Septins from the phytopathogenic fungus Ustilago maydis are required for proper morphogenesis but dispensable for virulence

Abstract

Background: Septins are a highly conserved family of GTP-binding proteins involved in multiple cellular functions, including cell division and morphogenesis. Studies of septins in fungal cells underpin a clear correlation between septin-based structures and fungal morphology, providing clues to understand the molecular frame behind the varied morphologies found in fungal world.

Methodology/principal findings: Ustilago maydis genome has the ability to encode four septins. Here, using loss-of-function as well as GFP-tagged alleles of these septin genes, we investigated the roles of septins in the morphogenesis of this basidiomycete fungus. We described that septins in U. maydis could assemble into at least three different structures coexisting in the same cell: bud neck collars, band-like structures at the growing tip, and long septin fibers that run from pole to pole near the cell cortex. We also found that in the absence of septins, U. maydis cells lost their elongated shape, became wider at the central region and ended up losing their polarity, pointing to an important role of septins in the morphogenesis of this fungus. These morphological defects were alleviated in the presence of an osmotic stabilizer suggesting that absence of septins affected the proper formation of the cell wall, which was coherent with a higher sensitivity of septin defective cells to drugs that affect cell wall construction as well as exocytosis. As U. maydis is a phytopathogen, we analyzed the role of septins in virulence and found that in spite of the described morphological defects, septin mutants were virulent in corn plants.

Conclusions/significance: Our results indicated a major role of septins in morphogenesis in U. maydis. However, in contrast to studies in other fungal pathogens, in which septins were reported to be necessary during the infection process, we found a minor role of septins during corn infection by U. maydis.

Figure 1. Septins localization throughout the cell cycle.

Cells expressing N-terminal GFP-tagged Sep1-4 under their native loci were grown to log phase at 28°C and observed with a widefield fluorescence microscope. Representative images were chosen to show the progression of septin localization throughout the cell cycle.

Figure 2. Septins subcellular localization.

A. DeltaVision captured images were deconvolved and cross-section images of the bud neck and the bud tip were obtained using the Imaris 6.0.1 software. B. Images of the complete volume of GFP-Sep4 cells were taken every 0.2 µm. A selection of images was chosen to show that the filaments run close to the cortex but no throughout the middle of the cytoplasm (middle section, z). C. GFP-Sep4 cells were grown to log phase and observed in a DeltaVision microscope. Representative images of different cell cycle stages are shown. Insets show a zoom in of the bud neck in which the filaments crossing through can be observed.

Figure 3. Septin deletion mutants display altered cell morphology and temperature sensitivity.

A. Spot test of tenfold serial dilutions of wild-type and sep1-4 deletion mutants grown at different temperatures. Septin deletion strains were unable to grow at 34°C. B. Double septin deletion mutants. sep4Δsep1Δ and sep4Δsep3Δ strains were lethal while the rest of possible combinations were viable. C. Wild-type and septin deletion mutant cells expressing a nuclear localisation signal tagged with GFP (NLS-GFP) were grown to log phase at 22°C (top panel) or 28°C (bottom panel) and stained with calcofluor (CF) to observe the cell wall. Widefield microscope images were captured. In comparison with wild-type cells, at 22°C a minor bud neck defect was observed in mutant cells (bendy (arrowhead) and wider (arrow) bud necks). However, at 28°C septin deleted cells showed a strong morphology defect characterised by a swollen region in the middle of the cell (asterisk) strongly stained by calcofluor. Cells became rounded at the centre but maintained some polar growth at their tips and divided by placing a septum across the middle of the cell (empty arrowhead).

Figure 4. Sensitivity of septin mutants to temperature and cell wall inhibitors. Rescue of phenotypes by 1M sorbitol.

A. Spot tests of tenfold serial dilutions of wild type and septin deletion mutants grown onto YPD with 1M sorbitol at different temperatures. The termosensitivity of septin mutants was rescued by the osmoregulator sorbitol. B. Wild type and septin deletion mutant cells were grown to log phase at 34°C and observed with a widefield fluorescence microscope. At 34°C septin mutant cells became rounded, placed a septum at the central region to divide and presented a cell separation defect. Finally they died by cell lysis. All these phenotypes were rescued by addition of 1M sorbitol. Bar: 10 µm. C. Spot test of tenfold serial dilutions of wild type and septin deletion mutants grown with the cell wall inhibitors calcofluor white (CFW), chlorpromazine (CPZ) and caffeine (Caff).

Figure 5. Septin deletion mutants are more sensitive to BFA.

A. Spot test of tenfold serial dilutions of wild type and septin deletion mutants grown at 22°C onto YPD with brefeldin A (BFA, 25 µM) or its solvent, methanol. Septin mutants were more sensitive than wild type cells to BFA. B. Wild type and septin deletion mutants cells were grown to log phase at 22°C in YPD and treated with BFA (200 µM) or its solvent methanol for 12 h. BFA treatment mildly affected wild type cell morphology. In contrast, septin mutant cells became rounded and lysated. In both cases a cell separation delay was observed. Bar: 10 µm.

Figure 6. Septin dependencies in the formation of the different septin structures.

A. GFP-Sep1 localization in sep2Δ, sep3Δ and sep4Δ mutant backgrounds. B. GFP-Sep2 localization in sep1Δ, sep3Δ and sep4Δ mutant backgrounds. C. GFP-Sep3 localization in sep1Δ, sep2Δ and sep4Δ mutant backgrounds. D. GFP-Sep4 localization in sep1Δ, sep2Δ and sep3Δ mutant backgrounds. Sep4 fibers were not observed in any of these strains suggesting that they required the presence of the other septins to assemble. A non-organized cortical matrix was observed in sep1Δ and sep2Δ cells (insets). In sep3Δ cells short filaments were scattered close to the cell cortex (inset). In all cases cells were grown to log phase at 22°C.

Figure 7. Sep4 filaments and the F-actin and microtubule cytoskeletons.

A. Fim1-GFP and GFP-Sep4 cells were grown to log phase and treated for 10 minutes with Latrunculin A (LatA, 10 µM) or its solvent DMSO. Fim1-GFP localization to F-actin patches was lost upon LatA treatment but GFP-Sep4 filaments were present as they were upon solvent treatment. B. Tub1-GFP and GSP-Sep4 cells were grown to log phase and treated for 20 minutes with Benomyl (BNM; 30 µM) or its solvent DMSO. Microtubule depolymerisation upon BNM treatment did not affect the maintenance of GFP-Sep4 filaments. C. Tub1-RFP GFP-sep4 cells were grown to log phase and red and green filter sets were used to capture sequential images over the complete volume of the cells. DeltaVision deconvolved images are shown alongside a Merge image. A partial co-alignment of Sep4 and microtubules was observed (arrow).

Figure 8. Virulence of septin mutants.

A. Leaves of plants inoculated either with wild-type or septin mutant strains. B. Quantification of tumor formation on infected maize plants

Figure 9. Septins are required for the proper formation of b-dependent filaments.

A. Crosses of control strains FB1 X FB2 and septin mutant strains in charcoal-containing agar plates. Note the gray appearance of mutant crosses indicating impairment in filament formation. B. Septin localization in b-dependent filaments. GFP-septin alleles were introduced into AB31 strain, which carries compatible bE and bW alleles under the control of crg1 promoter and forms b-dependent filaments upon shift to minimal medium with arabinose as carbon source. Images were obtained after 6 h of incubation in inducing conditions. C. Morphology phenotype of septin null mutants in AB31 background. Observe that septin null mutants showed impaired hyphal growth after 6 hours of incubation in inducing conditions, as well as bipolar filamentation. Bar: 15 µm. D. Graph indicating the percentage of cells that grew bipolarly 6 hours after filament induction. Error bars indicate s.d., more than 50 filaments were analysed for each strain.

Figure 10. Septins are required for the proper germination of teliospores.

A. Images of control and septin mutant teliospores germinated on CM-glucose-containing agar slides after 24 h of incubation at 22°C and 34°C. Note that wild-type teliospores extend a promycelium, from which haploid progeny are generated. In mutant teliospores aberrant germination is observed. Bar: 20 µm. B. Graph indicating the percentage of teliospores able to germinate after 24 h at two different temperatures (22°C and 34°C). Teliospores which germination produced germ tubes swelled or that were abnormal in shape, as well as teliospores producing more than one promycelium were considered as aberrant. Error bars indicate s.d., more than 100 teliospores were analysed for each strain. C. Graph indicating the percentage of teliospores displaying one or more than one germination tube after 24 h at 22°C. Error bars indicate s.d., 100 teliospores were analysed for each strain.