MoDnm1 Dynamin Mediating Peroxisomal and Mitochondrial Fission in Complex with MoFis1 and MoMdv1 Is Important for Development of Functional Appressorium in <i>Magnaporthe oryzae</i>

<div><p>Dynamins are large superfamily GTPase proteins that are involved in various cellular processes including budding of transport vesicles, division of organelles, cytokinesis, and pathogen resistance. Here, we characterized several dynamin-related proteins from the rice blast fungus <i>Magnaporthe oryzae</i> and found that MoDnm1 is required for normal functions, including vegetative growth, conidiogenesis, and full pathogenicity. In addition, we found that MoDnm1 co-localizes with peroxisomes and mitochondria, which is consistent with the conserved role of dynamin proteins. Importantly, MoDnm1-dependent peroxisomal and mitochondrial fission involves functions of mitochondrial fission protein MoFis1 and WD-40 repeat protein MoMdv1. These two proteins display similar cellular functions and subcellular localizations as MoDnm1, and are also required for full pathogenicity. Further studies showed that MoDnm1, MoFis1 and MoMdv1 are in complex to regulate not only peroxisomal and mitochondrial fission, pexophagy and mitophagy progression, but also appressorium function and host penetration. In summary, our studies provide new insights into how MoDnm1 interacts with its partner proteins to mediate peroxisomal and mitochondrial functions and how such regulatory events may link to differentiation and pathogenicity in the rice blast fungus.</p></div

MoDnm1, MoMdv1, and MoFis1 are important for peroxisomal and mitochondrial morphology.

<p>(A) Transmission electron microscopy (Hitachi H-7650) observation of peroxisomes in conidia of the indicated strains. Bar = 0.5 μm. P, peroxisome; M, mitochondria; N, nucleus. (B) Transmission electron microscopy (Hitachi H-7650) observation of mitochondria in hyphae of the indicated strains. Bar = 1 μm. M, mitochondria.</p

MoMdv1 functions as an adaptor linking MoDnm1 to MoFis1.

<p>(A) Yeast two hybrid assays for the interaction between MoDnm1, MoFis1 and MoMdv1. The AD and BD plasmids were co-transformed into yeast AH109, and transformants were plated on SD-Leu-Trp for 3 d and on selective SD-Leu-Trp-His-Ade with 1 mM X-gal and 5 mM 3-AT (3-amino-1,2,4-triazole) for 5 d. (B) GST pull down assays for MoDnm1, MoFis1 and MoMdv1. His₆-Mdv1, His₆-Fis1, GST-Dnm1 and GST-Fis1 were expressed and purified by affinity chromatography. Bound proteins were separated by SDS-PAGE in duplicate and analyzed by Western blotting with monoclonal anti-His (Mouse; M20001; Abmart) and anti-GST antibodies (Mouse; M20007; Abmart). Asterisks indicate GST-Dnm1.</p

Comparison of mycological characters among Guy11 and deletion mutant strains.

<p>Comparison of mycological characters among Guy11 and deletion mutant strains.</p

The InsB motif, DYNc and GED domains of MoDnm1 are important for Pathogenicity and peroxisomal fission.

<p>(A) Disease symptoms on rice seedlings sprayed with conidial suspensions at 7 dpi. (B) Observation of peroxisomal morphology in the indicated strains by confocal fluorescence microscope (Leica TCS SP8, 100x oil). Bar = 5 μm. (C) Statistical analysis of peroxisome numbers of different strains. The number of peroxisomes was counted by Image-pro plus software (Media Cybernetics Inc., Shanghai, China). ±SD was calculated from three repeated experiments and asterisks indicate statistically significant differences (Duncan's new multiple range test, <i>p</i><0.01).</p

Working model of MoFis1, MoMdv1 and MoDnm1complex in <i>M</i>. <i>oryzae</i>.

<p>The MoDnm1, MoFis1, and MoMdv1 complex mediates peroxisomal and mitochondrial fission to regulate pexophagy and mitophagy required for growth, conidiation and pathogenicity. In parallel, the complex could be involved in the regulation of autophagy that is also important for growth, conidiation, and pathogenicity.</p

MoDnm1, MoMdv1 and MoFis1 are required for normal endocytosis.

<p>Hyphae of the indicated strains were cultured in liquid CM for 40 h, then stained with FM4-64 and examined under confocal fluorescence microscope (Zeiss LSM710, 63x oil). Bar = 5 μm.</p

MoFis1 and MoMdv1 are important for virulence in <i>M</i>. <i>oryzae</i>.

<p>(A) Rice (<i>Oryza sativa</i> cv. CO39) seedlings were sprayed with conidial suspensions and examined 7 dpi. (B) Quantification of lesion types (per 1.5 cm²) on susceptible rice cultivar CO39 spayed with conidia of Guy11, Δ<i>Mofis1</i>, Δ<i>Momdv1</i>, Δ<i>Mofis1/MoFIS1</i>, and Δ<i>Momdv1/MoMDV1</i> strains. Asterisks represent significant differences (Duncan's new multiple range test, <i>p</i><0.01). (C, D) Detailed observation and statistical analysis for infectious growth in rice sheath cells at 36 hpi. Appressorium penetration sites (n = 100) were observed and invasive hyphae were rated from type 1 to 4. Error bars represent ±SD from three independent experiments. Bar = 5 μm. Asterisks indicate IH extended to surrounding cells. (E) Invasive hyphal growth of the Δ<i>Mofis1</i> and Δ<i>Momdv1</i> mutants in rice sheath cells at 48 hpi. Asterisks indicate IH extended to surrounding cells.</p

MoDnm1, MoMdv1 and MoFis1 are involved in mitophagy and pexophagy.

<p>(A) The indicated strains were cultured in liquid CM for 40 h, then shifted to BM-O for 20 h and starved in MM-N for 12 h. Cells were collected and the cell lysates were subjected to immunoblot analysis with the anti-Pex14 antibody. The amount of stable Pex14 was monitored by relative ratios of Pex14 and Actin (underneath the blot). Densitometric analysis was performed by Image-pro plus (Media Cybernetics Inc., Shanghai, China). (B) The indicated strains were cultured in liquid CM for 40 h, then shifted to BM-G for 30 h and starved in MM-N for 12 h. Mycelia were harvested and the cell lysates were subjected to immunoblot analysis with the anti-Porin antibody. The amount of stable porin was represented by relative ratios of porin and actin (underneath the blot). Densitometric analysis was performed by Image-pro plus (Media Cybernetics Inc., Shanghai, China).</p

MoPex11A, MoDnm1, MoMdv1, and MoFis1 all have functions on peroxisomal fission.

<p>(A) a, Disease symptoms on the rice seedlings which were sprayed with conidial suspensions at 7 dpi. b, Disease symptoms on the detached barley which were drop-inoculated with conidial suspensions and examined at 24 hpi. Bar = 10 μm. (B) Observation of peroxisomal morphology in the indicated strains using confocal fluorescence microscope (Leica TCS SP8, 100x oil). Bar = 5 μm.</p