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Vol. 16, Issue 6, 2746-2758, June 2005
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Max-Planck-Institut für terrestrische Mikrobiologie, D-35043 Marburg, Germany
Submitted March 1, 2005;
Revised March 31, 2005;
Accepted April 1, 2005
Monitoring Editor: David Drubin
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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50 µm in length and the nucleus fails to migrate into the hypha. A similar phenotype is found in dynein mutants that have a nuclear migration defect and stop hyphal elongation at 50 µm. These results demonstrate that microtubules are dispensable for polarized growth during morphological transition, but become essential in long-distance hyphal growth, which is probably due to their role in nuclear migration. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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). Considering the increasing world population, a better understanding of the molecular basis of phytopathology and important pathogens is therefore required. Pathogenic development of many fungi requires a yeast-hyphal transition, which enables the pathogen to be passively spread or to actively invade substrates by polar hyphal growth (overview in Gow, 1995). Morphological changes during this morphological switch involve polarized growth that is thought to depend on the cytoskeleton, which provides tracks for the delivery of supplies to the expanding hyphal tip (review in Geitmann and Emons, 2000). Indeed, numerous studies demonstrate a crucial role of F-actin in polarized fungal growth (reviewed by Harold, 1990) and fungal endocytosis (reviewed in Engqvist-Goldstein and Drubin, 2003), whereas a few reports confirm the importance of F-actin in the dimorphic transition (Yokoyama et al., 1990; Akashi et al., 1994). In contrast, the importance of microtubules (MT) is much less clear. MTs are involved in nuclear migration (reviewed by Xiang and Fischer, 2004), but they have no obvious role in tip growth of Candida albicans (Yokoyama et al., 1990) and Saccharomyces cerevisiae (Huffaker et al., 1988). In contrast, disruption of MTs drastically reduced tip growth rates in Aspergillus nidulans (Horio and Oakley, 2005) and led to branching in Schizosaccharomyces pombe (Sawin and Nurse, 1998). Despite the fact that the role of MTs in plant pathogenic fungi is even less understood, the tubulin cytoskeleton is the major target for some commonly used fungicides (summarized in Holloman et al., 1997), which is due to the crucial role of tubulin in mitosis. However, many fungal pests apparently do not undergo any cell division on the plant surface (Fischer and Holton, 1957; Heath and Heath, 1979), which might be due to a cell cycle arrest (Garcia-Muse et al., 2003). Thus, it is an important question whether MTs have additional roles in polarized growth during pathogenic development.
During the last decade, the fungus Ustilago maydis became a model system for analysis of the molecular basis of fungal plant pathogenicity (Basse and Steinberg, 2004; Kahmann and Kämper, 2004). This basidiomycete belongs to the smut fungi, an important group of plant pathogens that can cause considerable grain yield loss and economic damage (Agrios, 1997). Consequently, U. maydis is considered to be a potential threat to the economy of North America and is listed by Ad Hoc Group of the Biological Weapons Convention (Madden and Wheelis, 2003). Recently, much progress has been made in understanding the organization of the cytoskeleton in yeast-like cells of U. maydis (Steinberg et al., 2001; Banuett and Herskowitz, 2002; Straube et al., 2003; Adamikova et al., 2004). However, very little is known about the structural basis of the yeast-hyphal transition during mating, cell-cell fusion, and subsequent polarized growth of b-dependent hyphae. Pathogenic development of corn smut starts with the recognition of mating pheromone secreted by yeasts of the opposite mating type (reviewed in Banuett, 1995). This induces a cell-cycle arrest (Garcia-Muse et al., 2003) and triggers cells to switch from their default budding program to filamentous growth, which results in the formation of long conjugation hyphae. These filaments grow toward each other and fuse at their tips. Combining the cytoplasm of compatible cells leads to the formation of the bE/bW transcription factor that triggers growth of the b-dependent hyphae on the surface of plant epidermis and is a major regulator of the following steps in pathogenic development of U. maydis (reviewed in Kahmann and Kämper, 2004).
Here we set out to elucidate the role of the cytoskeleton in yeasts, pheromone-induced conjugation hyphae, cell-cell fusion in mating, and growth of b-dependent hyphae. We demonstrate that F-actin is essential for polarized growth in all stages and is required for cell-cell fusion. In contrast, MTs are dispensable for mating and growth of conjugation and b-dependent hyphae up to a length, when nuclei start to migrate into the hyphae. However, MTs become essential for extended hyphal growth and nuclear migration.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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) supplemented with 1% glucose (CM-G) to an OD600 = 0.6. Formation of conjugation hyphae was induced by the addition of 0.5 µl synthetic pheromone (pheromone; a1 or a2, stock 2.5 µg/µl dimethyl sulfoxide [DMSO]), final concentration 2.5 x 10-3 µg/µl; Szabo et al., 2002) to 500 µl of FB2, FB2Peb1Y cell suspension in a 2-ml reaction tube and incubation for 5–7 h at 22°C, 200 rpm. Growth of b-dependent filaments was monitored in strains AB33, AB33GT, and AB33GFP3Myo5_RT that contain the filament-inducing bE/bW transcription factor under the control of the nitrate inducible nar promoter (Banks et al., 1993). b-dependent filamentous growth was induced by growing cells overnight in CM-G to OD600 0.8 x10 ml cell suspension was centrifuged at 3000 rpm for 3 min and resuspended in 10 ml nitrate minimal medium supplemented with 1% glucose (NM-G; Holliday, 1974). Cells were analyzed after 6–7 h of growth in NM-Glucose at 28°C.
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Inhibitor Studies
For all inhibitor experiments with haploid yeast-like cells 500 µl cell suspension were incubated in a 2-ml reaction tube and either benomyl at 0.1–20 µM (stock 10 mM in DMSO, Sigma-Aldrich, Steinheim, Germany), latrunculin A (LatA) at 0.1–10 µM (stock 20 mM in DMSO, kindly provided by Dr. Karen Tenney, University of California, Santa Cruz) was added for 1–8 h with gentle shaking. In control experiments the corresponding amount of the solvent DMSO was used. To investigate the effect of these drugs on growth of conjugation hyphae, cells were first treated for 1.5 h with synthetic pheromone (see above) before LatA, benomyl, or DMSO was added and cells were incubated for additional 5.5 h. To investigate the role of the cytoskeleton in b-dependent hyphae strain AB33 was grown overnight to a cell density of OD600 = 0.8 in CM-G. Cells were harvested by centrifugation and resuspended in the same volume of fresh hyphal growth-inducing NM-medium and incubated at 28°C (200 rpm), after 2.5 h 500 µl cell suspension were transferred into a 2 ml reaction tube and DMSO, benomyl, or LatA at a final concentration of 0.1–10 µM was added. Cells were analyzed after additional 6–8 h at 28°C. In the subsequent quantitative analysis on the influence of benomyl on growth of b-dependent hyphae 8 ml of AB33 (preincubated for 2 h in NM) cell culture was incubated with 20 µM benomyl or the equivalent amount of DMSO in a culture flask at 28°C, 200 rpm. Cells were sampled every hour and cell length was measured using MetaMorph (Universal Imaging) software. To disrupt MTs in b-dependent hyphae of strain AB33GT 500 µl cell suspension was incubated with 10 µM benomyl or the corresponding amount of DMSO for 30 min at 28°C (200 rpm). For indirect detection of F-actin 500 µl of a suspension of AB33GFP3Myo5_RT hyphae (7h NM) were treated with 100 µM carbonyl cyanide m-chlorophenyl-hydrazone (CCCP) for 10 min and rigorous binding of GFP-Myo5 to F-actin was immediately monitored. To confirm that these filaments are indeed F-actin, cell were incubated in 50 µM LatA for 30 min, subsequently treated with 100 µM CCCP for 10 min, and microscopically analyzed.
Mating on Water Agar and Confrontation Assays
The ability to undergo cell fusion was assayed on 2% water agar. Fifty microliters of water agar, supplemented with either 20 µM benomyl, 50 µM LatA, or the equal amount of DMSO, were placed on a coverslip and kept in a wet chamber until use. Strains FB1YFP and FB2CFP that express cytoplasmic YFP or CFP, respectively, were grown to a density of OD600 = 0.8, centrifuged at 3000 rpm for 3 min, and concentrated to a final cell density of OD600 = 5.0. Two milliliters of each cell suspension were combined and incubated for 1 h at room temperature at 150 rpm. Subsequently, 3 µl of the cell mixture was placed on top of the water agar droplet and slides were incubated in a moist chamber for 13, 17, and 25 h at 22°C. Cell fusion was analyzed by detecting CFP and YFP signals. Structures that contained both fluorescent proteins were counted as products derived from fusion of two cells, whereas cells with only CFP or YFP signals were considered to be single. Confrontation assays were performed as described by Snetselaar et al. (1996). In brief, slides were covered with 200 µl of 2% water agar, containing either 20 µM benomyl, 50 µM LatA, or the equal amount of the solvent DMSO. Strains FB1mG and FB2mG that contain GFP under the control of the pheromone-sensitive mfa1-promoter (Spellig et al., 1996) were grown to a cell density of OD600 = 0.8, centrifuged at 3000 rpm, and concentrated to OD600 = 5. One microliter of each cell suspension was placed confronting the other on the water agar film in a distance of 200 µm or less. Confrontation assays were covered with 1 µl of paraffin oil and incubated in a moist chamber for 16–22 h at 22°C.
Stimulation with Synthetic Pheromone to Assay Pheromone Perception
For pheromone stimulation strain FB1mG that contains GFP under the control of the pheromone-sensitive mfa1-promoter (Spellig et al., 1996) was grown to a cell density of OD600 = 0.5. 500 µl cell suspension was supplemented with DMSO as control, 50 µM LatA for 1 h at 22°C, horizontally shaking at 200 rpm. Subsequently 0.5 µl of synthetic pheromone (a2, stock 2.5 ng/µl DMSO; Szabo et al., 2002) was added and cells were incubated in a 2-ml Eppendorf tube for 2 h at 200 rpm, 22°C. Finally, GFP expression as a read out for pheromone perception was monitored microscopically.
Nuclear Staining with DAPI
For 4,6-diamidino-2-phenylindole (DAPI; Sigma-Aldrich; D-9542) staining of nuclei in conjugation hyphae, FB2 cells were first stimulated with synthetic pheromone. After 1.5 h of incubation 20 µM benomyl or the equal volume of DMSO was added to the cells. For the same experiment Dyn2ts cells were pheromone stimulated as described above and after 3 h of incubation at 22°C were shifted to permissive growth temperature, 32°C. Samples were taken after 4 and 7 h of incubation. Cells were fixed with 3% formaldehyde for 10 min, washed in phosphate-buffered saline (PBS) twice, and resuspended in 50 µl ddH2O. Samples were mounted on poly-L-lysine cover slips incubated for 3 min and dried at 60°C. Coverslips with fixed cells were washed in PBS several times and stained with DAPI (0.5 µg/ml) for 10 min at 60°C. Coverslips were washed several times in PBS before analysis.
Microscopy, Image Processing, and Quantitative Analysis
Cells from logarithmically growing cultures were dropped on a thin 2% agarose-layer and immediately observed using a Zeiss Axioplan II microscope (Carl Zeiss, Oberkochen, Germany). Epifluorescence of GFP was observed using standard FITC filters. For colocalization studies, YFP and CFP were analyzed with specific filter sets (YFP: BP500/20, FT515, BP535/30; CFP: BP436, FT455, BP480–500). Frames were taken with a cooled CCD-camera (CoolSNAP HQ, Photometrics, Tucson, AZ) that was controlled by the Meta-Morph (Universal Imaging, West Chester, PA) software. Measurements and image processing, including adjustment of brightness, contrast, and gammavalues were performed with MetaMorph (Universal Imaging) and Photoshop (Adobe, San Jose, CA). To determine the length of hyphae, the distance between the tip of the hyphae and the mother cell (excluding the mother cell) was measured; in case of the bipolar growing hyphae only the length of the longer filament was determined.
Statistical analysis by two-tailed t test at 
= 0.05 was carried out using Prism (GraphPad, San Diego, CA). All values are given as means ± SD unless otherwise stated.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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). To visualize MTs in hyphae we expressed GFP- tubulin in strain AB33 (AB33GT), which grows filamentously upon shift to nitrate containing medium. The resulting b-dependent hyphae contain long MTs that reach into the hyphal apex and the basal septum that separates the empty parts of the growing hyphae from the living tip cell (Figure 1, arrow). Unfortunately, all attempts to visualize F-actin filaments including staining with rhodamine–phalloidin and expression of GFP-tropomyosin failed in U. maydis. However, treatment of a strain that expressed GFP-myosin 5 (AB33GFP3Myo5_RT) with 100 µM carbonyl cyanide m-chlorophenyl-hydrazone (CCCP), an uncoupler of the oxidative chain that rapidly depletes ATP from the cytosol (Azarkina and Konstantinov, 2002) led to the appearance of GFP-Myo5–bound filaments that were sensitive to 50 µM of the actin inhibitor LatA (Figure 1B), suggesting that they are F-actin cables. The existence of both MTs and F-actin cables in hyphal cells confirm previous results on yeast-like cells (Steinberg et al., 2001; Banuett and Herskowitz, 2002), indicating that both cytoskeletal elements participate in morphogenesis and polar growth in the various developmental stages of U. maydis.
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; Ayscough et al., 1997), inhibited bud growth and affected the shape of the cell (Figure 2A; LatA). Interestingly, in the presence of LatA both daughter as well as older mother cells changed shape and swelled, indicating that the older cell walls are still flexible. Benomyl, 20 µM, disrupted MTs in these cells (Straube et al., 2003) and led to an accumulation of large budded cells (Figure 2, C1, Benomyl) that contained condensed nuclei within the mother cell (unpublished data), indicating that benomyl treatment arrests cells in mitosis. In addition, extended incubation also resulted in 5% cells that showed lateral budding (up to 2.5%) or grew bipolar or slightly irregular (Fig: 2, C2), indicating that MTs have a role in morphogenesis and polarity of haploid cells. A quantitative analysis of these effects revealed that yeast-like cells are highly sensitive to LatA treatment, because half of the cells showed aberrant growth and morphology at 1–1.5 µM LatA (IC50: 1.3 µM). In comparison, cells were able to tolerate up to 1 µM benomyl (IC50: 1.0 µM) before the number of arrested cells increased.
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). We made use of strain AB33 (Brachmann et al., 2001) in order to investigate the role of the cytoskeleton in infective b-dependent hyphae. This strain contains both parts of the bE/bW transcription factor under the control of the nitrate inducible nar promoter (Banks et al., 1993). On shift to nitrate containing medium AB33 cells start to form straight hyphae until the living tip cell leaves behind empty sections at the subapical region (Figure 4A, control, arrow) that are thought to allow b-dependent hyphae to grow on the plant surface without access to nutrients (Steinberg et al., 1998). Consistent with the results on yeast-like cells and conjugation hyphae, treatment with 10 µM LatA impaired growth of b-dependent hyphae (Figure 4B, LatA). Growth inhibition was already seen at very low amounts of LatA (IC50: 0.1 µM) (Figure 4D). However, in the presence of LatA 20% of the cells were slightly elongated (Figure 4B), which might be due to that fact that hyphal growth of AB33 cells was induced for 2.5 h in drug-free medium, before LatA was applied. In contrast, treatment with 20 µM benomyl did not abolish growth of b-dependent hyphae (Figure 4C, Ben). However, in the absence of MTs hyphae grew irregular and often at both cell poles (Figure 4C, Ben, arrowheads). More strikingly, benomyl-treated hyphae remained shorter than control hyphae (Figure 4C, benomyl). In addition, without MTs empty sections were not formed, but the apices of older hyphae were often filled with vacuoles (Figure 4C, benomyl, inset).
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These results indicated that shorter b-dependent hyphae are formed in the absence of MTs. It was recently described that disruption of MTs significantly slowed down the growth rate of A. nidulans hyphae (Horio and Oakley, 2004). Therefore, we considered it possible that the shorter hyphae of benomyl treated cells in U. maydis are also a consequence of reduced tip growth rates and that long b-dependent hyphae are formed after extended growth in nitrate-containing medium. Indeed, quantitative analysis of hyphal growth velocities in the presence of the solvent DMSO (Figure 5A, control) and 20 µM benomyl (Figure 5A, benomyl) revealed that disruption of MTs lowered the elongation rates by 60% (control: 8.23 ± 0.49 µm/h; benomyl: 3.61 ± 0.40 µm/h). However, even after >14 h of growth in benomyl, b-dependent hyphae remained shorter (Figure 5B, + ben, ON), indicating that MTs are required for growth of b-dependent hyphae above 50–60 µm. Interestingly, a similar result was found in conjugation hyphae. These hyphae only rarely formed empty sections but reach up to 100 µm in length (Figure 5B). Again, in the presence of benomyl conjugation hyphae remained shorter (Figure 5B) and reach a maximum length of 50 µm.
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; Wedlich-Söldner et al., 2002). In pheromone-induced conjugation hyphae of strain FB2Peb1Y, plus-end binding Peb1-YFP moves to the apex in 83.5 ± 5.0 cases (n = 2 experiments, 27 hyphae), indicating that conjugation hyphae also focus their MT plus-ends toward the growing hyphal tip (Figure 5D). Consequently, pheromone-treated dynein mutants formed hyphae. However, nuclei never migrated into the hyphae and tip growth also stopped at 50 µm length (Figure 5, C and B; Dyn2ts), whereas cells formed new hyphae at the opposite cell pole (Figure 5C; Dyn2ts, arrow marks bipolar hyphae, asterisk indicates nucleus). Interestingly, the length of dynein mutant hyphae also did not exceed a length of 45 ± 8.97 µm(n = 30) when incubated overnight (Figure 5B, Dyn2ts ON). This phenotype of dynein mutant hyphae was remarkably similar to that of benomyl-treated cells. Therefore, we consider it possible that hyphal elongation beyond 50–60 µm requires nuclear migration in order to maintain communication between the nucleus and the expanding hyphal tip.
F-actin Participates in Pheromone Secretion but not in Perception
We finally set out to analyze the role of the cytoskeleton in the perception of pheromone. We made use of strain FB1mG that expresses GFP under the control of the mating pheromone a-promoter (mfa). This promoter is induced shortly after recognition of compatible pheromone, which allows monitoring of pheromone perception by analyzing GFP expression (Spellig et al., 1996). Under natural conditions cells have to recognize a pheromone gradient that is produced by the compatible mating partner. This situation is given in confrontation assays, where compatible strains are placed on water agar facing each other and are overlaid by paraffin oil that allows diffusion of secreted pheromone (Snetselaar et al., 1996). Indeed, in these assays FB1mG and FB2mG cells recognized each other, which is indicated by mfa1-promoter driven GFP expression, and formed long conjugation hyphae that bridge the gap between both strains (Figure 6A). In the presence of benomyl both strains were stimulated, but only short conjugation hyphae were formed (Figure 6B, inset). Consistent with the results described above these hyphae did not elongate any further (Figure 6B, compare inset at 16 and 22 h), but were able to bridge smaller gaps between the compatible strains (Figure 6B, right panel). In contrast, disruption of F-actin by 50 µM LatA not only abolished filament formation but also impaired pheromone perception in these assays (Figure 6C). Interestingly, the ability to perceive pheromone was dependent on the distance between the partners, with strong GFP expression when compatible cells were in very close contact (Figure 6C). This indicated that F-actin was required to percept very low amounts of secreted pheromone. To test this hypothesis, we incubated liquid cell cultures with synthetic pheromone. At a pheromone concentration of 2.5 ng/µl >95% of both LatA- and DMSO-treated cells showed GFP expression, confirming that F-actin is not essential for pheromone perception at high concentrations (Figure 6E). However, cells treated with highly diluted synthetic pheromone (Figure 6E; 2.5 and 1 x 10–2 ng) in the presence of DMSO and LatA showed comparable levels of pheromone stimulation. At this low concentration only 20–40% of the control cells were stimulated (Figure 6E). This argues against a need of F-actin for perception of pheromone traces. Alternatively, we considered it possible that LatA-treated cells are defective in pheromone secretion, which would also result in reduced perception and, consequently, less GFP expression in confrontation assays.
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Cell-Cell Fusion Requires F-actin but not Microtubules
Our data demonstrate that shorter conjugation hyphae are formed in the absence of MTs, whereas F-actin is crucial at this stage. This suggested that short MT-independent conjugation hyphae confer cell-cell fusion. We therefore developed an assay that allowed the quantitative analysis of fusion of compatible cells that express either CFP or YFP. When plated on water agar strain FB1Y and FB2C recognized each other and started to form conjugation hyphae that grew toward the mating partner (Figure 7A; in overlay CFP in red, YFP in green). After 7 h mating partners occasionally found each other and the tips of the conjugation hyphae were curled around the compatible partner cell (Figure 7B; arrow), whereas the older parts of the cell became vacuolated (Figure 7B; arrowheads). These structures contain both CFP and YFP (Figure 7B, overlay in yellow), demonstrating that both compatible cells fused and mixed their cytoplasm. However, in order to circumvent the initial steps of cell-cell recognition, we incubated compatible strains at high density (OD600 = 5) in liquid culture for 1 h. Under these conditions, strains FB1mG and FB2mG that express GFP as a result of pheromone recognition (Spellig et al., 1996) are stimulated but do not show conjugation tube formation (Figure 7C). After similar pretreatment of strains FB1Y and FB2C cells were plated on water agar supplemented with cytoskeletal inhibitors. This assay allowed us to monitor the need of MTs and F-actin for cell-cell fusion. Thirteen hours after incubation, cells that either contained YFP (Figure 7D, control, green) or CFP (Figure 7D, control, red) were found. In addition, b-dependent hyphae were seen that contained both YFP and CFP (Figure 7D, control, yellow). Fusion products were also found in the presence of 20 µM benomyl (Figure 7D, yellow, arrow), although the resulting hyphae remained shorter and grew irregularly. In contrast, in the presence of LatA no structures containing both CFP and YFP were detected, indicating that F-actin is essential for cell-cell fusion. The portion of fused cells increased over time in both DMSO-(Figure 7E, control; Figure 7F) and benomyl-treated cells (Figure 7E, benomyl, and 7F), whereas no fusion was found in LatA-treated cells. This demonstrates that F-actin is essential for this important step in the pathogenic development, whereas MTs are without detectable role in cell-cell fusion.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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; Kahmann and Kämper, 2004). We found that F-actin is of key importance in all these steps. In fungi, actin exists as cortical patches and filaments (Harold, 1990; Heath, 1995), and it is thought that filaments deliver wall vesicles to the expanding growth region (Harold, 1990; Ayscough et al., 1997). This notion is supported by the finding that myosin-V, which is known to be a processive organelle motor in other systems (Langford, 2002), participates in filamentous growth of U. maydis (Weber et al., 2003). Interestingly, in S. cerevisiae it was reported that actin cables are not essential for the formation of mating projections, whereas cortical actin patches are crucial (Read et al., 1992). Actin patches might be sites of endocytosis (reviewed in Engqvist-Goldstein and Drubin, 2003), and endocytosis also participates in polar hyphal growth of U. maydis (Wedlich-Söldner et al., 2000). Consequently, LatA-treatment might inhibit both cable-based exocytosis, as well as patch-dependent endocytosis, and this could explain why F-actin is of crucial importance in U. maydis. In this respect it is worth to notice that our evidence for actin cables in U. maydis hyphae is circumstantial. However, F-actin cables exist in yeast-like cells (Banuett and Herskowitz, 2002) and the decoration of filaments with myosin under low ATP conditions make the existence of F-actin cables likely.
The Actin Cytoskeleton Is Important for Pheromone-based Cell-Cell Communication
In confrontation assays the induction of mfa promoter-driven GFP expression indicated that control cells are able to detect the secreted pheromone over a distance of more than 150 µm. Under similar conditions LatA treatment abolished GFP expression, but the mating partner was recognized at shorter distances. In S. cerevisiae LatA treatment resulted in reduced pheromone recognition, which is thought to be due to a mislocalization of the pheromone receptor (Ayscough and Drubin, 1998). However, we applied synthetic pheromone to liquid cultures at various concentrations, but could not find any reduction in the reaction to pheromone due to F-actin disruption. In confrontation assays, as well as under natural conditions on the plant surface cells have to secrete their own pheromone. This raises the possibility that LatA-treated cells might be defective in pheromone secretion, which in turn results in less perception in the partner cell. Actin-dependent exocytosis of pheromone has been described for S. cerevisiae (reviewed in Kurjan, 1992), and our results argue for a similar situation in U. maydis.
MTs Do Not Participate in Cell-Cell Fusion
In this manuscript we demonstrate that cell-cell fusion in U. maydis depends on intact F-actin, whereas MTs are not involved in this process. In contrast, MTs are important for cell-cell fusion in the fission yeast S. pombe (Petersen et al., 1998). This is surprising, as both fission yeast and U. maydis contain prominent MT arrays (Hagan, 1998; Steinberg et al., 2001) that are expected to participate in similar processes. It was speculated that the need for MTs in mating is due to MT-based transport of unknown components toward the projection tip (Petersen et al., 1998). Alternatively it was suggested that the effect of MTs on cell-cell fusion could be indirect via defects in the actin cytoskeleton (Petersen et al., 1998). We have preliminary evidence that benomyl treatment has no long-term effect on actin patch organization and actin cables (our own unpublished results), which might explain why U. maydis MTs are not involved in cell-cell fusion.
Defects in MT-based Nuclear Migration Might Be Responsible for impaired Hyphal Growth
In the absence of MTs hyphal growth in U. maydis continues at a rate of 3.6 µm/h. Interestingly, in A. nidulans disruption of the tubulin cytoskeleton reduced the elongation velocity 10-fold to a rate of 3.2 µm/h (Horio and Oakley, 2005), a velocity that is surprisingly similar to that of benomyl-treated U. maydis hyphae. Thus, slow hyphal growth of both fungi might be mediated by similar mechanisms that could involve actin-dependent myosins. Indeed, it was shown that class V myosins participate in polarized hyphal growth in U. maydis (Weber et al., 2003) and preliminary evidence exists for a cooperation of the MT- and actin-based transport machinery in hyphal growth (I. Manns and G. Steinberg, unpublished results). Unexpectedly, MT-independent growth ceased at a hyphal length of 50–60 µm. In control hyphae, this is exactly the distance between the nucleus and the tip, suggesting that nuclear migration and tip growth are linked. Conditional mutants in the Ustilago dynein have defects in nuclear migration in yeast-like cells (Straube et al., 2001) and hyphae (this study), but the minus-directed dynein is most likely not directly involved in tip-growth, as the MT plus-ends are directed to the hyphal tip. Nevertheless, hyphal growth of dynein mutants also stopped at 50–60 µm length, again supporting the notion that nuclear migration is required for extended hyphal growth. Such a connection was found in tip-growing root hairs in A. thaliana (Ketelaar et al., 2002) and might also underlie the growth inhibition of Aspergillus mutants defective in NUDF, a dynein regulator that has highest homology with the human LIS1 gene (Xiang et al., 1995). Furthermore, LIS1-mediated nuclear migration might be essential for neuronal cell migration and brain development (Morris et al., 1998), indicating that the role of MTs in nuclear migration is of general importance in long-distance growth and migration of eukaryotic cells.
The Microtubule Cytoskeleton as a Target for Fungicides?
The actin cytoskeleton performs essential roles in all early steps of pathogenic development of U. maydis. Thus, any compound that interferes with the organization or function of F-actin should efficiently inhibit fungal infections. However, to our knowledge no currently used fungicide addresses components of the actin cytoskeleton, which might be due to the high degree of conservation in eukaryotic actins. On the other hand, actin-associated proteins, such as fungal specific myosin-chitin synthase fusion proteins are known to play essential roles in fungal pathogenicity (Madrid et al., 2003; Liu et al., 2004) and therefore might be potential targets for novel fungicides. In contrast to actin, microtubules are affected by numerous benzimidazole and phenylcarbamate fungicides (overview in Hollomon et al., 1997), which might be due to their essential roles in fungal mitosis. However, good indication exists that important plant pathogens, including U. maydis arrest their cell cycle during early infection steps (Garcia-Muse et al., 2003). In Uromyces phaseoli and U. maydis infectious hyphae consist of a single tip cell that leaves behind vacuolated parts while growing on the plant surface (Heath and Heath, 1979; Steinberg et al., 1998). Thus, in these fungi anti-tubulin fungicides are expected to interfere with MTs in hyphal growth, rather than targeting mitotic events. We demonstrate here that MTs have nonessential roles in early hyphal growth, but they are essential for extended hyphal elongation, which is due to their role in nuclear migration. Therefore, anti-tubulin drugs are expected to be ineffective in early infection, but are supposed to inhibit the infection at later stages.
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ACKNOWLEDGMENTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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Footnotes |
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Abbreviations used: MT, microtubule; F-actin, filamentous actin; GFP, green fluorescent protein; CFP, cyan-shifted fluorescent protein; YFP, yellow-shifted fluorescent protein; aa, amino acids.
Address correspondence to: Gero Steinberg (Gero.Steinberg{at}staff.uni-marburg.de).
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REFERENCES |
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