Efficient acquisition of sucrose by a plant pathogenic fungus.

<p>Model for the uptake of sucrose by the biotrophic plant pathogenic fungus <i>Ustilago maydis</i> based on a new study by Wahl and colleagues <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1000308#pbio.1000308-Wahl1" target="_blank">[15]</a>. <i>U. maydis</i> possesses a high-affinity sucrose H+-symporter, Srt1, which is present in the plasma membrane of its invasive hyphae. The plant–fungus interface is established by invagination of the plant plasma membrane during intracellular invasive growth by the fungus. Srt1 competes for apoplastic sucrose with the plant SUC (or SUT) sucrose transporters <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1000308#pbio.1000308-Sauer1" target="_blank">[17]</a>,<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1000308#pbio.1000308-Carpaneto1" target="_blank">[18]</a> and with plant invertases, which result in glucose and fructose generation. This reduces direct uptake of sucrose by plant cells or hexose uptake by the STP transporters, allowing the fungus to derive its primary carbon source from living plant cells without eliciting plant defence mechanisms. Adapted from Figure 8 of Wahl et al. <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1000308#pbio.1000308-Wahl1" target="_blank">[15]</a>.</p

Genome assemblies of Magnaporthe oryzae isolated from Bangladesh in 2016 and 2017

<div>Genomic DNA from nineteen isolates of <i>Magnaporthe oryzae</i> sampled from barley, wheat and torpedo grass in Bangladesh were sequenced using Illumina HiSeq 2500 producing 125 base paired-end reads. Reads were assembled ‘de novo’ using Spades.</div

Photographs to show growth test of M. oryzae on differnet lipid sources.

<p>The wild type strain Guy11 and isogenic <i>Δfar1</i>, <i>Δfar2</i>, and <i>Δfar1Δfar2</i> double mutants were grown on minimal medium with fatty acids as the sole carbon sources. The following carbon sources were added to minimal medium; acetate (50 mM) propionate (0.1%), butyrate(10mM), olive oil (1%w/v), oleic acid (50mM) and triolein (50mM) in agar plates, inoculated with a <i>M.oryzae</i> mycelial plug and incubated at 24°C for 12 days.</p

Characterization of Far1 and Far2 from <i>M. oryzae</i>.

<p><b>A.</b> Schematic diagram to show domains found in protein sequences of Far1 and Far2 identified using Pfam (accession numbers shown in brackets in key) and Prosite scans. Amino acid positions are marked on the diagram. <b>B</b>. Multiple sequence alignment (using clustal) showing Zn₂-Cys₆ binuclear cluster domain in selected sequences from <i>FAR1</i> and <i>FAR2</i> clade. <b>C</b>. Maximum likelihood phylogenetic tree created using PhyML. Bootstrap support values of 70 or greater are indicated on tree.</p

Bar charts to show gene expression profiles of <i>PEX6</i>, <i>PTH2</i>, <i>MFP1</i> and <i>ICL1</i> genes in <i>M. oryzae</i> mutants incubated in A. oleic acid and B. triolein as sole carbon source.

<p>Total RNA was extracted from fungal mycelium that was grown in complete medium for 48h and then transferred to minimal medium containing oleic acid or triolein for 24; <i>PEX6</i> (peroxisomal biogenesis), <i>MFP1</i> (fatty acid β-oxidation), <i>PTH2</i> (acetyl-CoA translocation), <i>ICL1</i> (glyoxylate cycle), were determined using quantitative real-time RT-PCR.</p

Bar charts to show gene expression profile of A. <i>ACS2</i> and B. <i>ACS3</i> acetyl CoA synthetase gene in Guy11 and the isogenic, <i>Δfar1</i> and <i>Δfar2</i> mutants.

<p>Total RNA was extracted from fungal mycelium that was grown in complete medium for 48h and then transferred to minimal medium containing oleic acid or triolein for 24<i>ACS2</i> and <i>ACS3</i> was determined using quantitative real-time RT-PCR.</p

Functional characterization of <i>FAR1</i> and <i>FAR2</i> genes of <i>M. oryzae</i>.

<p><b>A</b>. <i>FAR1</i> and <i>FAR2</i> are expressed during appressorium development in <i>M. oryzae</i>. Gene expression was evaluated using supersage analysis and a heat map is shown in which gene expression at 4h, 6h, 8h, 14h, and 16h of appressorium development is compared to expression in mycelium grown in complete media. The transcription initiation factor TFIID subunit 14 (MGG_05204) is included to indicate relative expression of a constitutively expressed gene. <b>B.</b> Epifluorescence micrographs to show nuclear localisation of FAR1-GFP, FAR2-GFP and histone H1-RFP gene fusions in <i>M. oryzae.</i> Bar = 10 µm. <b>C.</b> Bar charts to show conidiogenesis in Δ<i>far1</i>, Δ<i>far2</i> and Δ<i>far1</i>Δ<i>far2</i> mutants compared to the wild type, Guy11. Significant differences (P<0.05 or P<0.01) are indicated by one or two asterisks, respectively. <b>D.</b> Rice blast symptoms produced by Δ<i>far1</i>, Δ<i>far2</i> and Δ<i>far1</i>Δ<i>far2</i> mutants after 5 days of inoculation. Bar charts to show lesion counts from 100 infected leaves. No significant difference was observed between the mutant strains and Guy11 (P >0.01).</p

Micrographs to show lipid body mobilisation in <i>M. oryzae</i> during a time course of appressorium morphogenesis.

<p><b>A.</b> Wild type strain Guy11, <b>B. </b><i>Δfar1Δfar2</i> double mutant and <b>C.</b> autophagy mutant <i>Δatg8</i>. Lipid droplets in germinating conidia and appressoria were visualized by staining with Bodipy 493/503 (Invitrogen). Conidia were inoculated onto plastic cover slips in a moist chamber at 24 °C and observed for appressorium formation and lipid body mobilization at intervals, by mounting directly in fresh Bodipy 493/503 solution for 15 min. (Scale bar = 10 µm).</p

<i>MoTSC13</i> is required for full virulence of <i>M. oryzae</i>.

<p>(<b>A</b>) Targeted deletion of <i>MoTSC13</i> results in reduced pathogenicity on rice CO-39. Lesion density and size was severely reduced in ΔMotsc13 rice infections compared to Guy11. Full pathogenicity of Δ<i>Motsc13</i> mutants was restored by introduction of <i>MoTSC13:GFP</i> (Compl.). Lesion density represents the lesion number per 5 cm of infected leaf area (n>40). (<b>B</b>) <i>MoTSC13</i> is necessary for penetration peg formation and invasive hyphae expansion during <i>in planta</i> growth. Scale bar = 10 µm.</p

Macroautophagy is necessary for nuclear degeneration during appressorium development in <i>M. oryzae</i>.

<p>(<b>A</b>) The Macroautophagy gene <i>MoATG1</i> is required for nuclear degeneration. Upper panel: time course live cell images showing nuclear division and nuclear degeneration during appressorium development in a <i>M. oryzae</i> Δ<i>Moatg1</i> mutant. Δ<i>Moatg1</i> conidia expressing <i>H1:RFP</i> were examined by epifluorescence microscopy at indicated time points. Lower panel: time series of bar charts showing the percentage of spores in Δ<i>Moatg1</i> containing 0 to 4 nuclei (mean ± SD, n>100, triple replications). (<b>B</b>) The Macroautophagy core gene <i>MoATG4</i> is required for nuclear degeneration. Upper panel: time course live cell images showing nuclear division and nuclear degeneration during appressorium development in <i>M. oryzae</i> Δ<i>Moatg4</i> mutant. Δ<i>Moatg4</i> conidia expressing <i>H1:RFP</i> were examined by epifluorescence microscopy at indicated time points during appressorium development. Lower panel: time series of bar charts showing the percentage of spores in Δ<i>Moatg4</i> containing 0 to 4 nuclei (mean±SD, n>100, triple replications). Scale bar = 10 µm.</p