Figure 1.
Filamentous growth of U. maydis on the plant surface.
The dikaryotic filament of U. maydis grows by inserting retraction septa at the distal end of the filament. For details see main text. Arrows indicate growth direction; asterisk marks a new assembled retraction septum and the cytoplasm-filled tip compartment is drawn in grey.
Figure 2.
drf1 expression is induced during filamentous growth and its deletion shows a cell separation defect.
A: Domain alignment of MmDrf1 (Mus musculus), UmDrf1 and UmSrf1. GBD: GTPase binding domain; FH1: Formin homology domain 1; FH2: Formin homology domain 2; DAD: Diaphanous autoregulatory domain; DID: Diaphanous inhibitory domain. B: Quantitative real time-PCR for drf1 and ppi (um03726) were performed on RNA prepared from budding cells (bud) and filaments (fil). Each column represents the mean ratio drf1/ppi from three independent experiments. ppi served as internal control. C: wt, Δdrf1 or Δcdc42 mutant cells were grown in rich media and stained with calcofluor white (CF). Abnormal chitin depositions are labelled with arrows. D: The secondary septa of wt and Δdrf1 mutant cells from logarithmic cultures were stained with calcofluor white and the intensities were measured using ImageJ. *: p<0.05, when compared to the wt strain. (Scale bars: 10 µm).
Figure 3.
Filaments of drf1 mutants do not form retraction septa.
A: The indicated haploid strains were spotted either alone or in combination on charcoal-containing medium. White colonies are indicative for the formation of aerial hyphae. Filamentous growth was analyzed one (1d) and two (2d) days after spotting the respective strains. B: Two-day-old filaments formed by the indicated crossings were stained with calcofluor white (CF; lower panel). The respective brightfield image is shown (upper panel). Filaments of drf1 deletion strains lack retraction septa. To depict more than 180 µm of the growing filament it was necessary to combine two images (Scale bars: 15 µm). C: Quantification of filament length. Using the same strains as in B, filament length was calculated using ImageJ. For each strain and time point 30 filaments were analyzed. Error bars indicate standard deviation.
Figure 4.
A: Compatible mixtures of the indicated haploid strains were spotted on charcoal-containing medium. Plates were incubated for two days. B: Pull-down experiment of GFP-Drf1-GBD: GST (lanes 1 and 2) or GST-Cdc42 (lanes 3 and 4) loaded with GDP (lanes 1 and 3) or GTPgS (lanes 2 and 4) were loaded with U. maydis protein extract containing GFP-Drf1-GBD (Ex). After pull-down immunoblotting (WB) was performed with anti-GFP antibody. Blots were stained with PoinceauS (PS) as loading control. GTP-bound Cdc42 could interact with the GBD of Drf1. C: Constitutive active Drf1ΔGBD was able to partially suppress the don1 deletion phenotype. Wt, Δdon1 and Δdon1+pETEF-Drf1ΔGBD were stained with calcofluor white (CF) (central columns). Arrows indicate secondary septum formation. For the colony morphology images (right column) single cells were grown for three days on YEPS with 1.3% Agar. (Scale bars: 10 µm for the left column, 2 µm for the magnifications and 1 mm for the colonies).
Figure 5.
Hyphal septation depends on CAR formation.
A: CAR formation in wt filaments is shown by the actomyosin ring marker Cdc15-GFP (green) and co-staining with calcofluor white (red). The magnification shows the formation of the first distal septum (red) while the actomyosin ring started to constrict (green). B: The same settings were used for the AB31Δdrf1 mutant expressing Cdc15-GFP. The region of the bud neck is magnified. C: The constitutive active variant Drf1ΔGBD-GFP (green) was expressed in AB31Δdrf1 and localized in filaments at the site of restored hyphal septation (red). (Scale bars: 15 µm).
Table 1.
Pathogenicity assay.
Figure 6.
Appressoria formation and virulence are reduced in the drf1, don1 and don3 deletion strains.
A: The indicated strains were injected into maize seedlings. One day after infection appressoria were quantified on the leaf surfaces using the appressorium specific marker AM1 (n>200). *: p<0.05, when compared to the SG200AM1 strain. The experiment was conducted in three biological replicates. Error bars indicate standard deviations. B: Disease symptoms caused by the solopathogenic SG200AM1 strain and its derivative drf1, don1 and don3 deletion strains (indicated below each column) were scored 12 days after infection. Symptoms were grouped into colour-coded categories according to Kämper et al. [9] depicted on the right. Average values of three independent experiments were expressed as percentage of the total number (n) of infected plants. ***: p<0.0001 using the Wilcoxon ranked sum test, when compared to the SG200AM1 strain.
Figure 7.
drf1 mutants are affected in appressoria formation in long filaments.
A: Appressoria formed by the solopathogenic SG200AM1 strain and its derivative drf1 deletion strain were quantified 20 hours after spraying cells on Parafilm in the presence of 16-hydroxyhexadecanoic acid. *: p<0.05, when compared to the SG200AM1 strain. B: Distribution of appressoria formed by filaments of different length was compared between SG200AM1 and SG200AM1Δdrf1. *: p<0.05, when compared to the SG200AM1 strain. Errorbars indicate standard deviation. C: Filaments were visualised using calcofluor white (red). Appressorium formation is indicated by expression of the AM1 marker (green). When filaments of drf1 mutants exceeded a certain length appressoria did not develop.
Figure 8.
Penetration efficiency from appressoria of drf1 mutants is reduced.
A: Confocal projections of colonized leaf tissue 18 hours after infection with the indicated strains. Fungal material on the leaf surface was stained with calcofluor white (red) and expression of the AM1 marker was monitored (green). “Unsuccessful penetration” describes appressoria that continued growth on the plant surface. “Undecided” describes appressoria, which have just been formed. Penetration events are divided in “no post-appressorial septum formation”, which is characterized by GFP-fluorescence in penetrating hyphae as well as in hyphal parts on the plant surface and “post-appressorial septum formation”, where GFP-fluorescence was exclusively found in penetrating hyphae. (Scale bars: 20 µm) B: The penetration efficiency was calculated by analyzing the indicated number of filaments from three independent experiments. *: p<0.05, when compared to the wt strain. Errorbars indicate standard deviation.
Figure 9.
Septum formation of drf1, don1 and don3 deletion mutants in planta.
Confocal projections of colonized leaf tissue three days after infection with the indicated strains. Fungal hyphae were stained by WGA-AF488 (green). Plant tissue was stained by propidium iodide (red). The formation of septa in intracellular growing hyphae is indicated by arrows. (Scale bars: 25 µm).
Figure 10.
The Cdc42 signaling network regulates septation events that are uncoupled to mitosis.
During the life cycle of U. maydis the coordination of septum formation can be either coupled to or uncoupled from mitosis. If septation is independent of mitosis it requires the signaling network consisting of Don1/Cdc42/Drf1 and Don3.