Journal of Fungi

Article Aspergillus fumigatus, One Uninucleate Species with Disparate Offspring

François Danion 1,2,3 , Norman van Rhijn 4 , Alexandre C. Dufour 5,† , Rachel Legendre 6, Odile Sismeiro 6, Hugo Varet 6,7 , Jean-Christophe Olivo-Marin 5 , Isabelle Mouyna 1, Georgios Chamilos 8, Michael Bromley 4, Anne Beauvais 1 and Jean-Paul Latgé 1,8,*,‡

1 Unité des Aspergillus, Institut Pasteur, 75015 Paris, France; [email protected] (F.D.); [email protected] (I.M.); [email protected] (A.B.) 2 Centre d’infectiologie Necker Pasteur, Hôpital Necker-Enfants Malades, 75015 Paris, France 3 Department of Infectious Diseases, CHU Strasbourg, 67000 Strasbourg, France 4 Manchester Fungal Infection Group, University of Manchester, Manchester M13 9PL, UK; [email protected] (N.v.R.); [email protected] (M.B.) 5 Bioimage Analysis Unit, Institut Pasteur, CNRS UMR3691, 75015 Paris, France; [email protected] (A.C.D.); [email protected] (J.-C.O.-M.) 6 Centre de Ressources et Recherches Technologiques (C2RT), Institut Pasteur, Plate-Forme Transcriptome et Epigenome, Biomics, 75015 Paris, France; [email protected] (R.L.); [email protected] (O.S.); [email protected] (H.V.) 7 Département Biologie Computationnelle, Hub de Bioinformatique et Biostatistique, Institut Pasteur, USR 3756 CNRS, 75015 Paris, France 8 Institute of Molecular Biology and Biotechnology FORTH and School of Medicine, University of Crete, 70013 Heraklion, Crete, Greece; [email protected] * Correspondence: [email protected] † Current addresses: Centre Scientifique et Technique Jean Féger, Total, 64000 Pau, France. ‡ Current addresses: Institute of Molecular Biology and Biotechnology FORTH,


University of Crete Heraklion, 70013 Heraklion, Greece.


Citation: Danion, F.; van Rhijn, N.; Abstract: Establishment of a fungal infection due to Aspergillus fumigatus relies on the efficient Dufour, A.C.; Legendre, R.; Sismeiro, germination of the airborne conidia once they penetrate the respiratory tract. However, the features O.; Varet, H.; Olivo-Marin, J.-C.; of conidial germination have been poorly explored and understood in this fungal species as well as in Mouyna, I.; Chamilos, G.; Bromley, other species of filamentous fungi. We show here that the germination of A. fumigatus is asynchronous. M.; et al. Aspergillus fumigatus, One If the nutritional environment and extensive gene deletions can modify the germination parameters Uninucleate Species with Disparate for A. fumigatus, the asynchrony is maintained in all germinative conditions tested. Even though the Offspring. J. Fungi 2021, 7, 30. causes for this asynchrony of conidial germination remain unknown, asynchrony is essential for the https://doi.org/10.3390/jof7010030 completion of the biological cycle of this filamentous fungus.

Received: 2 December 2020 Keywords: Aspergillus fumigatus; conidium; germination; asynchrony; transcriptome; aspergillosis Accepted: 29 December 2020 Published: 6 January 2021

Publisher’s Note: MDPI stays neu- tral with regard to jurisdictional clai- 1. Introduction ms in published maps and institutio- Aspergillus fumigatus is a saprotrophic fungus which lives in the soil in decaying vege- nal affiliations. tal material and upon inhalation of airborne conidia can cause life-threatening infections. Conidia are ubiquitous in the air and continuously transported through the air current. Encountering an aqueous nutritive environment leads to conidial germination. Morpho- logically, germination can be classically divided into three stages. First, a lag phase occurs Copyright: © 2021 by the authors. Li- without any morphological modification but with an active intracellular expression of censee MDPI, Basel, Switzerland. genes [1,2], preparing the synthesis of protein necessary for germination but not requiring This article is an open access article distributed under the terms and con- de novo RNA synthesis. The second stage is the isodiametral growth of germinating coni- ditions of the Creative Commons At- dia consecutive to an intracellular increase in osmotic pressure. The third stage corresponds tribution (CC BY) license (https:// to a polarized growth with the formation of a germ tube [3,4] after the first mitosis. If the creativecommons.org/licenses/by/ association between the polarized growth and mitotic events has been well established in 4.0/). Aspergillus [4–6], the characterization of the early stages of germination has been poorly

J. Fungi 2021, 7, 30. https://doi.org/10.3390/jof7010030 https://www.mdpi.com/journal/jof J. Fungi 2021, 7, 30 2 of 22

understood. Even though an Aspergillus colony originates from one conidium with a single nucleus, heterogeneity in the physiological activities in different regions of a colony or in different hyphal compartments within a single hyphae has been repeatedly noticed [7]. This heterogeneity is also observed for a conidial population and linked to resistance to antifungal drugs [8]. Bleichroldt et al. [9] identified distinct subpopulations of A. fumigatus conidia showing different patterns of heterogeneity at the cell wall level that manifest differential fitness to the antifungal caspofungin. However, the exact mechanisms linking single cell heterogeneity to this drug susceptibility status are not understood. Although germination is the primary event at the origin of human infections, exit of quiescence and germination of the conidia have been poorly documented. This is especially true for the pre-mitotic events leading to the swelling of the conidium. Moreover, even though germination is highly dependent on the nutritional environment [10], the role of different nutrients during early germination stages has not been precisely investigated yet. The aim of our study was to characterize the heterogeneity and asynchrony of the early steps of the germination process of the filamentous fungi A. fumigatus in different nutritional environments.

2. Methods 2.1. Strains The A. fumigatus strain used in this study was CEA17∆akuBKU80, which originated from the clinical isolate CBS 144–89 [11,12]. This strain was maintained on 2% malt-agar slants at ambient temperature. Preliminary experiments showed that the percentage of germination and the size of the swollen conidia when the germ tube was issued were not significantly different with 2 to 4 weeks old conidia (data not shown). Accordingly, conidia were harvested from 21-day-old malt-agar slants using 0.05% Tween–water, filtered (40 µm), washed three times with 0.05% Tween–water and then counted using the LUNA Automated Cell Counter. Several cell wall mutants of the Aspergillus unit of the Institut Pasteur and a library of transcription factor mutants previously constructed and provided by the Bromley labo- ratory were also used in this study. The cell wall mutants were affected in their content of melanin, α1,3 glucan, rodlets, galactomannan and chitin [13–17].

2.2. Germination Conditions Conidia were suspended in different media at a concentration of 5 × 105 spores/mL in a volume of 220 µL in 8-well uncoated Ibidi slides (80821) and incubated at 37 ◦C. Four media were used: (1) D-glucose 1% (G); (2) D-glucose 1% with ammonium tartrate dibasic 0.92 g/L (GA); (3) D-glucose 10g/L with ammonium tartrate dibasic 0.92 g/L and KH2PO4 1.52 g/L (GAP); (4) Minimal medium (MM). Minimal medium contains D-glucose 10 g/L, ammonium tartrate dibasic 0.92 g/L, KH2PO4 1.52 g/L, MgSO4 0.52 g/L, KCL 0.52 g/L, trace element solution (Na2B4O7, CuSO4, MnSO4, Na2MoO4, ZnSO4). All 4 media were supplemented with 34.5 g/L MOPS (morpholinepropanesulfonic acid; pH 7) to eliminate the possible change in pH during germination. Ammonium, glucose and phosphate are the three essential macronutrients for the synthesis of all fungal macromolecules and hence required for Aspergillus growth. However, their separate or joint impact has not been assessed during the germination of A. fumigatus. This is the reason for the selection of the G, GA and GAP media in comparison with the complete defined medium MM. In addition, RPMI-1640 medium is classically used in the testing of the effect of antifungal drugs and selected for its composition, which is closer to the host environment, so it has been also used [18–20].

2.3. Video Microscopy The germination process was followed over a 24-h time period under a Nikon TI microscope equipped with a 37 ◦C incubator and a 40× objective, with 1 picture captured every 4 min. Data were analyzed using ICY software [21–23]. We set up a specific program J. Fungi 2021, 7, 30 3 of 22

to automatically detect the conidia and measure their size over time. The size was evaluated as the area measured in pixels and was kept as such since pixels were the true measures recorded by the software. For a better understanding of the size of the fungal cell, pixels were sometimes translated into µm2 with a value of 0.0256 µm2 per pixel.

2.4. Nucleus and Septum Staining Staining for nucleus and septum was performed on unfixed material. For nuclear staining, supernatant of the germinating medium was removed and Hoechst (Molecu- lar probes/Thermofischer 33342) was added at a concentration of 1:2.000 in 220 µL in phosphate-buffered saline (PBS) for 10–15 min, then washed and resuspended in PBS. The number of nuclei was counted with an Evos microscope using bright field and UV filters. For septum staining, CalcoFluor White (CFW) was added at a concentration of 5 µg/mL for 10–15 min, washed and then evaluated with an Evos microscope using bright field and UV filters. Incubation time was 24 h in G, GA and GAP media and 11 h in MM medium to obtain the same morphology with short germ tubes.

2.5. Transcriptome The amount of DNA extracted from germinating conidia was very low in G and GA media, with a mean of 2 ng in GA and 28 ng in G for 108 conidia germinating at a 40 ± 10% ratio, and did not permit us to run a proper RNA-seq experiment. Accordingly, the tran- scriptome analysis was only performed in GAP medium and MM medium. Even though the composition of the culture media remained the same, the experimental conditions were slightly different from the conditions used for the video microscopy to be able to recover enough mRNA to perform the RNA-seq experiment. For the transcriptome experiments, the fungus was grown in square plastic dishes of 120/120 mm (Greiner, Kremsmünster, Austria) with 35 mL of liquid medium, with a time of incubation of the conidia in the GAP medium and MM medium of, respectively, 30 h of incubation in GAP medium and 11 h for MM medium to maintain a similar fungal morphology in both conditions. Moreover, the number of germinated conidia presenting a germ tube was 40 ± 10% in both media. However, in MM medium, the fungus continued to grow after 11 h, whereas in GAP medium, growth stopped after 30 h. Three biological replicates were performed. Conidia were collected in phenol and then disrupted with 0.5-mm diameter glass beads in a volume of 500 µL. RNA was isolated using the Ambion TURBO kit. Total RNA was checked on the Bioanalyser system (Agilent) for its quality and integrity. Ribosomal RNA depletion was performed using the Bacteria RiboZero kit (Illumina, San Diego, CA, USA). From rRNA- depleted RNA, directional libraries were prepared using the TruSeq Stranded mRNA Sam- ple Preparation Kit, following the manufacturer’s instructions (Illumina, San Diego, CA, USA). Libraries were checked for quality on Bioanalyser DNA chips (Agilent, Santa Clara, CA, USA). Quantification was performed with the fluorescent-based quantitation Qubit dsDNA HS Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA). Sequencing was performed as an SRM run (SR: single read, M: multiplexed samples) for 65-bp sequences on a HiSeq 2500 Illumina sequencer (65 cycles). The multiplexing level was 6 samples per lane. Quality control of FastQ files was performed using FastQC and trimmed with Trimmomatic (sliding window, averaging over 4 bases with average quality of >20) [24]. Trimmed reads were aligned to the A. fumigatus A1163 genome (assembly ASM15014v1, Ensembl Fungi release 48) via HISAT2 (standard parameters) and counted with FeatureCounts with the En- sembl Fungi v48 gene annotation [25–27]. Differential expression analysis was performed with DESeq2 [28]. A generalized linear model was set in order to test for the differential expression between the intracellular persisters and control conditions. Raw P values were adjusted for multiple testing according to the Benjamini and Hochberg procedure and genes with an adjusted p value lower than 0.05 were considered differentially expressed. RNA-seq datasets are available in the NCBI Gene Expression Omnibus under accession number GSE152682 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE152682). J. Fungi 2021, 7, 30 4 of 22

2.6. Epigenetic Inhibitors Epigenetic inhibitors were selected from previous publications, some of them having already been shown to be active against A. fumigatus [29–38]. All inhibitors were added to G, GA, GAP and MM media with successive dilutions from the starting solution. Epigenetic inhibitors were RG108 (diluted in DMSO at 70 mg/mL), Vorinostat diluted in DMSO at 2 mg/mL, Bromidespin in DMSO 10 mg/mL, Zebularine in DMSO at 50 mg/mL, Trichostatin in DMSO at 5 mM, Nanaomycin A in DMSO at 13 mg/mL, JQ1 in DMSO at 5 mg/mL, Terbinafin in DMSO at 10 mg/mL 3,-deazaneplanocin in water at 3 mg/mL, GSK1324726A: in DMSO at 50 mg/mL. Conidial germination as well as its asynchrony was assessed under a light microscope.

2.7. Statistical Analysis Data were analyzed using one or two-way ANOVA on 20–100 conidia measured per factor. Least mean square classification was used to rank average data with a Student t test and means not connected by the same letter are significantly different. Analysis was run on JMP software (SAS Institute, Cary, NC, USA).

3. Results 3.1. Characterization of Conidial Germination in A. fumigatus Germination of fungal conidia is initiated by an isodiametral growth stage resulting in the formation of a spherically enlarged, “swollen” conidium. This is followed by a polarized phase leading to the production of a germ tube. As previously reviewed by other authors and ourselves [1,4], and from data presented herein, it is clear that germination of A. fumigatus conidia is asynchronous. In MM medium, swelling of the first conidia started after around 1 h, whereas others did not reach this stage until 10 h post-incubation (Figures1 and2). During the swelling phase, conidial size increased from a mean area of 254 ± 17 pixels (6.5 µm2) for resting conidia to an area of 757 ± 283 pixels (19.4 µm2) after 8 h. Average size and SD were computed on 100 conidia to analyze the asynchrony of the conidial germination. Among the conidial population, the first germ tube was formed 7 h after the beginning of the incubation in MM medium, whereas the last one appeared after 18 h. After 11 h, more than 90% of conidia had germ tubes. The number of germ tubes per conidium also varied among conidia: 16 h after the beginning of germination, 4% of conidia had one germ tube, 51% had two germ tubes, 44% had three germ tubes and 2% had four germ tubes. a b

Figure 1. Asynchrony of conidial germination after 6 h (a) and 9.30 h (b) of incubation in the MM medium. See the resting Figure 1. Asynchrony of conidial germination after 6 h (a) and 9.30 h (b) of incubation in the MM medium. See (arrow heads), swollen (arrows) and germinated conidia the resting (arrow heads), swollen (arrows) and germinated conidia. J. Fungi 2021, 7, 30 5 of 22

a

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Figure 2. Asynchrony of conidial germination estimated by the measurement of the conidial volume of 100 conidia after incubation in the MM medium; (a) scatter plot of the volume of each conidia measured every 4 min; (b) box plot of the volume of all conidia measured every 60 min. Volume (estimated as pixels) was measured only on conidia without germ tubes.

The kinetic of growth from the beginning of the swelling to the emergence of the first germ tube was exponential (i.e., proportional to er), with an intrinsic growth rate r of Figure 2. Asynchrony of conidial germination estimated by the measurement of the conidial volume of 0.0035 ± 0.00049 (standard deviation) every 4 min. In our experimental conditions, the lag 100 conidia afterphase incubation had a mean in ofthe 167 MM min medium. (±85 for Volume standard (estimated deviation). as The pixels) value was of themeasured intrinsic only growthon rate is an inherent property of each conidium which is not influenced by the length of the conidia withoutlaggerm phasetubes. or the time at which the germ tube emerged (Figure2; Supplementary Figure S1), indicating that the germination characteristics are an intrinsic property of each conidium.

3.2. Does the Nutritional Environment Modify the Conidial Germination Asynchrony? To assess if asynchrony of germination is linked to specific nutrients in the growth media, germination of the conidia was investigated in four different culture conditions: glucose alone (G), glucose and ammonium (GA), glucose, ammonium and phosphate (GAP) and minimal medium (MM) (Table1; Figure3; Supplementary Figures S2–S6). Even though the value of the different germination parameters was directly dependent on the J. Fungi 2021, 7, 30 6 of 22

environmental culture conditions (Table1), the asynchrony of the conidial germination was conserved in the G, GA, GAP, MM (Figure3) and RPMI media.

Table 1. Germination parameters in different media G, GA, GAP and MM.

G GA GAP MM Conidia area at T0, mean, px 263 ± 9 a 272 ± 13 a 253 ± 21 a 254 ± 17 a Conidia area at 6 h, px 355 ± 10 b 378 ± 13 b 416 ± 21 a 519 ± 17 a Conidia area at 8 h, px 395 ± 10 c 412 ± 13 c 518 ± 13 b 757 ± 21 a Conidia area at 16 h, px 471 ± 10 c 460 ± 16 b 1078 ± 24 a NA Conidia area at first germ tubes appearance, px 620 ± 52 c 545 ± 29 c 1435 ± 39 a 1184 ± 16 b Time at first germ tube appearance, min 1223 ± 51 a 834 ± 28 b 843 ± 38 b 528 ± 15 c Measurements are recorded in pixels (px). Mean ± standard error are presented. Levels not connected by same letter are significantly different. NA: not appropriate because, in the MM medium, 93% of conidia have germinated and produced mycelium by this time.

G GA

100µM 100µM GAP MM

100µM 100µM

FigureFigure 3. 3.Morphology Morphology ofof thethe conidiaconidia germinating in G G (Glucose), (glucose), GA GA (glucose (glucose ++ ammonium ammonium tartrate), tartrate), GAP GAP (glucose(glucose + ammonium + ammonium tartrate tartrate + phosphate) + phosphate) and complex and MMcomplex for 24MM h. for 24 h. Conidial swelling was not correlated with the percentage of germinating conidia. The largest increase in the size of the swelling conidia was seen in MM medium but also in GAP medium, while the percentage of germination was the lowest in GAP medium (Figure4 ; Supplementary Figure S6; Table1). In G medium, the size of conidia at 8 h was small, with 395 ± 10 px (10.1 µm2). At 16 h, mean size of swollen conidia was 471 ± 10 px (12.1 µm2). In GA, conidia were also small, with a mean size of 412 ± 13 px (10.5 µm2) at 8 h of incubation and 460 ± 16 px (11.8 µm2) at 16 h in swollen conidia. In GAP medium, J. Fungi 2021, 7, 30 7 of 22

the phenotype was completely different, with conidial swelling similar to that seen in MM medium. At 8 and 16 h, mean sizes of conidia (without germ tubes) in GAP were 518 ±13 px (13.3 µm2) and 1078 ± 24 px (27.6 µm2), respectively. The size of the conidia after 6 h incubation in RPMI was 704 ± 27 px and the first germ tube appeared the earliest of all media (375 ± 7 min), with a mean size of 906 ± 25 px.

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Figure 4. Quantification of the conidium germination in G, GA, GAP and MM media. (a) Average size in the different media until the emission of the first germ tube (computed as conidial surface estimated by pixels); (b) heterogeneity of germination with 100 swollen conidia (until the emission of Figure 4. Quantification of the conidium germination in G, GA, GAP and MM media. a, average size in the the first germ tubes) randomly selected; (c) percentage of germinated conidia with germ tubes. different media until the emission of the first germ tube (computed as conidial surface estimated by pixels); b, heterogeneity of germination with 100 swollen conidia (until the emission of the first germ tubes) randomly selected; c, percentage of germinated conidia with germ tubes. J. Fungi 2021, 7, 30 8 of 22

The first germ tube was seen after 7 h of incubation in MM, 8 h in GA, 11 h in GAP and 18 h in G (Figure4). With the exception of GAP, the number of germinated conidia slowly increased over time to reach more than 80% after 10 h in MM and 2 days in GA and G. In GAP, the emergence of new germ tubes unexpectedly stopped after 24 h and never reached a percentage above 20% of germinated conidia in the experimental conditions used for video microscopy. The morphology of the germ tube was also different in the different media (Figure3). Germ tubes were thin in G and GA media, with a diameter of 0.9 µm, compared to 2.6 µm in the GAP and MM media. In G medium, germ tubes were short, with an average length of 7.5 µm and 12 µm after, respectively, 24 and 48 h. In GA, germ tubes were longer than in G medium (average length of 17 and 29 µm after 24 and 48 h). In GAP, germ tubes had an average length of 14 and 15 µm after, respectively, 24 and 48 h due to growth arrest after 30 h. The largest germ tubes with a diameter of 2.6 µm were similar in MM and GAP media (Table1). Interestingly, the analysis of the germ tube size also shows heterogeneity of the population of the germinated conidia (Figure5). Additional experiments in a medium with glucose and phosphate without ammonium tartrate indicated that increased width of the germ tube was associated with the presence of phosphate (data not shown).

Glucose Glucose Ammonium

M = 7.2 M = 10.8 σ2 = 14.76 σ2 = 62.34

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6… 9… 1… 1… 2… 3… 3… 4… 4… 5… 6… 7… 7… 8… 9… 1… 1… 1… 1… 1… 1… 1… 1… Figure 5. Heterogeneity in6… the population of germinated conidia estimated by the size of the germ tubes (in m) in the different media. Incubation time was 24 h in G, GA, GAP and 11 h in MM. Median values and variance are shown in the upper rightFigure corner 5. Heterogeneity of each plot. in the population of germinated conidia estimated by the size of the germ tubes (in µm) in the different media. Incubation time was 24 hours in G, GA, GAP and 11 hours in MM. Median values and variance are shown in the upper right corner of each plot. J. Fungi 2021, 7, 30 9 of 22

After 24 h of incubation in the medium, the number of germ tubes per conidium was influenced by the composition of the medium. Only single germ tubes were seen in G. In GA and GAP media, 3.9% and 13.7% of germinated conidia displayed two germ tubes after 24 h. In MM medium, 25% of the germinated conidia had two germ tubes after 11 h of incubation in the medium (data not shown).

3.3. Nuclear Division and Septum Formation in Different Nutritive Environments In G and GA media after 24 h of germination, all conidia, even when they had a germ tube, had a single nucleus which eventually migrated into the germ tube (Figure6 ). After 3 days of incubation in G and GA (at a time when growth had stopped), only 4% of the germinated conidia had two and 2% had three nuclei, indicating that mitosis was inhibited. This limited increase in the number of nuclei was independent of the length of the germ tube (data not shown). At this time, the nucleus stayed in >80% of the germinated conidia in the original conidium and did not migrate into the germ tube, indicating that the nuclear migration was inhibited concomitantly with the repression of mitosis. In the remaining germinated conidia, one nucleus was seen in the germ tubes. In contrast to G and GA media, mitosis was active in GAP and MM media; the number of nuclei started to increase during the conidial swelling and continued to expand during germ tube elongation. Interestingly, the number of nuclei per conidium before germ tube initiation or in germinated conidia in GAP (mean of 3 nuclei) was higher than in MM (mean of 2 nuclei). Later during germ tube emergence, an average of 7 to 8 nuclei was observed in 30 to 35 µm germ tubes in MM (after 11 h incubation), whereas 10 nuclei were present in germ tubes of similar size in GAP (after 24 h incubation) (data not shown). No septa were seen in germ tubes produced in G medium after 24 h (Figure7). Even though germinated conidia had only one nucleus in GA after 24 h, 6.6% of the germ tubes, which ranged in size from 15 to 35 µm, had a septum. In GAP (24 h) and MM (11 h), 11% and 12% of the germ tubes, respectively, had septa, even though the average length of the germ tubes (12 to 60 µm in both media) was similar to those in the GA medium (Figure7 ) and even though the numbers of nuclei were much higher in the GAP and MM media (see above).

3.4. Growth Arrest in G, GA and GAP Media Is Caused by Exhaustion of Nutrients Present in the Conidia In our experimental conditions, germ tube growth stopped after 24–30 h in GAP media and 48 h in G media and GA media, while the germ tube continued to grow in MM medium. After the arrest of growth, the replacement of G, GA and GAP medium by the same respective fresh medium did not induce renewed growth. However, when G, GA and GAP media were replaced by MM medium, growth was immediately restored (Supplementary Figure S7). These results indicate that the growth arrest in G, GA and GAP was not due to a starvation event resulting from the total consumption of the components of the G, GA or GAP media but to the exhaustion of essential nutrients originally present in the conidium. Data presented above show that conidial swelling, rate of germination, germ tube morphology, septum formation and nuclear division were separate morphogenetic events differently influenced by the nutritional composition of the environment. J. Fungi 2021, 7, 30 10 of 22

Light microscopy Hoechst

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Figure 6. Nuclei in germinating conidia. Germinated conidia in G and GA media only displayed one nucleus per conidium after 24 h of germination. Active nuclear division in MM and GAP media after respectively 11 h and 24 h germination. Staining with Hoechst of germinating conidia; Nucleus staining by Hoechst.

Figure 6. Nuclei in germinating conidia. Germinated conidia in G and GA media only displayed one nucleus per conidium after 24 h of germination. Active nuclear division in MM and GAP media after respectively 11 h and 24 h germination. Staining with Hoechst of germinating conidia; Nucleus staining by Hoechst. J. Fungi 2021, 7, 30 11 of 22

a b

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Figure 7. Septa shown by CalcoFluor staining of germinating conidia in different media. (a) G; (b) GA; (c) GAP; (d) MM. Figure 7. Septa shown by Calcofluor staining of germinating conidia in different media. a, G; b, GA; c, GAP; d, MM. 3.5. Transcriptome Analysis Since the growth of germ tubes stopped after 30 h in GAP medium and the growth continued in MM medium, a transcriptome analysis was undertaken in these two media to attempt to identify which genes were essential for germ tube growth (Figure8 and Supplementary Figure S8 and Table S1). A gene ontology analysis showed that, as expected, the growth arrest in GAP was directly associated with a reduced expression of the genes coding for ribosome and protein synthesis. Interestingly, the genes involved in the synthesis of B vitamin were all downregulated (Supplementary Table S1). As expected, the nucleotide sugar synthesis pathway and the alpha and beta glucan synthases were downregulated in the GAP medium. Deficit in growth was strongly associated with a downregulation of all pathways, leading to the production of GAG, which is a hallmark of A. fumigatus mycelial growth. In the GAP medium, nutrient starvation induced growth arrest and the fungal metabolism was tuned for nutritive anabolism. The search for food was correlated to the high expression of plant glycosyl hydrolases (such as cellulase, pectinase, hemicellulase) able to degrade plant material, indicating that the “grass eater” A. fumigatus was starved in this medium and actively looking for a carbon source. Interestingly, transcriptome data indicated that Zn was lacking during growth in GAP but not iron. The high expression of cell wall hydrolases such as alpha and beta glucanases as well as chitinases indicated that this fungus undertook autolysis to survive in this medium. The attempt of this fungus to survive in the GAP medium was also evidenced by the initiation of pathways to enter quiescence such as trehalose synthesis and a reduction in the membrane ergosterol synthesis. The entry into quiescence in the GAP medium was confirmed by the easy restart of growth when MM medium was added to the fungus incubated in GAP (supplementary Figure S7). The development of survival programming was associated with increased upregulation of many secondary metabolite clusters. With the exception of cluster 6, the genes controlling the formation of secondary metabolites from clusters 9, 12, 14, 16, 20, 31, 33 coding for unknown molecules but also clusters controlling the production of Endocrocin, Helvolic acid, Gliotoxin, Pyripyropene A and Neosartoricin Fumitremorgin were upregulated. These secondary metabolites are all putatively involved in the resistance Figure 8. Heat maps showing significative examples of differentially regulated A. fumigatus genes during germination in GAP and MM media. Level of expression of genes for a ratio GAP/MM : a, down regulation of ergosterol synthesis and upregulation of trehalose synthesis suggesting the entrance in quiescence of conidia germinating in GAP medium; b, ribosome and protein synthesis pathways down regulated in absence of growth in GAP medium;;c, Down regulation of cell wall synthases and up regulation of autolytic cell wall glycosyl hydrolases indicating that the germ tubes are starved in GAP medium; d, high expression of genes coding for plant material degrading glycosylhydrolases indicating the starvation status of GAP cells and the attemps to its quest for food.

J. Fungi 2021, 7, 30 12 of 22

of A. fumigatusAFUB_001190 ribosomal toprotein harmful S13p/S18e co-inhabitants of3 the soil, such as protozoa, bacteria, insects and AFUB_001520 60S ribosomal protein L31e AFUB_001750 cytosolic large ribosomal subunit protein L7A 2 fungiAFUB_002590 [39–43 60S]. ribosome biogenesis protein Brx1, putative AFUB_003490 ribosomal protein/carboxylic ester hydrolase (Ppe1), putative AFUB_003520 60S ribosomal protein L29, putative 1 AFUB_003800 60S ribosomal protein L12 AFUB_004560 50S ribosomal protein YmL27 AFUB_004610 30S ribosomal protein, putative 0 a AFUB_004660 40S ribosomal protein S8e AFUB_004740 37S ribosomal protein Mrp10, mitochondrial AFUB_004870 Ribosomal L18ae protein family −1 AFUB_005000 60S ribosomal protein L15 AFUB_005690 40S ribosomal protein S19 AFUB_005760 ribosome biogenesis protein Kri1 −2 AFUB_005840 40S ribosomal protein S12 AFUB_006000 40S ribosomal protein S3, putative AFUB_006380Ergosterol ribosomal protein L16a −3 Trehalose AFUB_006610 ribosome biogenesis protein Ssf2, putative AFUB_006720 60S ribosomal protein L27e 3 AFUB_006950AFUB_000550 40S ribosomal erg10A protein S2, putative 3 AFUB_006980 60S ribosome biogenesis protein Rrp14, putative AFUB_007150AFUB_083570 40S ribosomal erg10B protein S26 AFUB_021090 alpha, alpha−trehalose phosphate synthase subunit AFUB_007210AFUB_080560 60S acidic r ibosomalerg13A protein2 P1 2 AFUB_007360AFUB_010660 30S ribosomal erg13B subunit S4, putative AFUB_008520 60S ribosomal protein L9, putative AFUB_008620AFUB_020770 ribosome biogenesis hmg1 protein1 Erb1, putative AFUB_021080 putative trehalose−6−phosphate synthase 1 AFUB_008630AFUB_010660 AAA family A hmg2TPase/60S ribosome export protein Rix7, putative AFUB_008860AFUB_064880 50S ribosomal erg12 protein L17 0 AFUB_008890 ribosomal protein S23 (S12) AFUB_043350 orlA trehalose 6−phosphate phosphatase 0 AFUB_009300AFUB_058300 37S ribosomal erg8 protein S11, putative AFUB_009800AFUB_064220 nonribosomal mvd1 peptide synthase Pes1 −1 AFUB_009940AFUB_077180 60S ribosomal idi1 protein L35 −1 AFUB_010410 ribosomal RNA processing protein, putative AFUB_062010 tpsC trehalose−6−phosphate synthase AFUB_010560AFUB_050980 60S ribosomal erg20 protein L6 AFUB_011140AFUB_087800 60S ribosomal erg9 protein L1 −2 −2 AFUB_011390 50S ribosomal subunit protein L15 AFUB_011530AFUB_055310 37S ribosomal erg1 protein Mrp17 AFUB_001790 tpsA trehalose−6−phsophate synthase AFUB_012200AFUB_052590 60S ribosomal erg7A protein L3 −3 −3 AFUB_012370AFUB_069030 60S ribosomal erg7B protein L5, putative AFUB_013950 60S ribosomal protein L17 AFUB_089470 putative alpha, alpha−trehalose phosphate synthase subunit AFUB_014580AFUB_099400 40S ribosomal erg6 protein S5, putative

AFUB_015260AFUB_063960 40S ribosomal erg11A protein S22 F

AFUB_015670AFUB_089270 ribosome assemb erg11Bly protein Noc2, putative C AFUB_019230 40S ribosomal protein S10a AFUB_019350AFUB_003560 40S ribosomal erg24A protein S14, putative AFUB_019380AFUB_006090 37S ribosomal erg24B protein Rsm22 AFUB_019810AFUB_084150 60S ribosomal erg25A protein L24b AFUB_020100 ribosomal protein L34 protein, putative AFUB_020670AFUB_098170 40S ribosomal erg25B protein S21 AFUB_020810AFUB_030680 exosome comple erg26Ax endonuclease 1/ribosomal RNA processing protein, putative AFUB_021200 40S ribosomal protein S11 AFUB_023110AFUB_033070 37S ribosomal erg26B protein S18, putative AFUB_023440AFUB_068520 60S ribosomal erg27 protein L18 AFUB_023990AFUB_005060 60S ribosomal erg2 protein L19 AFUB_024460 50S ribosomal protein L14 AFUB_024770AFUB_093140 exosome comple erg3Ax endonuclease 2/ribosomal RNA processing protein, putative AFUB_025080AFUB_017380 60S ribosomal erg3B protein L30, putative AFUB_025090AFUB_085530 60S ribosomal erg3C protein L10 AFUB_025900 40S ribosomal protein S15, putative AFUB_025910AFUB_004350 60S acidic r ibosomalerg5 protein P2/allergen Asp F 8 AFUB_026110AFUB_062080 40S ribosomal erg4A protein S17, putative AFUB_026240 40S ribosomal protein S11 AFUB_026290AFUB_007490 40S ribosomal erg4B protein S9 AFUB_026300 50S ribosomal protein L13 AFUB_026900 50S ribosomal protein L4 AFUB_027590 60S ribosomal protein L3 AFUB_027920 mitochondrial large ribosomal subunit protein L16, putative b AFUB_029210 ribosome biogenesis protein (Bms1), putative AFUB_032040 60S ribosomal protein L32 AFUB_032560 60S ribosomal protein L37a AFUB_032650 50S ribosomal subunit L30 AFUB_032720 60S ribosome biogenesis protein Nip7, putative AFUB_035880 40S ribosomal protein S0 AFUB_036860 60S ribosomal protein L22, putative AFUB_037800 mitochondrial large ribosomal subunit L49, putative 3 AFUB_038210 telomere and ribosome associated protein Stm1, putative AFUB_001190 ribosomal protein S13p/S18e AFUB_038430 40S ribosomal protein S7e AFUB_001520 60S ribosomal protein L31e AFUB_040630 60S ribosomal protein L35Ae AFUB_001750 cytosolic large ribosomal subunit protein L7A 2 AFUB_041020 40S ribosomal protein S8e, putative AFUB_002590 60S ribosome biogenesis protein Brx1, putative AFUB_042090 40S ribosomal protein S9 AFUB_003490 ribosomal protein/carboxylic ester hydrolase (Ppe1), putative AFUB_042100 60S ribosomal protein L21, putative AFUB_003520 60S ribosomal protein L29, putative 1 AFUB_042210 40S ribosomal protein S4, putative AFUB_003800 60S ribosomal protein L12 AFUB_042280 60S ribosomal protein L37 AFUB_004560 50S ribosomal protein YmL27 AFUB_042410 40S ribosomal protein S27, putative AFUB_004610 30S ribosomal protein, putative AFUB_043390 60S ribosomal protein L27a, putative 0 AFUB_043540 60S ribosomal protein L20, putative AFUB_004660 40S ribosomal protein S8e AFUB_051530 60S ribosomal protein L4, putative AFUB_004740 37S ribosomal protein Mrp10, mitochondrial AFUB_052090 mitochondrial large ribosomal subunit protein L1, putative AFUB_004870 Ribosomal L18ae protein family −1 AFUB_052500 proteasome maturation ans ribosome synthesis protein Nop10, putative AFUB_005000 60S ribosomal protein L15 AFUB_052540 50S ribosomal protein L27, putative AFUB_005690 40S ribosomal protein S19 AFUB_053010 40S ribosomal protein S3Ae AFUB_005760 ribosome biogenesis protein Kri1 −2 AFUB_053170 60S ribosomal protein L23 AFUB_005840 40S ribosomal protein S12 AFUB_053890 60S ribosomal protein L8, putative AFUB_006000 40S ribosomal protein S3, putative AFUB_053970 50S ribosomal subunit L7, putative AFUB_006380 ribosomal protein L16a −3 AFUB_054060 ribosome biogenesis GTPase Lsg1, putative AFUB_006610 ribosome biogenesis protein Ssf2, putative AFUB_054940 60S ribosome biogenesis protein Sqt1, putative AFUB_006720 60S ribosomal protein L27e AFUB_055900 37S ribosomal protein S16 AFUB_006950 40S ribosomal protein S2, putative AFUB_057040 mitochondrial ribosomal protein, putative AFUB_006980 60S ribosome biogenesis protein Rrp14, putative AFUB_058350 37S ribosomal protein S12 AFUB_007150 40S ribosomal protein S26 AFUB_058440 ribosome biogenesis protein Urb1, putative AFUB_059030 mitochondrial ribosomal protein S19, putative AFUB_007210 60S acidic ribosomal protein P1 AFUB_059110 37S ribosomal protein S5 AFUB_007360 30S ribosomal subunit S4, putative AFUB_059310 mitochondrial ribosomal protein L23, putative AFUB_008520 60S ribosomal protein L9, putative AFUB_059380 mitochondrial ribosomal protein L11, putative AFUB_008620 ribosome biogenesis protein Erb1, putative AFUB_060400 nonribosomal peptide synthase, putative AFUB_008630 AAA family ATPase/60S ribosome export protein Rix7, putative AFUB_060470 50S ribosomal protein L4 AFUB_008860 50S ribosomal protein L17 AFUB_060480 mitochondrial large ribosomal subunit YmL35, putative AFUB_008890 ribosomal protein S23 (S12) AFUB_061170 60S acidic ribosomal protein P0, putative AFUB_009300 37S ribosomal protein S11, putative AFUB_061190 mitochondrial ribosomal protein L43, putative AFUB_009800 nonribosomal peptide synthase Pes1 AFUB_061220 ribosome biogenesis protein Ria1, putative AFUB_009940 60S ribosomal protein L35 AFUB_063110 50S ribosomal protein L3 AFUB_010410 ribosomal RNA processing protein, putative AFUB_063370 ribosome biogenesis (Nop4), putative AFUB_010560 60S ribosomal protein L6 AFUB_063470 ribosomal biogenesis protein Gar2 AFUB_011140 60S ribosomal protein L1 AFUB_064340 37S ribosomal protein Rsm25 AFUB_064530 60S ribosomal protein L36 AFUB_011390 50S ribosomal subunit protein L15 AFUB_064660 60S ribosome biogenesis protein Mak11, putative AFUB_011530 37S ribosomal protein Mrp17 AFUB_064694 60S ribosomal protein L34 AFUB_012200 60S ribosomal protein L3 AFUB_064730 40S ribosomal protein S13 AFUB_012370 60S ribosomal protein L5, putative AFUB_064830 60S ribosomal protein L11 AFUB_013950 60S ribosomal protein L17 AFUB_064950 60S ribosomal subunit Rpl38, putative AFUB_014580 40S ribosomal protein S5, putative AFUB_065290 ribosome biogenesis protein Pescadillo, putative AFUB_015260 40S ribosomal protein S22 AFUB_066100 60S ribosomal protein L25, putative AFUB_015670 ribosome assembly protein Noc2, putative AFUB_066860 50S ribosomal protein L12 AFUB_019230 40S ribosomal protein S10a AFUB_067580 50S ribosomal protein L30, putative AFUB_019350 40S ribosomal protein S14, putative AFUB_067820 50S ribosomal protein L24 AFUB_019380 37S ribosomal protein Rsm22 AFUB_067880 40S ribosomal protein S6 AFUB_019810 60S ribosomal protein L24b AFUB_069150 50S ribosomal protein L2 AFUB_020100 ribosomal protein L34 protein, putative AFUB_069730 50S ribosomal protein L36, putative AFUB_020670 40S ribosomal protein S21 AFUB_070330 37S ribosomal protein Rsm24, putative AFUB_073330 40S ribosomal protein S8 AFUB_020810 exosome complex endonuclease 1/ribosomal RNA processing protein, putative AFUB_075010 60S ribosomal protein L6, putative AFUB_021200 40S ribosomal protein S11 AFUB_076370 ribosome biogenesis protein (Rrb1), putative AFUB_023110 37S ribosomal protein S18, putative AFUB_077280 ribosomal protein L26 AFUB_023440 60S ribosomal protein L18 AFUB_078620 50S ribosomal protein Mrp49 AFUB_023990 60S ribosomal protein L19 AFUB_078660 40S ribosomal protein S10b AFUB_024460 50S ribosomal protein L14 AFUB_078720 40S ribosomal protein S29, putative AFUB_024770 exosome complex endonuclease 2/ribosomal RNA processing protein, putative AFUB_082110 ribosome biogenesis protein Noc4, putative AFUB_025080 60S ribosomal protein L30, putative AFUB_082720 ribosome biogenesis protein, putative AFUB_025090 60S ribosomal protein L10 AFUB_083630 ribosome recycling factor, putative AFUB_025900 40S ribosomal protein S15, putative AFUB_088030 ribosomal protein S5 AFUB_025910 60S acidic ribosomal protein P2/allergen Asp F 8 AFUB_088700 37S ribosomal protein S24 AFUB_026110 40S ribosomal protein S17, putative AFUB_088760 ribosomal small subunit assembly protein, putative AFUB_026240 40S ribosomal protein S11 AFUB_089970 mitochondrial ribosomal protein DAP3, putative AFUB_089980 ribosome biogenesis protein (Rrs1), putative AFUB_026290 40S ribosomal protein S9 AFUB_090030 Ribosomal protein S28e AFUB_026300 50S ribosomal protein L13 AFUB_090870 40S ribosomal protein S13 AFUB_026900 50S ribosomal protein L4 AFUB_093070 60S ribosomal protein L28 AFUB_027590 60S ribosomal protein L3 AFUB_093740 37S ribosomal protein S9 AFUB_027920 mitochondrial large ribosomal subunit protein L16, putative AFUB_094040 ribosomal large subunit biogenesis protein MAK16, putative AFUB_029210 ribosome biogenesis protein (Bms1), putative AFUB_094470 ribosomal protein L14 AFUB_032040 60S ribosomal protein L32 AFUB_095890 ribosomal S30/ubiquitin fusion AFUB_032560 60S ribosomal protein L37a AFUB_095900 60S ribosomal protein L24a AFUB_032650 50S ribosomal subunit L30 AFUB_098550 60S ribosome protein L13, putative AFUB_032720 60S ribosome biogenesis protein Nip7, putative AFUB_099140 60S ribosomal protein L7 AFUB_035880 40S ribosomal protein S0 AFUB_099370 ribosome associated DnaJ chaperone Zuotin, putative AFUB_036860 60S ribosomal protein L22, putative AFUB_037800 mitochondrial large ribosomal subunit L49, putative AFUB_038210 telomere and ribosome associated protein Stm1, putative AFUB_038430 40S ribosomal protein S7e AFUB_040630 60S ribosomal protein L35Ae Figure 8. Cont. AFUB_041020 40S ribosomal protein S8e, putative AFUB_042090 40S ribosomal protein S9 AFUB_042100 60S ribosomal protein L21, putative AFUB_042210 40S ribosomal protein S4, putative AFUB_042280 60S ribosomal protein L37 AFUB_042410 40S ribosomal protein S27, putative AFUB_043390 60S ribosomal protein L27a, putative AFUB_043540 60S ribosomal protein L20, putative AFUB_051530 60S ribosomal protein L4, putative AFUB_052090 mitochondrial large ribosomal subunit protein L1, putative AFUB_052500 proteasome maturation ans ribosome synthesis protein Nop10, putative AFUB_052540 50S ribosomal protein L27, putative AFUB_053010 40S ribosomal protein S3Ae AFUB_053170 60S ribosomal protein L23 AFUB_053890 60S ribosomal protein L8, putative AFUB_053970 50S ribosomal subunit L7, putative AFUB_054060 ribosome biogenesis GTPase Lsg1, putative AFUB_054940 60S ribosome biogenesis protein Sqt1, putative AFUB_055900 37S ribosomal protein S16 AFUB_057040 mitochondrial ribosomal protein, putative AFUB_058350 37S ribosomal protein S12 AFUB_058440 ribosome biogenesis protein Urb1, putative AFUB_059030 mitochondrial ribosomal protein S19, putative AFUB_059110 37S ribosomal protein S5 AFUB_059310 mitochondrial ribosomal protein L23, putative AFUB_059380 mitochondrial ribosomal protein L11, putative AFUB_060400 nonribosomal peptide synthase, putative AFUB_060470 50S ribosomal protein L4 AFUB_060480 mitochondrial large ribosomal subunit YmL35, putative AFUB_061170 60S acidic ribosomal protein P0, putative AFUB_061190 mitochondrial ribosomal protein L43, putative AFUB_061220 ribosome biogenesis protein Ria1, putative AFUB_063110 50S ribosomal protein L3 AFUB_063370 ribosome biogenesis (Nop4), putative AFUB_063470 ribosomal biogenesis protein Gar2 AFUB_064340 37S ribosomal protein Rsm25 AFUB_064530 60S ribosomal protein L36 AFUB_064660 60S ribosome biogenesis protein Mak11, putative AFUB_064694 60S ribosomal protein L34 AFUB_064730 40S ribosomal protein S13 AFUB_064830 60S ribosomal protein L11 AFUB_064950 60S ribosomal subunit Rpl38, putative AFUB_065290 ribosome biogenesis protein Pescadillo, putative AFUB_066100 60S ribosomal protein L25, putative AFUB_066860 50S ribosomal protein L12 AFUB_067580 50S ribosomal protein L30, putative AFUB_067820 50S ribosomal protein L24 AFUB_067880 40S ribosomal protein S6 AFUB_069150 50S ribosomal protein L2 AFUB_069730 50S ribosomal protein L36, putative AFUB_070330 37S ribosomal protein Rsm24, putative AFUB_073330 40S ribosomal protein S8 AFUB_075010 60S ribosomal protein L6, putative AFUB_076370 ribosome biogenesis protein (Rrb1), putative AFUB_077280 ribosomal protein L26 AFUB_078620 50S ribosomal protein Mrp49 AFUB_078660 40S ribosomal protein S10b AFUB_078720 40S ribosomal protein S29, putative AFUB_082110 ribosome biogenesis protein Noc4, putative AFUB_082720 ribosome biogenesis protein, putative AFUB_083630 ribosome recycling factor, putative AFUB_088030 ribosomal protein S5 AFUB_088700 37S ribosomal protein S24 AFUB_088760 ribosomal small subunit assembly protein, putative AFUB_089970 mitochondrial ribosomal protein DAP3, putative AFUB_089980 ribosome biogenesis protein (Rrs1), putative AFUB_090030 Ribosomal protein S28e AFUB_090870 40S ribosomal protein S13 AFUB_093070 60S ribosomal protein L28 AFUB_093740 37S ribosomal protein S9 AFUB_094040 ribosomal large subunit biogenesis protein MAK16, putative AFUB_094470 ribosomal protein L14 AFUB_095890 ribosomal S30/ubiquitin fusion AFUB_095900 60S ribosomal protein L24a AFUB_098550 60S ribosome protein L13, putative AFUB_099140 60S ribosomal protein L7 AFUB_099370 ribosome associated DnaJ chaperone Zuotin, putative J. Fungi 2021, 7, 30 13 of 22

c

3 AFUB_018960 chitin synthase A AFUB_052270 class III chitinase ChiA1

AFUB_098840 chitin synthase B AFUB_044700 nitroreductase f amily protein 2 AFUB_049230 chitin synthase C AFUB_090720 class III chitinase , putative

AFUB_012070 chitin synthase D AFUB_085870 class III chitinase ChiA2

Synthesis of chitin AFUB_029080 chitin synthase ChsE AFUB_085200 class V chitinase ChiB1 1

AFUB_029070 chitin synthase , putative AFUB_052460 class V chitinase Chi100 AFUB_081920 chitin synthase F Chitinases AFUB_054400 class V chitinase , putative 0 AFUB_034810 chitin synthase G AFUB_041890 conser ved hypothetical protein AFUB_037900 class V chitinase , putative −1 AFUB_078400 1,3−beta−glucan synthase catalytic sub unit FksP Synthesis of AFUB_049900 class V chitinase , putative AFUB_072830 Rho GTPase Rho1 AFUB_092800 class V chitinase , putative −2 beta(1,3)glucan AFUB_056090 Rho guanyl nucleotide exchange factor (Rom2), putative AFUB_075830 class V chitinase , putative Synthesis of AFUB_047490 alpha−1,3−glucan synthase , putative AFUB_080200 chitinase, putative −3 AFUB_027030 alpha−1,3−glucan synthase , putative AFUB_082480 beta−N−acetylhe xosaminidase NagA, putativ e alpha(1,3)glucan AFUB_014990 alpha−1,3−glucan synthase , putative AFUB_003760 conser ved hypothetical protein

AFUB_018250 1,3−beta−glucanosyltr ansferase Gel1 AFUB_021050 alpha−1,3−glucanase/m utanase, putative

AFUB_077400 1,3−beta−glucanosyltr ansferase Gel2 AFUB_023240 alpha−1,3−glucanase/m utanase, putative Modification of AFUB_028470 1,3−beta−glucanosyltr ansferase Gel3 Alpha-1,3-glucanases AFUB_052440 mutanase AFUB_022370 1,3−beta−glucanosyltr ansferase, putative AFUB_080210 alpha−1,3 glucanase/m utanase, putative

beta(1,3)glucan AFUB_084480 1,3−beta−glucanosyltr ansferase, putative AFUB_081470 mutanase

AFUB_036000 1,3−beta−glucanosyltr ansferase, putative AFUB_097510 alpha−1,3−glucanase/m utanase, putative AFUB_078410 1,3−beta−glucanosyltr ansferase, putative AFUB_096090 glucokinase GlkA, putativ e

AFUB_041210 UDP−glucose 4−epimer ase, putative AFUB_032000 glucokinase , putative

AFUB_041250 glycosyl tr ansferase, putative UDP glucose AFUB_019200 phosphoglucom utase, putative

Synthesis of GAG AFUB_041240 extracellular serine−rich protein, putative AFUB_088400 UTP−glucose−1−phosphate ur idylyltransferase Ugp1, putative AFUB_041220 cell surface spherulin 4−like protein, putative AFUB_072250 glucosamine−fr uctose−6−phosphate aminotr ansferase AFUB_041230 endo alpha−1,4 polygalactosaminidase , putative AFUB_095880 glucosamine 6−phosphate acetyltr ansferase, putative

AFUB_004590 endo−1,3−beta−glucanase Engl1 UDP N-acetyl glucosamine AFUB_006590 N−acetylglucosamine−phosphate m utase

AFUB_061710 endo−1,3(4)−beta−glucanase , putative AFUB_088730 UDP−N−acetylglucosamine p yrophosphor ylase AFUB_045140 endo−1,3(4)−beta−glucanase , putative AFUB_043280 aldose 1−epimer ase, putative AFUB_000230 endo−1,3(4)−beta−glucanase , putative AFUB_035960 aldose 1−epimer ase, putative AFUB_061880 beta−glucanase , putative UDP galactopyranose AFUB_038850 galactokinase AFUB_084070 conser ved hypothetical protein Endo beta(1,3)glucanases AFUB_027310 galactose−1−phosphate ur idylyltransferase AFUB_050810 endo−1,3(4)−beta−glucanase , putative and AFUB_036480 conser ved hypothetical protein AFUB_029980 GPI anchored endo−1,3(4)−beta−glucanase , putative furanose AFUB_058380 UDP−glucose 4−epimer ase AFUB_070280 endo−1,3(4)−beta−glucanase , putative AFUB_071250 UDP−glucose 4−epimer ase AFUB_005640 endo−1,3(4)−beta−glucanase , putative AFUB_072510 phosphomannom utase (Sec53), putative AFUB_004010 exo−beta−1,3−glucanase (Exg1), putativ e AFUB_045220 phosphoglucom utase, putative AFUB_075220 exo−beta−1,3−glucanase , putative GDP mannose AFUB_073580 GDP−mannose pyrophosphorylase A AFUB_091190 glucanase, putative AFUB_068530 mannose−1−phosphate guan ylyltransferase AFUB_025210 endo−beta−1,6−glucanase , putative

AFUB_001500 exo−beta−1,3−glucanase , putative

AFUB_077960 exo−beta−1,3−glucanase , putative

AFUB_013990 exo−beta−1,3−glucanase Exg0

AFUB_041600 exo−beta−1,3−glucanase , putative

AFUB_017490 exo−beta−1,3−glucanase , putative

AFUB_099700 exo−beta−1,3−glucanase , putative Exo beta(1,3)glucanases AFUB_101620 exo−1,3−beta glucanase , putative AFUB_006160 beta−glucosidase , putative

AFUB_091720 beta−D−glucoside glucoh ydrolase

AFUB_086800 beta−glucosidase , putative

AFUB_016780 beta−glucosidase , putative

AFUB_084510 beta−glucosidase , putative

AFUB_000280 beta−glucosidase , putative

AFUB_054750 beta−glucosidase

AFUB_077900 glycosyl h ydrolase family 3, putative

AFUB_074660 beta glucosidase , putative

Figure 8. Cont. J. Fungi 2021, 7, 30 14 of 22

d

3 AFUB_000230 endo−1,3(4)−beta−glucanase, putative AFUB_001130 endo−1,4−beta−xylanase, putative AFUB_001500 exo−beta−1,3−glucanase, putative 2 AFUB_004010 exo−beta−1,3−glucanase (Exg1), putative AFUB_004590 endo−1,3−beta−glucanase Engl1 1 AFUB_005070 endoglucanase, putative AFUB_005640 endo−1,3(4)−beta−glucanase, putative AFUB_012030 endoglucanase, putative 0 AFUB_013990 exo−beta−1,3−glucanase Exg0 AFUB_017490 exo−beta−1,3−glucanase, putative −1 AFUB_025210 endo−beta−1,6−glucanase, putative AFUB_025390 endoglucanase/cellulase, putative −2 AFUB_030110 endoglucanase, putative AFUB_030160 endoglucanase, putative AFUB_034010 endo−1,4−beta−xylanase, putative −3 AFUB_041450 pectin methylesterase, putative AFUB_041600 exo−beta−1,3−glucanase, putative AFUB_044010 endoglucanase, putative AFUB_044090 endo−1,4−beta−glucanase, putative AFUB_047990 endo−1,4−beta−xylanase, putative AFUB_048040 acetyl xylan esterase (Axe1), putative AFUB_048130 endo−1,4−beta−xylanase (XlnA), putative AFUB_048180 GPI−anchored cell wall beta−1,3−endoglucanase EglC AFUB_050360 extracellular endoglucanase, putative AFUB_050810 endo−1,3(4)−beta−glucanase, putative AFUB_056130 endo−1,4−beta−xylanase, putative AFUB_061710 endo−1,3(4)−beta−glucanase, putative AFUB_061880 beta−glucanase, putative AFUB_062240 cellulase family protein AFUB_064960 endoglucanase, putative AFUB_066600 extracellular endo−1,4−beta−xylanase, putative AFUB_070280 endo−1,3(4)−beta−glucanase, putative AFUB_073450 endoglucanase, putative AFUB_075220 exo−beta−1,3−glucanase, putative AFUB_075590 endoglucanase, putative AFUB_077620 endoglucanase, putative AFUB_077960 exo−beta−1,3−glucanase, putative AFUB_078210 endo−1,4−beta−xylanase (XynG1), putative AFUB_080500 pectin methylesterase family protein AFUB_080680 beta−1,6−glucanase, putative AFUB_080890 pectin methylesterase, putative AFUB_080950 endoglucanase, putative AFUB_081200 acetyl xylan esterase, putative AFUB_084270 endoglucanase, putative AFUB_085090 pectin methylesterase, putative AFUB_085120 endoglucanase, putative AFUB_088110 endo−1,4−beta−glucanase AFUB_091730 endoglucanase, putative AFUB_092320 endoglucanase, putative AFUB_096550 endoglucanase, putative AFUB_099700 exo−beta−1,3−glucanase, putative AFUB_101610 endoglucanase/cellulase, putative AFUB_101620 exo−1,3−beta glucanase, putative

Figure 8. Heat maps showing significative examples of differentially regulated A. fumigatus genes during germination in GAP and MM media. Level of expression of genes for a ratio GAP/MM: (a) down regulation of ergosterol synthesis and upregulation of trehalose synthesis suggesting the entrance in quiescence of conidia germinating in GAP medium; (b) ribosome and protein synthesis pathways down regulated in absence of growth in GAP medium; (c) Down regulation of cell wall synthases and up regulation of autolytic cell wall glycosyl hydrolases indicating that the germ tubes are starved in GAP medium; (d) high expression of genes coding for plant material degrading glycosylhydrolases indicating the starvation status of GAP cells and the attemps to its quest for food. J. Fungi 2021, 7, 30 15 of 22

3.6. Trying to Modify the Asynchrony of Conidial Germination in A.fumigatus None of the nutritional conditions tested lead to modified synchronicity of germina- tion. Moreover, all the mutants analyzed to date, including the 40 cell wall mutants of the Aspergillus unit and the over 400 transcription factor null mutants of the COFUN library [16], displayed an asynchronous germination. As an example, the germination parameters of the rodlet, melanin, α-(1,3)-glucan and rodlet/melanin/α-(1,3)-glucan mutants [14] were slightly modified by the respective deletions without abrogating asynchrony (Supple- mentary Table S2). Heterogeneity in germination was also seen when the germination experiments were undertaken with conidia of different ages or at different concentrations (Supplementary Figure S9 and Table S3). At the highest concentration (107), half of the conidia remained quiescent whereas the other half were swollen conidia or germ tubes, suggesting that self-inhibitors were released by high concentrations of ungerminated coni- dia [44,45]. However, these self-inhibitors did not modify the asynchrony of conidial germination (Supplementary Figure S9). The analysis of the germination of each conidium in conidial chains was also very informative. Such observation was facilitated in β1,3 glucanase mutants [46], which had defects in conidial separation. Therefore, during germination of these conidial chains, all conidia germinated differently, irrespectively of the age of the conidium (Figure9). This result suggested that the asynchrony of germination was a characteristic inherent to the conidium which was established intracellularly prior to the release of the conidia into the atmosphere. Changing the environment did not abolish germination asynchrony, even though it can modify the values of the germination parameters.

10mm

Figure 9. Asynchrony of conidial germination in a conidial chain of a glucanase mutant characterized Figure 9. Asynchrony of conidial germination in a conidial chain of a glucanase mutant characterized by a defect by a defect in conidial separation. Note that the presence of resting, swollen or germinating conidia in conidial separation. Note that the presence of resting, swollen or germinating conidia is is totally independent of the conidium age. totally independent of the conidium age. The use of various epigenetic inhibitors (RG108, Vorinostat, Romidespin, Zebular- ine, Trichostatin, Nanaomycin A, JQ1 Terbinafin, 3-deazaneplanocinA) added to G, GA, GAP and MM media did not modify the asynchrony of the germination (data not shown). Interestingly, one of these inhibitors, the tetrahydroquinoline GSK1324726A, stimulated conidial germination (30% and 76% germination in G or GA vs. G or GA + GSK1324726A, J. Fungi 2021, 7, 30 16 of 22

respectively, after 24 h), whereas the growth was similar in GAP and MM with or without GSK1324726A. The maximal size of the germ tube in G + GSK1324726A reached 98 µm after 24 h, whereas the maximal size was 25 µm in G after 24 h. Interestingly, in the G medium, the stimulation of growth by the GSK compound was associated with an increase in the nu- clear division: 30% of germ tubes had five nuclei (Figure 10 and Supplementary Figure S10). However, none of them induced any modification of the germination heterogeneity, germ tube diameter or germ tube size/nucleus number correlation.

Figure 10. Stimulation of the germ tube growth by the GSK1324726A compound in the G or GA medium. The elongation of the germ tube was associated to an increase in nuclear divisions. Note the high number of nuclei per germinated conidia while in absence of drug the growth of the germ tube was stopped and contained only one nucleus per germ tube.

Figure 10. Stimulation of the germ tube growth by the GSK1324726A compound in the G or GA medium. The elongation of the germ tube was associated to an increase in nuclear divisions. Note the high number of nuclei per germinated conidia while in absence of drug the growth of the germ tube was stopped and contained only one nucleus per germ tube. J. Fungi 2021, 7, 30 17 of 22

4. Discussion Early transcriptome analyses have shown that active transcripts from one third of the genome are present in conidia. The analysis of these transcripts suggested that conidia were undergoing a fermentative metabolism at low noise [1,47]. These data suggest that resting conidia, resembling a time bomb, are ready to explode and germinate as soon as they encounter a favorable aqueous nutritive environment without any need for de novo transcription to initiate germination [2,48]. This has been confirmed by the present study, where germination was induced by the presence of a stimulatory environmental carbon and nitrogen source in absence of de novo RNA transcription. As there was no nuclear division in media containing glucose and (glucose + ammonium), the amount of RNA extracted was very limited and only reached in these two media 1% of the amount extracted when the fungus was grown in MM medium. The occurrence of low RNA levels in the G and GA media also confirmed that the transcripts present in the resting conidia were sufficient without de novo transcription to produce enough proteins to initiate germination after glucose ± ammonium was sensed [1]. In contrast, nuclear division was active in the germinating conidia of both GAP and MM media. RNA-seq analysis of the transcripts regulated during germ tube emergence showed that the genes upregulated during germination were highly similar to those upregulated during vegetative growth in other studies [47–49]. Interestingly, the blockade of growth during germination was associated with the stimulation of the expression of genes induced by starvation, such as the glycoside hydrolases responsible for the hydrolysis of plant polysaccharides, which represent a natural substrate for this fungus, living in the soil on decaying plant material. The follow-up of the expression of secondary metabolite genes also suggested that these metabolites may have a yet undiscovered role in the fungus search of energy. Sensing of the exogenous nutrients by the conidium was enough to trigger germination, while the follow-up of polarized germ tube growth was fully dependent on the presence of appropriate exogenous nutrients. The mechanisms implemented by a conidium to sense the environment to initiate germination remain, however, unknown. Our study confirmed that nuclear division and cytokinesis are not closely coupled during conidial germination. This is also true in apicomplexan parasites, where replication in these organisms relies on the mechanisms in place to correctly segregate complete sets of organelles [50]. Tip growth in hyphae is a continuous process of extension which is not coupled to nuclear division in the same manner that bud formation and mitosis are connected in yeast. When Neurospora crassa and Podospora anserina, A. nidulans or Ashbya gossypii hypha extend, the population of nuclei increases through asynchronous or parasynchronous mitosis, and the formation of new cells, or hyphal compartments, does not seem tightly coordinated to nuclear division [51–54]. Experimental results in mold research are commonly represented as average measure- ments across whole populations of conidia whilst ignoring what is happening at the single cell or subpopulation level within these populations [55]. The present study has, however, shown the heterogeneity of germination patterns of individual conidia in a conidial pop- ulation. Bleichrold et al. [9] already showed that the conidial cell wall is heterogeneous in composition among different conidia originating from a single conidium and that this heterogeneity is modified by germination. Heterogeneity can be a result of the cell cycle, cell ageing, epigenetic regulation [56,57], cellular metabolism [58] and gene expression stochasticity [59]. This stochasticity itself can also be regulated [60]. In A. fumigatus, the fact that CFW labeling levels of individual conidia were not inherited in their offspring suggested that many genes that are directly involved in cell wall synthesis are differentially regulated, resulting in cell wall heterogeneity [9]. The fact that the progeny of a colony issued from a quick or slow germinating conidium displayed the same germinating asyn- chrony as their parental conidia suggests that this asynchrony is not fixed genetically since all nuclei that originate from single mitotic events should have identical DNA packages. The concept of nuclear autonomy is now accepted for multinucleate organisms [51,61–63] but has not been properly investigated in A. fumigatus. The pu- J. Fungi 2021, 7, 30 18 of 22

tative role of epigenetics on germination asynchrony should be investigated, especially since the genome of A. fumigatus has been shown to be rich in epigenetic markers and in DNA modification enzymes (http://www.fungidb.org)[64]. Epigenetic processes have been shown to be involved in heterogeneous expression of hyphae [65]; however, our study with inhibitors does not suggest a role for epigenetics in germination asynchrony. The impact of the GSK1324726A molecule on conidial germination was even unexpected. This molecule belongs to a family of inhibitors of Bromodomain and extraterminal (BET) family proteins known to interfere with transcriptional initiation after binding to lysine acetylated histones and elongation, used to inhibit the growth of cancer cells. Moreover, the fungal BET proteins in yeast are global transcriptional regulators which regulate the transcription of >500 genes and which are essential for fungal life. However, JQ1, which is another member of the same inhibitor family, has no effect on A. fumigatus, and the human BET inhibitors do not seem to have an effect in yeast [66,67]. The reasons for the stimulatory effect (germination and germ tube length) of the GSK compound on A. fumigatus in nutrient deficient media (G and GA) remain unknown. In A. gossypii, it has been shown that nuclei display independence in a common cyto- plasm [53]. The mechanism to produce spatially variable transcripts and heterogeneous cell behaviors is at least partly associated with the segregation of non-translating mRNA molecules and their associated proteins into messenger ribonucleoprotein (mRNP) gran- ules [68–70]. The best characterized of these mRNPs are processing bodies (P-bodies), which are liquid droplet-like compartments that lack a limiting membrane and contain specific sets of proteins and mRNAs [71–73]. It has been proposed that P-bodies con- tain translationally repressed mRNAs in combination with signaling molecules important for the proper control of cell proliferation, environmental response and morphological switching. A putative role of these mRNPs in the asynchrony in germination is currently being investigated. Why would a multinucleated cell evolve mechanisms to ensure asynchronous nuclear division? Phenotypic heterogeneity in conidial survival has been observed during envi- ronmental stress [74–77]. Interestingly, a delay in germination has been associated with drug tolerance [9,16]. Slow-growing cells are better in tolerating adverse environments. This is consistent with non- or slow-growing bacteria that tolerate both complement and antibiotics [78–80]. A nuclear autonomous cycle also enables nuclei in a specific subcellular location to divide, such as near a branch point or at a site rich in nutrients, without having to duplicate or transport more distant nuclei. It may also allow differential resistance and be responsible for the source of latent infections. The role of conidial asynchrony during infection has not been explored yet.

Supplementary Materials: The following are available online at https://www.mdpi.com/2309-608 X/7/1/30/s1, Figure S1: Absence of correlation between the isotropic swelling rate and the time at the beginning of the swelling (a) or at the emission of the germ tube (b) on twenty conidia in minimal medium; Figures S2–S5: Videos showing the conidial germination in different media (see below); Figure S6: Characteristics of the conidial germination of A. fumigatus conidia in the different media glucose (G), glucose-ammonium (GA), glucose-ammonium-phosphate (GAP), minimal medium (MM) and RPMI; Figure S7: Germinating conidia were alive after they stopped growth in a 1% Glucose medium; Figure S8: Heat map of expression of genes coding for secondary metabolite clusters differentially regulated during germination in GAP and MM media; Figure S9: Increase in conidial concentration reduces the germination; Figure S10: Stimulation of the germination by the GSK1324726A drug. Table S1: transcriptome analysis of differentially regulated A. fumigatus genes during germination in GAP and MM media; Table S2: Germination parameters of the conidia of parental strains (KU80) and alpha 1,3 glucan mutant (A), Melanin mutant (P) rodlet mutant (R) and quintuple mutant without melanin, rodlets and alpha 1,3 glucan (APR); Table S3: Germination of conidia recovered after 1 (W1), 2 (W2), 4 (W4) and 8 (W8) weeks of growth. Video S1: Germination of conidia in G medium, Video S2: Germination of conidia in GA medium, Video S3: Germination of conidia in GAP medium, Video S4: Germination of conidia in MM medium. J. Fungi 2021, 7, 30 19 of 22

Author Contributions: Conceptualization, F.D., A.B. and J.-P.L.; software, A.C.D. and J.-C.O.-M.; validation, A.B. and J.-P.L.; data analysis, F.D., A.C.D., N.v.R. and J.-P.L.; investigation, F.D., N.v.R., R.L., O.S., H.V., G.C., J.-C.O.-M., I.M., M.B., A.B. and J.-P.L.; writing original draft preparation, F.D. and J.-P.L.; writing, review and editing, all authors; supervision, J.-P.L.; funding acquisition, F.D. and J.-P.L. All authors have read and agreed to the published version of the manuscript. Funding: This research was partly funded by the French national reference center for Primary Immunodeficiencies (CEREDIH, Hôpital Necker-Enfants Malades, Paris, France), grant 2017 And the APC was funded by CEREDIH. This research was also funded by la Fondation pour la Recherche Médicale (DEQ20150331722 LATGE Equipe FRM 2015). Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: The data presented in this study are available on request from the corresponding author. Conflicts of Interest: The authors declare no conflict of interest.

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