Endocytosis Is Essential for Pathogenic Development in the Corn Smut Fungus Ustilago maydis

Characterization of Wild-Type and Conditional yup1^ts Mutant Strains in Plant Infection Assays.

(A) Maize plants were infected with wild-type control strains and yup1^ts mutants at 22 and 34°C.

(B) Quantification of tumor formation on infected maize plants at 22 and 34°C

(C) Cross of control strains FB1 × FB2 and yup1^ts mutant strains FB1Yup1^ts × FB2Yup1^ts on charcoal-containing agar plates at 22 and 34°C.

(D) Filamentous growth and formation of conjugation hyphae were induced in strain FB1 and in FB1Yup1^ts at 34°C. DMSO was used as a solvent control.

(D1) Pheromone perception was visualized using the mfa1 promotor–GFP fusion construct as a reporter at 34°C. Both conjugation hyphae formation and pheromone perception were quantified at 22 and 34°C. Bars = 5 μm.

(D2) Quantitative analysis of conjugation hyphae formation at 22 and 34°C.

(D3) Quantification of cells expressing pmfa-GFP after pheromone stimulation.

Localization of Pra1-GFP.

(A) Strain FB1PraG was treated with 10 μM of the actin inhibitor LatA (A2) or with the solvent DMSO (A1) for 45 min before washout of LatA (A3). Insets highlight the amounts of Pra1-GFP in the membrane. Bar = 3 μm.

(B) Line-scan analysis of Pra1-GFP signal intensities. Scanning lines (dashed lines) are indicated in (A). a.u., arbitrary units.

(C) Quantitative analysis of the intensity of Pra1-GFP in the tip of conjugation hyphae of strain FB1Pra1G in the presence or absence of LatA.

(D) Incubation of yeast-like cells of strain FB1PraG with synthetic pheromone for 3 h induced the formation of conjugation hyphae (DIC [differential interference contrast]). Pra1-GFP (green) localized to the tip of the hyphae (arrowhead), where it formed an apical cap (overlay, right inset). In addition, Pra1-GFP localized in subapical organelles (Pra1-GFP, arrows) that costained with CellTracker Blue (red; celltracker), indicating that they were vacuoles (overlay, left inset; colocalization results in yellow). Bar = 5 μm.

(E) Analysis of Pra1-GFP signal intensities at the tip of conjugation hyphae at different times after treatment with LatA.

(F) Colocalization of Pra1-GFP (green) with the double RFP-tagged EE markerYup1 (red) on small motile organelles (yellow in overlay). Bar = 1 μm.

(G) Time-lapse microscopy of Yup1RFP₂ (red) with Pra1GFP (green), resulting in yellow in the merged images. Arrowheads indicate moving vesicles. Bar = 1 μm.

Localization of Pra1-GFP in the yup1^ts Mutant Background.

(A) Localization of Pra1-GFP in strain FB1Yup1^tsPra1G and the control strain FB1Pra1G that were stimulated with pheromone for 2 h at 22°C before the shift to 34°C for 2 h. Arrows indicate primary endocytic vesicles. The arrowhead points to the accumulation of Pra1-GFP at the tip. Bar = 5 μm.

(B) Colocalization of vacuoles stained with CellTracker Blue (red) and Pra1-GFP (green) in strains FB1Pra1G (control) and FB1Yup1^tsPra1G at 34°C. Colocalization results are in yellow. Bar = 1 μm.

(C) Double labeling experiments in yup1^ts mutants demonstrate that Pra1-GFP (green) and endocytic membranes stained with FM 4-64 (red) colocalize in the apical cytoplasm in yup1^ts cells. Bar = 1 μm.

(D) Pra1-GFP–containing vesicles obtained from the protein extracts of pheromone stimulated and shifted the control strain and FB1Yup1^tsPra1G at 34°C. The merged image represents vesicles isolated from FB1Yup1^tsPra1G at 34°C, which was also incubated with FM 4-64. Bars = 1 μm.

(E) Analysis of the localization of Pra1-GFP (green) with respect to the plasma membrane (red) in control cells and yup1^ts mutant hyphae at 34°C. A line scan of the intensities demonstrates that FM4-64 and Pra1-GFP colocalized at the edges of the cell ([E2], arrows; scanning line indicated in [E1], overlay). In yup1^ts cells, Pra1-GFP was depleted from the plasma membrane and accumulated within the cytoplasm in the hyphal tip ([E3] and [E4]). Bars = 1 μm.

(F) Immunolocalization of Pra1-GFP in control (F1) and yup1^ts mutant (F2) cells. Bars = 0.5 μm.

Quantitative Analysis of Pra1-GFP Recycling.

(A) Pra1-GFP signals in the tip of conjugation hyphae. Cells were treated with water (control) or cycloheximide (+cyclo) for 45 min. Hyphae were incubated for an additional 120 min with LatA/cycloheximide or DMSO/cycloheximide. Bar = 1 μm.

(B) Quantitative analysis of corresponding Pra1-GFP signals in (A). *** P < 0.001. a.u., arbitrary units.

Complementation of yup1^ts Mutants.

(A) Protein gel blots showing levels of Pra1-GFP in cell extracts of the yup1^ts mutant background at native levels and Pra1-GFP expressed under the control of the constitutive otef promotor.

(B) Localization of Pra1-GFP in yup1^ts (FB1Yup1^tsPraG) and with additional expression of Pra1-GFP under the control of the constitutive otef promotor (FB1Yup1^tsoPra1GFP) at 22°C. High levels of pra1-gfp expression increased the amount of receptor in the plasma membrane (inset). Bars = 2 μm.

(C) Strain FB1Yup1^tsmRoPraG was grown at 34°C and stimulated with synthetic pheromone or DMSO as a control. The inset highlights the increased membrane signal of Pra1-GFP. Bar = 10 μm.

(D) Quantification of the response to synthetic pheromone of control and yup1^ts cells treated with synthetic pheromone or synthetic pheromone and LatA. Bars represent the percentage of cells that show mfa promoter–induced RFP expression. Error bars represent values ± sd.

Mating and Fusion Ability of the yup1^ts Mutant Constitutively Expressing Pra1.

(A) Conjugation hyphae formation of wild-type and yup1^ts cells expressing otef Pra1 with synthetic pheromone to analyze the ability to form conjugation hyphae.

(B) Confrontation assay of mating partners expressing otef Pra1 as well as cytoplasmic GFP or RFP to identify their mating types. Asterisks in the control panel indicate the nuclei in the haploid conjugation hyphae. Their fusion site to form the dikaryotic filament is marked by the arrow. Fused hyphae express both GFP and RFP, resulting in yellow. yup1^ts cells did not fuse (inset).

(C) Assay for the fusion of GFP- or RFP-labeled mating partners on charcoal plates after incubation overnight. Fused hyphae express both RFP and GFP, resulting in yellow. The top insets show the overall colony morphology observed after overnight incubation at 34°C. The bottom left inset in the yup1^ts panel shows a rare fusion event of mutant cells. The bottom right inset depicts hyphae that have been incubated for 3.5 d. Bars = 10 μm.

(D) Quantification of tumor formation after infection of maize plants with control and yup1^ts mutant strains both expressing additional Pra1/Pra2 under the control of the constitutive otef promotor. Error bars represent values ± sd.

Analysis of yup1^ts Mutants in Planta.

(A) Control and yup1^ts hyphae stained with calcofluor after incubation at 22°C for 14 h and subsequent shift to 34°C. Appressoria that penetrate the plant surface are formed by control and yup1^ts hyphae (insets). Bar = 5 μm.

(B) Images of Chlorazole Black E–stained control and yup1^ts hyphae after 1 d of incubation at 22°C and subsequent shift to 34°C for 1 to 2 d. Bar = 10 μm.

(C) Quantitative analysis of tumor formation in plants infected with control strains and yup1^ts strains after incubation at 22°C for 3 d and subsequent shift to 34°C for a total of 14 d after infection. Error bars represent values ± sd.

(D) Whole tumors of infected plants were harvested 3 weeks after infection. Wild-type tumors contain black teliospores (arrow) that form at the edge of the tumor (control, inset). By contrast, yup1^ts tumors are devoid of teliospores in the center as well as at the edge of the tumor (yup1^ts). Bar = 1 cm.

(E) Higher magnification of tumor tissue confirmed the presence of teliospores in wild-type tumors (control), whereas teliospores were absent in yup1^ts tumors and only hyphal fragments were visible (yup1^ts). Bar = 10 μm.

Teliospore Germination.

(A) Quantification of teliospore germination at the permissive temperature (22°C) and the restrictive temperature (34°C) for control and yup1^ts spores. At 34°C, germination of yup1^ts teliospores is greatly impaired and only slightly increases after incubation (yup1^ts+) for 3 d. Germination of control teliospores is set to 100%.

(B) Images of control and yup1^ts teliospores germinated at 22°C on CM-glucose–containing agar slides. Bar = 10 μm.

(C) and (D) Images of control and yup1^ts teliospores germinated at 34°C. Bars = 5 μm.

(C) Yup1^ts spores after incubation for 1 d at 34°C (inset) and 3 d.

(D) When yup1^ts teliospores managed to germinate, they again showed morphological alterations (left). Control teliospores germinated at 34°C after 1 d of incubation (right).