The authors have declared that no competing interests exist.
Nematode-trapping fungi (NTF) are a diverse and intriguing group of fungi that live saprotrophically but can switch to a predatory lifestyle when starving and in the presence of nematodes. NTF like
Pathogenic microorganisms are living at the expense of their host organisms and immediate killing of the host may be disadvantageous. Therefore, many bacterial or fungal pathogens developed an arsenal of small-secreted proteins during the colonization to modulate their host for instance to suppress its defense reactions. This allows a biotrophic phase, at least for some time. Some higher eukaryotic “pathogens” also live at the expense of their hosts but are often predators. In this case, quick killing is followed by digestion. In the case of predatory fungi, one could expect a similar situation, quick killing followed by digestion. However, the genome of such fungi encodes many putative small-secreted proteins, and we show here that one of them indeed appears to be secreted into the host and contributes to virulence. The protein is produced in the trapping devices of the fungus and especially in the penetration peg right after entering the nematode. Heterologous expression in
Nematode-trapping fungi (NTF) are carnivorous microorganisms that can trap and digest nematodes with sophisticated trapping structures. Most fungi in this versatile group can live saprotrophically and are able to switch to a predatory lifestyle while others are obligate pathogens [
Nematodes cause agricultural losses of 80 billion US dollars worldwide [
Most NTF belong to the monophyletic group in the order Orbiliales (Ascomycota), and the co-evolution with their nematode prey dates back 400 million years. Their occurrence in many taxonomic groups is an indicator that they evolved independently serval times during evolution [
Secretion of the SSPs can follow the conventional secretion pathway or an alternative route, as it was shown in the rice-blast fungus
Although the necessity of SSPs in predatory interactions is not obvious, many genes of NTF encode such small proteins, and many of the genes are transcriptionally upregulated during the nematode attack and are found in the pathogen-host interaction (PHI base) database e.g. in
In this paper, we analyzed a
The analysis of the
If
To obtain further evidence for the upregulation of
To investigate the localization of CyrA during the infection, the protein was fused to GFP. The fusion protein was fully functional, because it rescued the virulence phenotype of a
Next, we constructed a strain expressing CyrA without its signal peptide fused to GFP
When nematodes were captured, the fusion protein (expressed from the
To check if CyrA secretion at the infection bulb is mediated by the conventional secretion pathway, the CyrA-mCherry expressing strain was treated with Brefeldin A (BFA) during the interaction
To further investigate the mechanism of virulence factor secretion in
To gain further insights into the molecular function of CyrA,
After the initial experiments, long-term observations of single captured nematodes were performed as described above. This experiment was carried out with wild type, the deletion mutant, and a re-complemented strain using
To investigate putative functions of CyrA during the infection and to identify possible targets of the virulence factor, we expressed
The
Therefore, we expressed the same constructs under the control of the hypodermal and adult-specific
Fungal pathogens secrete virulence factors or effector proteins to overcome host defenses and to facilitate the infection [
The localization of the fusion protein CyrA-mCherry revealed its accumulation at the infection bulb. This round structure is formed during the penetration, which requires high osmotic pressure and the secretion of lytic enzymes [
Most likely many of the proteins secreted at this site are crucial to overcome the first line of host defenses. Micrographs of later time points of the attack show no fusion protein surrounding the trophic hyphae that develop from the infection bulb. This enforces the hypothesis that CyrA is relevant during the early steps of the infection. Proteins that are important during the initial steps of the penetration before the formation of the bulb will most likely accumulate at the infection site rather than in the infection bulb. This observation shows that fluorescent protein fusions allow to specify the spatiotemporal occurrence of proteins secreted by
Before a nematode has entered the trap, the fusion protein was observed in dynamic foci with accumulation at the inner side of the empty trap. Cytoplasmic vesicles and electron-dense particles were observed in early electron microscopical studies of
When expressed under the constitutive
The octameric exocyst complex tethers secretory vesicles to the plasma membrane before fusion. After this exocyst guided docking, fusion is mediated by SNARE-proteins [
These results show that the exocyst complex plays a role in virulence factor secretion in NTF and they indicate that membrane identity is a crucial part of the spaciotemporal organization of proteins secreted during the interaction.
The
Like other invertebrates
The fact that the reduction in lifespan was only apparent in the N2 strain background and not in the coelomocyte deficient strain could be due to their role in signal transduction. A role of the coelomocytes has already been described during the starvation response. During starvation, ASI neuron signaling leads to increased endocytosis by coelomocytes which in turn leads to NLP-7 signaling in neurons and induction of downstream genes in the intestine leading to lifespan extension. The coelomocytes are required for this lifespan extension during starvation [
When CyrA was expressed in
Taken together, the coelomocytes may play a role in the
This is, to our knowledge, the first description of a role for a small-secreted protein in the interaction of the nematode-trapping fungus
Strain | Genotype | Reference |
---|---|---|
N2 | Wild type | University of Freiburg |
Ban126 | Dr. D. Bano, DZNE Bonn | |
GS2478 | arIs37 I; |
Caenorhabditis elegans center, University of Minnesota |
KIT01 | This work | |
KIT02 | This work | |
KIT03 | This work | |
KIT04 | This work | |
KIT09 | This work | |
KIT10 | This work | |
KIT14 | This work | |
KIT18 | This work | |
KIT21 | This work | |
KIT22 | This work | |
KIT23 | This work | |
KIT24 | This work | |
KIT25 | This work | |
KIT26 | This work | |
KIT27 | This work | |
KIT28 | This work | |
KIT29 | This work | |
KIT30 | This work | |
KIT31 | This work | |
KIT32 | This work |
Strain | Genotype | Reference |
---|---|---|
sNH08 | This work | |
sNH11 | This work | |
sNH14 | This work | |
sNH16 | This work | |
sNH21 | This work | |
sNH23 | This work | |
sNH25 | This work | |
sNH27 | This work | |
sNH29 | This work | |
sNH30 | This work | |
sNH41 | This work | |
sNH42 | This work | |
sNH52 | This work | |
sNH66 | This work | |
sNH22 | This work | |
sNH47 | This work | |
sNH60 | This work | |
sNH65 | This work | |
sNH74 | This work |
For trap induction around 1x104
Protoplast transformation was carried out as described [
For RNA extraction traps were induced by incubating 106
For extraction of
Total RNA was extracted with Trizol reagent (Invitrogen, Karlsruhe, Germany). DNase digestion was performed using the Turbo DNA-free Kit (Invitrogen, Karlsruhe, Germany) and the RNA was diluted to 50 ng/μl. The Luna One Step Kit (NEB, Germany) was used for the qRT-PCR analysis on an CFX Connect Real-Time PCR Detection System (Bio-Rad, Munich, Germany). Each reaction mixture contained 0.2 μM oligonucleotides
Name | Sequence (from 5’ to 3’) | Description |
---|---|---|
Backbone_Efi_for | TTAATTAACCGGGATCCAAGTG | Backbone amplification |
Backbone_Efi_rev | GAATTCACTGGCCGTCGTTT | Backbone amplification efmov |
h2b_NLS_for | ATGCCACCAAAAGCCGCC | Backbone amplification |
ClaHp_tglucOL_for | ATGCTCTTTCCCTAAACTCCCCCCATCTCGGTTCCTCCCGCTT | ClaH-GFP fusion |
ClaH_gfpOL_rev | GATTACTTACCTCACCCTTGGAAACGAATCCACGATAACCAGTAG | ClaH-GFP fusion |
CyrA_genomic_for | AATTGGCAGCATCGATACTCG | Cornfirmation of the KO |
CyrA_genomic_rev | CCATAACGCCAAGCGCTT | Cornfirmation of the KO |
trpcT_rev | TGGGGGGAGTTTAGGGAAAG | C-terminal |
cyraP_trpctOL_for | ATGCTCTTTCCCTAAACTCCCCCCATGCATTCCAATCACTCAACCC | C-terminal |
cyraP_cyraOL_rev | GAACGATAGTGCTGAGGAGCTGCATTTTCGACAGTATTTGTGAAAAGAAGT | C-terminal |
CTCCATCACATCACAATCGATCCAAATGAACCCCAATGTATACGACTCAT | C-terminal CyrA wo SP | |
GATTACTTACCTCACCCTTGGAAACGTAGCACTTTTCGCACAAAGT | C-terminal CyrA wo SP | |
CyrA_rev | CTTAATTAAGTAGCACTTTTCGCA | C-terminal mCherry fusion of |
tGluC_for | CGTATGTAGATAAGATGTATGATT | C-terminal mCherry fusion of |
cherry_cyraOL_for | GTGCGAAAAGTGCTACTTAATTAAGATGGTAAGCAAGGGCGAGGT | C-terminal mCherry fusion of |
cherry_glucTOL_rev | TAATCATACATCTTATCTACATACGCTAAGCGGCCGCTTTGTAGA | C-terminal mCherry fusion of |
mcherry_h2bOL_rev | TTTCGGCGGCGGCTTTTGGTGGCATAGCGGCCGCTTTGTAGAG | C-terminal_promoter fusion |
h2b_cherryOL_for | GGATGAACTCTACAAAGCGGCCGCTATGCCACCAAAAGCCGCC | C-terminal_promoter fusion |
h2b_tubtOL_rev | CAAAGTAGGAATGACATCAGATATCTATTTGGCAGACGAGGAAGAGTA | C-terminal_promoter fusion |
BroA_asc_for | TATGGCGCGCCATGGCCGGACTTCACCAAG | C-terminal GFP fusion BroA |
BroA_pac_rev | GCGTTAATTAATCTCCAAGATTGAAGACCTGAC | C-terminal GFP fusion BroA |
hsp16.48P_rev | TTCTTGAAGTTTAGAGAATGAACAG | |
Scar_for | GTC AGC AAG GGA GAG GCA | |
unc54_for | GAGCTCCGCATCGGCCGC | |
miniPPF_rev | GGGCCCTGTGAAATTGTTATCCG | |
hspP_ppfOL_for | AGCGGATAACAATTTCACAGGGCCCGCTGGACGGAAATAGTGGTAA | |
hspP_uncOL_rev | TAACTGCCTCTCCCTTGCTGACCATTTCTTGAAGTTTAGAGAATGAACAG | |
scr_hspPol_for | CTGTTCATTCTCTAAACTTCAAGAAATGGTCAGCAAGGGAGAGG | |
scr_uncOL_rev | GACAGCGGCCGATGCGGAGCTCTTACTTGTAGAGCTCGTCCATTC | |
hspP_cyraOL_rev | GAACGATAGTGCTGAGGAGCTGCATTTCTTGAAGTTTAGAGAATGAACAG | |
CTGTTCATTCTCTAAACTTCAAGAAATGCAGCTCCTCAGCACTAT | ||
TGATAACTGCCTCTCCCTTGCTGACGTAGCACTTTTCGCACAAAGTC | ||
scr_cyraOL_for | TAAGACTTTGTGCGAAAAGTGCTACGTCAGCAAGGGAGAGGCA | |
ppf37_MCS_rev_new | CTCGAGGAATTCCTGCAGG | |
TTTAGAAAGACATCAGTTCATCAACATGCAGCTCCTCAGCACTAT | ||
emGFP_ |
TAAGACTTTGTGCGAAAAGTGCTACTCAGGTGGATCTGGAGGC | |
emGFP_UTRol_rev | GATGACAGCGGCCGATGCGGAGCTCTCATTTGTAAAGTTCATCCATTCC | |
TTTAGAAAGACATCAGTTCATCAACATGAACCCCAATGTATACGAC | ||
CyrA_stop_uncOL_rev | GATGACAGCGGCCGATGCGGAGCTCTTAGTAGCACTTTTCGCACAAAGT | |
col19_mcsOL_for | AGATATCCTGCAGGAATTCCTCGAGACGTACCATTATTCGAGACAAC | |
col19_cyraSPOL_rev | GAACGATAGTGCTGAGGAGCTGCATGTTGATGAACTGATGTCTTTCTAA | |
ppf37_MCS_rev | CCTGCAGGAATTCCTCGAG | |
SP |
GGGGTTGGCCATGGCGAC | |
dflGPD_bbOL_for | TCGAGTTTTTCAGCAAGATGGATCCGGTATCTATTACATTGCATTGC | |
dflGPD_g418OL_rev | CGTGCAATCCATCTTGTTCAATCATTTTGAATTATTGACTTTTGTCGAG | |
G418_for | ATG ATT GAA CAA GAT GGA TTG CA | |
GATGGCTCGAGTTTTTCAGCAAGATTTTCCTAAAGCCAAGTGTTCC | Deletion of |
|
GACCTCCACTAGCATTACACTTATCGTAGCCATTGTTGTCATAG | Deletion of |
|
CTATGACAACAATGGCTACGATAAGTGTAATGCTAGTGGAGGTC | Deletion of |
|
CAAAGTCTTAATACCACTGACACAGATGTTTGGGGGGAGTTTAGGGAAAG | Deletion of |
|
GCTCTTTCCCTAAACTCCCCCCAAACATCTGTGTCAGTGGTATTAAG | Deletion of |
|
ATTGTAGGAGATCTTCTAGAAAGATTGGAATCCCGGCTCGTTTATTC | Deletion of |
|
CyrA_woSP_asc_for | GGCGCGCCATGAACCCCAATGTATACGAC | GFP fusions CyrA |
CyrA_pac_rev | TTAATTAATTAGTAGCACTTTTCGCACAAA | GFP fusions CyrA |
CyrA_asc_for | GGCGCGCCATGCAGCTCCTCAGCACTA | GFP fusions CyrA |
CyrA_nostop_pac_rev | TTAATTAAGTAGCACTTTTCGCACAAAGT | GFP fusions CyrA |
AATGGCGCGCCATGCAGCTCCTCAGCACTAT | Laccase-Assay; CyrA - Laccase; |
|
ATAGCTAGCGTAGCACTTTTCGCACAAAGT | Laccase-Assay; CyrA - Laccase; |
|
mCherry_Tub(t)OL_rev | GCAAAGTAGGAATGACATCAGATATCTAAGCGGCCGCTTTGTAGAG | Promotorfusion full h2b |
bcmCherry_for | ATGGTAAGCAAGGGCGAGG | Promotorfusion h2b-mCherry |
h2b_cyrapOL_for | CTTCTTTTCACAAATACTGTCGAAAATGCCACCAAAAGCCGCC | Promotorfusion h2b-mCherry |
h2b_mcherryOL_rev | GATTACTTACCTCGCCCTTGCTTACCATTTTGGCAGACGAGGAAGAGT | Promotorfusion h2b-mCherry |
dfh2b_for | ATGCCACCAAAAGCCGCC | Promotorfusion h2b-mCherry |
TCTTGCCGTCCCTCTTTTCA | qRT-PCR |
|
TCCGGTATCTTTGGCACCAT | qRT-PCR |
|
rab_asc_for | ATGGCGCGCCATGGCTGAAGGCGGTCCA | RabA-GFP |
rab_pac_rev | GCTTAATTAACTAACAAGCACAACCGTCCT | RabA-GFP |
GATGGCTCGAGTTTTTCAGCAAGATTTTCCTAAAGCCAAGTGTTCC | Recomplementation |
|
GTTGACCTCCACTAGCATTACACTTTGGAATCCCGGCTCGTTTA | Recomplementation |
|
pjetBB_rekomb_rev | ATCTTGCTGAAAAACTCGAGC | Recomplementation |
trpcP_for | AAGTGTAATGCTAGTGGAGGT | Recomplementation |
cyra_mid_rev | TGTTGTCATAGGCCTTGTTG | Sequencing of CyrA fusion constructs |
AGTTACGTATCTCGTGAGCGA | Southern-analysis probe; RB |
|
ATCTTTTATGCTGTACGTCCAAC | Southern-analysis probe; RB |
|
olicP_efiOL_for | TCACAATCGATCCAACCGGCGCGCCATGCGCGGTCTCCTCACTTA | SP-CyrA |
cyraSP_GFPol_rev | GATTACTTACCTCACCCTTGGAAACGGGACCGGCGAGGGCC | SP-CyrA |
olicP_efiOL_for | TTGTAAAACGACGGCCAGTGAATTCTGCAGCTGTGGAGCCGCATT | SP-GFP fusion |
cyrASP_GFPol_rev | TACTTACCTCACCCTTGGAAACCATGGGGTTGGCCATGGCGAC | SP-GFP fusion |
Exo_LBol_for | GATGGCTCGAGTTTTTCAGCAAGATTACCATTGGATGATGACGTTGTT | |
Exo_Lbol_rev | GTTGACCTCCACTAGCATTACACTTGGTCGGTGCTTTTTATTGTCC | |
hph_exoOL_for | GAGGGGACAATAAAAAGCACCGACCAAGTGTAATGCTAGTGGAGGT | |
hph_exoOL_rev | AAGCAGAAGAGACAAAACCCCAAGATGGGGGGAGTTTAGGGAAA | |
Exo_Rbol_for | ATGCTCTTTCCCTAAACTCCCCCCATCTTGGGGTTTTGTCTCTTCT | |
Exo_RBol_rev | ATTGTAGGAGATCTTCTAGAAAGATGGCTACGCATGCTATGAAG | |
Exo_LB_for | TAC CAT TGG ATG ATG ACG TTG | |
Exo_RB_rev | GGCTACGCATGCTATGAAG |
All plasmids are listed in
Name | Description | Reference |
---|---|---|
pCFJ90T | myo-2p::tdtomato | A. Dillin, UC Berkeley |
pMyo-2-EGFP | myo-2p::GFP | R. Baumeister, Uni. Freiburg |
pNH10 | This work | |
pNH12 | This work | |
pNH21 | This work | |
pNH22 | This work | |
pNH24 | This work | |
pHN29 | This work | |
pHN30 | This work | |
pNH32 | This work | |
pNH38 | This work | |
pNH20 | This work | |
pNH46 | This work | |
pNH61 | This work | |
pNH62 | This work | |
pNH72 | This work | |
pNH48 | This work | |
pVW23 | [ |
|
pNH58 | This work | |
pNH59 | This work | |
pNH97 | This work | |
pNH106 | This work | |
pNH103 | This work | |
pNH104 | This work | |
pNH13 | This work | |
pNH38 | This work | |
pNH90 | This work | |
pNH63 | This work | |
pNH109 | This work |
To create the promotor fusion of the
For the C-terminal GFP fusion protein of CyrA the gene was amplified with a forward primer containing an
For the localization of the different organelles, pNH21 was used as a backbone and the respective gene sequences were inserted via Gibson assembly (BroA (dfl_002479), ClaH (dfl_009034), RabA (dfl_005994)).
The
To reintroduce the functional
For the expression of
Plasmids harboring the
For the ABTS-assay 1x104 spores of
For the Brefeldin A (BFA, Sigma-Aldrich) treatment
For the long-term observation spores were incubated on LNA microscopy slides for 24 h together with a mixed population of
For the lifespan assay 30 L4 stage nematodes were transferred to NGM plates supplemented with 150 μM 5-fluoro-2′-deoxyuridine (FUdR). The plates were cultured at 20°C and the number of live and dead nematodes was determined with a dissecting microscope every day. The assay was conducted with at least 100 worms in two independent experiments. Survival rates were calculated using the Kaplan-Meier method and survival rates were evaluated using the log-rank test (P<0.05) using OASIS (
P-values were calculated with GraphPad Prism applying the student’s t-test. Experiments were conducted in three technical and biological replicates.
For microscopical analyses 4x104 spores were inoculated on thin LNA slides and incubated for at least 12 h at 28°C. Conventional fluorescence images were captured at room temperature using a Zeiss Plan-Apochromat 63x/1.4 Oil DIC, EC Plan-Neofluar 40x/0.75, EC Plan-Neofluar 20x/0.50, or EC Plan-Neofluar 10x/0.30 objective with a Zeiss AxioImager Z.1 and AxioCamMR. For Confocal microscopy, the Zeiss LSM 900 with Airyscan 2 was used. Images were collected using ZEN 2012 Blue Edition.
Stereomicroscopy was performed using a Zeiss Lumar.V12 with AxioCam HRc and NeoLumar S 1.5x objective. Images were collected with the AxioVision software.
For the long-term observation virulence assay of
The fungal cell wall was visualized by Calcofluor-white (CFW, fluorescent brightener 28, Sigma Aldrich) as described [
The CyrA-GFP expressing strain sNH30 was co-incubated with
(MP4)
The
(MP4)
The
(MP4)
The
(MP4)
We are grateful to Dr. Daniele Bano of DZNE Bonn for providing the