Autophagy-related protein MoAtg14 is involved in differentiation, development and pathogenicity in the rice blast fungus Magnaporthe oryzae

Abstract

Autophagy is the major intracellular degradation system by which cytoplasmic materials are delivered to and degraded in the vacuole/lysosome in eukaryotic cells. MoAtg14 in M. oryzae, a hitherto uncharacterized protein, is the highly divergent homolog of the yeast Atg14 and the mammal BARKOR. The MoATG14 deletion mutant exhibited collapse in the center of the colonies, poor conidiation and a complete loss of virulence. Significantly, the ΔMoatg14 mutant showed delayed breakdown of glycogen, less lipid bodies, reduced turgor pressure in the appressorium and impaired conidial autophagic cell death. The autophagic process was blocked in the ΔMoatg14 mutant, and the autophagic degradation of the marker protein GFP-MoAtg8 was interrupted. GFP-MoAtg14 co-localized with mCherry-MoAtg8 in the aerial hypha. In addition, a conserved coiled-coil domain was predicted in the N-terminal region of the MoAtg14 protein, a domain which could mediate the interaction between MoAtg14 and MoAtg6. The coiled-coil domain of the MoAtg14 protein is essential for its function in autophagy and pathogenicity.

Figure 1

(A) The amino acid sequence of the N-terminal motif containing the conserved cysteine residues in the ascomycetes fungi. The conserved cysteine residues are in the box. The green line indicates the start of the conserved coiled-coil region. GgAtg14, accession No. XP_009224438; CgAtg14, accession No. EQB48915; CoAtg14, accession No. ENH80301; TvAtg14, accession No. XP_013959553; TrAtg14, accession No. XP_006966865; FoAtg14, accession No. EMT61395; FgAtg14, accession No. XP_011316371; BgAtg14, accession No. EPQ63265; AoAtg14, accession No. BAE65502; AfAtg14, accession No. XP_747209; PrAtg14, accession No. CDM36188. (B) The domains of the yeast ScAtg14 and M. oryzae MoAtg14. Boxes in grey indicate the coiled-coil domains. (C) The expression profiles of the MoATG14 gene in development, pathogenicity and starvation stress. qRT-PCR assays were carried out with RNA samples obtained from different stages of the wild-type strain Guy11, including vegetative hyphae, conidia (CO), appressoria, invasive hyphae (IH) and nitrogen starved hyphae (MM-N). Gene expression levels were normalized using the β-tubulin gene as an internal standard. Data are representative of at least two independent experiments with similar results, and the error bars represent the standard deviations of three replicates (P < 0.01). Different letters indicate a significant difference.

Figure 2. Targeted gene deletion of MoATG14 in M. oryzae.

(A) The MoATG14 locus and gene deletion vector. Arrows 1–8 indicate the primers ATG14up-1/2, ATG14dn-1/2, HPH-1/2 and ATG14-N1/2. (B) Southern blot analysis of ΔMoatg14 mutants 1 and 2, and the wild-type strain Guy11. Genomic DNA was digested with NcoI and separated on a 0.7% agarose gel. The DNA was hybridized with the probe (indicated in A) amplified from genomic DNA of Guy11.

Figure 3. Characteristics of M. oryzae strains.

(A) Guy11, the ΔMoatg14 mutant, and the complemented strain Moatg14c were grown on CM, V8, OMA, and MM medium for 8 days. (B) Few conidia were produced by the ΔMoatg14 mutant, in contrast to Guy11 and Moatg14c. Error bars represent one standard deviation (P < 0.01). Different letters indicate a significant difference in the conidiation of the ΔMoatg14 mutant, Guy11, and Moatg14c. (C) Development of conidia on conidiophores observed under cover slips with a light microscope 24 h after induction of conidiation. Few conidia developed in the ΔMoatg14 mutant. Scale bar = 50 μm. (D) Conidiation in the ΔMoatg14 mutant grown on CM medium and CM medium supplemented with 10 g/L maltose, 6.25 g/L sucrose, 10 g/L glucose, 1 mM G1P and 0.5 mM G6P. Error bars represent one standard deviation (P < 0.01). Different letters indicate a significant difference. (E) The MoAtg14 deletion mutant is nonpathogenic. Disease symptoms on cut leaves of barley inoculated with mycelial plugs from Guy11, the ΔMoatg14 mutant, and Moatg14c. Typical leaves were photographed 4 days after inoculation. Two-week-old rice seedlings were inoculated by spraying with 1 × 10⁵ conidia/ml conidia suspensions from Guy11, the ΔMoatg14 mutant, and Moatg14c. Lesion formation on the rice leaves was evaluated 7 days after inoculation.

Figure 4. Conidia of the wild-type strain, the ΔMoatg14 mutant, and the complemented mutant Moatg14c were allowed to form appressoria on plastic coverslips at 0 h, 4 h, 8 h and 24 h after inoculation.

(A) Cellular distribution of glycogen. Samples were stained with KI/I₂ solution. Microscopically, the glycogen appears as dark brown deposits. Scale bar = 10 μm. (B) The proportion of the conidial cells containing glycogen stained by KI/I₂ solution during appressorium development in the Guy11, the ΔMoAtg14 mutant, and Moatg14c. Error bars represent one standard deviation (P < 0.01). Different letters indicate a significant difference. (C) Cellular distribution of lipid droplets. Samples were stained with Nile red and observed in the dark with UV epifluorescence. The lipid droplets show a red signal fluorescence. Scale bar = 10 μm. (D) Collapse of appressoria. Conidia were allowed to form appressoria on plastic coverslips 24 h after inoculation, and the collapsed appressoria were assessed after exposure to 2 M or 3 M glycerol solution for ten minutes. Arrows indicate the collapsed portions of the conidia. Scale bar = 10 μm. (E) The turgor pressure of the appressoria was measured by incipient cytorrhysis assays. The proportion of the collapsed appressoria after exposure to 2 M or 3 M glycerol solution for ten minutes are shown. Error bars represent one standard deviation (P < 0.01). Different letters indicate a significant difference.

Figure 5. Autophagy was blocked in the ΔMoatg14 mutant.

(A) Autophagy was triggered in the starved mycelia. Numerous autophagic bodies were detected in the vacuoles of Guy11 and Moatg14c under starvation conditions. The mycelia were treated with 4 mM PMSF under nitrogen starvation for 4 h. No autophagic bodies were evident in ΔMoatg14 mutant vacuoles under the same conditions. Arrows indicate the vacuoles. (B) Normal GFP-MoAtg8 localization was impaired in the ΔMoatg14 mutant. Conidia were collected from the Guy11 and ΔMoAtg14 strains expressing GFP-MoAtg8. Appressoria: 1 × 10⁴ conidia of Guy11 and ΔMoAtg14 expressing GFP-MoAtg8 were inoculated on the hydrophobic cover slip and incubated for 8 h. Mycelia: aerial hyphae of Guy11 and ΔMoAtg14 expressing GFP-MoAtg8. The strains Guy11 and ΔMoAtg14 expressing GFP-MoAtg8 were grown in liquid CM medium at 25 °C for 48 h (N+), and shifted to liquid MM-N medium with 4 mM PMSF for 4 h (N−). Scale bar = 10 μm. (C) Conidial autophagic cell death assays of strains. The conidial cells of the wild-type strain and Moatg14c showed conidial autophagic cell death during appressoria development. The ΔMoAtg14 mutant had defects in conidial autophagic cell death. (D) The proportion of the conidial cells containing an FDA signal during the development of the Guy11, ΔMoAtg14 and Moatg14c strains. Error bars represent one standard deviation (P < 0.01). Different letters indicate a significant difference.

Figure 6. Co-localization of GFP-MoAtg14 and mCherry-MoAtg8. Scale bar = 10 μm.

Figure 7

(A) Yeast two-hybrid assays. The interactions between MoAtg14 and MoAtg14 CCD as bait and MoAtg6 and MoAtg6 CCD as the prey were assessed. Yeast transformants grown on the SD/-Ade/-Leu/-Trp/-His plates were assayed for β-galactosidase activity. (B) Schematic representation of deleted variants of MoAtg14. (C) The coiled-coil domain in MoAtg14 is essential to maintain normal colony morphology. Strains were grown on CM for 10 days. The asterisk indicates the collapse of the colony. (D) The coiled-coil domain in MoAtg14 is essential to maintain normal pathogenicity. Disease symptoms of rice inoculated with 1 × 10⁵ conidia from Guy11, the ΔMoatg14 mutant, Moatg14c, Moatg14-ΔC, Moatg14-ΔN, and Moatg14-ΔCCD. The lesions formed on the rice were photographed 7 days after inoculation.