The Cell End Marker Protein TeaC Is Involved in Growth Directionality and Septation in Aspergillus nidulans

Strains, plasmids, and culture conditions.Supplemented minimal medium (MM) and complete medium for A. nidulans were prepared as described earlier, and the standard strain construction procedures have also been described elsewhere (13). A list of A. nidulans strains used in the present study is given in Table 1. Standard laboratory Escherichia coli strains (XL1-Blue and Top10F′) were used. Plasmids are listed in Table 2.

Molecular techniques.Standard DNA transformation procedures were used for A. nidulans (37) and E. coli (26). For PCR experiments, standard protocols were applied using a Biometra TRIO Thermoblock for the reaction cycles. DNA sequencing was done commercially (MWG Biotech, Ebersberg, Germany). Genomic DNA was extracted from A. nidulans with the DNeasy plant minikit (Qiagen, Hilden, Germany). DNA analyses (Southern hybridizations) were performed as described previously (26).

Deletion of teaC.Flanking regions of teaC were amplified by PCR using genomic DNA and the primers TeaC-left-for (5′-GAA CAG TTG CCT TTC GAA AT-3′) and TeaC-left-rev-SfiI (5′-TGG TGG CCA TCT AGG CCG TAG CAG GAT GTT CAA AGG-3′) for the upstream region of teaC and the primers TeaC-right-for-SfiI (5-AAT AGG CCT GAG TGG CCC CGA CTG GCA CCA CTA C-3′) and TeaC-right-rev (5′-AGA GGC TGG ATT CCT TCT-3′) for the downstream region and cloned into pCR2.1-TOPO to generate plasmids pYH32 and pYH33, respectively. The SfiI restriction sites are underlined. In a three-fragment ligation, the N. crassa pry-4 gene was released from plasmid pSCI with SfiI and ligated between the two teaC-flanking regions, resulting in the vector pYH14.

Transformants of strain TN02A3 were screened by PCR for the homologous integration event. Single integration of the construct was confirmed by Southern blotting (see Fig. S1 in the supplemental material). A total of 20 strains were analyzed, of which 8 showed the teaC deletion pattern. All eight deletion strains displayed the same phenotypes. One teaC deletion strain was selected for further studies and named SYH21. Coupling of the observed phenotypes with the gene deletion event was confirmed by crosses, recomplementation with teaC-derived clones, and downregulation of the gene through the inducible alcA promoter.

Tagging of proteins with GFP and mRFP1.To create an N-terminal green fluorescent protein (GFP) fusion construct of TeaC, a 1.2-kb N-terminal fragment of teaC (starting from ATG) was amplified from genomic DNA with the primers teaC-AF(5′-TAG GCG CGC CGA TGG CTA GAC CTA GAA TGG-3′) and teaC-PR(5′-CTT AAT TAA TTC TTC AAC AGC CTT AGT TTT-3′) and cloned into pCR2.1-TOPO, yielding pYH34. The restriction sites are underlined. The AscI-PacI fragment from pYH34 was subcloned into the corresponding sites of pCMB17apx, yielding pYH06. To create an N-terminal mRFP1 fusion construct of TeaC, the AscI-PacI fragment from pYH06 was subcloned into the corresponding sites of pDM8, yielding pYH24. To produce TeaC N-terminally tagged with mRFP1 expressed from the native promoter, a 1.5-kb fragment of the teaC putative promoter region was amplified from genomic DNA with the primers TeaCp-pro-AvrIII (5′-ACC TAG GTG ACC TTG GGT ATC GTT G-3′) and teaC-pro-KpnI (5′-AGG TAC CAC GAA TTA TGT AGC AGG AT-3′), digested with AvrI and KpnI, and ligated with AvrI-KpnI-digested pYH24, yielding pYH30 (alcA promoter replaced with the teaC promoter in pYH24). Using the same approach as for TeaC, N-terminal GFP fusion constructs of TeaC were created. All plasmids were transformed into the uracil-auxotrophic strain TN02A3 (ΔnkuA). The integration events were confirmed by PCR and Southern blotting (results not shown).

For bimolecular fluorescence complementation (BiFC) analyses, the N-terminal half of YFP (YFP^N) or the C-terminal half of YFP (YFP^C) was fused to the N terminus of the protein of interest. YFP^N (154 amino acids of YFP and 5 amino acids linker) was amplified with the primers fwd-Kpn-YFP-N (5′-CGG TAC CAT GGT GAG CAA GGG CGA GGA GCT G-3′) and rev-YFP-N-Li-Asc (5-CGG CGC GCCCGT GGC GAT GGA GCG CAT GAT ATA GAC GTT GTG GCT GTT GTA G-3′). YFP^C (86 amino acids of YFP and 17 amino acids linker) was amplified with the primers fwd-Kpn-YFP-C (5′-CGG TAC CAT GGC CGA CAA GCA GAA GAA CGG CAT CAA GG-3′) and ev_YFP-C_Li_Asc (5′-CGGCGCGCCGTGGTTCATGACCTTCTGTTTCAGGTCGTTCGGGATCTTGCAGGCCGGGCGCTTGTACAGCTCGTCCATGCCGAGAGTGATCCC-3′). The KpnI-AscI fragment of YFP^N or YFP^C was ligated into KpnI- and AscI-digested pCMB17apx, yielding pDV7 (GFP replaced with YFP^N in pCMB17apx) and pDV8 (GFP replaced with YFP^C in pCMB17apx). To create an N-terminal YFP^C fusion construct of TeaC, the AscI-PacI fragment from pYH06 was subcloned into the corresponding sites of pDV8, yielding pYH08. To create N-terminal YFP^N fusion constructs teaA and sepA fragments from pNT1, pNT9, were subcloned into the corresponding sites of pDV7, yielding pYH01 and pYH03.

Yeast two-hybrid analysis.The yeast two-hybrid analysis was performed by using the MatchMaker3 Gal4 two-hybrid system (BD Clontech). For bait generation, fragments of teaC cDNA corresponding to the N-terminal half of TeaC (356 amino acids) with the primers TeaC-EF (5′-GGC CGA ATT CAT GGC TAG ACC TAG AAT GG-3′) and TeaC-BMR (5′-GGA TCCTTA CAG TAG GTT CGG AGT GAG-3′) or corresponding to the C-terminal half of TeaC (428 amino acids) with the primers TeaC-EMF (5′-GAA TTC GAA AAG CCG CGC TCA AG-3′) and TeaC-BR (5′-GGC CGG ATC CTT ATT GAC TCG TCG ACC TG-3′) were amplified and cloned into the pGBK7 vector, which contains the Gal4 DNA-BD and the TRP1 marker, yielding pYH17 and pYH19 (BD Clontech). The fragments of teaC cDNA corresponding to the N-terminal half and C-terminal half of TeaC from pYH17 and pYH19 were amplified and cloned into the pGADT7 vector, which contains the GAL4 DNA-AD and the LEU2 marker (BD Clontech), yielding pYH16 and pYH18. pGBK7-associated plasmids were transformed into yeast Y187 (mating type MATα), and pGADT7-associated plasmids were transformed into yeast AH109 (mating type MATa). The system utilizes two reporter genes (HIS3 and lacZ) under the control of the GAL4-responsive UAS. The β-galactosidase activity was analyzed by liquid culture assays using ONPG (o-nitrophenyl-β-d-galactopyranoside; Sigma) as a substrate. The activity was calculated in Miller units (20). Experiments were repeated three times.

Light and fluorescence microscopy.For live-cell imaging of germlings and young hyphae, cells were grown on coverslips in 0.5 ml of MM plus 2% glycerol (derepression of the alcA promoter and thus moderate expression of the gene), MM plus 2% threonine (activation of the gene thus high expression levels), or MM plus 2% glucose (repression of the alcA promoter). Cells were incubated at room temperature for 1 to 2 days. For pictures of young hyphae of each gene deletion strain, the spores were inoculated on microscope slides coated with MM plus 2% glucose plus 0.8% agarose and grown at 30°C for 1 day. Images were captured at room temperature by using an Axioimager microscope (Zeiss, Jena, Germany). Images were collected and analyzed by using the AxioVision system (Zeiss).

FM4-64, Hoechst 33342, calcofluor white, benomyl, and cytochalasin A treatment.FM4-64 was used at a concentration of 10 μM in the medium. Coverslips were incubated for 5 min and washed. Benomyl [methyl 1-(butylcarbamoyl)-2-benzimidazole carbamate; Aldrich] was used at a final concentration of 2.5 μg/ml in the medium from a stock solution of 1 mg/ml in ethanol. Cytochalasin A (Sigma) was used at a final concentration of 2 μg/ml in the medium from a stock solution of 100 mg/ml in dimethyl sulfoxide. Calcofluor white was used at a final concentration of 60 μg/ml in the medium. Hoechst 33342 (Molecular Probes) used at a final concentration of 10 μg/ml in the medium.

Protein extracts and Western blotting.For induction of the alcA promoter, A. nidulans cultures ware shaken in MM containing 2% threonine or 2% glycerol for 48 h. The mycelium was harvested by filtration through Miracloth (Calbiochem, Heidelberg, Germany), dried by pressing between paper towels, and immediately frozen in liquid nitrogen. After the mycelium was ground in liquid nitrogen, the material was resuspended in protein extraction buffer (50 mM Tris-HCl [pH 8], 150 mM NaCl) supplemented with protease inhibitors (1 mM phenylmethylsulfonyl fluoride, 10 μM leupeptin, 1 μM pepstatin). Protein extracts ware clarified twice by centrifugation (centrifuge 5403; Eppendorf, Hamburg, Germany) at 13,000 rpm at 4°C for 10 min. The protein concentration was quantified with a Roti-Quant kit (Roth, Karlsruhe, Germany). After denaturation of the samples, protein extracts were loaded on a 7.5% sodium dodecyl sulfate-polyacrylamide gel. For blotting, nitrocellulose membranes from Schleicher & Schuell (Dassel, Germany) were used. For Western blotting, we used rabbit polyclonal anti-GFP (Sigma-Aldrich, Munich, Germany) at a dilution of 1:4,000 and polyclonal anti-rabbit antibodies coupled to peroxidase (product A0545; Sigma-Aldrich) at a dilution of 1:4,000.

Isolation and deletion of teaC.We searched the A. nidulans genome at the Broad Institute (Cambridge, MA) (http://www.broad.mit.edu/annotation/genome/aspergillus_group/MultiHome.html) for proteins with similarity to the cell end marker protein Tea4 from S. pombe and identified one open reading frame (AN1099.3) (11). DNA and cDNA sequences were confirmed, and five introns with lengths of 66, 60, 66, 46, and 45 bp were determined. We named the gene teaC, although sequence similarity between the 687-amino-acid A. nidulans protein and the 810-amino-acid S. pombe protein was restricted to a conserved SH3 domain (Fig. 1A). Within this 86-amino-acid domain the identity was 63% with an e-value of 6e−27. Outside this region, the homology was very low, and the overall identity was only 12.5%. The sequence similarity to the S. cerevisiae Bud14 protein, which shares some functional relationship with Tea4, was even lower. In other aspergilli, proteins with high identities to A. nidulans TeaC were found (Fig. 1B). The identity to corresponding proteins from other ascomycetous fungi was again much lower, e.g., Penicillium chrysogenum (43%) and Magnaporthe grisea (only 22%). Sequence identity to basidiomycetous species was less than 10%. These results indicate that TeaC sequences are only poorly conserved within fungi.

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FIG. 1.

Comparison of TeaC with related proteins from other fungi. (A) Scheme of A. nidulans TeaC, S. pombe Tea4, and S. cerevisiae Bud14, indicating the location of the SH3 domain. The alignment of the three SH3 domains is displayed below the scheme. (B) Alignment of TeaC homologues from four Aspergillus species as indicated. The SH3 domain is highlighted with the red line below the sequences. The alignments were done with CLUSTAL W with standard parameters. Identical amino acids are shaded in black, and chemically conserved amino acids are shaded in gray using the program box shade. aa, amino acids.

To analyze the function of teaC in A. nidulans, we constructed a teaC deletion strain (SYH21; see Materials and Methods) and compared it to a ΔteaA deletion strain constructed previously (31). Both strains produced compact and small colonies (75 to 85% of the diameter) compared to the wild type. The growth defect was slightly more severe in the ΔteaA deletion strain (Fig. 2A). In both strains hyphae displayed a zig-zag growth phenotype compared to straight hyphae in the wild type (Fig. 2B). The effect on the maintenance of growth directionality was also stronger in the ΔteaA strain. The mutant phenotype was mainly seen in germlings.

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FIG. 2.

Phenotypic comparison of wild-type with ΔteaC and ΔteaA strains. (A) Colonies of wild-type (TN02A3), ΔteaA (SSK91), and ΔteaC (SYH21) strains. Strains were grown on MM glucose agar plates for 2 days. (B) Hyphae of wild-type, ΔteaA, and ΔteaC strains were grown for 1 day on microscope slides coated with MM with 2% glucose and 0.8% agarose. Images were taken with differential interference contrast. Hyphae are 3 to 4 μm in diameter. (C) Z-stack image of wild-type (TN02A3) and ΔteaC (SYH21) strains grown on MM with 2% glucose for 1 day and stained with calcofluor white. (D) Z-stack image of wild-type (TN02A3) and ΔteaC (SYH21) strains grown on glucose MM and stained with calcofluor white and Hoechst 33343. The arrow points to a short, anucleate hyphal compartment. Hyphae are 3 to 4 μm in diameter. (E) Quantification of the effect of different gene deletions on second germ tube formation. Conidia of each strain were germinated in MM with glucose and analyzed for the emergence of the second germ tube. For each strain, 100 germlings were counted. WT, wild type.

The ΔteaC mutants also showed an increased number of septa and branches, probably due to the reduced extension rate of the hyphae and not specific for the teaC deletion (Fig. 2C and see Fig. S2 in the supplemental material). In addition, we noticed some small compartments (5 μm; 8% [n = 100]), which did not contain any nucleus (Fig. 2D). This did not occur in the wild type or in slower-growing A. nidulans strains.

Another polarity defect has been detected in the way the second germ tube emerges from a conidiospore. In the wild type, the second hypha emerges opposite to the first germ tube (bipolar) in 83% of the spores. In contrast, the second hyphae emerged at random positions in 56% of the spores in the ΔteaC mutant strain. A similar defect was observed in 55% of the spores in the ΔteaA mutant (n = 100) (Fig. 2E). This suggests that teaA and teaC are required for the selection of the site of polarity initiation.

Overexpression of TeaC inhibits septum formation.We next tested whether overexpression of TeaC would also cause any morphological phenotype. We expressed teaC using the control of the alcA promoter under inducing conditions (threonine; see Materials and Methods). When the GFP-TeaC strain was grown on threonine MM colonies appeared smaller than the ones from wild type (Fig. 3A). The high expression level of TeaC was shown by Western blotting and an increase in GFP fluorescence (Fig. 3B and C; see also below). Under inducing conditions, we often observed lysis at the hyphal tips (Fig. 3D) and rarely branching of hyphae. Furthermore, the number of conidia that produced a second germ tube was reduced from 66% to 17% (n = 100) (Fig. 3E). Some conidia without a second germ tube grew continuously isotropic up to 20 μm in diameter after 2 days. Some hyphae appeared to be empty, probably because of leakage of the cytoplasm. This was visualized by calcofluor white staining of the cell wall and GFP staining of the cytoplasm. Whereas the spore on the right side in Fig. 3F showed fluorescence in both channels, the left spore showed only fluorescence of the cell wall, indicating an empty spore.

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FIG. 3.

Effect of overexpression of teaC. (A) Comparison of colonies of SYH03 [alcA(p)-GFP-teaC] and wild-type strains on glycerol- or threonine-containing medium after 4 days of growth at 37°C. (B) Western blot of hyphae of SYH03 grown on MM with glycerol or threonine as a carbon source. Portions (10 μg) of total protein extract were loaded onto the gel and processed for the Western blot as described in Materials and Methods. (C) Fluorescence microscopic picture of a hypha of SYH03 after induction with threonine for 1 day. (D) Germinated conidia of SYH03 grown on MM with threonine as a carbon source for 48 h observed using differential interference contrast. The hyphal tip had lysed. (E) Quantification of the effect of teaC overexpression on the emergence of the second germ tube. For each strain, 100 germlings were counted. (F) Germinated conidia of SYH03 grown on MM with threonine as carbon source for 48 h observed using differential interference contrast, stained with calcofluor white (CW), or observed in the GFP channel. WT, wild type; DIC, differential interference contrast.

When we analyzed the number of septa, we found that teaC overexpression repressed almost completely septation (Fig. 4A). Because TeaC interacts with SepA and possibly regulates the localization and/or the activity (see below), we sought to determine which phenotype would be caused by overexpression of TeaC, together with the overexpression of SepA. The displayed phenotype was similar to the phenotype of teaC overexpression, namely, very few septa (Fig. 4B and C). In addition, conidia were swollen, hyphae had an irregular shape, and hyphal tips appeared very large. To quantify this effect, we counted the number of septa in germlings, which were between 100 and 300 μm in length (n = 50) (Fig. 4C). Overexpression of SepA alone did not cause a reduction of the number of septa, and the morphology was less severely affected (data not shown).

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FIG. 4.

Effect of overexpression of teaC on septation. (A) Strains SYH17 [teaC(p)-mRFP1-teaC] (WT) and SYH03 [alcA(p)-GFP-teaC] grown on threonine MM and stained with calcofluor white. The red arrows point to the stained septa. Hyphae are 3 to 4 μm in diameter. (B) Strain SYH26 [alcA(p)-GFP-sepA and alcA(p)-mRFP1-teaC] grown on MM with threonine as carbon source for 48 h and stained with calcofluor white. The ringlike structure is a small branch observed from the top. (C) Quantification of the number of septa in strains SYH17 (considered as the wild type), SYH03, SNT28 [alcA(p)-GFP-sepA], and SYH26 grown in threonine MM. A total of 50 germlings were counted for each strain. WT, wild type; CW, calcofluor white.

Localization of TeaC at the hyphal tip depends on microtubules (MTs) but not on the kinesin KipA.To investigate the subcellular localization of TeaC, we constructed an A. nidulans strain expressing a GFP-TeaC fusion protein under the control of the alcA promoter (see above and Materials and Methods). The construct was transformed into strain TN02A3, and a transformant was selected in which the construct was integrated into the teaC locus (SYH03). This leads to duplication of the 5′ end of the gene under the control of the endogenous promoter and the GFP-teaC fusion construct under the control of the alcA promoter. Under repressing conditions, the strain displayed the ΔteaC deletion phenotype (see above). Under derepressed conditions, wild-type hyphal morphology was obtained. This experiment showed that the observed phenotypes in the ΔteaC deletion strain were indeed caused by the deletion and not by another mutation, and it showed that the GFP-TeaC fusion protein was functional. GFP-TeaC localized to one bright point at all hyphal tips and at newly forming septa (Fig. 5A). In addition, some weaker points on the side of the tip were observed. The GFP-TeaC points always localized close to the plasma membrane or appeared attached to it. Furthermore, some spots appeared in the cytoplasm, which could represent the MT plus ends (see also Fig. 6A).

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FIG. 5.

TeaC localization. (A) Hyphae of strain SYH03 [alcA(p)-GFP-teaC] grown on MM with glycerol as a carbon source. The upper row shows differential interference contrast (DIC) pictures; the lower row shows same hyphae in the GFP channel. GFP-TeaC localized to one point in the hyphal apex (enlarged in the inset), at the branching site, and at the septa. (B) Localization of TeaC (GFP) and the Spitzenkörper (visualized with FM4-64). (C) mRFP1-TeaC expressed under the control of the native promoter (SYH17). Fluorescence signals were observed at the hyphal tip and new branches and at the septa. Hyphae are 3 to 4 μm in diameter.

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FIG. 6.

Relationship between TeaC and MTs. (A) Observation of GFP-labeled MTs and mRFP1-labeled TeaC (SYH22). The arrows point to the MT plus ends. The same hyphae was observed at time zero and after 2.5 s, as indicated in the images. (B) Effect of benomyl on the localization of TeaC in strain SYH22 [teaC(p)-mRFP1-teaC]. A. nidulans was grown in MM with glycerol for 1 day and then shifted to medium containing 2.5 μg of benomyl/ml for 30 min. (C) In the ΔkipA mutant SYH25 TeaC moved away from the center of the apex to the side of the tip (arrow). (D) Effect of cytochalasin A on TeaC localization. SYH17 was grown in MM with glycerol for 1 day and shifted to medium containing 2 μg of cytochalasin A/ml for 2 min. Hyphae are 3 to 4 μm in diameter.

In growing tips, the Spitzenkörper has an important role as vesicle supply center. To compare the localization of TeaC with the Spitzenkörper, we stained hyphae with FM4-64, which has been used in several fungi to stain the Spitzenkörper (9, 23, 31). The Spitzenkörper appeared to colocalize with GFP-TeaC (Fig. 5B), although from these pictures it was not unambiguously clear whether TeaC is part of the Spitzenkörper or only attached to the membrane and therefore very close to the Spitzenkörper.

To confirm TeaC localization and to prove that the observed localization was not an artifact of the expression under alcA promoter control, we constructed a strain (SYH17) producing mRFP1-TeaC under the control of the native promoter. SYH17, in which mRFP1-TeaC is the only source of TeaC, did not show the phenotype observed in the ΔteaC mutant, indicating that mRFP1-TeaC at this expression level was also biologically functional. Although signals of mRFP1 appeared much weaker, mRFP1-TeaC still localized to one point, and weaker signals were observed along the apex and at forming septa (Fig. 5C). The single spot of mRFP1-TeaC normally localized at the center of the hyphal apex (Table 3). At a small number of tips (13%, n = 100), mRFP1-TeaC localized along the tip membrane. For further experiments, we normally used strains producing mRFP1-TeaC under the native promoter, when we analyzed the localization of TeaC.

To test whether TeaC localization at the hyphal tip depends on MTs, we studied a strain with GFP-labeled α-tubulin and mRFP1-TeaC (SYH22) and found that TeaC localized to the plus end of the MTs, although the signal intensity was close to the detection limit (Fig. 6A). Next, we used the MT-destabilizing drug benomyl. After this treatment (2.5 μg of benomyl/ml), almost all fluorescence of GFP-MTs was diffused into the cytoplasm within 5 min (data not shown), and the mRFP1-TeaC point at the tips was sometimes divided into several points and disappeared after 30 to 40 min from >80% of the tips (n = 100) (Fig. 6B and Table 3). The delay between the disappearance of the TeaC spot and the disassembly of the MTs suggests that TeaC is rather stable at the cortex.

To analyze whether TeaC is transported in a kinesin-7-dependent way to the MT plus end, we studied TeaC localization in a ΔkipA mutant. mRFP1-TeaC still localized to one point at nearly 80% of the tips (n = 100), indicating KipA-independent polarization. However, the mRFP1-TeaC point did not localize to the center of the apex and moved away to the side of the hypha (Fig. 6C and Table 3). We further studied this effect by localization of TeaC in a KipA^rigor mutant, in which KipA (fused to GFP) harbors a point mutation in the ATP-binding domain (16, 38). GFP-KipA^rigor binds but does not move along MTs, and thus decorates them. In this strain (SYH23) mRFP1-TeaC still localized at 90% of the tips, but the mRFP1-TeaA point often moved away to the side of the apex and sometimes divided into two points (Table 3). These results indicate that TeaC can be transported independently of KipA to the tip and that other KipA-transported proteins are probably required for exact positioning of TeaC. We are assuming that the key for further understanding of the cell end marker protein complex lies in the characterization of cargoes of the KipA motor protein.

We investigated also the effect of cytochalasin A (2 μg/ml), an inhibitor of actin polymerization, on TeaC localization. mRFP1-TeaC dispersed around the tips within 50% of the tips (n = 100) by 2 to 10 min after the treatment (Fig. 6D and Table 3) compared to the control strain only treated with 0.02% dimethyl sulfoxide. Taken together, our results suggest that TeaC localization depends on the MT and the actin cytoskeleton.

TeaC connects TeaA and SepA.To investigate whether A. nidulans TeaC colocalizes with TeaA, we constructed a strain (SYH13) expressing mRFP1-TeaA and GFP-TeaC and compared their localization patterns. The mRFP1-TeaA point at the apex colocalized with that of GFP-TeaC. If mRFP1-TeaA localized at two points (10%, n = 100) TeaC also colocalized to the same spots (Fig. 7A). Likewise, we tested colocalization of TeaC with SepA. We constructed a strain (SYH18) expressing GFP-SepA and teaC(p)-mRFP1-teaC. TeaC and SepA did not obviously colocalize at the hyphal apex. When SepA localized at the hyphal apex, TeaC appeared adjacent to the SepA signal (11%, n = 100). When TeaC localized at the hyphal apex at one point, SepA did not localize at the hyphal apex (89%, n = 100) (Fig. 7B).

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FIG. 7.

Interaction of TeaC with TeaA and SepA. (A) Colocalization of TeaC and TeaA in strain SYH18; (B) visualization of TeaC and SepA in strain SYH13 at the hyphal tip; (C) partially colcalization of TeaC and SepA in strain SYH13 at the septa; (D) localization of SepA in strain SNT28; (E) localization of TeaC in strain SYH03; (F) interaction of TeaC with SepA (strain SYH05) shown in the bimolecular fluorescence complementation assay; (G) interaction of TeaC with TeaA (strain SYH06) shown in the bimolecular fluorescence complementation assay. Hyphae are 3 to 4 μm in diameter.

We also found no obvious colocalization at septa (Fig. 7C). It appeared that TeaC surrounded SepA (Fig. 7C). While doing time course experiments, we found that SepA and TeaC follow a constricting ring but that TeaC constriction was slightly delayed in comparison to SepA (Fig. 7D and E).

Next, we sought to determine whether TeaA/TeaC and TeaC/SepA would interact. This was studied by BiFC. The N-terminal half of YFP (YFP^N) was fused to SepA and TeaA, and the C-terminal half of YFP (YFP^C) was tagged with TeaC. Strains expressing only YFP^N-SepA, YFP^N-TeaA, or YFP^C-TeaC showed no YFP fluorescence. In contrast, in the strain expressing both YFP^N-SepA and YFP^C-TeaC produced YFP signals as a single point and along the apex and at hyphal septa (Fig. 7F). In the case of the combination of YFP^N-TeaA and YFP^C-TeaC, YFP signals were detected only at the hyphal tip (Fig. 7G). We also tested YFP^N-TeaR (the membrane anchor protein) and YFP^C-TeaC interaction, but no YFP fluorescence was observed (data not shown). The observed interactions were analyzed with the yeast-two hybrid system. TeaA showed self-interaction and interaction with TeaC. In both cases, the C termini of TeaA and TeaC were important for the interaction (Fig. 8). Interaction of TeaC and SepA could not be detected in this assay, because SepA alone already induced the expression of the yeast two hybrid reporters (data not shown).

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FIG. 8.

Yeast two-hybrid assay to confirm the interactions described in Fig. 7. (A) Colonies of the yeast strains. The proteins indicated on the left side were fused to the binding domain (BD), and the proteins indicated above the picture were fused to the activation domain (AD). The mated yeasts were selected on SD−Leu−Trp plates and grown on SD medium (SD−Leu−Trp−His) supplemented with 1 mM or 10 mM 3AT (3-amino-1,2,4-triazole). (B) The β-galactosidase activity was analyzed from liquid cultures using ONPG as substrate and is expressed as Miller units. The data are expressed as the means of three independent experiments. The standard deviation is indicated.

Localization dependency of TeaC and TeaA.We analyzed TeaC localization in a ΔteaA mutant and found that the correct positioning of TeaC depends on TeaA (Fig. 9A and Table 3). In the absence of TeaA, TeaC appeared dispersed in the cytoplasm. We also analyzed TeaA localization in a ΔteaC mutant. mRFP1-tagged TeaA did not concentrate in one point in the apex but appeared as several points along the membrane (Fig. 9B).

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FIG. 9.

Localization dependency of TeaC and TeaA. (A) TeaC localization in the ΔteaA mutant (SYH19); (B) TeaA localization in the ΔteaC mutant (SYH24). Hyphae are 3 to 4 μm in diameter.