Synchronization of oscillatory growth prepares fungal hyphae for fusion
- Karlsruhe Institute of Technology - South Campus Institute for Applied Biosciences Dept. of Microbiology, Germany
- Karlsruhe Institute of Technology – North Campus Institute for Automation and Applied Informatics, Germany
- Karlsruhe Institute of Technology – North Campus Institute of Biological and Chemical Systems – Biological Information Processing, Germany
- Karlsruhe Institute of Technology – South Campus Zoological Institute Dept. of Systems Biology / Bioinformatics, Germany
Figures
Figure 1 with 2 supplements
Oscillatory recruitment of signaling proteins during hyphal growth of A. flagrans.
(a, b) Time course of SofT-GFP and mCherry-ChsB localization at hyphal tips. Arrows indicate the accumulation of the proteins at the hyphal tips. Dotted circles indicate an area of 6×6 pixels which …
Figure 1—figure supplement 1
Localization of SofT-GFP at hyphal tips of A. flagrans.
(a) SofT-GFP is present in three of the four hyphae. Dashed lines outline the shape of one hypha without SofT-GFP recruitment. The arrow indicates an accumulation of SofT-GFP near the nucleus of one …
Figure 1—figure supplement 2
The N-terminal disordered region and WW domain of SofT are sufficient to perform vegetative hyphal fusion in A. flagrans.
(a) Domain structure of the A. flagrans SofT and prediction of disordered protein regions using iupred2a (https://iupred2a.elte.hu). The protein contains 1213 amino acids and harbors a WW domain …
Figure 2 with 1 supplement
A mathematical model demonstrates the emergence of the temporal synchronization of two hyphae approaching each other.
(a) In the model, each hyphal cell contains an excitatory system with an activator () and inhibitor (). This excitatory system is triggered by extracellular signaling molecules () and, in …
Figure 2—figure supplement 1
Example simulation containing a single cell to illustrate the oscillatory dynamics of all model variables.
To illustrate the time course of all model variables, a simulation with a single cell was implemented by setting the cell distance to infinity ().
Figure 3 with 2 supplements
Decoupling of of SofT and MakB oscillations entering the cell-to-cell dialogue.
(a, b) Time course of SofT-GFP during a hyphal fusion event. Selective micrographs of the time course are shown and are divided into an orientation phase (orange frame) and a cell-to-cell dialogue …
Figure 3—figure supplement 1
Oscillatory recruitment of MakB-mCherry during hyphal growth of A. flagrans.
Time course of MakB-mCherry at a hyphal tip. Arrows indicate the accumulation of the protein at the nucleus. The circle indicates an area of 15×15 pixels which was used to measure the fluorescent …
Figure 3—figure supplement 2
The oscillating extension of hyphae is synchronized during hyphal fusion in A.flagrans.
(a) Time course of mCherry-ChsB during a hyphal fusion event. A maximum-intensity projection was generated from the time-lapse sequence. It was further bleach-corrected using the bleach correction …
Figure 4 with 2 supplements
The stepwise extension of hyphae is coordinated during the cell-to-cell dialogue stage of hyphal fusion.
Maximum-intensity projection of a time course of R-GECO during a hyphal fusion event. Stars indicate the fluorescent excitation of R-GECO in the presence of Ca2+ inside the hyphae. The changes in …
Figure 4—figure supplement 1
Extracellular Ca2+ is necessary for hyphal fusion and pulse-like exocytosis.
(a) Hyphae showed close physical contact without hyphal fusion after incubation with the Ca2+ chelating agent EGTA (5 mM). (b) No pulses of R-GECO were observed in hyphae on low-nutrient agar (LNA) …
Figure 4—figure supplement 2
Sufficient concentration of Ca2+ is necessary to produce oscillations.
In a simulation containing only a single cell, at a Ca2+ concentration of 0 (), no oscillations are observed. Around a Ca2+ concentration of 1 (), the cell generates regular oscillations (as …
Figure 5 with 3 supplements
Scheme of the cell-to-cell dialogue.
(a) Cell fusion of two hyphal cells is mediated by the synchronization of oscillatory growth. Vesicles needed for hyphal growth and communication accumulate at the hyphal tip and are released to the …
Figure 5—figure supplement 1
Localization of Soft-GFP in A. nidulans.
Arrows indicate accumulations of Soft-GFP inside hyphae. (Top) Dashed lines outline the hypha grown on low-nutrient agar (LNA) for 16 hr at 37°C. (Bottom) Hyphae were grown on Aspergillus minimal …
Figure 5—figure supplement 2
Gene deletion of figA and figB in A. flagrans.
(a) The A. flagrans ∆figA gene deletion mutant can perform vegetative hyphal fusion and trap C. elegans. (b) The A. flagrans ∆figB gene deletion mutant can perform vegetative hyphal fusion and trap C…
Figure 5—figure supplement 3
Gene deletion of hamA leads to loss of hyphal fusion.
(a) Growth of the A. flagrans ∆hamA gene deletion mutant grown on potato dextrose agar (PDA) after 7 days at 28°C. (b) Deletion of hamA leads to loss of vegetative hyphal fusion. (c) Kymograph of a …
Videos
Video 1
Localization of GFP-SofT during hyphal tip growth.
Video 2
Time course of GFP-SofT during a hyphal fusion event.
Video 3
Localization of mCherry-ChsB during hyphal tip growth.
Video 4
Co-localization of GFP-SofT (depicted in green) and mCherry-ChsB (depicted in magenta) during hyphal tip growth.
Video 5
Time course of GFP-SofT during a hyphal fusion event.
Video 6
Time course of MakB-mCherry during hyphal tip growth.
Video 7
Co-localization of GFP-SofT (depicted in green) and MakB-mCherry (depicted in magenta) during hyphal tip growth.
Video 8
Co-localization of GFP-SofT (depicted in green) and MakB-mCherry (depicted in magenta) during a hyphal fusion event.
Video 9
Time course of Lifeact-GFP during a hyphal fusion event.
Video 10
Time course of R-GECO during a hyphal fusion event.
Video 11
Time course of GFP-ChsB (depicted in green) and R-GECO (depicted in magenta) during a hyphal fusion event.
Video 12
Time course of mCherry-RimA during hyphal tip growth.
Tables
Table 1
A. flagrans and A. nidulans strains used in this study.
| Strain number | Genotype | Origin |
|---|---|---|
| CBS 349.94 | Wild type | Youssar et al., 2019 |
| sVW16 | tubA(p)::lifeact::GFP::gluC(t); trpC(p)::hph::trpC(t) | Wernet et al., 2022 |
| sVW01 | sofT::hph (∆soft) | Youssar et al., 2019 |
| sVW20 | ∆sofT; sofT(p)::sofT::gluC(t); neo; makB(p)::makB::makB(t); ntc | Haj Hammadeh et al., 2022 |
| sVW25 | tubA(p)::lifeact::GFP::gluC(t); trpC(p)::hph::trpC(t) | Wernet et al., 2022 |
| sVW29 | sofT(p)::sofT::GFP::sofT(t); trpC(p)::hph::trpC(t) | This study |
| sVW40 | soft(p)::sofT (∆1–504 AA N-terminus) gpdA(p)::neo::trpC(t) | This study |
| sVW41 | soft(p)::sofT (∆534–1213 AA C-terminus) gpdA(p)::neo::trpC(t) | This study |
| sVW43 | soft(p)::sofT (∆1–540 AA N-terminus, no WW domain) gpdA(p)::neo::trpC(t) | This study |
| sVW51 | tubA(p)::mCherry::chsB::chsB(t); gpdA(p)::neo::trpC(t) | This study |
| sVW52 | sofT(p)::GFP::sofT; tubA(p)::mCherry::chsB::chsB(t);trpC(p)::hph::trpC(t); gpdA(p)::neo::trpC(t) | This study |
| sVW65 | h2b(p)::r-GECO::gluC(t); gpdA(p)::neo::trpC(t) | This study |
| sVW69 | h2b(p)::r-GECO::gluC(t);tubA(p)::GFP::chsB::chsB(t); gpdA(p)::neo::trpC(t); trpC(p)::hph::trpC(t) | This study |
| sVW75 | sofT::hph (∆soft); tubA(p)::mCherry::chsB::chsB(t); gpdA(p)::neo::trpC(t); trpC(p)::hph::trpC(t) | This study |
| sVW76 | figA::hph (∆figA) | This study |
| sVW77 | figB::hph (∆figB) | This study |
| sVW78 | tubA(p)::mCherry::rimA::chsB(t); gpdA(p)::neo::trpC(t) | This study |
| sVW44 | hamA::hph (∆hamA), trpC(p)::hph::trpC(t) | This study |
| sVW80 | sVW51, hamA::hph (∆hamA), trpC(p)::hph::trpC(t) | This study |
| RPA 33 | yA1, riboB2, pyrG89, pyroA4, pabaA1, nkuA::argB+ | |
| VW-a01 | yA1, riboB2, pyrG89, (AnSoft-GFP::Afpyro) pyroA4, pabaA1, nkuA::argB+ | This study |
Table 2
Plasmids used in this study.
| Name | Description/genotype | Reference |
|---|---|---|
| pVW57 | Plasmid backbone containing trpC(p)::hph::trpC(t) | Wernet et al., 2022 |
| pVW92 | Plasmid backbone containing gpdA(p)::neo::trpC(t) and tubA(p) | Wernet et al., 2021 |
| pNH57 | Plasmid containing GFP::hph | Nicole Wernet (Karlsruhe) |
| pVW106 | sofT(p)::sofT::GFP::sofT(t); trpC(p)::hph::trpC(t) | This study |
| pVW118 | tubA(p)::mCherry::chsB::chsB(t);gpdA(p)::neo::trpC(t) | This study |
| pVW132 | tubA(p)::GFP::chsB::chsB(t); gpdA(p)::neo::trpC(t) | This study |
| pVW120 | tubA(p)::r-GECO::gluC(t); gpdA(p)::neo::trpC(t) | This study |
| pVW125 | h2b(p)::r-GECO::gluC(t); trpC(p)::hph::trpC(t) | This study |
| pVW131 | h2b(p)::r-GECO::trpC(t); trpC(p)::hph::trpC(t) | This study |
| pVW132 | figA::hph (∆figA) | This study |
| pVW133 | figB::hph (∆figB) | This study |
| pVW134 | tubA(p)::mCherry::rimA::chsB(t); gpdA(p)::neo::trpC(t) | This study |
| pVW113 | hamA::hph (∆hamA) | This study |
| pVW-a68 | AnSoft-GFP::Afpyro | This study |
| pVW108 | soft(p)::sofT (∆1–504 AA N-terminus) gpdA(p)::neo::trpC(t) | This study |
| pVW109 | soft(p)::sofT (∆534–1213 AA C-terminus) gpdA(p)::neo::trpC(t) | This study |
| pVW112 | soft(p)::sofT (∆1–540 AA N-terminus, no WW domain) gpdA(p)::neo::trpC(t) | This study |
Table 3
Oligonucleotides used in this study.
| Name | Sequence (5’ to 3’) | Description |
|---|---|---|
| chsB_fwd | gaatggatgaactctacaaa atggcacagcaaggaggtt | mCherry-ChsB |
| chsB_rev | aggagatcttctagaaagatgatggggcgttaaggtttc | mCherry-ChsB |
| pJet_fwd | atctttctagaagatctcctacaatattc | mCherry-ChsB |
| mCherry_rev | tttgtagagttcatccattccac | mCherry-ChsB |
| tubP_rev | gatgaattatatttcgtcaagaag | GFP-ChsB; tubA(p)::r-geco |
| chsb_fwd | atggcacagcaaggaggtt | GFP-ChsB |
| gfp_chsb_fwd | ttgacgaaatataattcatcatggtttccaagggtgagg | GFP-ChsB |
| gfp_chsb_rev | taacctccttgctgtgccatagcggccgctttgtaaagtt | GFP-ChsB |
| R_geco_pOL_fwd | ttgacgaaatataattcatcatggtcgactcatcacgtc | R-GECO (tubA(p)) |
| r-geco_tOL_rev | atacatcttatctacatacgctacttcgctgtcatcatttg | R-GECO (tubA(p)) |
| tgluC_gOL_fwd | aaatgatgacagcgaagtagcgtatgtagataagatgtatgattag | R-GECO (tubA(p)) |
| tgluC_geco_rev | aggagatcttctagaaagatatcttgttggggggaaggg | R-GECO (tubA(p)) |
| h2b_p_trpCOL_fwd | ctttccctaaactccccccaggagaagaaaggagcaaaatc | R-GECO (h2b(p)) |
| h2b_p_gecoOL_rev | cgacgtgatgagtcgaccattttgaaatttgttttttgtttgggtag | R-GECO (h2b(p)) |
| trpC_rev | tggggggagtttagggaaag | R-GECO (h2b(p)) |
| r_geco_fwd | atggtcgactcatcacgtc | R-GECO (h2b(p)) |
| soft_gfp_locus_fwd | ctcgagtttttcagcaagattaccgtcctcagtacaacatg | SofT-GFP |
| soft_gfp_locus_rev | acctcacccttggaaaccatatacccatactcgcatctgg | SofT-GFP |
| soft_GFPcas_fwd | ccagatgcgagtatgggtatatggtttccaagggtgagg | SofT-GFP |
| soft_GFPcas_rev | caaccgcccggacgaatcattggggggagtttagggaaag | SofT-GFP |
| soft_Term_fwd | ctttccctaaactccccccaatgattcgtccgggcggtt | SofT-GFP |
| softterm_gfp_rev | attgtaggagatcttctagaaagattgggacgagtgggatttaaaatgga | SofT-GFP |
| figA_lb_fwd | ctcgagtttttcagcaagatTGTCGCTTGGGCTTGATAG | Deletion figA |
| figA_lb_rev | CCTCCACTAGCATTACACTTATCTGCTAACGTAACTAGACG | Deletion figA |
| figA_hph_fwd | GTCTAGTTACGTTAGCAGATAAGTGTAATGCTAGTGGAGG | Deletion figA |
| figA_hph_rev | CTAACAGGCCTATCGGAGTTTGGGGGGAGTTTAGGGAAAG | Deletion figA |
| figA_rb_fwd | CTTTCCCTAAACTCCCCCCAAACTCCGATAGGCCTGTTAG | Deletion figA |
| figA_rb_rev | aggagatcttctagaaagatCGGAGGTCGTCAAGAAGC | Deletion figA |
| figA_up_fwd | AGGAAGACCGATTACGAAAC | Deletion figA |
| figA_down_rev | CGATATACGATCCGAAGGTC | Deletion figA |
| figA_lb_g418_rev | AATGCAATGTAATAGATACCATCTGCTAACGTAACTAGACG | Deletion figA |
| figA_g418_fwd | GTCTAGTTACGTTAGCAGATGGTATCTATTACATTGCATTGCG | Deletion figA |
| figB_lb_fwd | ctcgagtttttcagcaagatGGCGAAGAGACTGGATTTATC | Deletion figB |
| figB_lb_rev | CCTCCACTAGCATTACACTTTTTGAAGTTTTGCTGATGATGTGAG | Deletion figB |
| figB_hph_fwd | ATCATCAGCAAAACTTCAAAAAGTGTAATGCTAGTGGAGGT | Deletion figB |
| figB_hph_rev | TTGTTCAGCTTTTTTCCCATTGGGGGGAGTTTAGGGAAAG | Deletion figB |
| figB_rb_fwd | CTTTCCCTAAACTCCCCCCAATGGGAAAAAAGCTGAACAAAAAAAATC | Deletion figB |
| figB_rb_rev | aggagatcttctagaaagatCGCCTGTGTAACGGCTTTTG | Deletion figB |
| figB_up_fwd | AGAGCCGCATGGTTTATTTAG | Deletion figB |
| figB_down_rev | AGCACAGAGTAACCTGGAC | Deletion figB |
| rimA_orf_fwd | GAATGGATGAACTCTACAAAATGGAAACCCCAGCTCCAG | mCherry-RimA |
| rimA_rb_rev | AGGAGATCTTCTAGAAAGATCGTTCTATGCCTGAAATCGG | mCherry-RimA |
| ham10_lb_fwd | ctcgagtttttcagcaagatGGCGGATATCAATCTTATCTTG | Deletion hamA |
| ham10_lb_rev | CCTCCACTAGCATTACACTTGGTGACCGAAATCGCCTTAT | Deletion hamA |
| ham10_hph_fwd | ATAAGGCGATTTCGGTCACCAAGTGTAATGCTAGTGGAGG | Deletion hamA |
| ham10_hph_rev | CACAAGGATGGCTTCCCATTTGGGGGGAGTTTAGGGAAAG | Deletion hamA |
| ham10_rb_fwd | CTTTCCCTAAACTCCCCCCAAATGGGAAGCCATCCTTGTG | Deletion hamA |
| ham10_rb_rev | aggagatcttctagaaagatCGTTCCGATTTACTCGTCG | Deletion hamA |
| ham10_up_fwd | GCCGAGACTACTAGCTAGG | Deletion hamA |
| ham10_down_rev | CTATATCGTTGGTTCGAGGG | Deletion hamA |
| soft_Nterm_tgluCOL_rev | ATACATCTTATCTACATACGTTAAATACCGGTCTCCGGTGC | N-terminal SofT truncation |
| soft_ntermnoWW_tgluCol_rev | ATACATCTTATCTACATACGTTATGGAAGAGGTGGGGGAGA | N-terminal SofT truncation, no WW domain |
| softProm_trpCOL_fwd | CTTTCCCTAAACTCCCCCCACAGAGTTCGAATAGCGTTGC | C-terminal SofT truncation |
| softProm_CtermOL_rev | ATACCGGTCTCCGGTGCCATTGTGGAGACGAAGGCAAAG | C-terminal SofT truncation |
| softCterm_fwd | CCTTTGCCTTCGTCTCCACAATGGCACCGGAGACCGGTATT | C-terminal SofT truncation |
| softCterm_tgluC_rev | ATACATCTTATCTACATACGTTAATACCCATACTCGCATCTG | C-terminal SofT truncation |
| VW230 | TTGTAAAACGACGGCCAGTGACACCAGAAATCTCTCCGAATG | Soft-GFP, A. nidulans |
| VW231 | GTGAAAAGTTCTTCTCCTTTCTTCCCATGCTCTAAACTCG | Soft-GFP, A. nidulans |
| VW232 | CGAGTTTAGAGCATGGGAAGAAAGGAGAAGAACTTTTCACTGG | Soft-GFP, A. nidulans |
| VW233 | TTAAAAAGACTCGGCATCAAgcgagtgtctacataatgaagg | Soft-GFP, A. nidulans |
| VW234 | ttcattatgtagacactcgcTTGATGCCGAGTCTTTTTAATGT | Soft-GFP, A. nidulans |
| VW235 | GACCATGATTACGCCAagctGGGTGACGGATTATTACCTCT | Soft-GFP, A. nidulans |
