Figures and data
Ppe1 is important for full virulence in M. oryzae. (A) The relative expression level of Ppe1 during infection of rice leaf sheaths assayed by qRT-PCR. (B) Fluorescence signals of the PPPE1-mCherry transformant in penetration assays with rice leaf sheaths. Scale bars = 10 μm. (C) Typical leaves of CO-39 seedlings sprayed with conidia of the wild type (70–15), PPE1 gene deletion mutants (Δppe1-45, Δppe1-101) and complement strains (Δppe1-com-3, Δppe1-com-8) examined at 7 days post inoculation (dpi). Boxplot below shows the number of disease lesions per 5 cm of individual inoculated leaf. n = 22, 23, 19, 19, and 22, respectively. (D) Wounded leaves of CO-39 drop-inoculated with the marked strains were examined at 10 dpi (upper panel) and measured for the lesion area (lower panel). n = 10, 9, 7, 5, and 4 respectively. (E) Rice leaf sheath penetration assays. Conidia suspensions from each strain were respectively injected into 4-5 weeks old rice leaf sheaths. Infection levels were observed at 32 hpi (hours post inoculation). The infection levels were grouped into four types: Type 1, appressorium formation without penetration; Type 2, having penetration peg or primary invasive hyphae; Type 3, having more than two branched invasive hyphae restricted in the first infected rice cell; Type 4, having invasive hyphae crossing to neighboring host cells. Scale bar = 10 μm. Statistical analyses of the infection types and percentages are also shown. Significant differences were determined by one-way ANOVA. Asterisks indicated significant differences (p<0.0001). ns indicated no significant difference (p>0.05).
Ppe1-mCherry forms a ring during appressorial penetration. (A) A ring of Ppe1-mCherry signals at the penetration site was observed at 22 hpi in leaf sheath penetration assays with the PPE1-mCherry transformant. Upper panel indicates the Ppe1-mCherry expressed in wild-type strain (70–15). Lower panel indicates the Ppe1-mCherry expressed in Δppe1 mutant. Scale bars = 2 μm. (B) The Ppe1-mCherry penetration ring was persist even after the growth of primary and secondary invasive hyphae. Scale bars = 5 μm. (C) The diameters of appressoria, Ppe1-mCherry penetration ring (ring), and its inner cycle. n= 21, 65 and 65 respectively. (D) The formation of Ppe1-GFP rings at the penetration site formed by the PPE1-GFP transformant. Scale bars = 2 μm. (E) The in-situ PPE1-mCherry transformant also formed a fluorescent ring at the penetration site in leaf sheath penetration assays. Scale bar = 2 μm. AP, Appressorium. Triangle arrows pointed to penetration rings.
Penetration ring formation during invasive hyphal penetration into neighboring cells. (A) Ppe1-mCherry forms rings at about 36 hpi. (B) Ppe1-GFP also forms rings similar to Ppe1-mCherry during penetration to neighboring cells. (C) Ppe1 ring formation is also observed during barley infection. Scale bars = 10 μm. Arrows heads point to penetration rings at the penetration sites of invasive hyphae. White squares indicate zoomed regions.
The penetration ring localizes on the periphery of penetration peg. (A) Ppe1 ring surrounds the Sep3 constricted ring. Scale bar = 2 μm. (B) Ppe1 ring also surrounds the Lifeact ring. Scale bar = 1 μm. (C) Ppe1 ring is formed around the narrow site between an appressorium and invasive hypha. Three-dimensional (3D) volume rendering images were constructed by confocal optical z-series images captured at 0.35 μm intervals, with a total of 22 steps. (D) Ppe1 ring partly colocalized with callose ring at the apoplastic papilla. Scale bar = 1 μm. Hollow triangles indicate the site of penetration peg. Line graphs were generated at the directions pointed by the white arrows.
Penetration ring formation requires the emergence of penetration peg. (A) Ppe1 ring expression and localization in 70-15, Δmst12, Δnox2 and Δnox1 strains at 24 hpi. Conidia suspensions from each strain were inoculated on rice leaf sheaths and observed at 24 hpi. (B) Ppe1 ring could not be formed on impenetrable coverslips. Conidia from Ppe1-mCherry strain were inoculated on hydrophobic coverslips and observed at 24 hpi. (C) Ppe1 ring was observed at 38 hpi on penetrable cellophane membrane. Scale bars = 2 μm.
Other secreted Ppe proteins also localize to the penetration ring. (A) Phylogenetic tree of Ppe1 constructed using neighbor-joining method. (B) Paralogs of Ppe1 in M. oryzae localize to the penetration ring. Ppe2-mCherry, Ppe3-mCherry and Ppe5-mCherry were driven by their respective native promoters. MoTPpe1-mCherry from Magnaporthe oryzae Triticum was expressed in M. oryzae (70–15), driven by its native promoter. Scale bars = 2 μm. (C) Comparison of the localization of penetration ring with those of some previously reported fungal effectors. Bas4-GFP and Slp1-GFP uniformly outlined the extra-invasive hyphal membrane (EIHM), while Pwl2-mCherry and AvrPiz-t-GFP accumulated in the biotrophic interfacial complex (BIC). Scale bars = 10 μm.
A proposed working model for the discovery of penetration ring in M. oryzae. Penetration ring forms at the periphery of peg and accumulates fungal secreted protein such as Ppe1 to promote M. oryzae invasion.
Ppe1 is dispensable for mycelial growth, conidiation and appressorium formation in M. oryzae. (A) Amino acid sequence of M. oryzae Ppe1. Signal peptides (green) were predicted by Signal-5. Cysteine residues are shown in red. (B) Detection of Ppe1 signal peptide secretion by yeast signal trap assay. The recombinant pSUC2-Ppe1SP and pSUC2-Pwl2SP constructs were transformed into the yeast strain YTK12 respectively. The pSUC2-Pwl2SP-expressing strain served as a positive control while pSUC2 empty vector was used as a negative control. (C) Targeted gene deletion strategy for PPE1 in M. oryzae 70-15. Schematic map showing targeted disruption of PPE1 gene via Agrobacterium tumefaciens-mediated transformation. The Southern blot result shows a successful replacement of PPE1 by single insertion of hygromycin phosphotransferase (HPH) resistance cassette at PPE1 ORF locus. (D) Colonies of the wild type (70–15), two independent Δppe1 transformants, and complemented strains. (E) Statistical analyses of the colony diameters of each strain after being cultured on complete medium (CM) at 25 °C for 10 days. n = 9. (F) Statistical analyses of the number of conidia produced by each strain after being cultured on CM at 25 °C for 10 days. n = 17. (G) Appressorium formation by the indicated strain observed at 24 hours on artificial hydrophobic surfaces. Scale bar = 50 μm. Significant differences were determined by one-way ANOVA. ns stands for no significant difference (p>0.05).
Detection of Ppe1-mCherry fusion protein by western blot. M, protein marker. CK, rice leaf sheaths injected with sterile double distilled water.
Ppe1 ring is formed simultaneously with peg penetration. (A-C) Spatiotemporal dynamics of Ppe1 ring and Sep3 ring during appressorium-mediated peg penetration into rice leaf sheaths. Scale bars = 2 μm. Hollow triangles show the sites of penetration peg. Line graphs were generated at the directions pointed by the white arrows.
Spatiotemporal dynamics of Ppe1-mCherry and Sep3-GFP on hydrophobic coverslips. Conidia were harvested from a strain co-expressing Ppe1-mCherry and Sep3-GFP and inoculated on hydrophobic coverslips and observed at each time point. Scale bars = 1 μm.
Ppe1 ring was also observed during transpressorium-mediated peg penetration. (A-D) The spatiotemporal dynamics of Ppe1-mCherry and Sep5-GFP during transpressorium-mediated cell-to-cell penetration. Red squares indicate the zoomed regions, respectively. Scale bars = 10 μm.
Fungal strains used in this study.
