A Class-V Myosin Required for Mating, Hyphal Growth, and Pathogenicity in the Dimorphic Plant Pathogen Ustilago maydis

Characterization of Δmyo5 and Temperature-Sensitive myo5^ts Mutants.

(A) Plate growth of FB2 (control) and myosin mutants at 20 and 34°C. At low temperature, the growth of control cells, Δmyo5, and myo5^ts is indistinguishable. However, both Δmyo5 and myo5^ts show growth defects at 34°C that can be rescued by the expression of a functional GFP-Myo5 fusion protein. Note that myo5 is not essential for the survival of U. maydis.

(B) Morphology defect of Δmyo5 cells. Wild-type cells are cigar shaped and form polar buds ([B1]). By contrast, deletion of myo5 leads to very thick and irregularly shaped cell aggregates. Most cells do not separate but form septa (arrow in [B2]). Interestingly, most Δmyo5 cells are elongated (arrowhead in [B3]), whereas older cells often lose polarity and are rounded (arrow in [B3]). Bars = 5 μm.

(C) Polar distribution of F-actin patches in Δmyo5 stained with anti-actin antibodies. Bar = 5 μm.

(D) Chitin distribution in Δmyo5. Rhodamine-labeled wheat germ agglutinin (WGA) detects newly synthesized chitin at cell tips (arrow), indicating that Δmyo5 mutants grow in a polar manner. Bar = 5 μm.

(E) Localization of chitin synthase detected with a cross-reactive antibody raised against Chs2p from S. cerevisiae (Sietsma et al., 1996) in Δmyo5 mutants. The antibody most likely detects chitin synthases at the growing tip of Δmyo5 cells, in agreement with the WGA staining of chitin. Bar = 5 μm.

(F) Morphology of temperature-sensitive myo5^ts mutants. At the permissive temperature (20°C, [F1]), FB2Myo5^ts strains grow by polar budding but are significantly thicker than control cells (see [B1] for comparison). At the restrictive temperature (34°C), mutant cells show a defect in dissolving the cell wall after cytokinesis (arrow in [F2]; 1.5 h at 34°C). At later stages at 34°C, they form septa (arrow in [F3]; 6 h at 34°C) and start branching. After extended growth at 34°C, myo5^ts mutants form large aggregates that are reminiscent of Δmyo5 cells. Bars = 5 μm in (F1) to (F3) and 10 μm in (F4).

(G) Chitin synthase distribution in myo5^ts cells. At the permissive temperature, anti-Chs2p antibodies detect punctate staining in the growing bud, a ring at the bud neck (arrow in [G1]), and the distal cell pole. At 34°C, this chitin synthase distribution is largely unaffected, although more anti-Chs2p signals are detected at the periphery (arrowhead in [G2]). Note that the distribution of anti-Chs2p signals is very much in agreement with chitin staining (not shown) and the expected localization of chitin syntheses at the growth region, suggesting that the cross-reactive antibody is specific for chitin synthase in U. maydis. Bar = 5 μm.

Localization of a Functional GFP-Myo5 Fusion Protein in Haploid Sporidia.

(A) GFP-Myo5 distribution during stages of the cell cycle. GFP-Myo5, expressed under the control of its native promoter, rescues the phenotype of Δmyo5 cells. In presumably nongrowing cells, GFP-Myo5 shows a dispersed localization at the cell periphery (A1). Before bud formation, cells begin to polarize and GFP-Myo5 concentrates at one cell pole (A2). In small-budded cells, GFP-Myo5 remains at the bud tip (A3), whereas in cells with medium-sized buds, the fusion protein localizes to the growing and the nongrowing ends of the cell (arrow in [A4]). Finally, it accumulates at the site of cell cleavage ([A5]; inset shows the equal signal intensity on both sides of the formed septa). Note that the distal localization of GFP-Myo5 (arrow in [A4]) coincides with the appearance of endosomes at this cell pole (Wedlich-Söldner et al., 2000, 2002), which suggests exocytotic activity at this cell end. Bar = 5 μm.

(B) Distribution of GFP-Myo5 and immunolabeled actin patches during the cell cycle of haploid U. maydis. During the early stages of budding, GFP-Myo5 (green) and actin (red) colocalize ([B1] to [B3]; overlay results in yellow). However, in large budded cells, actin appears at the bud neck (arrows in [B4] and [B5]), whereas most GFP-Myo5 still localizes to the growing bud tip. During cytokinesis, both GFP-Myo5 and actin concentrate at the cleavage site (B6). Bar = 5 μm.

(C) Upon oxygen depletion caused by prolonged observation, GFP-Myo5 localizes to filamentous structures. The inset shows a large bud that contains filaments that are in contact with the GFP-Myo5 accumulation at the tip. Bar = 5 μm.

(D) GFP-Myo5 localization after ATP depletion. In untreated cells expressing high levels of GFP-Myo5 (FB2oGMyo5), the tip signal intensity increases (D1). After treatment with CCCP, which inhibits the mitochondrial respiration chain and therefore depletes ATP, GFP-Myo5 stains long filaments (D2). These structures can be disrupted by the actin inhibitor cytochalasin D (CD [D3]), suggesting that they are F-actin cables. Bars = 5 μm.

(E) Localization of GFP-Myo5 after the disruption of microtubules, F-actin, or inhibited myosin activity. Treatment with the solvent DMSO (not shown) or benomyl (Ben [E1]) at concentrations that were shown to disrupt all microtubules (20 μM) (Straube et al., 2003) does not significantly affect GFP-Myo5 distribution. However, 50 μM of the potent F-actin inhibitor latrunculin A (LatA [E2]) as well as 10 mM of the myosin inhibitor BDM (E3) abolish the polar accumulation of GFP-Myo5, suggesting that actin-dependent transport underlies the tip localization of Myo5. Bar = 5 μm.

Localization of the GFP-Myo5 Fusion Protein in Hyphae.

(A) GFP-Myo5 localizes to the tip of dikaryotic hyphae derived from crossing FB2mGM and FB1 on charcoal plates. Note that no signal is detected at the septum (arrows) and that the tip staining varies from pointed dots to dispersed gradients (not shown). Bar = 10 μm.

(B) GFP-Myo5 in monokaryotic hyphae from liquid culture. Because of genetic manipulation, filamentous growth of strain AB33oGM can be induced by shifting cells to liquid medium containing nitrate. In these cells, GFP-Myo5 is restricted to a bright dot at the very tip of the hyphae. Note that prolonged observation results in dispersed distribution and filament staining, suggesting that these localization patterns reflect suboptimal growth conditions. Bar = 5 μm.

(C) Treatment with the solvent DMSO only slightly affects GFP-Myo5 distribution (C1) in AB33oGM. The addition of 20 μM benomyl (Ben) results in dispersed gradients of the fusion protein that lost the pointed localization (C2). By contrast, gradients are almost absent with 10 mM BDM (C3) and disappear completely in the presence of 50 μM latrunculin A (LatA [C4]). Bar = 5 μm.

(D) Quantitative analysis of GFP-Myo5 distribution in inhibitor-treated AB33oGM hyphae.

(E) Distribution of GFP-Myo5 in hyphae lacking Kin2, a conventional kinesin from U. maydis. On charcoal plates (E1), the control strain SG200 forms white colonies, whereas the kin2 deletion mutant that expresses GFP-Myo5 (Δkin2oGM) rarely forms filaments, thereby appearing gray on charcoal plates (Lehmler et al., 1997). In liquid culture, these hyphae contain an apical GFP-Myo5 dot (E2), demonstrating that conventional kinesin is not involved in Myo5 transport to the hyphal apex. Bar = 5 μm.

Analysis of the Formation of Dikaryotic myo5^ts Hyphae on Charcoal-Containing Plates.

Wild-type strains (WT × WT, 28°C) formed white colonies that consist mainly of dikaryotic hyphae. Crossing of wild-type and Δmyo5 strains shows drastically impaired filament formation (WT × Δmyo5, 28°C) that is restored by expression of the functional GFP-Myo5 fusion protein (WT × Δmyo5GFPMyo5). Mating reactions of wild-type and temperature-sensitive myo5^ts strains show no defects in filament formation at permissive and restrictive temperatures (WT × Myo5^ts, 20 and 28°C). The formation of dikaryotic hyphae is impaired in crosses of myo5^ts cells under permissive conditions (Myo5^ts × Myo5^ts, 20°C) and is abolished completely at the restrictive temperature (Myo5^ts × Myo5^ts, 28°C). However, filamentation is restored by incubation at the permissive temperature for 2 days followed by growth at the restrictive temperature (Myo5^ts × Myo5^ts, 20 to 28°C). This finding suggests that myo5^ts strains fail to form filaments at an early stage of the mating process but hyphae appear after the initial fusion is complete.

Defects of myo5^ts Mutants during Early Steps of Mating.

(A) At the beginning of mating and before conjugation tube formation, secreted pheromone of the opposing mating partner is recognized and mfa1 expression increases. In myo5^ts and control strains, GFP was put under the control of the mfa1 promoter, which allows the visualization of pheromone perception. In addition, the ability to form conjugation tubes was monitored in confrontation assays, in which adjacent mating partners bridge large distances by the formation of conjugation hyphae. In a negative control of cells of the same mating type, tubes are not formed and mfa-GFP expression is not detected (A1). In compatible confrontations of compatible strains, long conjugation tubes are formed, but only the mfa-GFP–containing strain shows GFP fluorescence (A2), whereas both the perception of pheromone and tube formation are detectable in confrontation assays of FB2mG and FB1mG at 20°C (A3) and 28°C (A4). By contrast, plating mfa-GFP–containing myo5^ts strains at the permissive temperature does not result in tube formation, nor are cells properly stimulated (A5). The mating reaction is improved after reducing the distance between the compatible myo5^ts strains, but only short hyphae are formed (A6). Filamentation of mutant cells is abolished at 28°C (A7). However, myo5^ts mutants are able to stimulate compatible control cells (A8). All images were taken after 14 h of incubation. Bar in (A1) = 50 μm.

(B) Treatment of strain FB2mG with synthetic a1-mating pheromone for 8 h at 20°C induces the expression of mfa-GFP and the formation of hyphal extensions that most likely resemble conjugation tubes (B1). Occasionally, cells without GFP expression are observed that also do not grow filamentously (B2). Bars = 20 μm.

(C) FB2Myo5^ts mG cells treated with pheromone are only partially stimulated and do not form any tubes after 8 h at 20°C (C1). After 14 h, only stimulated cells, identified by their GFP signals, form short and irregular extensions that resemble those depicted in (A6). Bars = 20 μm.

Analysis of the Role of Myo5 in Dikaryotic Hyphae.

(A) Expression of yellow- and cyan-shifted derivatives of GFP (YFP and CFP) allows the identification of mating partners that contain compatible mating loci. Signals are given in false colors. Bar = 5 μm.

(B) Fusion of compatible haploid wild-type strains expressing either YFP or CFP results in dikaryotic hyphae that contain both fusion proteins (YFP in red; CFP in green). Bar = 5 μm.

(C) Crossing CFP and YFP expressing compatible myo5^ts strains at the permissive temperature resulted in straight but slightly thicker dikaryotic hyphae, as confirmed by the detection of both YFP (red) and CFP (green) in a single cell. Bar = 5 μm.

(D) After mating at the permissive temperature and growth for another day at the restrictive temperature, dikaryotic mutant hyphae are thicker and grow irregularly. Note that this image is given at a lower magnification than that in (C). Bar = 5 μm.

(E) Chitin staining in myo5^ts hyphae under restrictive conditions. In contrast to wild-type hyphae, which accumulate chitin at the growing hyphal tip (not shown), WGA staining reveals the irregular composition of the mutant cell wall. This finding suggests that the abnormal morphology of myo5^ts hyphae is attributable to defects in the cell wall. Bar = 5 μm.

(F) At 28°C, myo5^ts hyphae show the temperature-dependent phenotype. Bar = 5 μm.

Myo5^ts Mutants Cells on the Epidermis of the Host Plant.

(A) Infections of young maize plants with a mixture of compatible wild-type cells results in a network of hyphae that can be detected on the plant surface after 2 days by Calcofluor staining of the fungal cell wall (A1). These filaments are derived from the fusion of mating partners, as confirmed by infection with CFP- and YFP-expressing compatible strains (A2). DIC, differential interference contrast. Bars = 10 μm.

(B) Infection of plants at 20°C with compatible myo5^ts strains that express YFP or CFP demonstrates that most mutant cells do not fuse with adjacent mating partners, and only rarely do two cells fuse, thereby expressing both CFP and YFP (B2). Consistent with the in vitro observation on charcoal plates, the fusion of compatible cells results in the formation of long dikaryotic infection hyphae (B3). Bars = 10 μm in (B1) and (B3) and 5 μm in (B2).