, exogenous addition of saturated DAG not only restored defect in appressorium formation but also complemented reduced virulence in both mutants. Taken together, our data indicate differential roles of lipid phosphate phosphatase genes and requirement of proper regulation of cellular DAGs for fungal development and pathogenesis.</p></div

Transcriptional profiling of <i>MoLPPs</i> in knockout mutant background.

<p>In each knockout mutant, relative transcript abundance of genes was measured for <i>MoLPP</i> genes</p

Restoration of appressorium formation and pathogenicity by addition of DAG.

<p>(A) Appressorium formation of wild-type and mutants in the presence of DAG and/or CaCl₂. Conidial suspension (2×10⁴ conidia/ml) was placed the hydrophobic side of cover slips and mixed with DAG and/or CaCl₂ to final concentrations of 20 µg/ml and 10 mM, respectively. Appressorium formation was observed under a microscope 8 and 12 h after incubation. (B) Spray inoculation (upper panels) and sheath assay (lower panels) with conidial suspensions supplemented with 20 µg/ml of DAG.</p

Transcript abundance of <i>MoLPP</i> genes in development stages in <i>Magnaporthe oryzae</i>.

<p>My, mycelia; Con, germinating conidia; App, appressoria; IF 72, early infection stage at 72 hour post inoculation (hpi); IF 148, late infection stage at 148 hour post inoculation (hpi).</p

Knock-out mutant generation, conidial germination and appressorium formation.

<p>(A) Targeted disruption of <i>MoLPP1</i>, <i>MoLPP2</i>, <i>MoLPP3</i> and <i>MoLPP5</i>. Schematic diagram of knockout of target gene (left) and Southern blot analysis of resulting mutant (right) are shown in pair for each gene. (B) Percentage of conidial germination and appressorium formation on hydrophobic surfaces with complemented strains. Asterisks indicate significant difference observed in the mutants, compared to the wild-type (Tukey test, p<0.05).</p

Multiple sequence alignments and Kyte-Doolittle hydropathy plots of PAP2 domain containing proteins in <i>M. oryzae</i>.

<p>(A) Alignments of three novel sequence motifs among eight PAP2 domain containing proteins using ClustalW algorithm. Different colors indicate the conserved sequence among eight proteins and numbers (left of motifs) show the starting position of three motifs. (B) Hydropathy plots of <i>MoLPP3</i> and <i>MoVAN</i> proteins showing potential transmembrane domains. Asterisks indicate the potential membrane-spanning domains. Hydropathy plot was generated using TopPred 2 (<a href="http://www.sbc.su.se/~erikw/toppred2/" target="_blank">http://www.sbc.su.se/~erikw/toppred2/</a>). Cutoff value (0.6) was indicated by dotted horizontal line.</p

Pathogenicity assay of knockout mutants.

<p>(A) Disease development after spraying conidial suspension onto rice leaves. Conidia (1×10⁵/ml) was sprayed onto the leaves and incubated for 7 days. (B) Comparison of diseased leaf area (DLA). The DLA was calculated relative to the total leaf area using the Axiovision image analyzer. (C) Invasive hyphal growth through sheath assay. Rice sheath was injected with 2×10⁴ conidia/ml and observed under microscope at 48 hour post inoculation (hpi). (D) Quantification of invasive growth. Number of cells that are invaded by the fungus was counted using sheath inoculation at 48 hour post inoculation (hpi).</p

Vegetative growth and asexual reproduction of <i>ΔMolpp1</i>, <i>ΔMolpp2</i>, <i>ΔMolpp3</i> and <i>ΔMolpp5</i> in <i>M. oryzae</i> with complementation.

a<p>Vegetative growth was measured at 12 dpi on complete agar medium and minimal agar medium. Data were presented as mean ± sd from three independent experiments.</p>b<p>Conidia was measured as the number of conidia from the culture flooded with 5 ml of sterilized distilled water. Data were presented as mean ± sd from three independent experiments.</p

Pathogenicity of wild-type, Δ<i>Moadh1</i> and Δ<i>Mosre1</i>.

<p>(A) The pathogenicity assay was performed by spraying a conidia suspension (5 × 10⁴ conidia/ml) of each strain onto susceptible rice seedlings. Photographs were taken 7 days after inoculation. (B) The pathogenicity assay via drop inoculation was examined. Conidia suspension (10⁵ conidia/ml) was dropped onto detached rice leaves and incubated at room temperature. dpi, days post inoculation.</p

Schematic diagram of SREBP regulation for adaptation to hypoxia.

<p>Sterols control activation of SREBP: it is inactive in the presence of and active in the absence of sterols. Growth in limited oxygen conditions inevitably resulted in a lack of sterols, activating SREBP. Increases in enzymes for sterol biosynthesis and other oxygen-dependent pathways produce more sterols, which can be used for mycelial growth.</p