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Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

Directed growth or movement is a common feature of microbial development and propagation. In polar growing filamentous fungi, directed growth requires the interaction of signal sensing machineries with factors controlling polarity and cell tip extension. In Neurospora crassa an unusual mode of cell–cell signalling mediates mutual attraction of germinating spores, which subsequently fuse. During directed growth of the two fusion partners, the cells co-ordinately alternate between two physiological stages, probably associated with signal sending and receiving. Here, we show that the Saccharomyces cerevisiae BEM1 homologue in N. crassa is essential for the robust and efficient functioning of this MAP kinase-based signalling system. BEM1 localizes to growing hyphal tips suggesting a conserved function as a polarity component. In the absence of BEM1, activation of MAK-2, a MAP kinase essential for germling fusion, is strongly reduced and delayed. Germling interactions become highly instable and successful fusion is greatly reduced. In addition, BEM1 is actively recruited around the forming fusion pore, suggesting potential functions after cell–cell contact has been established. By genetically dissecting the contribution of BEM1 to additional various polarization events, we also obtained first hints that BEM1 might function in different protein complexes controlling polarity and growth direction.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

Microbial growth and propagation in specific habitats often depend on tropic cellular responses upon environmental cues. Examples include tropic movement or growth towards nutrient sources or light, but also cell aggregation or the directed growth of pathogens towards entry sites of their host organism (Armitage, 1999; Weijer, 2004; Brand and Gow, 2009). Filamentous fungi grow as thread-like filaments, the hyphae. Hyphae typically grow by apical tip extension and belong to the most highly polarized cells. Polarity establishment and maintenance require the co-ordinated interaction of several multi protein complexes, residing at the cortex of growing hyphal tips, including the polarisome, Spitzenkörper proteins and the exocyst (Harris, 2006; Riquelme et al., 2011). During different developmental stages polar hyphal growth is strictly directed, that is, hyphae respond to environmental cues either by attraction or by repulsion (Brand and Gow, 2009). In Neurospora crassa and many other filamentous fungi conidial germ tubes attract each other and fuse (germling fusion), resulting in a network of germlings, which further develops into the mycelial colony (Roca et al., 2005a; Read et al., 2010). Similarly, hyphal branches within the older parts of a mature colony exhibit tropic responses towards each other, which also result in fusion (hyphal fusion) and increased interconnectedness within the mycelial unit (Hickey et al., 2002; Fleißner et al., 2008). During sexual development of N. crassa, specialized female reproductive hyphae, so-called trichogynes, orient their growth along gradients of mating pheromones released by potential mating partners (Bistis, 1981).

The described examples of directed growth require the interplay of factors and signalling pathways involved in recognition of environmental cues with the general molecular machinery required for polarity establishment/maintenance and cell extension. While the molecular signals and receptors mediating communication between mating partners have been described in N. crassa (Kim and Borkovich, 2004; 2006; Kim et al., 2012), communication and attraction of vegetative germ tubes and hyphae is less well understood. Recently, we identified a novel signalling mechanism between fusion germlings, in which the two partner cells co-ordinately switch between two physiological stages, possibly between signal sending and receiving (Fleißner et al., 2009b). Essential factors of this unusual cellular interplay are the MAP kinase MAK-2 and the SO protein. MAK-2 is the homologue of the fus3p/kss1p MAP kinases of the pheromone response and filamentation pathways in Saccharomyces cerevisiae (Pandey et al., 2004). SO is a protein of unknown molecular function, which is only present in filamentous ascomycete fungi (Fleißner et al., 2005). Subcellular localization of MAK-2 and SO revealed that both proteins are recruited to the plasma membrane of fusion tips in an alternating manner (Fleißner et al., 2009b). When MAK-2 is present at the tip of the first fusion partner, SO is present at the tip of the second fusion cell. Three to six minutes later the roles are reversed. MAK-2 disappears from the first tip and SO accumulates, while SO disperses from the second tip and MAK-2 becomes present. These co-ordinated oscillating protein dynamics continue for three to six times until the cells achieve physical contact. Then, both proteins concentrate at the contact site. These dynamic recruitment patterns, especially of the MAP kinase MAK-2, suggest functions in controlling the growth direction of the germ tube in response to the postulated signal from the partner cell (Fleißner et al., 2009b; Read et al., 2009; Goryachev et al., 2012). In S. cerevisiae fus3p is recruited to the tip of polar growing mating projections (shmoos), where it activates the formin bni1p, which controls actin nucleation, and thus polarization of the cytoskeleton (Matheos et al., 2004). Recruitment of fus3p to the shmoo tip is promoted by the scaffolding protein bem1p. bem1p physically interacts with the MAP kinase scaffold ste5p, which carries the MAP kinase fus3p, as well as the upstream MEK ste7p and MEKK ste11p (Leeuw et al., 1995; Lyons et al., 1996). Recruitment of this module is promoted through the physical interaction of bem1p with cdc24p, which in turn interacts with membrane-bound cdc42p (Elion, 2000). In addition, bem1p is also involved in symmetry breaking and polarization during bud formation, where it appears to constitute a parallel mechanism to actin-based cell polarization (Kozubowski et al., 2008).

In the filamentous fungi Aspergillus nidulans and Epichloë festucae, bem1p homologous proteins localize to growing hyphal tips, consistent with a conserved role as a polarity component. In contrast to yeast, however, loss of function mutations had only minor effects on general hyphal polarity in both species (Leeder and Turner, 2008; Takemoto et al., 2011). In their elegant study employing E. festucae, Takemoto et al. found the bem1p homologue BemA to be part of NADPH oxidase complexes and to be essential for normal tip localization of the NADPH oxidase regulator NoxR (Takemoto et al., 2011). Yeast does not contain NADPH oxidase proteins, suggesting that BEM1 might have different or additional functions in filamentous fungi. So far, however, the different molecular roles of BEM1 for growth and development of filamentous fungi remain mostly unclear.

Here, we show that in N. crassa, BEM1 is essential for efficient MAP kinase-based co-ordination of cellular behaviour and directed growth resulting in vegetative cell fusion. Phenotypic comparison of different bem1 mutants together with subcellular localization of BEM1 further suggests the existence of different BEM1 complexes mediating the different polarization events in the life cycle of N. crassa.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

Identification of the N. crassa bem1 homologue

By using blast analysis (Altschul et al., 1990) we identified gene NCU06593 (bem1) as the N. crassa homologue of bem1 of S. cerevisiae. According to the most recent annotations of the Broad Institute (http://www.broadinstitute.org/annotation/genome/neurospora/MultiHome.html) and MIPS (http://mips.helmholtz-muenchen.de/genre/proj/ncrassa/) the bem1 open reading frame (ORF) consists of 1847 bp and contains two introns of 58 bp each (position 115–172 bp and 914–971 bp). We verified these predictions by cloning and sequencing of bem1 cDNA. The predicted gene encodes a protein of 576 amino acid residues, with a predicted molecular weight of 63.0 kDa.

The N. crassa BEM1 protein matched the S. cerevisiae bem1p (EGA59743.1) in an NCBI protein blast search with an e-value of 2−66. Both proteins have a similar domain structure consisting of two SH3 domains present in the N-terminal half of the protein followed by a PX-domain and a PB1 domain in the C-terminal half (Fig. 1). To determine conservation of the amino acid sequence of the four domains, corresponding amino acid sequences of N. crassa and S. cerevisiae were aligned using the Needleman-Wunsch alignment tool of the European Bioinformatics Institute (http://www.ebi.ac.uk/Tools/psa/) (Table S3).

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Figure 1. Domain structure of N. crassa BEM1. SH3, Src homology 3 domain; PX, Phox homology domain; PB1, Phox and Bem1p homology domain.

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Construction of bem1 knockout mutants

To determine the phenotype of a bem1 knockout mutant we replaced the entire bem1 ORF with a hph hygromycin resistance cassette in the wild-type strain FGSC 9719 and the histidine auxotrophic strain FGSC 9720. Replacement of the bem1 gene and the absence of additional heterologously integrated transformation cassettes were confirmed by Southern blot analysis. Homokaryotic mutant strains were isolated by single spore isolation from primary transformants and were verified by PCR analysis (data not shown).

On slant tubes or in culture flasks the Δbem1 mutants 2-A1 and 21-A1 exhibit macroscopic phenotypes significantly different from the wild-type reference strain FGSC 9719. Production of aerial hyphae is reduced and the conidiation pattern is altered in the mutant strains, such that in Δbem1 sporulation occurs evenly at the entire colony surface, while in wild-type conidia are predominantly formed at the colony periphery (Fig. 2A and B). The overall number of produced conidia is decreased by about 90% in Δbem1 mutant strains (Fig. 2C). The linear hyphal growth rate of the mutant 21-A1 is reduced by around 24% compared with the wild-type FGSC 9719 (Fig. 2D).

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Figure 2. The Δbem1 mutant is defective in the formation of aerial hyphae, linear hyphal growth and sporulation, but hyphal polarity is unaffected.

A. Wild-type FGSC 9719 and Δbem1 21-A1 in Erlenmeyer flasks on Vogel's minimal medium, 7 dpi.

B–D. Graphs indicate aerial hyphal growth, sporulation and linear hyphal growth, respectively, for FGSC 9719, Δbem1 21-A1, Δbem1 2-A1 and complemented strain 2208. Error bars indicate standard deviations from at least three independent experiments.

E. Nomarski images of vegetative hyphal tips of FGSC 9719 and Δbem1 21-A1 growing on Vogel's minimal medium. Scale bars, 5 μm.

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Δbem1 is not affected in general hyphal polarity

In S. cerevisiae bem1p is essential for the proper establishment of polarized growth during vegetative budding as well as for shmoo formation during mating. To test potential defects in general hyphal polarity in the N. crassa Δbem1 mutant the overall hyphal morphology was determined. Strain 21-A1 (Δbem1) and as a control strain FGSC 9719 were inoculated on minimal agar. After 24 h of incubation at 30°C, hyphae were observed by light microscopy. The shape of Δbem1 mutant hyphae was comparable to wild type (Fig. 2E). Hyphal trajectory appeared normal and the diameter of growing hyphal tips was also similar to the wild type (FGSC 9719: 16 ± 3 μm, 21-A1: 16 ± 2 μm, n = 50). To determine potential defects in branching the distance between branch points were determined. No significant differences were found (FGSC 9719: 110 ± 67 μm, 21-A1: 116 ± 61 μm, n = 50).

To test if BEM1 is involved in polarity establishment we determined germination time and frequency of conidia of 21-A1 (Δbem1). No differences between the wild type and the mutant were observed. Three hours after inoculation 81 ± 6% of 21-A1 conidia (n = 100) and 76 ± 7% of FGSC 9719 (n = 100) conidia had germinated.

In the course of these investigations we noticed that bem1 conidial germlings are significantly larger than wild-type ones (Fig. 3A–C). To test if this increase in size is caused by a prolonged initial period of isotropic, apolar growth before the onset of polar germination or if bigger spores are produced at the conidiophore, we determined the size of freshly harvested spores before swelling and initial isotropic growth. In addition, we analysed the morphology of wild-type and mutant conidiophores. In order to determine the number of nuclei within each spore, strains expressing GFP tagged Histone-1 were employed. Δbem1 spores had already a larger diameter and carried more nuclei than the wild type, while still in conidial chains in the conidiophore (Fig. 3D–F). Thus, the bigger size of the mutant spores is not caused by a prolonged period of apolar growth, but by a defect during spore formation.

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Figure 3. The Δbem1 mutant produces enlarged spores and germlings.

A and B. Nomarski images of spores (A) and germlings (B) from wild-type FGSC 9719 and Δbem1 21-A1. Scale bars, 10 μm (A) and 5 μm (B).

C. Germ tube diameter of FGSC 9719 and Δbem1 21-A1, error bars indicate standard deviations, n = 50.

D. Spore size of FGSC 9719, Δbem1 21-A1, Δbem1 2-A1 and complemented strain 2208, error bars indicate standard deviations of three independent quantifications (n = 50, each).

E. Merged fluorescent images of conidiophores of h1–gfp expressing wild type (WT-HG) and Δbem1 (2-HG). Cell walls were stained with Calcofluor white (false coloured in red). Scale bar: 5 μm.

F. Frequency distribution of number of nuclei per spore for strain WT-HG (grey) and Δbem1 2-HG (black), n = 157.

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Taken together these data indicate that BEM1 is dispensable for general hyphal polarity establishment and maintenance, but is essential for normal spore shape. The observed larger spore size of the Δbem1 mutant prompted us to revisit conidial germination to test, if these large structures might form higher numbers of germ tubes compared with the wild type. However, no differences between wild-type FGSC 9719 and the mutant 21-A1 were observed. In both cases, more than 98% of spores formed only a single germ tube (FGSC 9719: 98.7 ± 1.2%, 21-A1: 98.7 ± 2.3%).

Since macroconidia formation is defective in Δbem1 strains, we analysed if microconidia formation, which is another budding-like process in N. crassa, similarly depends on BEM1. However, no quantitative or qualitative differences in the production of microconidia were observed in wild type or Δbem1 (Fig. S1).

BEM1 promotes germling and hyphal fusion

In order to test a potential function of BEM1 in cell communication and/or directed cell growth related to vegetative cell fusion, we conducted germling fusion assays of strains 21-A1 (Δbem1) and FGSC 9719 (wild type). In the wild type, mutual attraction of conidial germlings, followed by cell–cell contact and fusion was abundant after 2.5 h of incubation at 30°C (Fig. 4A). Hyphal bridges were either formed by fusion of small branches emanating from the spore body or the germ tube, so-called conidial anastomosis tubes (CATs) (Roca et al., 2005b), or by direct fusion of germ tube tips, followed by the establishment of a new polar growing tip. Both, fusion between CATs and germ tube tips were observed in wild type. In contrast, no cell–cell interactions between germlings were observed in Δbem1 (Fig. 4A). After additional 2.5 h of incubation, fusion bridges appeared in a low frequency in the mutant. Interestingly, these rare fusion bridges appeared morphologically more uniform than wild-type fusions. In the mutant, only short bridges connecting spores in very close proximity were observed. Long distance interactions requiring repeated adjustment of the growth direction towards the fusion partner, as typically observed in germ tube tip fusion in wild type, were mostly absent in the Δbem1 mutant (Fig. 4B–D).

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Figure 4. Germling interactions are highly reduced in Δbem1.

A and B. Tropic interactions of germlings from wild-type FGSC 9719 and Δbem1 21-A1 after 2.5 h post inoculation (hpi) and 5 hpi respectively. Asterisks indicate tropic interactions; x indicate spores. Scale bars: 5 μm. Error bars indicate standard deviations of three independent experiments each of n = 50.

C. Frequency distribution of distances between tropically interacting germlings of FGSC 9719 and Δbem1 21-A1, n = 100.

D. Percentage of fusion events, which involved at least one tropically growing germ tube compared with those involving only CATs. Error bars indicate standard deviations of three independent experiments each of n = 30.

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So far, all N. crassa mutants affected in germling fusion are also deficient in fusion between mature hyphae within the mycelial fungal colony (if tested) (Read et al., 2010). Similarly, no anastomoses were detected in the mycelia of Δbem1 21-A1, while typical fusion bridges were readily observed in the reference strain FGSC 9719 (Fig. S2), indicating that BEM1 is essential for anastomosis formation in mature colonies.

The interaction of fusion tips is instable in the absence of BEM1

Mutual attraction and fusion of wild-type germlings requires co-ordination of cell behaviour over a spatial distance, indicated by the co-ordinated alternating recruitment of the MAP kinase MAK-2 and the SO protein from the cytoplasm to the plasma membrane (Fig. 5A and B). To test if this co-ordinated behaviour requires BEM1, subcellular dynamics of MAK-2-GFP and SO-GFP were compared between the wild type and the Δbem1 mutant. In wild-type AF-M512 robust oscillating MAK-2 recruitment to the plasma membrane and accumulation of the protein at the fusion site after cell–cell contact were observed (Fig. 5A). In non-interacting Δbem1 germlings (strain 2-MG), no recruitment of MAK-2 to sites at the plasma membrane occurred. Similarly, even in the rare fusion tips no visible concentration of MAK-2-GFP to the cell tips became apparent. In rare cases, accumulation of MAK-2-GFP around the fusion pore was found (Fig. 5C–E).

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Figure 5. Dynamic plasma membrane recruitment of MAK-2 and SO is not observed in Δbem1.

A and B. MAK-2-GFP (A) (strain AF-M512) and SO-GFP (B) (strain AF-SoT8) in tropically interacting wild-type germlings. Time in hours and minutes. Scale bar: 2 μm.

C–E. MAK-2-GFP expressing Δbem1 germlings (strain 2-MG). (D and E) Arrows indicate sites of cell–cell contact. Scale bars, 5 μm.

F–H. SO-GFP mis-localizes in Δbem1 (strain 2-SG). Arrows indicate fusion tips and sites of cell–cell contact. Scale bars: 5 μm.

I. Western blot analysis for detection of phosphorylated MAK-2. Proteins were extracted form germinating spores at the indicated time points.

J. Nomarski images of germinating conidia at these time points. Asterisks indicate tropic interactions. Scale bar: 5 μm.

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Similarly, no wild-type typic oscillating recruitment of SO-GFP to the plasma membrane was observed in Δbem1. Instead in most Δbem1 fusion pairs (strain 2-SG) SO-GFP was simultaneously present at the plasma membrane in both fusion partners, but was not concentrated at the tip region, suggesting aberrant or aborted cell–cell interaction (n > 100) (Fig. 5F–H). Consistent with this hypothesis, none of the tropically interacting cell pairs maintained mutual attraction or completed fusion during observation, suggesting that Δbem1 interactions are highly unstable and are readily aborted during experimental manipulation.

An earlier study reported a temporal correlation between conidial germination and fusion and the activation of the MAK-2 MAP kinase module, indicated by phosphorylation of MAK-2 (Pandey et al., 2004). To test if the lack of BEM1 influences the activation of MAK-2, we compared phosphorylation of MAK-2 in a wild-type and Δbem1 germination time-course using a phospho-specific antibody (Fig. 5I and J). Consistent with the earlier report (Pandey et al., 2004), increased amounts of phosphorylated MAK-2 were detected in wild-type FGSC 9719 with the onset of germination (2 h) and peaked at a time point when highest frequencies of tropic interactions and fusion are usually observed (3–4 h). After fusion of most germlings was completed (5–6 h) no phosphorylated MAK-2 was detected. In contrast, in Δbem1 (21-A1) no detectable amounts of the phosphorylated MAP kinase were present during the early time points, when conidia germinated and germ tubes extended, while no tropic interactions occurred. At a later time point (4.5 h) when rare and aberrant fusion events became apparent, phosphorylation of MAK-2 was detected (Fig. 5I and J). As described above, fusion in Δbem1 has a highly reduced frequency and occurs in a much narrower time frame than in wild type. Phosphorylation of MAK-2 was only detected within this time frame. Together, these data suggest that activation of the MAK-2 MAP kinase module is not related to germination but solely to cell–cell attraction and fusion. Absence of BEM1 results in a lack of MAK-2 activation at early time points.

BEM1 localizes to growing hyphal tips and is recruited to sites of plasma membrane fusion

To determine the subcellular localization and dynamics of BEM1 we constructed BEM1–GFP fusion constructs. The gfp encoding sequence was fused to the 3′ end of the bem1 open reading frame. This construct was expressed under the control of either the native bem1 promoter (strain 2-BG) or the ccg-1 promoter (strain 2208), which is routinely used for heterologous expression in N. crassa (Freitag et al., 2004). At the time when these experiments were conducted, an alternative annotation of the bem1 open reading frame was present in the MIPS database, which determined an ATG 141 bp upstream of the start codon predicted by the Broad Institute, as a potential start site. This longer open reading frame was also amplified and fused to the ccg-1 promoter and gfp.

Expression of both short constructs in the Δbem1 mutant resulted in restoration of the wild-type phenotype (Figs 2 and 3, and data not shown), indicating that the fusion proteins were fully functional. In contrast, expression of the long construct resulted in a strong hyphal polarity defect, even in a wild-type background (Fig. S3A and B, and data not shown). Interestingly, while hyphal polarity was severely affected and germinating spores frequently established more than just one polarity axis and germ tube, germling fusion appeared to be normal (Fig. S3C). By Western blot analysis using an anti-GFP antibody, synthesis of full-length proteins and the relative amount of BEM1–GFP, produced on the basis of the three different expression constructs, was tested. For both strains expressing the short constructs, a band corresponding to a protein weight of c. 90 kDa was detected, which is the predicted size of the BEM1–GFP fusion protein. Expression under the control of the ccg-1 promoter resulted in a c. 1.9 times higher protein amount compared with expression under the native promoter (Fig. S4). Expression of the long construct controlled by the ccg-1 promoter resulted in two distinct protein bands (Fig. S3D). One of the size of the normal short BEM1–GFP fusion construct and a less intense band corresponding to the predicted size of the longer construct. This suggests that although the long construct is expressed under the control of the ccg-1 promoter, translation still preferably starts at the second ATG. In strains expressing bem1–gfp under control of the native promoter, the longer protein variant was not detected, although here the first ATG is present, too. Together, these observations indicate that the short variant is the naturally occurring one. In all cases, BEM1–GFP showed comparable localization patterns. However, in case of the long expression construct, fluorescence microscopy could not distinguish the short and long protein variant (Fig. S3E–I). In all subsequent localization studies only strains expressing the short variant were analysed.

BEM1–GFP localized to growing tips of germ tubes in interacting as well as in non-interacting hyphae (Figs 6A and B and S5), thus, not allowing a direct conclusion concerning a specific function of the protein in the mutual attraction of the fusion partners. However, after physical contact of two fusion tips, the localization pattern changed, such that the protein, which had localized in a crescent shape at the growing tips, now concentrated in the area of physical contact. Deconvolution microscopy and subsequent 3D reconstruction revealed that BEM1 localizes in a ring-like structure potentially around the forming fusion pore (Fig. 6C), suggesting a potential function of BEM1 in fusion pore formation. After completion of fusion the BEM1–GFP signal disappeared.

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Figure 6. BEM1–GFP localizes to germling tips and is recruited to the site of cell–cell contact.

A. Time series of tropically interacting BEM1–GFP expressing germlings (strain 2208). Time in h and min. Scale bar, 2 μm. Upper panel: Nomarski image, lower panel: GFP.

B. Non-interacting germling (strain 2208). Scale bar, 2 μm. Left: Nomarski image, right: GFP.

C. Nomarski image (left) and 3D reconstruction of a stack of fluorescence images (middle and right) of a BEM1–GFP expressing germling pair (strain 2208). Right: 3D reconstruction slightly rotated. Scale bar, 2 μm.

D. FRAP time series of fluorescence images. The BEM1–GFP signal at the site of cell–cell contact was bleached in the area indicated by the square. Time in minutes and seconds. Strain 2208. Scale bar, 2 μm.

E. Recovery of the BEM1–GFP signal after photobleaching at the site of cell–cell contact. Error bars indicate standard deviations from three independent experiments.

F and G. successfully fused (F) and unfused (G) Δbem1 germling pairs indicated by green and red cytoplasmic mixing or separation respectively. Arrows indicate site of cell–cell contact. Green channels (middle left). Red channels (middle right). Merged channels (right). Scale bar, 5 μm.

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We hypothesized that potential functions of BEM1 after cell–cell contact require active recruitment of the protein to sites of plasma membrane fusion. To distinguish this hypothesis from the possibility that the presence of BEM1 at the fusion pore is just caused by residual protein complexes from the time of tropic growth, we applied FRAP (fluorescence recovery after photobleaching) analysis. Signal intensity was rapidly recovered after photobleaching of the cell–cell contact area, indicating highly dynamic exchange and recruitment of BEM1 to the region of fusion pore formation (Fig. 6D and E). To test if the presence of BEM1 is essential for plasma membrane fusion, quantitative fusion assays testing cytoplasmic mixing after membrane merger were conducted. In these assays, spores expressing GFP were mixed with equal numbers of conidia expressing cytoplasmic mCherry. Fusion pairs consisting of a red and a green fluorescent cell were further analysed. If cell fusion was successful, red and green fluorescence was detected in both cells (Fig. 6F), if fusion did not take place the two colours remained separated (Fig. 6G). As a result, no significant reduction in cell fusion was detected in Δbem1bem1 92% fused, wild type 94% fused, n = 50), indicating that BEM1 is dispensable for cell wall removal and plasma membrane fusion (Fig. 6F and G).

The localization pattern and dynamics of BEM1 around the fusion pore are reminiscent of the MAP kinase MAK-2. We hypothesized that the two proteins might colocalize at this stage. To test this hypothesis localization of dsRED-BEM1 and MAK-2-GFP were analysed in a heterokaryon consisting of strains 2-BR and AF-M512 expressing the two fusion constructs. During directed growth of two germlings towards each other MAK-2 showed the typical oscillating tip recruitment reported before. BEM1–GFP continuously remained at the cell tips. Clear colocalization of the two proteins was not seen (data not shown).

In mature hyphae BEM1–GFP accumulated at growing hyphal tips in a crescent shape similar to its localization pattern in germlings (Fig. 7A). In addition to its tip localization the fluorescent BEM1–GFP fusion protein was frequently detected at septa. Deconvolution microscopy and subsequent 3D reconstruction revealed that BEM1 localizes in a ring-like structure around the septal pore (Fig. 7B). These findings led us to revisit the phenotypic characterization of Δbem1 hyphae. To determine potential defects in septation the average distance between septa within hyphae was determined. No significant differences between wild-type strain FGSC 9719 (84 ± 44 μm) and the Δbem1 isolate 21-A1 (85 ± 43 μm) were found, indicating that BEM1 is dispensable for septa formation. Subsequent FRAP analyses revealed that the tip as well as the septa localization of BEM1 is highly dynamic (Fig. 7C–E), suggesting a rapid exchange of membrane-bound BEM1 and a cytoplasmic pool of this protein.

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Figure 7. BEM1–GFP localizes to hyphal tips and septa of vegetative hyphae.

A. Vegetative hypha of BEM1–GFP expressing strain 2208. Scale bar, 2 μm.

B. BEM1–GFP at the septal pore (strain 2208). Nomarski image (upper left), fluorescence image (upper right), merged images (lower left) and 3D reconstruction of fluorescence image stack, rotated 90° (lower right). Scale bar, 2 μm.

C–E. FRAP time series of fluorescence images. The BEM1–GFP signal at the hyphal tip (C) and at the septa (D, E) was bleached in the area indicated by the square. (D and E) Bleaching of the septal signal was only successful after a larger cytoplasmic region was photo bleached. Time in minutes and seconds. Strain 2208. Scale bars, 8 μm (C) and 5 μm (D, E).

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BEM1 promotes directed growth during vegetative but not sexual cell–cell interactions

The results of the germling and hyphal fusion assays indicate that Δbem1 mutant strains are deficient in directed growth during vegetative development. During sexual development N. crassa exhibits also strictly directed hyphal growth. As female reproductive structures trichogynes sense pheromones secreted by spores of the opposite mating type, and grow towards these potential mating partners. By conducting trichogyne assays (see Experimental procedures), we analysed if directed growth of trichogynes is also affected in Δbem1. The crosses analysed included 21-A1/FGSC 2489 and FGSC 988/FGSC 2489 as a control. In both cases, trichogynes grew towards conidia and curled around the mating partner after physical contact, in a manner indistinguishable between wild type and the mutant (Fig. S6). These observations indicate that BEM1 is dispensable for the communication between the mating partners and directed growth of the trichogyne towards the fertilizing spores.

Similar to mutations in the MAK-2 MAP kinase module lack of bem1 results in lethality of sexual progeny

Most previously described germling/hyphal fusion mutants of N. crassa are female sterile. This observation led to the hypothesis that hyphal fusion is essential for the formation of functional female fruiting bodies. To further investigate this question, we analysed fertility of the Δbem1 mutant. For this purpose following crosses were conducted: FGSC 2489/21-A1, 21-A1/FGSC 2489, 21-A1/2-A1 and FGSC 2489/FGSC 988. Between seven and 10 days after inoculation of crossing plates with the female strains, Δbem1 protoperithecia appeared in size, shape and abundance indistinguishable from wild-type protoperithecia (Fig. S7). Seven days after fertilization crosses were analysed for the formation of ascospores. In every combination abundant ascospores were formed and ejected from the ripe perithecia. No difference between wild-type crosses or crosses containing one or two Δbem1 mating partners were observed. Thus, BEM1 is dispensable for either male or female fertility during sexual development of N. crassa. However, when we analysed the progeny of these crosses, it became apparent that almost no Δbem1 isolates could be recovered. From heterozygous crosses only 1–2% of the recovered progeny were Δbem1 (n = 200). In homozygous Δbem1 crosses less than 0.2% of the progeny were viable (n > 1500). Analysis of asci from these crosses showed that each ascus contained eight black ascospores (data not shown). Taken together, these data indicate that Δbem1 ascospore development after fertilization is normal, but that these progeny are unable to germinate, a phenotype termed ascospore lethality. Comparable defects are caused by loss of function mutations in components of the MAK-2 MAP kinase module (Pandey et al., 2004). The molecular role of this signalling pathway in ascospore activation and germination is so far unknown.

The PX domain is dispensable for localization and function of BEM1

BEM1 contains four conserved protein domains (Fig. 1). To test their role for BEM1 function each domain was individually deleted and the resulting strains were tested for sporulation, spore size, growth rate and germling fusion.

To analyse the role of the PX domain a region of 112 aa (position 306–417) was deleted. Analysis of the respective strain ΔPX showed that expression of the ΔPX construct in the Δbem1 strain 2-A1 resulted in restoration of the wild-type phenotype (Fig. 8A–D), indicating that the PX domain is dispensable for BEM1 function. Since the PX domain is a phosphoinositide-binding structural domain involved in targeting of proteins to cell membranes we also tested if its deletion alters BEM1 localization by fusing the deletion construct with GFP. The observed localization pattern in the strain ΔPX-G was indistinguishable from wild-type BEM1 (Fig. S8A–D), indicating that the PX domain is not essential for plasma membrane targeting of BEM1.

figure

Figure 8. Phenotypic analysis of bem1 domain knockout mutants. Tropic interactions after 2.5 h post inoculation (A), linear hyphal growth in race tubes (B), sporulation (C) and spore size (D) of the mutants were compared with wild-type strain FGSC 9719 and Δbem1 21-A1. Error bars for (A) indicate standard deviations of three independent experiments (n = 50, each). Error bars in (B) and (C) indicate standard deviations (n = 3). Error bars for (D) indicate standard deviations (n = 50 spores).

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The PB-1 domain is essential for spore shape but dispensable for germling fusion

To analyse the role of the PB1 domain a region of 76 aa (position 501–576) was deleted. Expression of this construct let to a partial restoration of the Δbem1 phenotype. Tropic interactions between ΔPB1 germlings after 2.5 h of incubation were significantly increased (Fig. 8A). The Δbem1 defects in growth rate, sporulation and spore size could not be complemented by the ΔPB1 construct (Fig. 8B–D). Together, these data indicate that different polarization events are differentially affected by the loss of this domain, suggesting the potential existence of different BEM1 complexes in different developmental types of hyphae.

The two SH3 domains have partially redundant functions

The two SH3 domains were deleted individually as well as simultaneously. In strain ΔSH3a and ΔSH3b amino acids 39–94 and 151–188 were deleted respectively. The double-deletion strain ΔSH3ab carried deletions of both regions. Individual deletion of either one of the two domains had no negative effect on the frequency of tropic interactions observed. However simultaneous deletion of both domains strongly reduced tropic interaction between germlings to a level similar to that observed in the mutant strain (21-A1) (Fig. 8A). These observations suggest that the two SH3 domains have redundant roles in BEM1 function during cell communication and directed growth. Similarly, sporulation was not significantly affected in strains ΔSH3a and ΔSH3b carrying the single deletions, but was strongly reduced in the double-deletion strain ΔSH3ab (Fig. 8C). All strains showed an intermediate phenotype concerning the average spore size. Growth rate was not affected by absence of the SH3a domain but was significantly reduced in strain ΔSH3b. Deletion of both domains did not worsen this phenotype (Fig. 8B and D). Together, these data indicate that SH3a is dispensable for reaching wild-type growth rates.

Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

In this study, we show that BEM1 in N. crassa is dispensable for general polar hyphal growth, but plays important roles in spore formation and during tropic growth involved in vegetative hyphal fusion. The highly orchestrated signalling process involved in germling fusion is greatly destabilized and long distance interactions are absent if BEM1 is lacking. BEM1 localizes to the tips of polar growing hyphae and is actively recruited to sites of membrane fusion. The PB-1 domain of BEM1 is essential for normal spore formation, but dispensable for tropic germling interactions, suggesting the involvement of different BEM1 complexes in these developmental processes.

BEM1 is dispensable for general hyphal polarity establishment and maintenance

Polarized growth is a hallmark of filamentous fungi. The life cycle of N. crassa includes numerous distinguishable polarization events, including spore germination, extension of vegetative hyphae, induction and tropic growth of fusion hyphae, conidia formation and tropic growth of trichogynes as mating structures (Riquelme et al., 2011).

Localization of BEM1 to the growing tips of different types of hyphae suggests that BEM1 is part of a conserved polarity machinery. However, our phenotypic characterization of Δbem1 mutants indicates that BEM1 is of different importance for the various polarization events in N. crassa. During spore germination or hyphal extension, its absence does not result in polarity defects. Similarly, in A. nidulans and E. festucae Δbem1 hyphae possess only minor growth defects, such as slight swelling of hyphal tips (Leeder and Turner, 2008; Takemoto et al., 2011). In A. nidulans, the formin SepA mislocalizes if BemA is absent, suggesting that BemA is required for Spitzenkörper organization (Leeder and Turner, 2008). However, when we localized the Spitzenkörper marker chitin synthase 1 (Sanchez-Leon et al., 2011) in the N. crassa Δbem1 mutant, no significant differences were observed (data not shown). Other proteins are likely to substitute BEM1 functions in general hyphal polarity, probably to a different extent in different species. Studies in yeast suggest that two positive feedback loops contribute in parallel to polarity establishment – one based on bem1p functions the other one on the actin cytoskeleton (Wedlich-Soldner et al., 2004). In the first one, active cdc42p locally recruits bem1p to the plasma membrane. bem1p interacts with cdc24p, which in turn activates more cdc42p. In the second one activated cdc42p leads to the formation of actin cables, resulting in the recruitment of vesicular cdc42p. In filamentous fungi, hyphal growth strongly depends on a highly polarized cytoskeleton. Thus, in general hyphal tip extension, positive cytoskeletal-based feedback loops might therefore be much more important than soluble signalling-based loops including BEM1.

In contrast to general hyphal growth, macroconidia formation and germling/hyphal fusion are severely affected in N. crassa Δbem1 mutants. Interestingly, macroconidiation and germling fusion in N. crassa share many similarities with yeast budding and shmoo formation respectively (Springer and Yanofsky, 1989; Glass and Fleißner, 2006; Read et al., 2009). Both processes of yeast development also depend highly on bem1p function (Chenevert et al., 1992; 1994; Kao et al., 1996; Lyons et al., 1996), suggesting that S. cerevisiae and N. crassa employ conserved polarity machineries in these related processes.

During macroconidiation in N. crassa the lack of BEM1 results in enlarged and misshaped spores, containing numerous nuclei. This phenotype hints to defects in the controlled re-initiation of apical budding after a spore has formed. These defects would then result in prolonged isotropic growth, given rise to the observed giant spores. In the absence of internal landmarks or external signals, S. cerevisiae cells spontaneously polarize. This phenomenon, known as symmetry breaking, led to a model, in which activated cdc42p-GTP, which is initially randomly distributed at the cell periphery, forms fluctuating clusters, which compete for rapidly diffusing bem1p. The winning polarity centre gets strongly reinforced by a positive feedback loop involving a bem1p–PAK–cdc24p complex, which interacts with active membrane-bound cdc42p-GTP. This competition/reinforcement leads eventually to a winner takes all situation resulting in the establishment of only one polarity axis (Atkins et al., 2008; Kozubowski et al., 2008). During macroconidiation of N. crassa lack of BEM1 potentially delays the successful establishment of one polarity centre, resulting in prolonged isotropic growth. In an alternative model, the increase in spore size would be a consequence of the higher number of nuclei and therefore of defective cytokinesis. However, this hypothesis appears less likely, since nuclear division and conidia formation are not clearly linked in N. crassa. Conidia are formed from multinucleated hyphae, which alternate between polar and apolar growth (Springer and Yanofsky, 1989). As a result a pearl necklace-like structure is formed. Then, secondary septa are introduced, resulting in the formation of individual spores with various numbers of nuclei (in wild type typically between one and four).

Formation of normal sized spores requires the PB-1 domain of the BEM1 protein. In contrast, this domain is dispensable for germling fusion. Similarly, expression of the 5′-extended bem1 open reading frame under control of the ccg-1 promoter resulted in strong defects in general hyphal polarity and establishment of a single polarity axis during germination. Strikingly, germling fusion appeared normal in these isolates. Together, these observations suggest that different parts of the BEM1 protein are of different importance for the various growth modes. BEM1 is probably part of different tip localized protein complexes, which have diverse molecular compositions and functions. These data also indicate that no correlation between spore size and fusion competence exist, thereby excluding the possibility that the fusion deficiency in Δbem1 might be a secondary effect of the conidial morphogenetic defects. The observation that expression of the extended construct has a strong dominant effect on hyphal polarity is best explained by a titration effect. The longer protein potentially binds certain interaction partners, but can then not fulfil its normal function, keeping the interacting factors captured. A similar observation was made in A. nidulans, when only a part of the BemA protein was expressed (Leeder and Turner, 2008).

In yeast mating, bem1p functions as a molecular adaptor connecting the MAP kinase module of the pheromone response pathway with the core polarity machinery, resulting in recruitment of the MAP kinase fus3p to shmoo tips (Leeuw et al., 1995; Lyons et al., 1996). In germling fusion in N. crassa, the fus3p homologous MAP kinase MAK-2 gets also recruited to fusion tips, however, in a highly dynamic oscillating fashion, in which recruitment alternates between the two fusion partners (Fleißner et al., 2009b). This highly orchestrated process likely involves positive and negative feedback loops, which partially might depend on BEM1. In S. cerevisiae the absence of bem1p leads to a substantially reduced sensitivity to α-factor, indicating that bem1p facilitates efficient pheromone signalling (Kao et al., 1996). Our finding that in Δbem1 germling fusion is reduced, delayed and takes place only over very short distances, suggests a similar promoting function in N. crassa. Long distance interactions of conidial germlings require the polarity axis to be quickly adjustable to incoming signals. If one assumes additive actions of a cytoskeletal-based and a soluble BEM1-dependent polarity mechanism, the latter one might be much more suited to allow highly dynamic tropic interactions. Thus, in contrast to general polarized hyphal growth, germling fusion might much stronger depend on BEM1 functions. Consistent with this hypothesis, general hyphal growth ceases after disintegration of the microtubular cytoskeleton, while tropic growth involved in germling fusion continues (Roca et al., 2010). Lack of BEM1 has the exact opposite outcome.

When we tried to analyse the subcellular dynamics of SO and MAK-2 in the Δbem1 mutant, the tropic interaction of germlings was frequently and quickly aborted, and SO became mislocalized in both fusion partners. In an earlier study, we found the exact same characteristic pattern of SO mislocalization after specific inhibition of MAK-2 activity by a kinase inhibitor (Fleißner et al., 2009b). Together these observations suggest that in Δbem1 MAK-2 activation is highly instable and that BEM1 is essential for robust signalling via the MAP kinase module. So far, it remains unclear where and how BEM1 might stabilize cell communication since no clear indications for colocalization of the proteins were found. Similarly, testing potential physical interactions of MAK-2 and BEM1 via yeast two-hybrid analysis rendered only negative results (data not shown). In yeast, bem1p functions as a scaffold connecting cdc42p with its activator cdc24p and the cdc42p effector ste20p, a p21-activated kinase (Elion, 2000). In addition to Cdc42, filamentous fungi contain the closely related Rac1. In Ustilago maydis interaction of this GTPase with Cdc24 is also promoted by Bem1 (Frieser et al., 2011). In addition, in a screen for hyphal fusion-deficient mutants in N. crassa, a rac-1 mutant was isolated (Fu et al., 2011). In mature hyphae, CDC42 and RAC1 both localize to hyphal tips. The localization pattern of CDC42 is highly reminiscent of BEM1. In germlings, both GTPases localize overall to the plasma membrane but are enriched at hyphal tips (Araujo-Palomares et al., 2011). Together, these observations support a model in which RAC1 and/or CDC42 are involved in activation of MAK-2, and this activation is promoted by BEM1. The clear function and relationship of these factors during germling fusion of N. crassa have to be determined in future studies.

BEM1 as a potential linker of ROS signalling and polar growth?

A recent publication reported the BEM1 homologue of E. festucae (BemA) as being physically interacting with NoxR, a regulatory component for NADPH oxidases, which produce ROS (Takemoto et al., 2011). Stable NoxR complex formation at hyphal tips is dependent on the presence of BemA, suggesting a direct link between ROS generating systems and polarity machineries. Interestingly, we recently showed that NoxR and the NADPH oxidase Nox1 are essential for germling fusion in the grey mould Botrytis cinerea (Roca et al., 2012). A comparable observation was reported for N. crassa (Read et al., 2012), suggesting a potential role of ROS signalling in germling fusion. Based on these observations, we hypothesize that BEM1 might function as a linker connecting ROS and MAP kinase signalling with cell polarity. Consistent with this hypothesis Δbem1, Δmak-2 and Δnor1noxr) of N. crassa share other characteristic phenotypes, such as inviable sexual progeny (ascospores) (Cano-Dominguez et al., 2008). Ascospore germination is thought to include the controlled production of ROS spikes, resulting in establishment of a polarity axis and germ tube formation. To detect potential differences in ROS production between wild type and Δbem1 we stained germlings with the ROS-sensitive Nitro blue tetrazolium salt (NBT). Similar to what we have reported for B. cinerea (Roca et al., 2012), ROS were detected at the tips of germ tubes, at fusion tips and at fusion points after cell–cell contact. However, no differences in these patterns were found in wild type and mutant (data not shown). Similarly, ΔbemA hyphae in E. festucae exhibited similar ROS patterns as wild-type ones (Takemoto et al., 2011). The general detection of ROS, independent of their origin, appears to be not specific enough to detect subtle, but potentially very significant differences in their spatial and temporal production in mutants and wild type.

Based on the presented data we propose a model for germling fusion in N. crassa, in which a fusion signal activates the MAP kinase MAK-2 probably through the production of ROS. Activated MAK-2 is recruited to the site of polar growth potentially promoted by BEM1. In addition, BEM1 promotes also the localized accumulation of the ROS producing systems and the polarity machinery. By linking these different activities, BEM1 enhances positive feedback loops, which result in robust but highly dynamic cell-to-cell signalling and tropic growth. Further analysis of the composition and activity of BEM1 complexes in different polarization events in N. crassa will extend our understanding of the molecular networks controlling polarized growth, tropic responses and cellular differentiation.

Experimental procedures

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

N. crassa strains and growth media

Strains used in this study are listed in Table 1. A detailed strain list is provided within the supplemental data (Table S1), as well as a list of the plasmids used for the creation of theses strains (Table S2). The list of plasmids includes short descriptions explaining the corresponding cloning strategies. Strains were grown on Vogel's minimal medium (Vogel, 1956). For strains carrying auxotrophic markers, required supplements were added to the medium. Crosses were performed on Westergaard's medium (Westergaard and Mitchell, 1947). To test the mating type of strains, they were crossed with mating type tester strains FGSC 4347 and FGSC 4317.

Table 1.  N. crassa strains used in this study.
StrainGenotypeOrigin or reference
  1. FGSC, Fungal Genetic Stock Center.

FGSC 9719Δmus52::bar+; a FGSC
21-A1Δbem1::hph; Δmus52::bar+; a This study
FGSC 9720Δmus52::bar+; his3; A FGSC
2-A1Δbem1::hph; Δmus52::bar+; his3; A This study
2208Δbem1::hph; his3+::Pccg1-bem1–gfp; Δmus52::bar+; A This study
AF-M512 his3+::Pccg1-mak2–gfp; A Fleißner et al. (2009b)
2-MGΔbem1::hph; his3+::Pccg1-mak2–gfp; Δmus52::bar; A This study
AF-SoT8 his3+::Pccg1-so–gfp; A Fleißner and Glass (1990)
2-SGΔbem1::hph; his3+::Pccg1-so–gfp; Δmus52::bar; A This study
ΔPB1Δbem1::hph; his3+::Pccg1-bem1ΔPB1; Δmus52::bar; A This study
ΔPXΔbem1::hph; his3+::Pccg1-bem1ΔPX; Δmus52::bar; A This study
ΔPX-GΔbem1::hph; his3+::Pccg1-bem1ΔPX–gfp; Δmus52::bar; A This study
ΔSH3aΔbem1::hph; his3+::Pccg1-bem1ΔSH3a; Δmus52::bar; A This study
ΔSH3bΔbem1::hph; his3+::Pccg1-bem1ΔSH3b; Δmus52::bar; A This study
ΔSH3abΔbem1::hph; his3+::Pccg1-bem1ΔSH3ab; Δmus52::bar; A This study
2-BGΔbem1::hph; his3+::Pbem1-bem1–gfp; Δmus52::bar; A This study
FGSC 6103 his3; A FGSC
335-BG his3+::Pccg1-bem1–gfp; A This study
WT-HG his3+::Pccg1-h1–gfp; Δmus52::bar+; A This study
2-HGΔbem1::hph; his3+::Pccg1-h1–gfp; Δmus52::bar+; A This study
N3-06 his3+::Pccg1–gfp; A This study
2-GΔbem1::hph; his3+::Pccg1–gfp; Δmus52::bar; A This study
N3-07 his3+::Pccg1-cherry; A This study
2-CΔbem1::hph; his3+::Pccg1-cherry; Δmus52::bar; A This study
2-BRΔbem1::hph; his3+::Pccg1-dsRED-bem1; Δmus52::bar+; A This study
FGSC 2489 A FGSC
FGSC 988 a FGSC
FGSC 4317 fl A FGSC
FGSC 4347 fl a FGSC
335-BG his3+::Pccg1-bem1–gfp; A This study
W0103 his3+::Pccg1-bem1–gfp; mat A This study
W0402 his3+::Pccg1-bem1–gfp; mat A This study
2210Δbem1::hph; his3+::Pccg1-bem1–gfp This study

Construction of N. crassa Δbem1 mutant

Neurospora crassa Δbem1 mutants were constructed using the ‘Neurospora Knockout Strain Kit’ from the FGSC (Colot et al., 2006). The transformation cassette was assembled using yeast recombinational cloning (Colot et al., 2006); primer sequence information was provided by the Neurospora Functional Genomics Project (http://www.dartmouth.edu/~neurosporagenome/). N. crassa strains FGSC 9719 and FGSC 9720 were transformed with the replacement cassette by electroporation of macroconidia (Margolin et al., 1997; R.L. Metzenberg and K. Black, pers. comm.). The obtained transformants were tested for homologous single copy integration of the replacement cassette by using Southern blot hybridization analysis. Homokaryotic Δbem1 strains were obtained from primary transformants through single spore isolation. Macroconidia were spread on Brockman & De Serres medium (Brockman and De Serres, 1963) containing 200 μg ml−1 hygromycin. After an overnight incubation at 30°C colonies derived from single spores were transferred to slant tubes containing minimal medium (MM) with 200 μg ml−1 hygromycin. The described purification was repeated up to three times for each transformant. The homokaryotic state of the final isolates was confirmed by using PCR analysis using bem1 and hygB-specific primers.

Construction of GFP fusion constructs and domain deletion mutants

To fuse the bem1 sequence with gfp and to express this construct under the ccg-1 promoter, the bem1 open reading frame was amplified by PCR. The employed oligo primers contained the restriction sites XbaI and PacI, which were used to clone the construct into the vector pMF272 (Freitag et al., 2004). To express the bem1–gfp fusion construct under the bem1 promoter the open reading frame plus a 1 kb fragment upstream of the start codon were amplified by PCR. The employed oligo primers contained the restriction sites NotI and PacI, which were used to clone the fragment into pMF272. Domain deletion constructs were assembled via yeast recombinational cloning and subsequent cloning into pMF272. The gfp sequence of the plasmid was removed in this cloning step. All domain deletion constructs contained an 800 bp terminator sequence downstream of the stop codon.

Sample preparation for microscopy

Growth medium for microscopy was either Vogel's minimal medium (Vogel, 1956) or minimal medium containing 1.2% sodium acetate as the sole carbon source. Germling samples were grown for 2–3 h at 30°C until points of fusion could be observed. Hyphal samples were grown overnight at 30°C and squares of 1 cm of agar were cut from the edge of the colony where hyphal density was low and fusion between hyphae could be observed.

Light and fluorescence microscopy

Samples were observed on a Zeiss Axiophot-2 microscope equipped with Nomarski optics using a Zeiss Plan-Neofluar 100×/1.30 oil immersion objective (440481). As a light source for fluorescence excitation an Osram mercury short arc bulb HBO 100 W/2 was used. Images were captured with a PCO Pixelfly camera which was controlled by a PC using software programmed in C++.

Deconvolution microscopy

Samples were observed on a Zeiss Axioplan-Imaging-2 microscope equipped with Nomarski optics using a Zeiss Plan-Neofluar 100×/1.30 oil immersion objective (1031-172). Stacks of images with an increment of 200 nm and up to 40 focal levels were captured with a PCO Sensicam. Both, the CCD camera and the internal focus drive to move the stage were controlled by a PC using software programmed in C++. As light sources for fluorescence excitation LEDs emitting the required wavelength were used. Image stacks were deconvolved using Huygens deconvolution software, Scientific Volume Imaging (SVI), in classic mode with up to 150 possible iterations. 3D reconstructions were done with the MIP renderer (Huygens, SVI).

Laser scanning microscopy and FRAP

LSM microscopy and FRAP analyses were performed on a Zeiss 510 Meta using a Zeiss C-Apochromat 40×/1.20 water immersion objective and an Argon-Laser as a light source for fluorescence excitation. In FRAP analyses, the recovered fluorescence intensity in the respective regions of interest (ROIs) was set for every time point in relation to the intensity prior to bleaching minus the intensity at the first time point after bleaching. Correspondingly, the relative intensity at a specific time point after bleaching (I t rel.) was calculated by the formula: I t rel. = (ItI b)/(I 0I b) × 100%, were It is the absolute intensity at time t, I b is the absolute intensity directly after bleaching and I 0 is the absolute intensity directly before bleaching. In a control ROI which was set in the same hypha and as far as possible away from the bleached area, general bleaching during image capturing was monitored. Relative intensities (I t rel.) were corrected for general bleaching according to: I t rel. corr. = (I 0 contr./I t contr.) × I t rel. with I 0 contr. being the absolute intensity in the control ROI directly before bleaching and I t contr. the absolute intensity in the control ROI at time t.

Quantitative germling fusion assay

Assays were performed as described in Fleißner et al. (2009a).

Quantification of aerial hyphal growth

Glass tubes containing 2 ml of liquid Vogel's minimal medium (Vogel, 1956) were inoculated with 100 μl cheese-cloth filtered conidial spore suspensions (107 ml−1). Tubes were plugged with cotton and incubated at 26°C for 4 days. After the first 24 h height of aerial hyphal growth was marked and set as a reference line. Three days later heights of aerial hyphal growth were determined by measuring the distance between the reference line and the reached height. The values were divided by three to obtain the average daily growth rate. A minimum of three samples was taken into account for each strain.

Trichogyne attraction assays

Assays were performed as described before (Bistis, 1981; Fleißner et al., 2005).

Quantification of tropic germling interactions

Strains were grown on Vogel's minimal media (Vogel, 1956) slant tubes for 4–6 days or until significant conidiation occurred. Conidia were harvested by vortexing of the slant tubes with 2 ml of ddH2O. The conidial suspension was filtered by pouring through cheesecloth. Conidia were diluted to a concentration of ∼ 2 × 106 conidia ml−1 and 300 ml were spread on a minimal media plate. The plates were incubated at 30°C for 3 and 5 h respectively. Squares of 1 cm were excised and observed with the standard microscopic set-up described above. Each germling involved in a tropic interaction was counted as one. Two germlings were counted each as non-interacting if no tropic growth was observed, their conidia were no further away from each other then 10 μm and their germ tubes were grown out with a length of at least 5 μm.

Western blot analysis

For determination of BEM-1-GFP expression assayed strains were grown in liquid culture at 30°C for 16 h. The mycelium was harvested by filtering through one layer of Miracloth (Calbiochem) and frozen in liquid nitrogen. Protein extraction and sodium dodecylsulphate-polyacrylamide gel electrophoresis were performed as described in Pandey et al. (2004). Membranes were subsequently incubated with anti-GFP mouse monoclonal antibody (clones 7.1 and 13.1; dilution, 1:1000; Roche) and anti-mouse (heavy and light chains) peroxidase-conjugated antibody (dilution, 1:4000; Invitrogen). To assess protein loading, blots were stripped and probed with anti-β-tubulin monoclonal antibody (clone TU27; dilution, 1:2000; Covance) and the secondary anti-mouse antibody (dilution, 1:4000, see above). Proteins were detected by using the super signal west pico chemiluminescent substrate (Thermo Scientific). Density of bands in greyscale images of scanned X-ray films were compared using the NIH ImageJ software gel analyser tool. The determination of the level of phosphorylated MAK-2 during tropic growth of the germlings was performed as described in Pandey et al. (2004). After electrophoresis, proteins were transferred by wet electroblotting (Bio-Rad) onto polyvinylidene fluoride membranes (Milipore). Blocking and hybridization was done with 5% bovine serum albumin in phosphate-buffered saline (+0.05% Tween20). The membranes were incubated with anti-phospho p44/42 MAP kinase antibody (1:1000 dilution, #9101, Cell Signaling Technology) and anti-rabbit peroxidase-conjugated IgG (1:100 000 dilution, #7074, Cell Signaling Technology). Protein loading was assessed using the anti-β-tubulin antibody as described above. Proteins were detected by using the highly sensitive super signal west femto chemiluminescent substrate (Thermo Scientific) and exposure of the luminescent membranes to X-ray films.

Analysis of conidiophore morphology

To analyse the morphology of conidiophores, strains were grown on 2% water agar. After 24–48 h of incubation at 30°C conidiophores were isolated with the help of an inoculation lancet and transferred to a droplet of water containing 100 μg ml−1 Calcofluor white for staining of the cell walls. Samples were analysed by light and fluorescence microscopy.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

We thank Ralf Schnabel, Christian Hennig and Robert Hänsch for their help with the microscopy. We thank Louise Glass for hosting TS during his initial experiments. We thank our students Daniela Heine and Ulrike Siegmund for assisting in some of the experiments. We thank Meritxell Riquelme for providing a plasmid. We greatly acknowledge use of materials generated by PO1 GM068087 Functional analysis of a model filamentous fungus. This work was in part supported by funding from the German Research Foundation (Grant FL 706/1-1) to A.F.

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  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information
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Supporting Information

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information
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