Retromer Is Essential for Autophagy-Dependent Plant Infection by the Rice Blast Fungus

We thank Dr. Yizhen Deng at the Temasek Life sciences Laboratory (TLL) for providing the RFP-MoAtg8 plasmid. We would like to thank Drs. Zhenbiao Yang (University of California, Riverside) and Xianying Dou (Fujian Agriculture and Forestry University) for helpful discussions.Author Summary The rice blast fungus Magnaporthe oryzae utilizes key infection structures, called appressoria, elaborated at the tips of the conidial germ tubes to gain entry into the host tissue. Development of the appressorium is accompanied with autophagy in the conidium leading to programmed cell death. This work highlights the significance of the Vps35/retromer membrane-trafficking machinery in the regulation of autophagy during appressorium-mediated host penetration, and thus sheds light on a novel molecular mechanism underlying autophagy-based membrane trafficking events during pathogen-host interaction in rice blast disease. Our findings provide the first genetic evidence that the retromer controls the initiation of autophagy in filamentous fungi.Yeshttp://www.plosgenetics.org/static/editorial#pee

Subcellular localization of the retromer subcomplex.

<p>MoVps35-GFP localizes to punctate structures at or near the vacuolar membrane, and partially colocalizes with FM4-64 that marks endosomal and vacuolar membranes in conidia (A) and mycelium (B). Shown are confocal epifluorescent micrographs. Scale bar = 10 μm. (C) Time-lapse video microscopy of MoVps35-GFP-labeled punctate structures. Arrowheads indicate the relative position of MoVps35-GFP-labeled compartments at each time point. Elapsed time is indicated in seconds. See also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005704#pgen.1005704.s017" target="_blank">S3 Movie</a>. Scale bar = 10 μm. (D) and (E) show localization of MoVps26-GFP and MoVps29-GFP to similar punctate structures at or near the vacuolar membranes. Scale bars = 10 μm.</p

A model for the retromer function in Atg8 retrieval.

<p>Autophagy requires the formation of double-membrane bound autophagosomes that associate with a set of evolutionarily conserved autophagy-related proteins, including Atg8. During the autophagocytosis, Atg8 has to be conjugated to the lipid phosphatidylethanolamine (PE), resulting in the expansion of the phagophore membrane and formation of autophgosomes. At the late stage, the autophagosomes fuse with the vacuoles to form autophagolysosomes to deliver the sequestered material for degradation and/or recycling. In yeast, the outer membrane-bound Atg8 was released into the cytosol by delipidation before autophagomes-vacuole fusion, presumably for reuse in subsequent rounds of autophagosome formation. In our study, we found that <i>M</i>. <i>oryzae</i> retromer core complex (MoVps35-MoVps26-MoVps29) localized at the late endosome/ prevacuolar membranes, where it interacts with cleaved and lipidated Atg8 thus regulating its trafficking to the phagophore or autophagosome and preventing its degradation in the lumen of vacuole.</p

MoVps35 is required for retrieving MoAtg8.

<p>(A) Close association between MoVps35-GFP (green) and RFP-MoAtg8 (red) in conidia. The enlarged inset highlights compartments showing co-localized MoVps35-GFP and RFP-MoAtg8. White arrow in the inset shows the path for fluorescence intensity distribution by line-scan analysis. Bar = 10 μm. (B) 3D images showing the association of MoVps35-GFP with RFP-MoAtg8 in conidia. (C) Images of a time-lapse sequence from <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005704#pgen.1005704.s024" target="_blank">S10 Movie</a> showing MoVps35-GFP-mediated retrieval of punctate RFP-MoAtg8. Arrowheads mark dynamic dissociation process of RFP-MoAtg8 and the colocalization between MoVps35-GFP and RFP-MoAtg8. The boxed regions (in white) highlight compartments showing dynamic retrieval of RFP-MoAtg8 by MoVps35-GFP. From top to bottom are images of MoVps35-GFP and RFP-MoAtg8 (merged), MoVps35-GFP (alone), and RFP-MoAtg8 (alone), Bars = 5 μm. Elapsed time is indicated in millisecond.</p

MoVps35 regulates appressorium turgor generation.

<p>(A) Appressorium turgor was measured by incipient cytorrhysis assays. Appressoria were allowed to form on plastic coverslips for 24 h or 48 h, and the collapsed appressoria assessed after exposure to 2 M glycerol solutions. White arrows indicate the appressoria in the wild type (WT) or <i>ΔMovps35</i> strain. Black arrows indicate the conidial morphology when appressoria formed after 24 h or 48 h. Wild-type strain showed typical collapsed conidia during appressorial maturation (24 hpi). However, the conidia from the <i>ΔMovps35</i> mutant were still intact and turgid. Bar = 20 μm. (B) Proportion of collapsed appressoria after exposure of conidia to 2 M or 3 M glycerol, double asterisks indicate statistically significant differences (P < 0.01).</p

MoVps35 is essential for host penetration during rice blast.

<p>(A) Infection-related morphogenesis assays. Normal morphology and melanized appressoria formed by the wild type, <i>ΔMovps35</i> mutant and complemented strains on plastic cover slips. (B) Penetration assays with onion epidermal cells. By 48 h after inoculation, invasive hyphae were evident in plant cells penetrated by WT and complemented strains but not by the <i>ΔMovps35</i> mutant. A, appressorium; C, conidium; IH, invasive hyphae. (C) Equal number of conidia from WT, <i>ΔMovps35</i> and complemented strains were inoculated on barley leaves. At 48 hpi, the papillary callose deposits (arrowheads) were evident in the barley leaves inoculated with conidia from the WT or complemented strains, but were absent in <i>ΔMovps35</i>-inoculated samples. Scale Bars = 20 μm. (D) and (E) Quantifications of penetration pegs and infection hyphae from three independent experiments at each time point, where values are indicated as percentages. The double asterisks indicate statistically significant differences (P < 0.01).</p

The retromer subcomplex MoVps35-MoVps26-MoVps29 is required for proper pathogenesis in the rice-blast fungus.

<p>(A) Pathogenicity assays on rice seedlings. Rice leaves (<i>Oryza sativa</i> cv. CO39) sprayed with conidia from the wild-type strain <i>ΔKu70</i>, <i>ΔMovps35</i> mutant and the complemented <i>ΔMovps35</i> strain. (B), (C), (D) are pathogenicity assays on barley leaves. Barley leaves were inoculated with mycelial plugs (B) or with 20 microliter of conidial suspension (ca 10⁵ conidia/mL) (C). (D) Barley infection using wounded/abraded leaves. Lesion formation on leaves was observed 7 days after inoculation with mycelial plugs from the indicated strains. (E) Yeast two-hybrid assay for the interaction between retromer components. (F) and (G) Mutant analysis in <i>MoVPS26</i> and <i>MoVPS29</i> suggests a role for cargo-specific retromer in <i>M</i>. <i>oryzae</i> pathogenicity. Conidia from the indicated strains were inoculated on the susceptible rice (CO39) seedlings and imaged 7 days post inoculation.</p