The fungal pathogen <i>Magnaporthe oryzae</i> chemically disables the jasmonic acid (JA)–mediated defense signaling in rice.

<p><i>M</i>. <i>oryzae</i> secretes the antibiotic biosynthesis monooxygenase (Abm) and its endogenously produced chemical effector 12-hydroxy jasmonic acid (12OH-JA) in a biphasic manner to suppress host immunity during establishment of the blast disease in rice. Loss of Abm function leads to activation of the host defense via jasmonate signaling and consequently blocks fungal invasion in rice plants. Brownish orange inclusions depict the sites of methyl JA-induced innate immunity that blocks the <i>abm</i>Δ strain of <i>M</i>. <i>oryzae</i> in the first invaded rice cell. A, appressorium; C, conidium; IH, invasive hypha; JA-Ile, isoleucine conjugate of JA; Jaz9, jasmonate-ZIM domain repressor protein 9; MeJA, methyl JA; WT, wild-type <i>M</i>. <i>oryzae</i>. Hypothetical receptors or transporters for the fungal JA derivatives have been depicted on the host cell surface. The schematic has not been drawn to scale.</p

Characterization of pexophagy-deficient mutants in <i>M. oryzae</i>.

<p>(A) GFP-SRL mis-localizes to the cytoplasm in the <i>pex14</i>Δ mutant. Scale bar equals 10 micron. (B) Pexophagy is normal (restored) in <i>M. oryzae </i><i>atg26</i>Δ, <i>PEX14</i>_<i>1-258</i>, and <i>snx41</i>Δ (<i>ScSNX42</i>) strains (in GFP-SRL background). Vacuolar staining with Lysotracker Red DND99 was performed 5 min before confocal microscopy. Arrowheads denote vacuolar GFP-SRL. Conidia of <i>PEX14</i>_{<i>61-361</i>} and <i>snx41</i>Δ with <i>ScSNX41</i> (all in GFP-SRL background) co-stained with Lysotracker Red DND99 were analyzed by confocal microscopy. Arrows denote vacuolar compartments without GFP-SRL, indicative of a block in pexophagy. Bar = 10 μm.</p

Characterization of conidiation and pathogenicity in pexophagy-deficient strains.

<p>(A) Detection of pexophagy defects by biochemical assay. WT, <i>atg26</i>Δ, <i>PEX14</i>_<i>1-258</i>, <i>PEX14</i>_61-361, <i>snx41</i>Δ and the two complemented strains were grown in peroxisome biogenesis (Pe) or pexophagy induction (Px) conditions. The total lysates from aforementioned strains were subjected to immunoblotting with anti-thiolase (Thi) antibody. The immunoblot was reprobed with anti-Porin antisera as loading control. Percentage denotes reduction of Thiolase in Px condition compared to the Pe condition, as an indicator of pexophagy efficiency. Percentage reduction was calculated using ImageJ [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0079128#B38" target="_blank">38</a>] as follows: Percentage reduction of Thiolase level = (Density_Pe-Density_Px)/Density_Pe. (B) Bar chart depicting quantification of conidiation in the wild type (WT), <i>atg26</i>Δ, <i>PEX14</i>_{<i>61-361</i>}, <i>PEX141</i>_<i>-258</i>, <i>snx41</i>Δ, <i>snx41</i>Δ (<i>ScSNX41</i>) and <i>snx41</i>Δ (<i>ScSNX42</i>) strains grown on PA medium containing lactose as the sole carbon source. Note that values present averages (±S.E.) from three independent experiments (n = 30 colonies for each sample). Total conidia counts were performed 5 d post photo-induction. (C) Barley leaf explants were spot inoculated with conidia (2,000, 1000, and 500 per droplet) from wild type (WT), <i>atg26</i>Δ, <i>PEX14</i>_{<i>61-361</i>}, <i>PEX141</i>_<i>-258</i>, <i>snx41</i>Δ (<i>ScSNX41</i>) and <i>snx41</i>Δ (<i>ScSNX42</i>) strains. Disease symptoms were assessed 7 d post inoculation. </p

Generation of pexophagy-deficient mutants in <i>M. oryzae</i>

<p>(A) Schematic illustration of pexophagy occurring during conidial germination and appressorial development in <i>M. oryzae</i>. Vacuolar accumulation of GFP-SRL (peroxisome) was first seen in the two non-germinating cells (1 and 2) of the conidium at the early stage, whereas peroxisomes were outside the vacuoles in the germinating cell (3) and in the germ tube (4). In the newly formed appressorium (5), pexophagy was absent as peroxisomes were not delivered into the vacuoles. In mature appressoria, pexophagy occurs again and most of the peroxisomes were delivered to the lumen of the vacuoles. Right panel depicts pexophagy-competent (WT) or pexophagy-deficient situation in such cell types. Red spherical or filamentous compartments, vacuoles (stained by DND99 Lysotracker-Red); green vesicles, peroxisome labeled with GFP-SRL. (B) Pexophagy-competence and pexophagy-deficiency as illustrated by WT and <i>snx41</i>Δ mutant, respectively. Conidia were inoculated on plastic cover slip for 6-8 h, and co-stained with Lysotracker Red DND99 to label the vacuolar compartments 5 min before confocal microscopy. Arrowheads denote vacuolar GFP-SRL signal, an indication of delivery of peroxisomes to the vacuoles for pexophagy. Arrows denote vacuoles lacking GFP-SRL signal, due to blocked pexophagy. Scale bar equals 10 micron. (C) Comparison of amino sequence and domains between ScSnx41, ScSnx42 and MoSnx41. Sequence alignment and domain prediction were performed by using the BLAST program on NCBI website (<a href="http://tinyurl.com/2bp99mm" target="_blank">http://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastp&BLAST_PROGRAMS=blastp&PAGE_TYPE=BlastSearch&SHOW_DEFAULTS=on&LINK_LOC=blasthome</a>). (D) Schematic drawing of the annotated domains within <i>M. oryzae</i> Pex14 polypeptide showing an SH3 domain, a hydrophobic loop and a coiled-coil domain. Precise mutant derivatives for Pex14 are depicted together with the coordinates representing amino acid residues. </p

Analysis of pexophagy during <i>M. oryzae</i> conidiation and pathogenic development.

<p>(A) Confocal microscopy of conidiating cultures grown on PA medium, from <i>M. oryzae</i> GFP-SRL/RFP-Atg8 strain shows the lack of pexophagy therein during asexual development. Images shown are representative of the developmental stage depicted by the majority of vegetative mycelia, aerial hyphae or conidiophores. Dashed outline depicts aerial hyphae and its connecting mycelium. Scale bar equals 5 micron. (B) Pexophagy is naturally induced during appressorium formation and function. Conidia from the GFP-SRL/RFP-Atg8 strain were inoculated on inductive surface and visualized by confocal microscopy, at the indicated stages of germination (2-4 hpi), appressorium initiation (5-8 hpi) and appressorium maturation (14-16 hpi). For the assessment of <i>in </i><i>planta</i> differentiation, conidia were inoculated on barley leaf explants and visualized by confocal microscopy at the indicated stages. Arrowheads denote vacuoles containing GFP-SRL (peroxisomes). Arrows denote vacuoles without GFP-SRL. Scale bar equals 10 micron.</p

<i>twl</i>∆ shows increased sensitivity to oxidative stress.

<p><i>twl</i>∆ shows increased sensitivity to oxidative stress.</p

Twilight, a Novel Circadian-Regulated Gene, Integrates Phototropism with Nutrient and Redox Homeostasis during Fungal Development

<div><p>Phototropic regulation of circadian clock is important for environmental adaptation, organismal growth and differentiation. Light plays a critical role in fungal development and virulence. However, it is unclear what governs the intracellular metabolic response to such dark-light rhythms in fungi. Here, we describe a novel circadian-regulated <i>Twilight</i> (<i>TWL</i>) function essential for phototropic induction of asexual development and pathogenesis in the rice-blast fungus <i>Magnaporthe oryzae</i>. The <i>TWL</i> transcript oscillates during circadian cycles and peaks at subjective twilight. GFP-Twl remains acetylated and cytosolic in the dark, whereas light-induced phosphorylation (by the carbon sensor Snf1 kinase) drives it into the nucleus. The mRNA level of the transcription/repair factor <i>TFB5</i>, was significantly down regulated in the <i>twl</i>∆ mutant. Overexpression of <i>TFB5</i> significantly suppressed the conidiation defects in the <i>twl</i>∆ mutant. Furthermore, Tfb5-GFP translocates to the nucleus during the phototropic response and under redox stress, while it failed to do so in the <i>twl</i>∆ mutant. Thus, we provide mechanistic insight into Twl-based regulation of nutrient and redox homeostasis in response to light during pathogen adaptation to the host milieu in the rice blast pathosystem.</p></div

A proposed model for Twl-Snf1 regulated phototropic response during <i>M</i>. <i>oryzae</i> conidiation.

<p><i>TWL</i> transcript peaks before sunrise and the Twl protein remains cytosolic and acetylated in the dark, while concomitant deacetylation and phosphorylation (by Snf1) of Twl occurs in response to light exposure. Phosphorylated Twl translocates into the nucleus and likely activates <i>TFB5</i>, which induces conidiation in <i>M</i>. <i>oryzae</i>. Snf1 is subjected to regulation by light (likely at the transcriptional level) and nutrient status (post-translational modification). Tfb5 transcription is likely regulated via Twl-mediated phototropic induction, and its nuclear localization directly influenced by host-derived nutrients.</p

Twl facilitates redox homeostasis during <i>M</i>. <i>oryzae</i> pathogenicity.

<p>(A) Barley leaf explants were inoculated with conidia from the wild type or <i>twl</i>Δ. Disease symptoms were assessed after 5 days. Inoculum size (total number of conidia per droplet) is indicated accordingly. (B) Rice leaves from the susceptible cultivar IR31917 were inoculated with conidial droplets from wild type or <i>twl</i>Δ. Symptoms were examined at day 7 post inoculation. (C) Microscopic observation of <i>twl</i>Δ invasive hyphae developing in rice leaf sheath at 48 hpi, stained with 1% acid fuchsin. Mean values ± SE represent percentage of appressoria that differentiate invasive hyphae. Bar = 10 μm. For GSH treatment, L-Glutathione reduced (at a final concentration of 5 mM in water) was added to conidial droplets on rice leaf sheath at 24 hpi. (D) Rice leaves from 3 week-old IR31917 (blast susceptible) seedlings were wounded prior to inoculation with conidial suspension from wild type or <i>twl</i>Δ. The blast disease symptoms were evaluated 7 days post inoculation.</p

Specific protein interaction partners for Twilight identified using mass spectrometry.

<p>Specific protein interaction partners for Twilight identified using mass spectrometry.</p