Figure 1.
Analysis of pexophagy during M. oryzae conidiation and pathogenic development.
(A) Confocal microscopy of conidiating cultures grown on PA medium, from M. oryzae 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 in planta 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.
Figure 2.
Generation of pexophagy-deficient mutants in M. oryzae
(A) Schematic illustration of pexophagy occurring during conidial germination and appressorial development in M. oryzae. 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 snx41Δ 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 (http://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastp&BLAST_PROGRAMS=blastp&PAGE_TYPE=BlastSearch&SHOW_DEFAULTS=on&LINK_LOC=blasthome). (D) Schematic drawing of the annotated domains within M. oryzae 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.
Figure 3.
Characterization of pexophagy-deficient mutants in M. oryzae.
(A) GFP-SRL mis-localizes to the cytoplasm in the pex14Δ mutant. Scale bar equals 10 micron. (B) Pexophagy is normal (restored) in M. oryzae atg26Δ, PEX141-258, and snx41Δ (ScSNX42) 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 PEX1461-361 and snx41Δ with ScSNX41 (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.
Figure 4.
Characterization of conidiation and pathogenicity in pexophagy-deficient strains.
(A) Detection of pexophagy defects by biochemical assay. WT, atg26Δ, PEX141-258, PEX1461-361, snx41Δ 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 [38] as follows: Percentage reduction of Thiolase level = (DensityPe-DensityPx)/DensityPe. (B) Bar chart depicting quantification of conidiation in the wild type (WT), atg26Δ, PEX1461-361, PEX141-258, snx41Δ, snx41Δ (ScSNX41) and snx41Δ (ScSNX42) 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), atg26Δ, PEX1461-361, PEX141-258, snx41Δ (ScSNX41) and snx41Δ (ScSNX42) strains. Disease symptoms were assessed 7 d post inoculation.