Figure 1.
pep1 is essential for pathogenic development of U. maydis.
A: Predicted structure of Pep1. The protein comprises 178 aa. Signal-P (http://www.cbs.dtu.dk/services/SignalP/) predicts a putative N-terminal secretion signal (aa 1–26). In the central part of the protein four cysteine residues are present (C59, 75, 94, 112). The C-terminal part is enriched in glycine residues (aa 141–178). B: Disease rating of Early Golden Bantam maize plants 12 dpi after infection with U. maydis strains SG200, SG200Δpep1 (Δpep1), SG200Δpep1-pep1 (Δpep1-pep1), SG200pep1:gfp (pep1:gfp) and SG200Δpep1-pep1:gfpIP (pep1:gfpIP). Numbers indicate the total number of plants infected in three independent experiments. For details of the disease rating see Materials and Methods.
Figure 2.
Microscopic analysis of early infection-related development of U. maydis Δpep1 strains.
Pathogenic development of SG200rfp and SG200Δpep1rfp was visualized 24 hpi on maize leaves expressing PIN1-YFP. A: SG200rfp (red) penetrated the epidermis (arrowhead) and shows hyphal branching inside epidermis cells. Open arrowheads: Empty section of penetrated hyphae on the leaf surface. B: SG200rfp grows intracellularly in the epidermal layer, being completely encased by the plant plasma membrane (green). C, D: SG200Δpep1rfp hyphae grow on the leaf surface but fail to invade epidermis cells. Mutant hyphae are arrested immediately upon penetration of the plant cell wall (arrowheads and inserts: hyphal tips of SG200Δpep1rfp invaginate the plant plasma membrane at attempted sites of penetration). Bars are given.
Figure 3.
Plant responses elicited by infection with SG200Δpep1.
A: Macroscopic symptoms on maize leaves 4 dpi with SG200 and SG200Δpep1. Red arrowheads mark necrotic regions in SG200Δpep1 infected leaf tissue. B: Papilla formation in maize cells attacked by SG200Δpep1. Upper panel: Cell wall autofluorescence. Lower panel: Bright field projection of the same cell. Bar: 20 µm. C: H2O2 accumulation at penetration sites was visualized by DAB staining; 48 hpi. Left panel: SG200 appressoria do not induce H2O2 accumulation. Right panel: Penetration attempts of SG200Δpep1 are accompanied by a local accumulation of H2O2 (red arrowheads). Some SG200Δpep1 hyphae display multiple penetration attempts (lower right panel). Since SG200Δpep1 cells penetrate the cell wall the DAB stain accumulates in a focal plane below the fungal cell while the hyphae are still on the leaf surface, which explains the limited sharpness of these images. Bars: 5 µm. D: Hierarchical clustering of differentially regulated maize transcripts 24 hpi with SG200Δpep1. Colors represent expression levels for each gene being above (red) or below (blue) the mean expression level (white) in mock infected tissue (a), SG200 infected tissue (b) or SG200Δpep1 infected tissue (c).
Figure 4.
Expression and secretion of Pep1.
A: Haploid sporidia of strain SG200pep1:gfpR grown in YEPSL express RFP while Pep1-GFP fluorescence is not detectable. Bar: 5 µm. B: SG200pep1:gfpR penetrating a maize epidermis cell; 24 hpi. The Pep1-GFP signal demarcates the point of penetration and becomes visible in the intracellular hyphal part (arrow). Bar: 5 µm; C: Intracellular growing hyphae of SG200pep1:gfpR showing Pep1-GFP secretion around the tip region; 48 hpi. Bar: 2 µm. D: Tip of intracellularly growing hypha of SG200pep1:gfpR during cell to cell passage. Pep1-GFP strongly accumulates at penetration sites. E: Left panel shows SG200pep1:gfpR during cell to cell passage, 48 hpi. Right panel shows the rupture of the cell wall of the same cell inflicted by the penetrating fungal hyphae (arrow); Bars: 2 µm. Pictures A, B and C are maximum projections of confocal stacks. Green: Pep1-GFP; red: RFP; grey: plant cell wall autofluorescence induced by UV-laser. In D a confocal snapshot of a single optical layer is shown.
Figure 5.
Secretion of Pep1-mCherry into the maize apoplast.
A, B: SG200pep1M growing intracellularly in epidermal cells of maize line ZmPIN1a-YFP, 48 hpi. A1, A2 and A3 show the same hyphae with PIN1-YFP (green), Pep1-mCherry (red) and the merged yellow signals indicating co-localisation (arrowheads) around fungal hyphae, respectively. At sites of cell-to-cell passages, Pep1-mCherry is spreading from the fungal hyphae (A2, insert; B). Bars: 5 µm. C, D: SG200pep1M growing intracellularly in epidermal cells of maize line ZmTIP-YFP, 48 hpi. Plasmolysis was induced by 1 M NaCl, collapse of vacuoles results in enlarged apoplastic spaces. In cells colonized by SG200pep1M, this space is filled by Pep1-mCherry (+) which is not the case in cells not colonized by the fungus (−). Bars: 15 µm.
Figure 6.
Immunolocalization of Pep1-HA in U. maydis infected maize tissue.
A, B: Confocal projections showing immunolocalization of Pep1-HA in maize tissue infected by SG200Δpep1-pep1HA. Pep1-HA is detected around intracellular hyphae (h), predominantly accumulating at sites of cell to cell passage (arrowheads). Bars are given.
Figure 7.
Intracellular growth of a strain expressing pep1 under control of the otef promoter 7 dpi.
A, B: Hyphae of SG200Δpep1otef:pep1-gfp grow intracellularly, demonstrating functionality of Pep1-GFP driven by the otef promoter. C, D: Insufficient expression of Pep1-GFP leads to intracellular entrapment of fungal hyphae, that show multiple, unsuccessful attempts to leave the infected cell (arrowheads). Bars: 5 µm.
Figure 8.
Pep1 is conserved among U. maydis and U. hordei.
A: Sequence alignment of U. maydis Pep1 (Um) and U. hordei Pep1 (Uh). Identical amino acids are highlighted in green. Red boxes: conserved cysteine residues; black box: putative N-terminal secretion signal; blue box: poorly conserved glycine-rich C-terminal region. B: Cumulative plot of synonymous (red line) / non-synonymous (green line) substitutions in Pep1 from U. maydis and U. hordei. Calculation was done using the SNAP software tool (http://www.hiv.lanl.gov/content/sequence/SNAP/SNAP.html). Whereas the N-terminal and C-terminal parts of the proteins show a high ratio of non-synonymous substitutions, the central part of Pep1 (hatched box) shows a preference for sequence conservation. C–E: Confocal maximum projections of U. hordei 4 dpi in Golden Promise barley plants. C: Hyphae of strains 4875-5 crossed with 8a inside the leaf tissue. Hyphae (stained by WGA-AF488; green) show directed growth towards a vascular bundle (stained by propidium iodide, red). D, E: Infection by U. hordei Δpep1 strains 4 dpi (8aΔpep1×4875-5Δpep1) reveals successful penetration into epidermal cell, collapse of the invaded epidermis cell and no further proliferation in the plant tissue. Hyphae were stained by WGA-AF488 (green); dead plant cells are stained by propidium iodide (red). Bars correspond to 25 µm. F: Disease rating of Early Golden Bantam maize plants 12 days after infection with U. maydis strains SG200, SG200Δpep1 (Δpep1), SG200Δpep1-pep1 (Δpep1-umpep1) and SG200Δpep1-uhpep1 (Δpep1-uhpep1). Abbreviations of the respective strain designations are given in brackets. Numbers indicate the total number of plants infected in three independent experiments. The categories for the disease rating are given above. For details of the disease rating see Materials and Methods.
Figure 9.
A: Disease rating of Early Golden Bantam maize plants 12 dpi with U. maydis strains SG200, SG200Δpep1-pep1 (Δpep1-pep1), SG200Δpep1-pep1CS59 (pep1CS59), SG200Δpep1-pep1CS75 (pep1CS75), SG200Δpep1-pep1CS59,75 (pep1CS59,75), SG200Δpep1-pep1CS59,75,94,112 (pep1CS), and SG200Δpep1-pep1Δ141–178 (pep1Δ141–178). Abbreviations of the respective strain designations are given in brackets. Numbers indicate the total number of plants infected in three independent experiments. The categories for the disease rating are given above. For details of the disease rating see Materials and Methods. B–E: Intracellularly growing hyphae of strain SG200-pep1:gfpCS59,75 (B–D) and strain SG200pep1:gfp (E), 48 hpi. Pep1CS59,75-GFP (D) is not secreted at the hyphal tip and accumulates inside the hyphae compared to Pep1-GFP (E). B, C: Confocal pictures showing an overlay of GFP signal (green) and bright field projection (grey). D, E: Confocal pictures showing an overlay of GFP signal (green) and UV-laser induced cell wall autofluorescence (grey). Bars correspond to 5 µm.
Table 1.
Strains used in this study.