The A. nidulans Fus3 MAP kinase of the mating pathway phosphorylates the velvet domain protein VeA, and VelB-VeA complex formation is reduced in Anfus3 deletion

S. cerevisiae Fus3 interacts with transcription factor Ste12 that activates the mating pathway. The A. nidulans MAP kinase AnFus3 [MpkB] also controls sexual development [25], [28], [32]. Tagged AnFus3 recruited the transcription factor AnSte12 [SteA] by tandem affinity purification (TAP) only when the fungus was induced for sexual development but not during vegetative filamentous growth or asexual development (Figure 1B, Table S1). Endogenously expressed AnFus3::sGFP was functional (Figure S1) and immunoprecipitation of the fusion protein was able to enrich the SteA protein in a sexually induced culture (Table S1). The AnFus3-SteA interaction was further verified by bimolecular fluorescence complementation (BiFC) and was observed in fungal nuclei (Figure 1C). This corroborates that the interaction between kinase and transcription factor is conserved from yeast to filamentous fungi.

Due to their similar roles in development and secondary metabolism [28]–[30], we examined whether AnFus3 interacts with the velvet domain proteins and LaeA. AnFus3 interacted in vivo in a BiFC assay with LaeA and subsequently with VeA, but not with VelB. In addition, AnFus3 interacted with VosA (Figure 1D). VosA is part of the VosA-VelB heterodimer which represses asexual development [31], [33]. These results suggest that distinct velvet domain proteins or LaeA may include targets of MAPK phosporylation.

AnFus3 was immunoprecipitated from vegetatively grown fungal cells as sGFP fusion protein (Figure 2A) to identify direct substrates of AnFus3 in in vitro kinase assays. VeA expressed and purified from E. coli was the only tested protein which could be specifically phosphorylated by AnFus3, whereas bacterially produced VosA, LaeA or VelB were not phosphorylated. Further phosphorylation experiments performed with phospho-specific serine and threonine antibodies further supported that VeA was phosphorylated by AnFus3 and treatment of phosphorylated samples with lambda protein phosphatase (λ-PP) resulted in loss of phosphorylation signal (Figure 2B).

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Figure 2. Phosphorylation of VeA by the MAP kinase AnFus3 [MpkB] and influence of AnFus3 on the interactions of the velvet complex.

(A) In vitro phosphorylation of VeA by AnFus3 in a radioactive kinase assay. Left panels; autoradiograph of dried SDS gel run for phosphorylation reactions (30 µl of total 45 µl reaction volume), Coomassie stain of the proteins from phosphorylation reaction (10 µl of total 45 µl reaction). VeA protein in AnFus3 reaction tube is shown with red rectangle. Right panels; ponceau staining of the immunoprecipitated (immobilized to the GFP trap sepharose) AnFus3::sGFP and only sGFP protein. Immunodetection of the fusion protein and free sGFP by the α-gfp. (B) Confirmation of specific VeA phosphorylation by a non-radioactive method. All recombinant proteins (10 µg each) were treated with both AnFus3 and GFP. AnFus3 treated samples were additionaly incubated with the lambda protein phosphatase (λ-PP). Proteins were immunodetected by α-Penta His, α-GST. After AnFus3 treatment, VeA showed a 3–5 kDA molecular weight shift (red arrow) that disappeared after L-PP treatment. Only VeA treated with MAPK was recognized by P-ser/thr specific antibody. (C) Protein levels of VeA in the wild type and mpkB mutant background. VeA::cTAP signals were normalized to the internal actin levels. VeA protein levels did not change in the absence of MpkB. (D) Reduced velvet complex formation in the mpkB mutant. The VeA-associated proteins from the cultures of the wild type and mpkBΔ strains grown in the darkness sexually at 30°C for 20 hours. Three independent experiments were performed and the associated proteins were identified. The ratio of the peptides from VelB and LaeA to the VeA protein drastically reduced in the MAPK mutant, whereas alpha importin KapA interaction slightly increased. Black bars represent the standard error. *LaeA was only found in one of the three purifications in mpkBΔ strain, thus no error bar is assigned.

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VeA bridges VelB and LaeA in the trimeric VelB-VeA-LaeA complex. We addressed whether AnFus3 activity affects complex formation. VeA protein levels (Figure 2C) were similar in wild type and mpkB mutant strains. velB RNA was unchanged whereas laeA transcripts were downregulated as previously reported (Figure S2A) [28]. TAP purification of natively expressed VeA::cTAP revealed that under conditions where sexual development was normally promoted, only significantly reduced amounts of VelB and LaeA proteins were enriched by tagged VeA in the absence of MpkB (Figure 2D, Tables S2 and S3). The MAP kinase does not affect VeA nuclear import, because the interaction of VeA with the importin KapA was not significantly affected in mpkB mutant. Consistently, nuclear import of the subunits of the trimeric VelB-VeA-LaeA complex was not affected in a mkkB mutant lacking the upstream MAP2K AnSte7 (Figure S2B). Lack of laeA normally causes enhanced VeA and VelB expression as well as enhanced complex formation [31]. This suggests that decreased VeA-VelB association is not a result of the reduced levels of LaeA in mpkB mutants.

These results suggest that AnFus3 phosphorylates VeA in vitro and interacts with VeA in vivo. Furthermore, AnFus3 is required for enhanced association of VeA with VelB which are components of the VelB-VeA-LaeA velvet complex.

MAP2K AnSte7 is required for sexual development of A. nidulans

MAPKKK (SteC) and MAPK (MpkB) are necessary for sexual development in A. nidulans [24], [25]. Yeast Fus3 receives the phosphorylation signal from MAP2K Ste7. The corresponding filamentous fungus homolog has not yet been described. The ANID_03422 (mkkB) locus of A. nidulans encodes a protein, which is conserved in different Aspergilli (Figure S3) and has 25% identity to yeast Ste7 [34]. AnSte7 [MkkB] is also related to N. crassa MAP2K [35] and human MAP2K1 [36]. Overexpressing the corresponding mkkB gene resulted in two fold increase in the number of fruiting bodies and supported a role in sexual development (Figure S4A–S4C). mkkB deletion mutants had a slow growth phenotype and were blocked in early sexual development, which resulted in nest-like structures containing clumps of Hülle cells (yellow arrows, Figure 3A, 3B). Hülle cells support sexual development as specialized nursing cells for the growing fruiting body [31].

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Figure 3. Loss of sexual fruiting bodies and heterokaryon formation in mkkBΔ strain lacking AnSte7.

(A) Sexual developments of a wild type, mkkBΔ, mkkB complementation (comp+), steCΔ [lacking AnSte11] strains, which were point inoculated (1×10⁴) and grown on minimal medium in dark conditions (5 days at 37°C). Small squares are the close-up stereomicroscopic images of the strains. Red arrows indicate the mature black fruiting bodies of the wild type and complementation strains and yellow arrows denote the premature nests produced by mkkBΔ and steCΔ strains. (B) Raster electron microscopy (REM) image of the strains from (A). The wild type fruiting body (cleistothecium: Cl) is surrounded by the globose Hülle cells. steC and mkkB mutants produce only dispersed groups of Hülle cells (yellow arrows) instead of mature fruiting bodies. (C) Monitoring hyphal fusions and heterokaryons via fluorescence microscopy. Strains bearing either cytoplasmic synthetic green fluorescent protein (sGFP) or nuclear red fluorescent protein fused histone 2A (mRFP) were used in different combinations. Only two wild types form green and red fluorescent combinations through hyphal fusions. (D) Isolated protoplasts from two wild types (yellow and green), steCΔ (green), mkkBΔ (yellow or green) were used for protoplast fusions as shown in combinations and plated on selective medium. wt/wt, wt/steCΔ, wt/mkkBΔ combinations produce fruiting bodies after 7–8 days, whereas mkkBΔ/mkkBΔ combination only produces nests.

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AnSte7 is required for hyphal fusion as one of the initial steps of fruiting body formation. Hyphal fusion of wild type strains marked with either synthetic cytoplasmic green fluorescent protein (sGFP) or with nuclear monomeric red fluorescent protein (mRFP) resulted in hyphae with green cytoplasm and red nuclei (heterokaryon) (Figure 3C). In contrast, a mkkB deletion strain was unable to fuse with the wild type strain. We found the same hyphal fusion defect for the steCΔ strain as in the mkkB mutant (Figure 3C, 3D). This further supports that AnSte11 and AnSte7 act in a common pathway.

The analysis of putative additional functions of AnSte7 in later phases of sexual development required a by-pass of initial hyphal fusions. Therefore, heterokaryons were artificially produced by fusing protoplasts. An intact mkkB copy of the wild type strain allowed the development of mature fruiting bodies (red arrows), when wild type and mkkB mutant protoplasts were fused. In contrast, two mkkB mutants forced to form heterokaryons were impaired in fruiting body maturation and produced only early structures of development (yellow arrow, Figure 3D). This suggests several functions of MAP2K AnSte7 during sexual development presumably in concert with AnFus3.

AnSte50-Ste11-Ste7-Fus3 form a physically interacting module that is required for sexual development

We determined whether the A. nidulans kinases may replace functions of its yeast counterparts. Plasmids containing Anste7 [mkkB] and Anfus3 [mpkB] genes expressed under yeast promoters were transformed into ste7 and fus3 deletion strains. mkkB and mpkB did not alleviate the defects in pheromone response of the yeast mutants (Figure S4D). However, MpkB moderately suppressed the defects in pheromone response of a fus3 kss1 double mutant, showing that the MpkB is partially able to take over functions of the MAP kinase pair Fus3/Kss1. This suggests a partial overlap of the functions of the MAPK pathways of these two organisms.

The A. nidulans MAP kinase mating module was further characterized by identifying interaction partners of AnSte7 [MkkB] by TAP purification from different developmental stages (only vegetative is shown, Figure 4A, 4B, Figure S5, Tables S4, S5). Tagged AnSte7 did not recruit AnFus3, but copurified AnSte11 [SteC] and AnSte50 [SteD], a protein sharing homology to S. cerevisiae Ste50.

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Figure 4. Identification of AnSte7 [MkkB], and AnSte50 [SteD] associated proteins and role of MAPK pathway on secondary metabolism.

(A) A silver stained 5–14% gradient SDS polyacrylamide gel of AnSte7 [MkkB]::cTAP from the wild type and steCΔ [lacking AnSte11] background grown for vegetatively at 30°C for 20 hours. (B) Identified peptides of the proteins from the excised lanes of the wild type and steCΔ strains (Table S4 and S5). AnSte11 [SteC] (AN2269) and AnSte50 [SteD] (AN7252) were found as interactors of AnSte7 [MkkB]. (C) Interaction partners of the AnSte50::cTAP fusion from vegetatively, asexually and sexually grown cultures at 30°C for 20 hours (Table S6) (M2B; MkkB, MB; MpkB, SD; SteD, SC; SteC). (D) Identified polypeptides from the bands. (E) Monitoring of the phosphorylation status of the MpkB by phospho-p44/42 MAPK (Thr182/Tyr184) antibody in the wild type as well as pheromone pathway mutants grown for 24, 48 and 72 hours vegetatively. VeA protein levels served as loading control. 80 µg protein extract was loaded on each lane. (F) Production of secondary metabolite sterigmatocystin (ST) in the mutants of pheromone pathway, Anste11 [steCΔ], Anste50 [steDΔ], Anste7 [mkkBΔ], Anfus3 [mpkBΔ], Anste12 [steAΔ], respectively. Developed TLC plates show sterigmatocystin production. Sts; Sterigmatocystin standard. (G) Quantification of the ST production from the TLC plates. Wild type ST levels served as 100% standard. (H) Expression of the developmental, secondary metabolite genes in the pheromone pathway mutants. laeA, aflR, stcU for ST production, tdiA and tdiB for terrequinone (TQ) production and veA, mkkB, steD, mpkB, steA for developmental purposes were monitored. Strains were grown in the liquid medium for 24, 48 and 72 hours and total RNA was isolated and blotted (20 µg). Glycolytic gene gpdA expression and ethidium bromide stained rRNA was used as loading control.

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Ste50 functions as an adaptor for membrane recruitment of Ste11 in yeast [12]. Deletion of the corresponding steD in A. nidulans caused a defect in fruiting body formation (Figure S1A). Similar to the other MAPK mutants, steD mutant could not produce heterokaryons in outcrossings (not shown). Thus, the adaptor AnSte50 is as important for accurate fungal development as the other components of the MAPK module. A. nidulans AnSte50 was enriched by AnSte7::TAP in wild type, but not in the steCΔ strain indicating that AnSte11 is required for the AnSte50-Ste7 interaction (Figure 4A, 4B). These data suggest a physical interaction of AnSte50 and two MAPK module components in a AnSte50-Ste11-Ste7 complex.

Interaction partners of AnSte50 were identified to explore the entire fungal MAPK mating module. A functional steD::ctap (Figure S1A, S1B) recruited the MAP3K AnSte11 and the MAPK AnFus3 but not the MAP2K AnSte7 (Figure 4C, 4D and Table S6). This further supports that AnSte50-Ste11-Ste7-Fus3 forms a module similar to yeast Ste5-Ste50-Ste11-Ste7-Fus3 with the exception that a counterpart for the yeast Ste5 scaffold is missing in A. nidulans.

AnSte50-Ste11-Ste7-Fus3 represents an active MAP kinase module required for sexual development and secondary metabolite synthesis

We analysed whether AnSte11 and AnSte7 act upstream of MAPK AnFus3. MAPK phosphorylation was monitored by a phospho-specific antibody against the MAPK Thr182XTyr184 motif. Phosphorylated AnFus3 was permanently detectable in vegetative wild type cultures (Figure 4E). In contrast, modified AnFus3 was absent in mutants lacking AnSte11 or AnSte7, whereas the absence of AnSte12 did not change levels of phosphorylated AnFus3. In the absence of AnSte50, reduced phosphorylation of AnFus3 indicates some residual activity of the untethered AnSte11-Ste7 complex. This supports an active A. nidulans MAPK module consisting of AnSte50-Ste11-Ste7-Fus3 which controls fungal sexual development.

The role of AnSte50-Ste11-Ste7-Fus3 for secondary metabolism was examined. Impaired secondary metabolism had only been described for the mpkB mutants [28]. The mycotoxin sterigmatocystin (ST) levels were drastically reduced in the sterile steC, steD, mkkB, or mpkB mutants whereas ST levels in the sterile steAΔ [AnSte12] were similar to wild type (Figure 4F–4G). Similarly, the expression of the biosynthesis genes for ST (stcU) and terrequinone (tdiA and tdiB), and the expression of laeA and the transcription factor encoding aflR, both required for expression of secondary metabolite genes, were distinctly reduced in each mutant of the MAPK module (Figure 4H). These data corroborate that active AnSte50-Ste11-Ste7-Fus3 MAPK is not only required for sexual development but also for secondary metabolite production.

The components of the fungal MAPK module exhibit distinct localization patterns at hyphal tip, nuclear envelope, and septa

The yeast mating MAPK module transmits a signal from the plasma membrane to the nucleus by releasing MAPK Fus3 from the Ste5 scaffold at the membrane [13], [37]. We analysed how the signal is transmitted through the filament of A. nidulans to nuclear factors as AnSte12 or VeA. Time course immunoblotting (Figure S1C) showed that AnFus3 was constantly expressed during development. The mkkB mRNA for the upstream MAPKK was also present throughout all stages (Figure S5C). The corresponding protein AnSte7::sGFP was present in vegetative as well as in the initial phases of asexual and sexual development, but decreased afterwards (Figure S5C). Similarly, the AnSte50::sGFP (Figure S1C) seems to be degraded because the protein disappeared during mid and late asexual development.

Confocal spinning disc microscopy revealed that functional AnSte7::sGFP fusion protein expressed under native locus promoter was localised during early phase of growth throughout the cytoplasm, but never found in the nucleus (not shown). After becoming competent for differentiation (16 hours after germination), AnSte7::sGFP accumulated not only at the hyphal tip but also at the plasma membrane and at the septa of hyphae or spore forming cells (white arrows in Figure 5A). The AnSte7 signal was also present on the nuclear envelope. The AnSte7 localization pattern did not change in the absence of the MAP3K AnSte11 (not shown). Like AnSte7, a functional Ste50::sGFP fusion never entered the nucleus. AnSte50 was cytoplasmic and accumulated at later stages of vegetative growth at the hyphal tip, the septa of spore forming cells, the plasma membrane and the nuclear envelope (arrows in Figure 5B, 5D).

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Figure 5. Subcellular localizations of AnSte7 [MkkB], AnSte50 [SteD], and AnFus3 [MpkB]::sGFP fusions during the development of the fungus.

(A) Subcellular location of the AnSte7 [MkkB]::sGFP fusion protein in vegetatively and asexually growing hyphae. mRFP::Histone2A fusion indicates the position of the nuclei and FM4-64 stains the plasma membrane and the septa of the hyphae. Arrows indicate the accumulation of the fusion protein at hyphal tip, membrane and septa of the vegetative hyphae and metulae (M) of the conidiophores. (V) indicates the swollen vesicle part of the conidiophore. (B) Localization of AnSte50 [SteD]::sGFP fusion in the hyphal cells. SteD protein is cytoplasmic and after competence time (16 hours) it is enriched at the hyphal tip, membrane and nuclear envelope (indicated by arrows). (C) Nucleo-cytoplasmic and hyphal tip distribution of endogenously expressed AnFus3 [MpkB]::sGFP kinase fusion protein in the fungal hyphae. AnFus3 is found in the cytoplasm and nucleus, in late hours of vegetative growth (after 16 hours) accumulates at the plasma membrane and hyphal tips. (D) Presence of SteD protein at the base of the metulae and in the septa between the metulae (M) and phialides (P). Nuclear and partial septal localization of the AnFus3 protein in the asexual structures (arrows). Scale bars represent 10 µm.

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A functional AnFus3::sGFP expressed under the native promoter accumulated at the hyphal tip and was as well present in the cytoplasm as in the nucleus in vegetative and spore forming cells (Figure 5C, 5D). This suggests a dynamic and complex distribution of MAPK module subunits from the fungal membrane to the nucleus. It also revealed that the MAPK AnFus3 is as yeast Fus3 the only subunit with the potential to enter the nucleus.

The entire MAPK module colocalizes and interacts at hyphal tip and nuclear envelope

AnFus3 [MpkB]::mRFP was expressed constitutively together with AnSte7 [MkkB] and AnSte50 [SteD]::sGFP fusions to validate whether all components of the MAPK module are colocalized within the fungal filament (Figure 6). Most of the GFP signals of AnSte7 and Ste50 merge with the RFP signal of MpkB at the fungal tip, the plasma membrane and at the nuclear envelope where they might form dynamic protein complexes. Exclusively at hyphal tips we found two types of co-localizations of kinase pairs. In addition to direct co-localizations, similar to plasma membrane or nuclear envelope, there were extended co-localization patterns at the hyphal tip. This could reflect that a fraction of kinases is localized in vesicles at the hyphal tip.

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Figure 6. Colocalizations of AnSte7 or AnSte50::sGFP with AnFus3::mRFP.

(A) Colocalizations of AnSte7 and AnFus3 proteins within the same fungal cell. White arrows indicate AnSte7::sGFP and AnFus3::mRFP colocalizations at the hyphal tip, membrane and on the nuclear envelope. (B) Colocalizations of AnSte50 and AnFus3 proteins within the same cell at the hyphal tip, plasma membrane and perinuclear district. Scale bars are 10 µm.

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Bimolecular fluorescence complementation (BiFC) [38], [39] was applied to examine whether there are direct transient in vivo interactions between AnSte7 and Fus3, which could not be found by TAP purification (Figure 7B). Similar to the yeast localization of the Ste5-Ste11-Ste7-Fus3 MAPK module at the membrane, AnSte11-Ste7 and AnSte7-Fus3 interacted at the plasma membrane and also at the hyphal tip (Figure 7). There was an additional strong interaction of AnSte11-Ste7 at septa which border cellular segments as well as at septa of spore forming cells and spores (Figure 7C–7E). Quantification of the fluorescence intensity from the bright enhanced yellow fluorescent protein (EYFP) spots of AnSte11-Ste7, Ste7-Fus3 pairs revealed that they emit upto 10 fold more yellow fluorescence than the single EYFP molecules (Figure S6), suggesting that the kinase pairs form multimeric complexes.

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Figure 7. Confirmation of the subcellular interactions of the kinase complexes AnSte11-Ste7 and AnSte7-Fus3 by BIFC system.

(A) Interaction of N-EYFP::AnSte11 [SteC] and C-EYFP::AnSte7 [MkkB] proteins in the hyphal cells. AnSte11-Ste7 kinase complexes are located at the plasma membrane, septal connections, the hyphal tip and partially nuclear envelope. The upper panel shows the localization of the YFP signal in comparison to nuclear mRFP::Histone2A fluorescence. Lower panel displays the YFP signal emitting cells stained with membrane dye FM4-64. (B) Physical interaction of N-EYFP::AnSte7 [MkkB] with C-EYFP::AnFus3 [MpkB] proteins in the fungal cells. (C) Quantification of the subcellular locations of the AnSte11-Ste7 complexes that are often present at the hyphal tip, plasma membrane, septum and nuclear envelope. N:50 fungal cells were counted in triplicate. Standard deviations are presented as vertical bars. (D) Subcellular locations of the AnSte7-Fus3 interactions. AnSte7-Fus3 complexes hardly localize to the septum and are found more on the nuclear envelope. (E) Assembly of the AnSte11-AnSte7 and AnSte7-AnFus3 complexes on the surface of vesicles of asexual conidiophores. Arrows indicate the growth directions of the metulae initials on the vesicles. V; vesicle, S; stalk. Size of the scale bars is 10 µm.

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Consistently to the yeast situation, the transcription factor AnSte12 as well as fungus specific factors VeA and LaeA specifically interacted with the MAPK Fus3 in the nucleus (Figure 1C, 1D). AnSte50 also interacted with the kinases at the plasma membrane and hyphal tip (Figure 8). Only AnSte11-Ste7 strongly interacted at the septa but there was hardly any interaction between AnSte7-Fus3 or between the AnSte50 and any of the kinases at the septa (Figure 7C and 7D, Figure 8D–8F).

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Figure 8. Subcellular locations of the AnSte50-Ste11, AnSte50-Ste7, and AnSte50-Fus3 complexes in vivo.

(A) Interactions of N-EYFP::AnSte11 [SteC] and C-EYFP::AnSte50 [SteD] proteins in the fungal cells. Upper panel shows the nuclear mRFP signal and lower panel FM4-64 in comparison to the split YFP signal. (B) Interactions of AnSte50 [SteD] protein with AnSte7 [MkkB]. (C) Interactions of AnSte50 protein with AnFus3 [MpkB]. (D–F) Measurement of the subcellular locations of the AnSte50-Ste11, -Ste7, and Fus3 complexes that are frequently found in the nuclear envelope, plasma membrane, at the hypal tip and less often in the septal locations. Quantification was performed and analyzed as described in Figure 7. (G) Detection of the AnSte50-Ste11, AnSte50-Ste7, and AnSte50-Fus3 complexes on the vesicle (V) of the asexual conidiophore structures. S; stalk. Scale bars represent 10 µm.

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The entire MAPK module components migrate to the nuclear envelope to deliver AnFus3 into the nucleus

Yeast Ste7-Ste5-Fus3 migrates to tips of mating projections in pheromone treated cells. Only Fus3 travels to the nucleus upon activation by Ste7 [13]. A. nidulans is a homothallic fungus, which does not require a mating partner. Time lapse images revealed that MAPK module components AnSte7 and Ste50::sGFP can move within the fungal cell along the membrane. During the cellular movements, these molecules shortly touched the membrane then hit the nucleus. Sometimes, fusion protein moved back after contacting the nucleus in the opposite direction. (Figure 9A and 9B, Videos S1 and S2).

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Figure 9. Cellular movements of AnSte7 and AnSte50::sGFP fusions and interactions of the AnSte11-Ste7, AnSte7-Fus3 complexes in the steDΔ mutant lacking AnSte50.

(A) Cellular movements of the AnSte7::sGFP fusion. Single focal plane pictures of a time-lapse study for the AnSte7 fusion (in minutes) (Video S1). The MAPKK protein (white arrow) leaves the first nucleus; shortly after touching the membrane it reaches the second nucleus (visualized by RFP). The yellow arrow indicates movement direction. (B) AnSte50::sGFP cellular movement in both directions (Video S2). AnSte50 (white arrow) moves within the filament back to the nucleus (yellow arrow), and bounces back in the opposite direction after touching the nucleus. (C) Interactions of AnSte11-Ste7 complex occur at the hyphal tip, plasma membrane and the septa in wild type (see arrows). Membrane localization of AnSte11-Ste7 kinases drastically decreases in Anste50 [steDΔ] strain. Hyphal tip and septa localizations are unaffected. AnSte7-Fus3 interact at the hyphal tip, membrane and partially nuclear envelope. (D) Quantification of the locations of the AnSte11-Ste7 and AnSte7-Fus3 complexes in steDΔ strain. N:50 fungal cells were counted in triplicates. Standard deviations were given as vertical lines. Scale bar, 10 µm. (E) A comparative depiction of the MAPK modules and their action in the single-cell yeast and filamentous fungus. In the yeast system, (MAP3K)Ste11-(MAP2K)Ste7-(MAPK)Fus3 kinase complex assembles on the scaffold protein Ste5 which tethers the complex close to the plasma membrane. Ste50 is additionally required for membrane recruitment of the Ste11. Activation of Fus3 by Ste7 phosphorylation results in entry of the active Fus3 into the nucleus where it phosphorylates Ste12 transcription factor for mating responses. In the filamentous fungus, AnSte50 is partly responsible for the membrane attachment of the AnSte11-Ste7-Fus3 complex, which migrates to the nuclear envelope presumably to keep the AnFus3 active. Finally, AnFus3 (MpkB) is released into the nucleus where it interacts with SteA (Ste12) for hyphal fusions and sexual development. It also phosphorylates the velvet A protein, which in turn leads to activation of secondary metabolism with development.

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The dynamics of the protein interactions of the BiFC expressing strains were further analysed by time lapse movies (Videos S3, S4, S5, S6). The AnSte7-Fus3 pair moved together along the plasma membrane (Video S3, Figure S7A) towards the first nucleus, then they advanced to the next nucleus while some other spots did not move distinctly. Likewise, AnSte50-Fus3 complexes left one nuclear envelope, touched the membrane and moved to the next nucleus (Video S4, Figure S7B). Similar movements were also observed for other complexes of the MAPK module (not shown). AnSte11-Ste7 can dissociate from the plasma membrane, cross the cytoplasm and reach the nuclear envelope (Videos S5 and S6, Figure S7C, S7D).

The major difference to the yeast situation is that the MAPK module of A. nidulans travels from the outer border of the fungal cell through the cytoplasm to the nuclear envelope. The AnSte7-Fus3 pair as well as pairs of AnSte50 with all three kinases interacted at the nuclear envelope (Figure 7 and Figure 8). These data suggest significant differences in the molecular mechanism how a MAPK signal is transmitted in yeast in comparison to a filamentous fungus.

AnSte50 is required for efficient membrane attachment of MAPK complexes

The interactions of the AnSte11-Ste7 and AnSte7-Fus3 complexes were examined in steDΔ strain to examine AnSte50 function for cellular location of the module. The interaction of the three kinases at the plasma membrane of wild type (Figure 7) was abolished for AnSte11-Ste7 and drastically reduced for AnSte7-Fus3 in the steD mutant (Figure 9C, 9D). Plasma membrane localizations of the AnSte7 and Fus3::sGFP fusions were also reduced in the steD mutant (not shown). Contrastingly, the localization of the entire module at the hyphal tip or for the partial module AnSte11-Ste7 at the septum seems to be mediated by a mechanism which is largely independent of AnSte50.

These data suggest that AnSte50 supports association of the A. nidulans MAPK module with the plasma membrane but it does not affect the hyphal tip and septum localizations.

Strains, media, transformation, and cultivation of the microorganisms

Fungal strains created and used in the course of this study are given in Table S7. Aspergillus nidulans strains; FGSCA4 (veA+), TNO2A3 (veA1) [41], AGB152 (veA+) [42], AGB154 (veA+), AGB506 (veA+) [31], AGB551 (veA+), AGB552 (veA+) served as wild type transformation hosts for the knock-out, epitope tagging, BIFC, and overexpression experiments. Further details for the strains transformed with various plasmids are given in Table S8. Culturing fungal strains were described in detail elsewhere [43]. DH5α and MACH-1 (Invitrogen) Escherichia coli strains were applied for recombinant plasmid DNA. Aspergillus and E. coli strains were cultured as described previously [30]. Fungal and bacterial transformations were carried out as given in detail [30].

Manipulation of nucleic acids

Circular and linear DNA molecules were created based on the standard recombinant DNA technology protocols in detail [30]. Plasmids and oligonucleotides applied and constructed in this study are given in Table S8 and Table S9. During polymerase chain reaction (PCR) different kind of DNA polymerase combinations including Pfu (MBI Fermentas), Phusion (Finnzymes), Platinum-Taq (Invitrogen) and Taq (Fermentas) were used. Linear and circular DNA constructs were created as given below.

Creation of Anste7 [mkkB] deletion cassette and complementation plasmid

For construction of Anste7 [mkkB] deletion fragment, 1.1 kb 5 UTR and 0.6 kb 3 UTR flanking regions of AN3422 locus were amplified with 3422-A/C and 3422-D/F, respectively. These two fragments were fused with ptrA marker (amplified from pPTRII) by fusion PCR (3422-B/E), creating 3.6 kb deletion fragment which was transformed into TNO2A3 (veA1), AGB154 (veA+), creating AGB586, AGB587 strains, respectively. Complementation plasmid pME3854 was constructed by amplifying 4.2 kb genomic Anste7 [mkkB] locus (Comp-A/B) and subsequent cloning into StuI site of pAN8-1 [44] plasmid carrying the phleomycin resistance marker. Deletion and complementation events were verified by the Southern hybridization (Figure S8A–S8B).

Overexpression of Anste7 [mkkB]

Primers OZG302/303 amplified the 1.6 kb cDNA of Anste7 [mkkB] from sexual cDNA library [45]. T4 Polynucleotide kinase (PNK) treated phosporylated amplicons were inserted into PmeI site of pME3160 [30] under nitrogen source inducible niiA promoter leading to pME3855 that was transformed into AGB152, which resulted in AGB662.

Generation of endogenous Anste7 [mkkB]::gfp and ctap gene replacement modules

For the purpose of substitution of the orinigal Anste7 [mkkB] locus by mkkB::gfp and ctap, mkkB promoter including mkkB ORF (2.85 kb) and terminator regions (0.6 kb) were PCR-amplified from genomic DNA (3422-A/OZG380 for gfp, 3422-A/OZG382 for ctap, and OZG314/3422-F). Finally, the fragments 3422-A/OZG380 and OZG314/3422-F were fused to sgfp::natR module (with oligos 3422-B/3422E) creating 5.4 kb mkkB::sgfp::natR fusion construct. Likewise, 3422-A/OZG382 and OZG314/3422-F, and ctap::natR were joined by fusion PCR (3422-B/3422E) resulting in mkkB::ctap::natR cassette for gene replacement. mkkB::sgfp::natR construct (5.2 kb) was transformed into TNO2A3 and SWH51 [24], which yielded AGB590 and 592, respectively (Figure S8D–S8E). Similarly, mkkB::ctap::natR was introduced into AGB551 and SWH51 resulting in AGB597 and 598.

Construction of Bimolecular Fluorescence Complementation (BIFC) plasmids

Anste11 [steC] ORF was amplified from gDNA (OZG389/OZG392) and fused to nyfp (OZG73/387) leading to nyfp::Anste11 [steC] fusion fragment which was cloned in PmeI site of pME3160 plasmid yielding pME3859 (nyfp::steC). Anste7 [mkkB] ORF was PCR-amplified (for nyfp, OZG389/303, for cyfp OZG390/303) from genomic DNA followed by fusion to nyfp and cyfp, which produced nyfp::Anste7 [mkkB] and cyfp::Anste7 [mkkB] fusions. Similar to Anste11 [steC] cloning, nyfp::mkkB fragment was inserted in PmeI site of pME3160 generating pME3861 plasmid.

To test AnSte11/AnSte7 interaction, cyfp::mkkB was cloned in SwaI site of pME3859 leading to pME3860 that was brought in AGB506, which generated AGB599. For AnSte7/AnFus3 interactions, Anfus3 [mpkB] cDNA was amplified from cDNA library (OZG404/403 for nyfp, OZG405/403 for cyfp). OZG404/403 and OZG405/403 were fused to nyfp (OZG73/387) and cyfp (OZG75/388) fragments yielding nyfp::mpkB and cyfp::mpkB. nyfp::mpkB fragment was inserted in PmeI site of pME3160, which led to pME3864 and cyfp::mpkB was cloned in SwaI site of pME3861 generating pME3862 (nyfp::Anste7 [mkkB]/cyfp::Anfus3 [mpkB]). The BIFC plasmid pME3862 was introduced in AGB506 in order to generate AGB600. Anste12 [steA] gDNA was amplified with oligos OZG400/401 and fused to cyfp by fusion PCR (OZG75/400).

cyfp::veA, cyfp::velB, cyfp::vosA and cyfp::laeA were produced as described in detail [31]. Insertion of cyfp::steA, cyfp::veA, cyfp::velB, cyfp::vosA and cyfp::laeA in SwaI site of pME3864 yielded following plasmids pME3865 (mpkB/steA, AGB601), pME3866 (mpkB/veA, AGB623), pME3867 (mpkB/velB, AGB625), pME3868 (mpkB/vosA, AGB624), pME3869 (mpkB/laeA, AGB622).

Anste50 [steD] cDNA was amplified from cDNA library (OZG500/501) and joined to cyfp fragment with oligos OZG75/OZG500. Finally, this fragment was cloned in SwaI sites of pME3859, 3861, 3864, leading to plasmids pME3870, 3871 and 3927, respectively.

Generation of Anste50 [steD] deletion, endogenous steD::sgfp, and steD::ctap replacement fragments

steD deletion, steD::sgfp and steD::ctap linear fragments were created in an identical manner to mpkB constructs. steD deletion; OZG470/472, ptrA, OZG473/475 were fused by using oligos OZG471/474 in a fusion PCR. steD::sgfp::natR (5.1 kb); OZG470/564, sgfp::natR, OZG566/475 were joined by oligos OZG471/474. steD::ctap::natR (4.9 kb); OZG470/565, ctap::natR, OZG566/475 were joined by oligos OZG471/474. steD deletion cassette was brought into AGB552 and 551 generating, AGB576 and 650, respectively. steD::sgfp and steD::ctap were used for gene replacement in AGB551, giving rise to AGB657 and 659, respectively (Figure S9A–S9C).

Construction of Anfus3 [mpkB] deletion, sgfp, ctap, and mrfp fusions

mpkBΔ::ptrA deletion fragment was constructed by amplification of 1.2 kb 5 and 3 flanking regions of mpkB with primers (for 5 UTR, OZG443/445, for 3 UTR OZG446/448). OZG443/445, ptrA marker, and OZG446/448 were fused by oligos OZG444/447 creating 3.9 kb mpkBΔ::ptrA construct. Consequently, mpkBΔ::ptrA fragment was transformed into AGB552, which generated AGB611. To create Anfus3 [mpkB]::sgfp and ctap linear fragments, mpkB promoter as well as ORF was amplified by (OZG443/560 for gfp fusion and OZG443/561 for ctap fusion). OZG443/560, gfp::natR, and OZG562/448 were co-fused by oligos OZG444/447 creating 5.3 kb mpkB::sgfp::natR fragment. OZ443/561, ctap::natR, and OZG562/448 linear DNAs were fused to make mpkB::ctap::natR gene replacement fragment (5.1 kb). mpkB::sgfp and mpkB::ctap were transformed into the AGB551 strain, which yielded AGB654 and 659, respectively (Figure S9D–S9G). For creation of constitutively expressed mpkB::mrfp fusion, gpdA promoter (OZG735/736), mpkB cDNA (OZG737/738), and mRFP::H2A terminator (OZG739/740) were amplified and fused together (OZG735/740). The fusion was cloned in the SwaI site of a pyrG marker bearing plasmid. The final plasmid pME3966 was introduced into AGB590 and AGB657 for co-localization studies.

Construction of the yeast complementation plasmids

Promoter and terminator regions of STE7 (promoter OZG679/680, terminator OZG683/684) and FUS3 (promoter OZG685/686, terminator OZG689/690) were amplified from the wild type yeast genomic DNA. These regions were either fused to the gDNA or cDNAs of Anste7 [mkkB] (681/682) and Anfus3 [mpkB] (687/688) genes. Resulting fusions ^proSTE7::mkkB gDNA::STE7^ter, ^proSTE7::mkkB cDNA::STE7^ter, ^proFUS3::mpkB gDNA::FUS3^ter and ^proFUS3:: mpkB cDNA::FUS3^ter were cloned in SmaI site of yeast centromeric plasmid pRS316 (URA3) [46] yielding pME3958, 3959, 3960 and 3961, respectively. STE7 (OZG679/684), FUS3 (OZG685/690), and KSS1 (OZG691/692) genomic loci were amplified and similarly cloned into SmaI site of pRS316 resulting in pME3962, 3963, and 3964 plasmids, respectively. These control and chimeric constructs were transformed into the appropriate ste7, fus3, and fus3/kss1 [47] deletion strains.

Hybridization techniques and analysis of nucleic acids

Southern and Northern hybridizations were carried out as explained in detail [30], [43] according to protocols [48], [49].

Immunoblotting

Immunoblotting experiments for recognition of GFP, TAP fusion, VeA, and actin in protein extracts was performed according to described protocols [31]. α-phospho 44/42 (4377, Cell Signaling Technology Inc) was used for detection of the phosphorylated AnFus3 [MpkB]. For the detection of the phosphorylated proteins, α-phosphoserine/threonine (ab17464, Abcam) was employed. Manufacturers protocols were followed for incubation times and buffer applications of phosphospecific antibodies.

Expression of recombinant proteins

Proteins were expressed in Rosetta 2 (DE3) using ZYM5052 [50] media supplemented with 30 µg/ml Chloramphenicol and 100 µg/ml Ampicillin (GST-LaeA91) or 30 µg/ml Kanamycin (Velvet proteins) at 16°C. Cells were harvested by centrifugation, resuspended in lysis buffer (30 mM HEPES pH 7.4, 400 mM NaCl, 30 mM Imidazol) and lysed by passing through a Microfluidics Fluidizer at 0.55 MPa. The lysate was cleared by centrifugation at 30000×g for 30 minutes. His-tagged proteins were purified with a 5 ml NiNTA-Sepharose (GE Healthcare) and GST-tagged LaeA91 with a 5 ml GSH-Sepharose (GE Healthcare) column connected to an ÄKTA Prime chromatography system. After washing with 10 column volumes with lysis buffer, proteins were eluted with elution buffer plus 400 mM Imidazol or 30 mM reduced Glutathione. Velvet proteins were desalted with a HiPrep Desalting 26/10 column (GE Healthcare) into storage buffer (10 mM HEPES pH 7.4, 400 mM NaCl). GST-LaeA91 was cleaved with PreScission Protease at 4°C for 16 h and further purified by gel-filtration using a Superdex 200 26/60 and a final 5 ml GSH-Sepharose column both equilibrated in gelfiltration buffer (10 mM HEPES pH 7.4, 150 mM NaCl). All proteins were shock-frozen in liquid nitrogen and stored at −80°C until further use.

Protein immunoprecipitation

In order to immunoprecipitate GFP fusion proteins, protein crude extracts were prepared from vegetatively grown cultures. 100 µl GFP-Trap sepharose (Chromotek) was washed twice with 1 ml protein extraction buffer (50 mM Tris pH 7.5, 100 mM KCl, 10 mM MgCl₂, 0.1% NP40, 10% Glycerol, 20 mM β-glycerophosphate, 2 mM Na₃VO₄, 5 mM NaF, 0.5 mM PMSF, 1 mM benzamidine, 1 mM EGTA, 1 mM DTT). 20 ml (150 mg total) protein crude extract was incubated with 100 µl GFP-Trap sepharose (Chromotek) at 4°C for 2 hours on a rotating platform. Afterwards, sepharose-extract mixture was centrifuged at 4000 rpm at 4°C for 1 min. Crude extract was removed with a 5 ml pipette. The sepharose was washed twice with 20 ml of protein buffer and centrifuged at 4000 rpm at 4°C for 1 min. This step was repeated one more time. Finally, 1 ml of protein buffer was added and the sepharose was resuspended. Each of the 200 µl sepharose buffer mixture was transferred into 1.5 ml eppendorf cups and centrifuged at 4000 rpm at 4°C for 1 min and supernatant was removed. Immunoprecipitated proteins were washed three times with 1 ml kinase reaction buffer (KRB; 20 mM Tris pH 7.5, 10 mM MgCl₂, 1 mM DTT, 1 mM benzamidine, 1 mM Na₃VO₄, 5 mM NaF, 0.1 µCi [³²P]-ATP).

In vitro phosphorylation and dephosphorylation assay

In vitro phosporylation assay was performed with modifications according to protocol given in [51]. For in vitro phosphorylation experiment, 30 µl KRB, containing 0.1 µCi [³²P]-ATP and 10 µg recombinant protein were added to the sepharose beads and incubated at 30°C for 35 minutes with the periodic resuspensions in every five minutes. Afterwards, reaction tubes were centrifuged at 4000 rpm at R/T for 1 min and supernatants containing phosphorylated proteins were transferred into new eppendorf cups. Supernatans and sepharose containing immunoprecipitated proteins were mixed with 3× protein loading dye (30 µl supernatant and 15 µl loading dye) and incubated at 95°C for 10 min. 30 µl of the supernatant fraction was run on 4–15% gradient SDS gel that was dried for 2 h and exposed to Kodak X-omat film for 5 hours. 10 µl of the reaction was used for visualization of the proteins with coomassie staining. 2 µl of sepharose was used for immunoblotting and ponceau staing for validation of equal immunoprecipitated target protein (MpkB or GFP). For non-radioactive kinase experiments, same KRB buffer containing 5 µM ATP was used. Supernatants were treated with 1000 units lambda protein phosphatase (New England Biolabs) in the presence of 1 mM MnCl₂ at 30°C for 1 hour. Samples were added with 3× loading dye and boiled at 95°C for 10 min. 3 µl of the samples were used for immunoblotting.

Tandem Affinity Purification (TAP) protocol and LC-MS/MS protein identification

For the TAP purification of the MkkB, MpkB, SteD, and VeA interacting proteins and further LC-MS/MS identification previously published protocols were applied [30].

Confocal spinning disc and fluorescence microscopy

A. nidulans strains expressing various fluorescence proteins (EYFP/sGFP/mRFP) were inoculated in the 8-well borosilicate coverglass system (Nunc) containing the liquid minimal medium. Widefield fluorescence photographs were taken with an Axiovert Observer. Z1 (Zeiss) microscope equipped with a CoolSNAP ES2 (Photometrics) digital camera. CSU-X1 A1 confocal scanner unit (Yokogawa) connected with QuantEM:512SC (Photometrics) digital camera was used for laser microscopy. The SlideBook 5.0 software package (Intelligent Imaging Innovations) was used for fluorescence and laser confocal image and movie recording as well as productions. We defined signals as plasma-membrane localized if we found the signals that are at the border of the silhouette of the fungal cell or even surmount the fungal cell; similarly, we defined signals as nucleus-associated when we found multiple signals at the border of the nuclear silhouette.

Quantification of the YFP fluorescence

The EYFP protein was purified by using GFP-Trap as described for GFP protein. EYFP molecules were allowed to attach to poly-L-lysine coated coverslips for 10 minutes, in PBS buffer. Fungal cultures were grown as described above. The preparations were imaged using a SP5 TCS STED microscope (Leica Microsystems), under 514 nm excitation (provided by an Argon laser), using a 100× oil-immersion objective (1.4 NA, Leica). The images were processed by a custom-written routine in Matlab (The Mathworks Inc.). Briefly, the spots were identified by the application of an automatic threshold based on the intensity of the background. We then used Gaussian fits to the spots to determine their intensity, and to correct for the background intensity, which provided the baseline of the fits.

Analysis of secondary metabolites

Extraction of sterigmatocystin (ST) and thin layer chromatography (TLC) was carried out as given in detail [43]. Penicillin levels were determined as published previously [52].