2.2. Multiple Sequence Alignment of Fungal Arp1

Multiple sequence alignments of protein sequences and neighbor joining phylogenetic analysis were performed with MAFFT version 7 [54]. A bootstrap analysis was conducted with 1000 iterations to test the tree for statistical significance. The tree was displayed with Archaeopteryx.js [55].

2.3. Construction of Plasmids

Plasmids were generated via homologous recombination in S. cerevisiae [45], as described previously [56], or the NEBuilder HiFi DNA Assembly Cloning Kit (New England Biolabs, Ipswich, MA, USA) according to the instruction manual. Information on all primer sequences and plasmids is provided in Tables S1 and S2, respectively.

For the construction of the pArp1-KO knockout plasmid, the Golden Gate cloning system according to Dahlmann et al., 2020, was used [57]. The 5′-(1311 bp) and 3′-(1791 bp) flanking regions of the Smarp1 gene were amplified from S. macrospora wt gDNA with the Arp1-ko-5f/Arp1-ko-5r and Arp1-ko-3f/Arp1-ko-3r primer pairs, respectively. Together with the donor vector pGG-hph and the destination vector pDest-Amp, the fragments were cloned via the Golden Gate procedure [57].

To generate the pSmarp1 plasmid, a fragment (5028 bp) containing the native promotor, coding sequence and native terminator of Smarp1 was amplified from S. macrospora wt gDNA with the Arp1-nat-5f/Arp1-nat-3r primer pair. The fragment was cloned into the XhoI-linearized pRS-nat vector [58] via homologous recombination using the PJ69-4A yeast strain [45].

The fragment containing the native promoter and coding sequence of Smarp1 was amplified from S. macrospora wt gDNA using the Arp1_promotor_f/Arp1-Neo_r primer pair for the generation of the pSmarp1-mNG plasmid. The amplification of mNG was done with the Neo_f/Neo_TtrpC_r primer combination using the pmn-xyl plasmid [59]. The terminator of the anthranilate synthase gene trpC of Aspergillus nidulans, TtrpC, was amplified from the p1783-1 plasmid [60] with the TtrpC_f/TtrpC_pRS_r primer pair. The three PCR products were cloned into XhoI-linearized vector pRS-nat [58] via homologous recombination in the PJ69-4A yeast strain [45]. To construct the pSmarp1-TagRFP-T_nat/_hyg plasmids, the Smarp1 promoter and coding sequence was amplified with the Arp1_5′f/Arp1_RFP_r primer pair from S. macrospora wt gDNA. The TagRFP-T sequence and the A. nidulans TtrpC were amplified using the primer pairs of RFP-f/RFP-r-trpC from pTagRFP-T_nat [61] and TrpC_F/pRS426GFPrev from p1783-1 [60], respectively. The S. cerevisiae strain PJ69-4A was transformed with the three amplicons and XhoI-linearized pRS-hyg [62] or pRS-nat [58] to generate recombinant plasmids via homologous recombination [45,46].

For the generation of the pCgarp1-TagRFP-T plasmid, three fragments were amplified for the assembly with pJet1.2 (Thermo Fisher Scientific, Waltham, MA, USA) using the NEBuilder HiFi DNA Assembly Cloning Kit (New England Biolabs). Cgarp1, including the 1 kb 5′ region, was amplified from CgM2 gDNA using the 5′CgArp1_pJet_fw/CgArp1_rev oligonucleotides. The coding sequence of TagRFP-T, including the TtrpC terminator of A. nidulans, was obtained from pSmarp1-TagRFP-T (TagRFP-T_CgArp1_fw/TtrpC_r). The neomycin phosphotransferase nptII resistance cassette, was amplified from the pII99 plasmid [63] using nptII_TtrpC_fw/PtrpC_pJet_rev. For further subcloning, EcoRV restriction sites were integrated in the oligonucleotide sequences of 5′CgArp1_pJet_fw and nptII_TtrpC_fw. Outgoing from pCgarp1-TagRFP-T, the pJet_gen plasmid harboring the nptII-resistance cassette was generated via hydrolysis with EcoRV and subsequent ligation of the plasmid backbone.

Primers were synthesized by Sigma-Aldrich Chemie GmbH (Taufkirchen, Germany). The DNA sequencing of the plasmids was performed by Seqlab Sequence Service Laboratories GmbH (Göttingen, Germany).

2.4. Generation of the Smarp1 Deletion Strain Smarp1

To delete the S. macrospora arp1 gene, the pArp1-KO knockout plasmid was used as a template to amplify the 4568 bp deletion cassette with the GG_KO_fw/GG_KO_rv primer pair containing the 5′- and 3′- flanking regions of Smarp1 and the hph cassette. After the PCR clean-up of the amplicon, the S. macrospora Smku70 strain was transformed with the deletion cassette to replace the Smarp1 ORF with the hph-cassette. Primary transformants were crossed with the spore-color mutant fus1-1 (S23442) [64], and single-spore isolates (ssi) of Smarp1 carrying hygromycin resistance were selected [51]. The verification of the absence of the Smarp1 gene and the integration of the hph-cassette at the desired locus were performed with the Arp1-v5f/h3 (1578 bp) and Arp1-v5f/Arp1-vORF5-r (2170 bp) primer pairs, respectively. To check the presence of the Smku70 gene in Smarp1 after crossing, the Smku70-v1-f/ku70-ko-v3f(R) (2851 bp) primer pair was used. The deletion of Smarp1 was verified via Southern hybridization hydrolyzing the gDNA of S. macrospora wt, Smku70, and Smarp1 with EcoRV. After electrophoresis, a capillary blot with a nylon membrane was performed at RT overnight. The 1791-bp 3′-probe was amplified from S. macrospora wt gDNA with the Arp1-ko-3f/Arp1-ko-3r primer pair. The labeling of probes for Southern blot experiments was done using the Amersham AlkPhos Direct Labelling and Detection Kit (GE Healthcare, Amersham RPN3680, Boston, MA, USA). Detection was performed according to the manufacturer’s manual. Signals were visualized on X-ray films (Amersham Hyperfilm TM ECL; GE Healthcare, Boston, MA, USA) using an “Optimax X-ray film processor” (PROTEC GmbH & Co. KG, Oberstenfeld, Germany).

The complementation of Smarp1 was performed by transformation with the pSmarp1-TagRFP-T_nat plasmid or its untagged Arp1 version, pSmarp1, resulting in the Smarp1::Smarp1-TagRFP-T or Smarp1::Smarp1 strains, respectively. Positive primary transformants were selected on BMM supplemented with nourseothricin and hygromycin. Crosses were performed as described previously [51].

2.5. Generation of S. macrospora Strains for Fluorescence Microscopy

Strains were generated by transforming wt S. macrospora, as described previously [47]. The ectopic integration of the pSmarp1-TagRFP-T and pSmarp1-mNG plasmids led to the selection of positive transformants on BMM supplemented with nourseothricin-dihydrogen sulphate (50 µg/mL) and/or hygromycin B (110 U/mL) (Merck, Kenilworth, NJ, USA). Primary transformants were grown over 7–9 d at 27 °C to obtain single-spore isolates, as described previously [51].

2.6. Generation of C. graminicola Strains

The pCgarp1-TagRFP-T and pSmarp1-mNG plasmids were linearized using NotI and NdeI, respectively, prior to transformation in CgM2. Protoplasts were obtained from oval conidia by cell wall digestion using a lysis enzyme of Trichoderma harzianum, as described previously [10]. To obtain homokaryotic transformants, colonies that had developed on CM plates supplemented with 400 µg/mL geneticin disulphate (pCgarp1-TagRFP-T) or 150 µg/mL nourseothricin-dihydrogen sulphate (pSmarp1-mNG) were allowed to conidiate on OMA. After single-spore isolation using the generated falcate conidia, resistant transformants were tested for the expression of the red or green fluorescent Arp1 fusion proteins by fluorescence microscopy.

2.7. Fluorescence Microscopy

Microscopic documentation was performed with the AxioImager M1 microscope (Zeiss, Jena, Germany) with differential interference contrast (DIC) and specific filter sets for the detection of the fluorescent signals. Image capturing was performed with a Photometrix CoolSNAP HQ camera (Roper Scientific, Photometrics, Tucson, AZ, USA), and images were processed using ZEISS ZEN Digital Imaging (version 2.3; Zeiss). To detect the mNG signal, a 49002 Chroma filter set (exciter ET470/40x, ET525/50m, beam splitter T495lpxr) was used, and for tdTomato/TagRFP-T/FM4-64 signals, a 49005 Chroma filter set (exciter ET545/30x, emitter ET620/60 m and beam splitter T570LP) was used. For each experiment, at least three biological replicates were analyzed more than three times.

For the investigation of S. macrospora hyphae, the strains were grown for 24 h on BMM-covered glass slides and 2 mL of liquid BMM on top under continuous light at 27 °C. For FM4-64 (Thermo Fisher Scientific, Waltham, MA, USA) staining, the Sm::Smarp1-mNG strain was grown on solid BMM supplemented with 1.5% agarose (Biozym Scientific GmbH, Hessisch Oldendorf, Germany) at 27 °C under continuous light conditions for 24 h. Then, 100 µL of an FM4-64 solution (1 µg/mL in distilled water) was applied to the mycelium on the agar-piece and incubated for 15 min at 37 °C. For nuclei staining, DAPI (AppliChem, Darmstadt, Germany) was dissolved in distilled water to a stock solution of 1 mg/mL and further diluted with methanol to a concentration of 1 µg/mL. Incubation took place in the dark at 37 °C for 2–3 min. For time lapse studies of growing hyphae, S. macrospora strains were grown on BMM or Sordaria Westergaard (SWG) fructification medium [48,49,50] supplemented with 1.5% agarose (Biozym Scientific GmbH, Hessisch Oldendorf, Germany) under continuous light conditions for 24 h at 27 °C. For the analysis of SmArp1-mNG localization during ascospore germination, the wt::Smarp1-mNG x fus::RH2B strain was grown on BMM over 7–9 d, and discarded spores were washed down with 0.02% Tween 20. Ascospores were then plated on BMM containing 1.5% agarose, and pieces were cut out and analyzed under the fluorescence microscope after 3–4 h. Recording interval of 5 s over 20 min were used for time lapse studies. To analyze Arp1-mNG localization at nuclei, the ratio of Arp1-mNG localization close to a nucleus to the total number of nuclei was estimated for the 36 single pictures, thus resulting in Video S2.

The recording of C. graminicola hyphae expressing the Arp1 of S. macrospora C-terminally fused to mNG was performed after incubation for 3 d at 23 °C on CM covered with cellophane. The staining of these hyphae with FM4-64 (Thermo Fisher Scientific, Waltham, MA, USA) was done by the application of 100 µL of a 1 µg/mL working solution on the growing mycelium, followed by incubation for 15 min at 37 °C. For the analysis of Cgarp1-TagRFP-T localization during early germination and germling fusion, 50 µL of oval conidia (c = 5 × 10⁷/mL) were spread on water agar (1% agarose, 1% Serva agar, and 25 mM NaNO₃). Incubation took place for 2 h (monitoring of young germlings) or 6 h (monitoring of CAT fusions and older germlings) at 23 °C. For time lapse studies of young germlings, a recording interval of 5 min was used. To track Cgarp1-TagRFP-T localization during plant penetration, a heat-inactivated onion epidermis covering water agar (1% agarose and 1% Serva agar) was inoculated with 10 µL drops containing 10³ oval conidia in 0.01% Tween for 29 h at 23 °C [68]. Different layers of an infected onion epidermis were recorded at a fixed distance of 0.5 µm. All experiments were performed with a minimum of three independent replicates.

2.8. Analysis of Chemotropic Growth Using the Arp1-TagRFP-T Marker

The application of Cgarp1-TagRFP-T for the analysis of chemotropic growth was performed by confronting germlings derived from C. graminicola oval conidia (c = 5 × 10⁵/mL) with a growing gradient of 50 mM glucose using a 3D-printed device, as described previously [69]. After 6 h of incubation at 23 °C with a minimum of 40 germlings/experiment, whether the tips of fungal germlings were growing to the established glucose gradient or to the solvent control (water) was determined. To allow for the discrimination of active growing germlings and to include non-germinated conidia with an established polar axis in the analysis, the localization of the Arp1-TagRFP-T maker was also tracked. In the final analysis, only germlings and conidia with a clear Arp1-TagRFP-T localization at the tip or a distinct cellular site were evaluated, respectively. In the case that one germling showed Arp1-TagRFP-T localization at two tips, only individuals with a preferred growth direction were included into the analysis. From the obtained numbers, the chemotropic growth rate was calculated as described previously [69]. All experiments were performed with a minimum of five independent replicates. For statistical analyses, the t-test for equal variances was used for all displayed experiments.

2.9. Protein Sample Preparation and Western Blot Hybridization

S. macrospora strains were cultivated in liquid BMM and grown for 3 d at 27 °C to extract proteins from fungal mycelium. After harvesting, drying, and grinding the mycelium in liquid nitrogen, 520 µL of lysis buffer (10 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.5 mM EDTA pH 8.0, 1 mM PMSF, 2 mM DTT, 0.5% NP-40, 1× protease inhibitor cocktail IV (1 tbl/50 mL, 04693132001, Mannheim, Germany), and 1× PhosSTOP™ (1 tbl/10 mL, Roche, Mannheim, Germany))/g mycelium powder were added. Together with about 200 µL of glass beads (Ø 0.25–0.5 mm; Roth GmbH, Karlsruhe, Germany), cells were lysed in the TissueLyser (Qiagen, Hilden, Germany) at 30 Hrz for 2 min. Subsequently, cells were separated from debris by centrifugation at 10,000 rpm for 15 min at 4 °C and were prepared for Western Blot analysis by adding 4× NuPAGE^® LDS-SB (Thermo Fisher Scientific, Waltham, MA, USA) and 1 M DTT to the crude extract. After heating of the samples for 10 min at 70 °C, 25 µL were loaded on a 12% SDS gel. As a protein standard, the Nippon Genetics Co. Europe blue star pre-stained protein marker (NIPPON Genetics Europe, Düren, Germany) was used.

Blotting was performed using the AmershamTM ProtranTM Nitrocellulose Blotting Membrane (GE Healthcare, Little Chalfont, UK) with 1× transfer buffer and a Mini Trans-Blot^® Cell device, as described by the manufacturer (Bio-Rad Laboratories, Hercules, CA, USA) [70]. The nitrocellulose membrane was blocked with 5% (w/v) skim milk powder in 1× Tris-buffered saline supplemented with 0.05% Tween 20^® (TBST) for 1 h at RT. To detect antigen–antibody reactions, a primary TagRFP-T (rabbit) -antibody (1:12,500, BioCat (Evrogen, Moscow, Russia), AB233-ev) solved in 5% skim milk/TBST was used. After removing the primary antibody, the membrane was washed three times with 1× TBST for 15 min, and a horse-radish peroxidase (HRP)-coupled secondary anti rabbit-antibody (1:5000, Thermo Fisher Scientific, Waltham, MA, USA) was applied to the membrane for 1 h at RT. To detect the HRP-coupled antibodies, the ImmobilonTM Western HRP Substrate kit (Merck, Kenilworth, NJ, USA) was used. Signals were visualized on X-ray films (Amersham Hyperfilm TM ECL, Marlborough, MA, USA) using an “Optimax X-ray film processor” (PROTEC GmbH & Co. KG, Oberstenfeld, Germany).

3.2. SmArp1 Localization Is a Dynamic Process at Growing Hyphal Tips and in Proximity to the Nucleus

Fluorescence microscopy was performed to determine the cellular localization of SmArp1-TagRFP-T. The fusion construct predominantly localizes as subapical structures in the tips of growing hyphae (Figure 1a). The application of a higher imaging contrast allowed for the further determination of dot-like structures apart from hyphal tips (Figure 1a). To confirm that the SmArp1 localization was independent of the used fluorophore, we replaced TagRFP-T by a C-terminal fused mNG, a monomeric green fluorescent protein derived from the lancelet Branchiostoma lanceolatum [59]. SmArp1-mNG localized dynamically to growing hyphal tips (Figure S5 and Video S1). To exclude probable side effects on S. macrospora due to the expressed fusion constructs, growth rates were determined using race tubes. No difference in growth was seen in Sm::Smarp1-TagRFP-T (3.5 ± 0.29 cm) or Sm::Smarp1-mNG (3.1 ± 0.31 cm) compared to the wild-type strain (3.4 ± 0.27 cm). The co-staining of Sm::SmArp1-TagRFP-T with DAPI (Figure 1b) and co-expression experiments with a red fluorescent histone marker (RH2B tdTomato) were performed to address the dot-like localization pattern (Figure 1c). Both experiments showed that SmArp1-TagRFP-T localized to small dots close to nuclei that moved retrograde from the tip to the inner hypha (Figure 1c and Video S2). Moreover, the evaluation of the 36 single pictures of Video S2 revealed that 6–46% of nuclei showed a close-by SmArp1-mNG localization, emphasizing that the association of Arp1 to nuclei is a highly dynamic process.

Open in a separate window

Figure 1

The localization of Arp1 in S. macrospora and the heterologous expression of pSmarp1-TagRFP-T or pSmarp1-mNG in wild-type S. macrospora hyphae: a recording of hyphae from growing strains on BMM-covered glass slides (a,b) or on BMM agar supplemented with agarose (c) after incubation for 24 h at 27 °C. (a) SmArp1-TagRFP-T shows subapical localization at the hyphal tip and as puncta-like structures in the hyphae. TagRFP-T expression served as the control. (b) The nuclear localization of SmArp1-TagRFP-T is marked by arrows. Nuclei were stained with DAPI. (c) Selected images of Video S2 showing the dynamic localization of SmArp1-mNG in growing hyphae after 24 h of incubation on BMM agar at 27 °C. The nuclear localization of SmArp1-mNG is indicated by arrows. Nuclei are labeled by histone 2B with tdTomato (RH2B), size bar = 10 µm.

3.3. Dynamic Localization of Arp1 in C. graminicola

To follow the hypothesis that Arp1 fused to a fluorescent protein can be used as a universal marker for active growing hyphae in fungi, we chose to transform the pSmarp1-mNG plasmid into the wild-type CgM2 strain of the plant pathogen C. graminicola. Comparable to S. macrospora, the expression of Smarp1-mNG resulted in a subapical localization at the hyphal tip (Figure 2a), indicating a conserved localization among ascomycetous fungi. For the detailed investigation of localization during C. graminicola development and pathogenicity, CgM2 was transformed with the C. graminicola arp1 gene (Cgarp1) fused to TagRFP-T under the control of its native promoter. The resulting CgM2::Cgarp1-TagRFP-T strain showed a highly dynamic CgArp1 localization to growing tips of germlings. Hyphae arrested in growth completely lost tip localization (Figure 2b). At the same time, new Cgarp1-TagRFP-T patches appeared on new branching sites, following the new polar axis and indicating the current growth direction (Figure 2b and Video S3). Similar to findings for S. macrospora, the expression of both Arp1 fusion proteins did not result in aberrant growth patterns of C. graminicola transformants (CgM2: 9.9 ± 1.7 cm²; CgM2::Smarp1-mNG: 9.8 ± 2.2 cm²; CgM2::Cgarp1-TagRFP-T: 9.2 ± 1.6 cm²).

Open in a separate window

Figure 2

The localization of Arp1 in C. graminicola: (a) the heterologous expression of pSmarp1-mNG in wild-type C. graminicola CgM2 hyphae. Recording of hyphae from growing colonies on CM covered with cellophane after incubation for 3 d at 23 °C: (b) selected images of Video S3 showing the dynamic localization of CgArp1-TagRFP-T in growing germlings after 2 h of incubation on water agar at 23 °C. Recording interval = 5 min; polar growth axis is indicated by white arrows. (c) Arp1-RFP-T localization in C. graminicola germlings attempting for germling fusion. Germlings derived from oval conidia incubated for 6 h at 23 °C on water agar (50 µL, c = 5 × 10⁷/mL). The localization of Arp1-TagRFP-T at the tip is indicated with *. Probable future fusion sites are indicated with black arrows; size bar = 10 µm.

3.4. Absence of CgArp1 localization to C. graminicola future fusion points

Germling fusion is a highly dynamic process resulting in the formation of conidial anastomosis tubes (CATs) that link germinating conidia during early colony development [72]. This process includes the recognition of a probable fusion partner by a so-far unknown signal and the stepwise growth towards the counterpart in the chemotropic phase of the process [73]. Due to the observed correlation between CgArp1 localization to the polar sites of the fungal cell, we speculated about a probable localization in chemotropically active germlings during the CAT formation process. However, we were never able to monitor a clear localization of CgArp1 during germling interactions (Figure 2c), indicating a fundamental difference between growing hyphae and the fusion of CATs.

3.5. Arp1 Localizes Subapical to SPK

The SPK, a highly organized structure at the hyphal apex that is mostly composed of secretory vesicles, is broadly used to determine active hyphal growth, growth direction, germination, and branching site selection [28,74,75,76,77]. To compare the localization of Arp1 and the SPK, we stained S. macrospora strains expressing SmArp1-mNG with the red fluorescent, membrane-selective dye FM4-64 [78,79]. As depicted in Figure 3, staining with FM4-64 predominantly visualized the SPK (a dense structure at the hyphal apex) and, to a lesser extent, intracellular membranes. The overlay with SmArp1-mNG revealed that Arp1 localizes just behind the SPK, verifying its subapical localization. An analogous experiment with CgM2::Smarp1-mNG showed the same cellular localization (Figure S6).

Open in a separate window

Figure 3

The localization of SmArp1-mNG together with FM4-64-stained hyphae in S. macrospora. The expression of pSmarp1-mNG in wild-type S. macrospora hyphae stained with FM4-64 (1 µg/mL in distilled water) and incubated for 15 min at 37 °C. The recording of hyphae was performed after growing on solid agarose-BMM for 24 h at 27 °C under continuous light conditions; scale bar = 10 µm.

3.6. S. macrospora Arp1 Shows Dynamic Localization during Ascospore Germination

Based on the localization of Arp1 in actively growing hyphae, its association with the nucleus, and its subapical localization, we assumed that it could serve as a complementing marker protein in the highly polar processes of fungal development and pathogenicity. To visualize the localization of Arp1 during the ascospore germination of S. macrospora, Smarp1-mNG and nuclei marked with tdTomato (RH2B) fused to histone 2B were recorded in a wild-type background (Video S4). After meiosis and a post-meiotic mitosis, the asci of S. macrospora were found to contain eight nuclei that were surrounded by the spore wall. Subsequently, iterate mitoses led to multiple nuclei in each ascospore. During germination, spores form one germination vesicle at one side of the ascospore. While the size of the germination vesicles increases, multiple nuclei of the ascospore are released to the vesicle. Finally, hyphae start growing form the germination vesicle [80].

During this highly dynamic germination process, we tracked SmArp1-mNG localization to four different sites and structures of the germination vesicle and the emerging hypha (Figure 4). First, Arp1 fluorescent signals appeared close to nuclei. Moreover, SmArp1-mNG localized at the growing hyphal tip, moving as dot-like structures in anterograde and retrograde directions. Third, a quickly emerging and disseminating fluorescent filamentous signal appeared in the germination vesicle. Additionally, a probable microtubule organization center (MTOC) was found to dominate the germination vesicle.

Open in a separate window

Figure 4

The dynamic localization of SmArp1-mNG in germinating S. macrospora ascospores. Selected images of Video S4, which tracks the germination of S. macrospora ascospores expressing Smarp1-mNG and red fluorescent histone 2B (RH2B tdTomato) on BMM-agar supplemented with agarose after incubation for 3–4 h at 27 °C. Arrows = localization of SmArp1-mNG close to nuclei; MTOC= putative microtubule organization center; al = SmArp1-mNG localization inside the ascospore; asterisk = SmArp1-mNG localization in fast appearing and dispersing linear structures; size bars = 10 µm; recording interval = 5 s.

3.7. Analysis of Chemotropic Growth Is Improved by the Usage of the Arp1-TagRFP-T Marker

Chemotropic growth to nutrient sources, mating partners, and hosts is a fundamental process in fungi [6]. However, the assessment of the chemoattractive signal as well as cellular factors involved in signal recognition is not trivial. Recently, we established a new, easily applicable method using a 3D-printed device based on the chemotropic assay developed by Turrà et al., 2015 [69,81]. In our approach, growing C. graminicola germlings are confronted with a signal gradient that can be sensed over time, resulting in growth re-direction. Using a hyphal tip projection angle to a theoretical vertical line, whether a germling is growing towards the signal gradient (1–179°), is repelled by it (181–359°), or shows a neutral orientation (0°/360° and 180° [69]) can be determined. The application of this method results in C. graminicola germlings with a strong chemotropic growth rate when 50 mM of glucose is used as chemoattractant, whereas the growth direction of germlings is uniform in control experiments with water, as depicted by a chemotropic rate of about 50% [69].

Despite its significance, this evaluation mode does not differentiate between germlings showing active growth re-direction due to signal sensing and germlings that stopped their growth during incubation time and are not actively sensing. Furthermore, in these experiments, we often observed germlings that grew bi-directionally, which could not be included in the evaluation since a clear readout was missing (Figure 5a).

Open in a separate window

Figure 5

Enhanced evaluation of C. graminicola chemotropic growth by the application of the Arp1 marker: (a) the evaluation of growth direction of germlings based on microscopic analyses without (−) and with (+) Arp1 monitoring. The tip localization of Arp1-TagRFP-T was used as an indication for active growth or established polar axis. Blue box = same evaluation of results in (−) and (+); red box = different evaluation results in (−) and (+); violet box = germlings or non-germinated conidia that can be included in the (+) evaluation in contrast to (−); size bar = 10 µm. (b) The chemotropic rates of germlings to gradients of water (H₂O) and 50 mM glucose (glc) after incubation for 6 h at 23 °C obtained from experiments without (−) and with (+) the monitoring of the fluorescent Arp1 marker. Chemotropic rate: <50% = repulsion; = 50% = no growth reaction; >50% = attraction; meridian is indicated in red; average is indicated by x; n ≥ 5; *, p < 0.05; ns = not significant.

Due to correlation between Arp1 tip localization and active growth or polarity axis establishment, we tested whether the usage of the Arp1 localization marker allowed us to improve the readout quality of the chemotropic assay. As depicted in Figure 5a, the localization of CgArp1-TagRFP-T to the tip often, but not always, corresponded to growth direction. In the case of bi-directional growing germlings, it allowed us to determine the current growth direction solely indicated by CgArp1-TagRFP-T localization to one hyphal tip. Interestingly, we also found that non-germinating conidia displayed a clear CgArp1-RFP-T localization to one cellular site or displayed a rather scattered localization of Arp1. Anticipating the clear targeted localization to mark the current polar axis, we were also able to include those conidia into the evaluation (Figure 5a and Figure S7, and Videos S5 and S6). To test whether the integration of the CgArp1-TagRFP-T marker changed the determined chemotropic rate, we used this strain in experiments with a 50 mM glucose gradient and experiments with no-signal control (Figure 5b). For five replicates in which at least 40 germlings were evaluated for their dominant growth direction, we monitored DIC and TagRFP-T channel information and saved them for further analysis. In a first evaluation, we referred to the information obtained from DIC channel and analyzed chemotropic growth as described earlier [69]. This quantification resulted in chemotropic rates of 50.7% ± 1.7% (water control) and 57.3 % ± 1.6 % (50 mM glucose). In a second step, we used the same microscopic pictures for a renewed evaluation, and we integrated the information gained from the Arp1 monitoring. The exclusive integration of germlings or conidia with a clear CgArp1-TagRFP-T signal in the tip or a distinct cellular site resulted in a significant increase of the chemotropic rate to 50 mM glucose (62.6% ± 3.7%; p = 0.01), whereas no significant changes were observed in the control experiments (50.4% ± 3.3%; p = 0.86). From these results, we conclude that application of the Arp1 marker can be used to increase sensitivity to analyses of chemotropic growth.

We are grateful to Gertrud Stahlhut and Gabriele Beyer for excellent technical assistance. We also thank Anja Raschke, Institute for Agricultural and Nutritional Sciences, Martin-Luther-University Halle-Wittenberg, for sharing the protocol for C. graminicola onion epidermis infection.