Disruption of the Aspergillus fumigatus RNA interference machinery alters the conidial transcriptome

Domain mapping and genomic analysis

Domain structures were created using UniProt annotations (https://www.uniprot.org; Q4WSH7, Q4WY64, Q4WEW6, Q4WWR4, Q4WUR8, Q4WC92, Q4WXX3, Q4WA22, and Q4WVE3) and created with the drawProteins (Brennan 2018) package using R version 4.1.0 (2021-05-18).

The genomic data analyzed in this study were generated in Barber et al. (2021) and are publicly available as raw reads and assembled genomes under PRJNA697844 and PRJNA595552. Isolates originated from environmental and clinical sources and displayed a global distribution. Presence/absence variation in the genomes was quantified using OrthoFinder using peptide sequences from 300 genomes. Given that no cases of gene loss or amplification were detected among the genome assemblies, analysis of sequence variation was performed by aligning raw sequencing reads to the Af293 reference genome (FungiDB release 39). PCR duplicates were marked using MarkDuplicate from Picard v.2.18.25. Variant calling to detect SNVs and short indels was performed using HaplotypeCaller and high-quality variants identified using the following filtering criteria: SNP: QD < 2.0||MQ < 40.0||FS > 60.0||MQ RankSum < −12.5||ReadPosRankSum < −8.0; indel: QD < 2.0||FS > 200.0||ReadPosRankSum < −20.0. Variant function was predicted using SnpEff v.4.3.

Strains and culture conditions

Aspergillus fumigatus strains were grown on Aspergillus minimal medium (AMM) agar plates (70 mM NaNO₃, 11.2 mM KH₂PO₄, 7 mM KCl, 2 mM MgSO₄, 1% [w/v] glucose and 1 µL/mL trace element solution at pH 6.5). The trace element solution was composed of 1 g of FeSO₄ • 7 H₂O, 8.8 g of ZnSO₄ • 7 H₂O, 0.4 g of CuSO₄ • 5 H₂O, 0.15 g of MnSO₄ • H₂O, 0.1 g of NaB₄O₇ • 10 H₂O, 0.05 g of (NH₄)₆Mo₇O₂₄ • 4 H₂O, and ultrafiltrated water to 1000 mL. Spores were harvested after 5 d (or 7 d for the evolution experiment) with 10 mL of sterile distilled water using a T-shaped inoculation spreader, and spore suspensions were filtered through a 30-µm cell strainer (MACS; Miltenyi Biotec GmbH) to exclude mycelium. A. fumigatus transformants were screened on AMM agar plates supplemented with 150 µg/mL hygromycin, 0.1 µg/mL pyrithiamine, or 160 µg/mL phleomycin for selection. For in vitro transcription and confocal laser scanning microscopy experiments, A. fumigatus AfS150 was grown on malt agar (Sigma-Aldrich) plates supplemented with agar to a final concentration of 2% (w/v) for 5 d. Escherichia coli strains were grown at 37°C on LB broth or agar supplemented with 100 µg/mL carbenicillin when necessary.

Genetic manipulation strategies

Knockout constructs were created using several strategies; namely, Fusion PCR or the Gibson cloning method (Szewczyk and Krappmann 2010). To make constructs by Fusion PCR, an antibiotic selection gene and ∼1 kb of DNA flanking regions (5′ and 3′) of the target gene were amplified. PCR fragments were purified using the GeneJET PCR purification kit (Thermo Fisher Scientific) and concentrations were measured by NanoDrop 2000 spectrophotometer. Next, equimolar amounts of the three fragments, two flanking regions, and selection gene, were combined using Fusion PCR with the 2× Phusion Flash High Fidelity Master Mix and 10 pmol of each forward and reverse primer for the 5′ flank and 3′ flank, respectively. Fusion PCR products were purified and validated by agarose gel electrophoresis. For Gibson assembly, a selection gene and ∼1 kb flanking regions of the target gene were amplified by PCR using primers that create overhangs (Supplemental Table S2). In addition, pUC18 vector backbone was also amplified from plasmid “pUChph Volker.” DNA fragments were assembled and the resulting plasmid was used to transform chemically competent E. coli following the NEBuilder HiFi DNA Assembly protocol (New England Biolabs). Knockouts of A. fumigatus were generated by homologous recombination following the transformation of protoplasts (Weidner et al. 1998).

Growth and viability assays

The growth capacity of different A. fumigatus strains in AMM media was investigated by determining the dry weight from shaking cultures. 1 × 10⁷ spores were inoculated in 50 mL of AMM and incubated at 37°C and 200 rpm for 24 h. Mycelia were harvested using Miracloth (Merck KGaA), washed with deionized water, and dried at 60°C for 3 d. The susceptibility of different A. fumigatus strains to stress conditions was investigated by spotting 5 µL of spore suspension with concentrations ranging from 10⁵ to 10²/mL on AMM agar plates supplemented with different stress-inducing agents for 2 d. All plates were incubated at 37°C.

Inverted-repeat transgene silencing assays

To silence A. fumigatus pksP, sense and antisense pksP PCR products (500 bp each) were amplified with primer pairs oARK0031 & 0032 and oARK0033 & 0034, respectively. These PCR products were then cloned into the two multicloning sites in the p-Silent1 plasmid vector, which already has a hygromycin resistant cassette. The trpC promoter upstream of the inverted-repeat region originally present from the p-Silent 1 vector was replaced by a tetracycline-inducible promoter (TetON). The resulting plasmid was then digested with restriction enzymes SacI and FspI to excise the IRT-expressing region and hygromycin (hph) resistant cassette, which was then cloned into a pUC18 vector backbone flanked by 5′ and 3′ regions (∼1 kb) of a nonfunctional pyrG locus in the CEA17ΔakuB^KU80 (A1163, wild type) strain to generate pSilent-pksP1. Because some of the RNAi knockout strains already have a hygromycin resistant cassette, we constructed pSilent-pksP2 by replacing the selection marker with a phleomycin (ble) resistant gene. Both plasmids were linearized by BtsI restriction enzyme before transformation of strains (Supplemental Table S3). To generate a para-aminobenzoic acid synthetase (pabA) silencing construct, pabA-IRT sequence plus a 3′ trpC terminator was synthesized. This synthesized DNA was then cloned using Gibson assembly into a PCR product amplified from pSilent-pksP2 with primer pair oARK0096 & oARK0038. The latter PCR product consist of the pUC18 backbone, pyrG flanks, phleomycin resistant cassette, and TetON promoter to drive the pabA-IRT expression. The plasmid was linearized by PstI restriction enzyme before transformation of CEA17ΔakuB^KU80 wild-type strain (da Silva Ferreira et al. 2006).

Southern blot

Target gene deletion was confirmed by Southern blot, as described previously (Southern 2006). Digested genomic DNA samples were separated in a 1% (w/v) agarose gel. Separated DNA fragments on gel were transferred onto a nylon membrane (pore size: 0.45 µm, Carl Roth GmbH). The DNA was crosslinked to the membrane by UV irradiation followed by overnight hybridization with a DIG-11-dUTP-labeled DNA probe at 65°C. The membrane was washed twice for 5 min at 65°C (1× SSC, 0.1 SDS), followed by two washes with wash buffer II (0.5× SSC, 0.1 SDS) for 20 min at 65°C. The membrane was next rinsed with maleic acid wash buffer, incubated with blocking solution for 30 min to prevent unspecific antibody binding, followed by another incubation with 20 mL of blocking solution plus 0.5 µL of anti-digoxigenin-AP, Fab fragments (Roche) for 30 min. The unbound antibodies were removed by washing twice with 50 mL of maleic acid wash buffer for 15 min. Finally, the membrane was equilibrated with detection buffer, developed with several drops of the chemiluminescent substrate CDP-Star, ready-to-use (Roche) and detected with a chemiluminescence imaging Fusion FX SPECTRA instrument (Vilber Lourmat).

In vitro transcription and confocal laser scanning microscopy

For in vitro dsRNA transcription, genomic DNA was isolated from mycelium of A. fumigatus CEA17ΔakuB^KU80 grown overnight in liquid cultures and used as a template for the amplification of 500 bp of the pabA sequence by PCR. The PCR reaction mix consisted of 200–300 ng of template DNA, 10 pmol of each primer, and 25 µL of 2× Phusion Flash high-fidelity PCR master mix (Thermo Fisher Scientific). Both the forward and the reverse primer were designed to contain a T7 promotor at their 5′ end for subsequent dsRNA transcription (Supplemental Table S2; T7_pabA_500_1_ for/T7_pab_A500_1_rev). PCR products were loaded onto a 1% agarose gel to check for the correct size and purified with the GeneJET PCR purification kit (Thermo Fisher Scientific). The products were then used as a template for in vitro dsRNA transcription using the MEGAscript RNAi kit (Invitrogen) according to the manufacturer's protocol with slight modifications. Briefly, the transcription reaction was assembled as specified with the addition of 0.5 µL fluorescein-labeled-UTP solution (Sigma-Aldrich). The reaction was incubated for 4 h at 37°C and the RNA products were annealed by heating the reaction for 5 min to 75°C and subsequently left to cool at room temperature. Digestion of DNA and single-stranded RNA and the purification of dsRNA was performed as described in the protocol with the exception that the purified dsRNA was eluted in two steps using a total of 100 µL of 95°C ultrapure H₂O. Quantity and quality of the dsRNA was assessed by fluorometric analysis using a Qubit 4 Fluorometer (Thermo Fisher Scientific). dsRNA was stored in the dark at −80°C until usage.

For fluorescence microscopy, 1 × 10⁴ freshly collected A. fumigatus AfS150 conidia (Lother et al. 2014) were seeded into each well of an eight-well chamber slide (µ-Slide 8 Well, Ibidi) in a total of 100 µL of RPMI medium (Sigma-Aldrich), treated with 2 µg of labeled dsRNA, and incubated for 16 h at 37°C and 5% CO₂ (v/v). The following day, old media was removed from the samples and fresh media containing calcofluor white was added to the fungus. After 30 min of incubation at room temperature in the dark, hyphae were carefully washed with sterile phosphate-buffered saline (PBS). An amount of 100 µL of RPMI was added to each chamber and samples were observed by confocal laser scanning microscopy with a Zeiss LSM 780 microscope (Carl Zeiss). Images were processed using the ZEN blue edition software (version 3.4, Carl Zeiss).

RNA isolation

Fungal mycelium was collected from liquid culture using Miracloth (Millipore) and disrupted in liquid nitrogen using a precooled mortar and pestle. Roughly 0.5 g of homogenized mycelium was transferred into a 2-mL Eppendorf tube. An amount of 800 µL TRIzol was added to the ground mycelia and vortexed vigorously. Tubes were frozen briefly for 5 sec in liquid nitrogen and allowed to thaw on ice. An amount of 160 µL of chloroform was added to the thawed mixture, vortexed, and centrifugated for 5 min at 4°C at full speed. The aqueous upper phase was transferred to a fresh 2-mL tube without disturbing the interphase. RNA extraction from aqueous phase was done with one volume of phenol/chloroform/isoamyl alcohol (25:24:1, v/v). Brief vortexing preceded centrifugation for 5 min at 4°C. Extraction was repeated until no more interphase was observable, and this was followed by another extraction with 400 µL of chloroform. RNA was precipitated using 400 µL of isopropanol for 20 min, followed by pelleting by centrifugation for 20 min at 4°C. The pellet was washed with 700 µL of 70% ethanol and air dried for 5 min at 37°C prior to resuspension in RNase-free water. The RNA isolation was followed by a DNase treatment using 2 units of TURBO DNase (Thermo Fisher) per 10 µg of RNA for 30 min at 37°C in 100 µL of total volume. Total RNA was collected using the RNA Clean and Concentrator-25 kit (Zymo Research) according to the manufacturer's instructions.

Quantitative RT-PCR (qRT-PCR)

RNA was isolated from cultures of CEA17ΔakuB^KU80, pabA-IRT, and pksP-IRT with and without doxycycline induction after 24 h. Complementary DNA (cDNA) was synthesized from RNA according to the iScript cDNA synthesis kit protocol (Bio-Rad Laboratories, Inc.), and the concentration was determined using a Qubit Flex Fluorometer (Thermo Fisher Scientific). A 20-µL reaction volume per well was prepared in a 96-well plate. The PCR reaction mix consisted of 10 ng of cDNA, 4 pmol of each primer pair, 1 µL of 20× Eva Green (Biotium), and 10 µL of 2× MyTaq HS Mix (Bioline). Specific primers (qRT_coxV_for / qRT_coxV_rev) for A. fumigatus cytochrome C oxidase subunit V (AFUA_5G10560) were used as internal controls to normalize the expression of target genes. The cDNAs from four biological samples were used for analysis, and all the reactions were run in triplicate. The reaction was carried out using the QuantStudio 3 Real-Time PCR system (Thermo Fisher Scientific) with the following thermal cycling profile: 20 sec at 95°C, followed by 40 cycles of amplification (5 sec at 95°C, 34 sec at 60°C). Relative expression was calculated according to the 2^−ΔΔC_T method (Livak and Schmittgen 2001).

mRNA seq and analysis

Total RNA was isolated from three biological replicates of CEA17ΔakuB^KU80, ΔppdA/B, ΔdclA/B, and ΔrrpA/B knockout strains from conidia, 24-, and 48-h-old mycelium. RNA was isolated from mycelia as described above. For A. fumigatus conidia, the spores were first homogenized in a bead beater (0.5-mm beads; FastPrep-24, MP Biomedicals) in TRIzol before proceeding with the first chloroform extraction step as described in the RNA isolation method above. The RNA isolation was followed by a DNase treatment using two units of TURBO DNase (Thermo Fisher) per 10 µg of RNA for 30 min at 37°C in 100-µL total volume. Total RNA was then collected using the RNA Clean and Concentrator-25 kit (Zymo Research) according to the manufacturer's instructions. Total RNA collected were then sent for sequencing. Poly(A) selection and directional sequencing of mRNA was performed by Novogene using paired-end 150-bp read sequencing on a NovaSeq 6000.

Preprocessing of raw reads including quality control and gene abundance estimation was done with the GEO2RNAseq pipeline (v0.9.12) (Seelbinder et al. 2019) in R (version 3.5.1). Quality analysis was done with FastQC (v0.11.8) before and after trimming. Read-quality trimming was done with Trimmomatic (v0.36). Reads were rRNA-filtered using SortMeRNA (v2.1) with a single rRNA database combining all rRNA databases shipped with SortMeRNA. Reference annotation was created by extracting and combining exon features from corresponding annotation files. Reads were mapped against the reference genome of A. fumigatus (Af293, ASM265v1) using HiSat2 (v2.1.0, paired-end mode). Gene abundance estimation was done with featureCounts (v1.28.0) in paired-end mode with default parameters. MultiQC version 1.7 was finally used to summarize and assess the quality of the output of FastQC, Trimmomatic, HiSat, featureCounts, and SAMtools. The count matrix with gene abundance data without and with median-of-ratios normalization (MRN) (Anders and Huber 2010) were extracted. Raw files are accessible under Gene Expression Omnibus accession number GSE223618.

Differential gene expression was analyzed using GEO2RNAseq. Pairwise tests were performed using four statistical tools (DESeq v1.30.0, DESeq2 v1.18.1, limma voom v3.34.6, and edgeR v3.20.7) to report P-values and multiple testing corrected P-values using the false-discovery rate method q = FDR(p) for each tool. Gene expression differences were considered significant if they were reported significant by all four tools (q ≤ 0.05 and |log₂ (MRN)| ≤ 1). GO-Slim analysis was performed on genes identified as significantly differentially expressed by all four methods described above using https://fungidb.org (Amos et al. 2022) with a P-value cutoff of 0.05 and limiting the search to only GO-Slim terms.

Proteome analysis

Spores were harvested from 5-d-old AMM agar plates incubated at 37°C. Spores were pelleted in 2-mL screw cap tubes and a 300-µL equivalent of 0.5-mm beads were added. Bead beating was done in lysis buffer (1% [w/v] SDS, 150 mM NaCl, 100 mM TEAB [triethyl ammonium bicarbonate], with the addition of each one tablet cOmplete ULTRA protease inhibitor cocktail and PhosSTOP [both Roche] per 10 mL of lysis buffer), three times for 20 sec with 2-min rest on ice in between. Harvested mycelium from 48-h cultures was disrupted by mortar and pestle with liquid nitrogen. Cell debris was then homogenized in lysis buffer. All samples were then incubated at 37°C in a water bath sonicator for 30 min with 0.5 µL of Benzonase nuclease (250 U/µL). Proteins were separated from insolubilized debris by centrifugation at 18,000g for 15 min. Each 100 µg of total protein per sample was diluted with 100 mM TEAB to gain a final volume of 100 µL. Subsequently, cysteine thiols were reduced and carbamidomethylated for 30 min at 70°C by addition of 2 µL each of 500 mM TCEP [tris(2-carboxyethyl)phosphine] and 625 mM 2-chloroacetamide (CAA). The samples were further cleaned up by methanol–chloroform–water precipitation using the protocol of Wessel and Flugge (1984). Protein precipitates were resolubilized in 5% trifluoroethanol of aqueous 100 mM TEAB and digested for 18 h with Trypsin + LysC (Promega) at a ratio of 25:1 protein:protease. Samples were evaporated in a vacuum concentrator (Eppendorf). Tryptic peptides were resolubilized in 30 µL of 0.05% TFA and 2% ACN in H₂O filtered through 10-kDa MWCO PES membrane spin filters (VWR) at 14,000g for 15 min and transferred to HPLC vials.

LC-MS/MS analysis and protein database search

An improvement in technology was implemented between the two LC-MS/MS proteomics experiments described in this manuscript. Each method is described in detail below for clarity. Method 1 (M1) is related to 48-h mycelial proteomics (three biological replicates) and method 2 (M2) to conidial proteomics (four biological replicates). Each biological replicate was measured in triplicate (three analytical replicates). LC-MS/MS analysis was performed on an Ultimate 3000 nano RSLC system connected to either a QExactive Plus (M1) or an Orbitrap Exploris 480 mass spectrometer (both Thermo Fisher Scientific) equipped with a FAIMS ion mobility interface (M2). Peptide trapping for 5 min on an Acclaim Pep Map 100 column (2 cm × 75 µm, 3 µm) at 5 µL/min was followed by separation on an analytical Acclaim Pep Map RSLC nano column (50 cm × 75 µm, 2µm). Mobile phase gradient elution of eluent A (0.1% [v/v] formic acid in water) mixed with eluent B (0.1% [v/v] formic acid in 90/10 acetonitrile/water) was performed using the following gradients. M1: 0–5 min at 4% B, 30 min at 7% B, 60 min at 10% B, 100 min at 15% B, 140 min at 25% B, 180 min at 45% B, 200 min at 65% B, 210–215 min at 96% B, 215.1–240 min at 4% B. M2: 0 min at 4% B, 20 min at 6% B, 45 min at 10% B, 75 min at 16% B, 105 min at 25% B, 135 min at 45% B, 150 min at 65% B, 160–165 min at 96% B, 165.1–180 min at 4% B. Positively charged ions were generated at spray voltage of 2.2 kV using a stainless steel emitter attached to the Nanospray Flex Ion Source (Thermo Fisher Scientific). The quadrupole/orbitrap instrument was operated in Full MS/data-dependent MS2 mode to trigger fragmentation of the top 15 precursor ions (M1). For M2 the number of data-dependent scans was related to the cycle time of 1.5 sec (M2). Precursor ions were monitored at m/z 300–1500 (M1) or m/z 300–1200 (M2) at a resolution of 140,000 (M1) or 120,000 (M2) FWHM (full width at half maximum) using a maximum injection time (ITmax) of 120 msec (M1) or 50 msec (M2) and an AGC (automatic gain control) target of 3 × 10⁶ (M1) and a normalized AGC target of 300% (M2), respectively. Precursor ions with a charge state of z = 2–5 were filtered at an isolation width of m/z 1.6 (M1) or m/z 4.0 (M2) for further fragmentation at 30% (M1) or 28% (M2) HCD collision energy. MS2 ions were scanned at 17,500 FWHM (ITmax = 120 msec, AGC = 2 × 10⁵) for M1 and 15,000 FWHM (ITmax = 40 msec, AGC = 200%) for M2. Three different compensation voltages were applied (−48 V, −63 V, −78 V) for the FAIMS interface (M2).

Tandem mass spectra were searched against the FungiDB database (https://fungidb.org/common/downloads/Current_Release/AfumigatusAf293/fasta/data/FungiDB-57_AfumigatusAf293_AnnotatedProteins.fasta) of Aspergillus fumigatus Af293 (July 9, 2021) for M1 and against the UniProt pan proteome databases (December 21, 2022) of Neosartorya fumigata/Aspergillus fumigatus (https://ftp.uniprot.org/pub/databases/uniprot/current_release/knowledgebase/pan_proteomes/UP000002530.fasta.gz) for M2 using Thermo Proteome Discoverer (PD) 2.4 (M1) or 3.0 (M2). The following database search engines and respective scoring thresholds (in parentheses) have been applied for the different methods. M1: Mascot 2.4.1 (>30), Sequest HT (>4), MS Amanda 2.0 (>300), and MS Fragger 3.2 (>8). M2: Mascot 2.8 (>30), Comet (>3), MS Amanda 2.0 (>300), Sequest HT with and without INFERYS Rescoring (>3), and CHIMERYS (>2). Two missed cleavages were allowed for the tryptic digestion. The precursor mass tolerance was set to 10 ppm and the fragment mass tolerance was set to 0.02 kDa. Modifications were defined as dynamic Met oxidation, phosphorylation of Ser, Thr, and Tyr (M2), protein N-terminal acetylation with and without Met loss as well as static Cys carbamidomethylation. A strict false discovery rate (FDR) of <1% (peptide and protein level) and aforementioned search engine score threshold values were required for positive protein hits. The Percolator node and a reverse decoy database was used for q-value validation of spectral matches. Only rank 1 proteins and peptides of the top scored proteins were counted. Label-free protein quantification was based on the Minora algorithm using the precursor abundance based on intensity and a signal to noise ratio >5. Normalization was performed by using the total peptide amount method. Imputation of missing quan values was applied by using abundance values of 75% of the lowest abundance identified. Differential protein abundance was defined as a fold change of >2, P-value/ABS(log₄ratio) ≤ 0.05 and at least identified in two of three (M1) or three of four replicates (M2) of the sample group with the highest abundance (Supplemental Data Sets S4, S5). Heat maps were created in R v. 4.2.2 using pheatmap v. 1.0.12 by first log₂-transforming normalized abundance data and then calculating Z-scores for proteins that exhibited at least a log₂(FC) = 2 and a P-value ≤0.05 in one of the conditions.

Library preparation for small RNA-sequencing and bioinformatic analysis

Total RNA was isolated from three separate biological experiments using A. fumigatus CEA10 conidia (5 d on AMM agar plates at 37°C) and mycelium after 24 and 48 h of growth in AMM liquid culture at 37°C using the Universal RNA Kit (Roboklon) with slight modifications. Namely, conidia and mycelia at different time points were harvested and resuspended in RL buffer followed by bead beating three times for 30 sec each using 0.5-mm glass beads (Carl Roth). The total RNA was then isolated according to the yeast protocol provided by the manufacturer (Roboklon) using an on-column DNase treatment (Lucigen Baseline-ZERO DNase). The RNA isolation was followed by an additional DNase treatment using four units of TURBO DNase (Thermo Fisher) for 30 min at 37°C in 100-µL total volume. The small RNAs were then collected using the RNA Clean and Concentrator-25 kit (Zymo Research) according to the manufacturer's instructions for small RNA enrichment.

Since the 5′ end of A. fumigatus small RNAs are not well-defined, the isolated RNA was treated with 1 µL (5 units) of RppH enzyme (New England Biolabs) in NEB buffer 2 (50 mM NaCl, 10 mM Tris-HCl, 10 mM MgCl₂, 1 mM DTT at pH 7.9) for 1 h at 37°C to facilitate removal of a pyrophosphate from the 5′ end of triphosphorylated RNA. The reaction was ended with EDTA for 5 min at 65°C followed by cleanup using the RNA Clean and Concentrator-25 kit (Zymo Research) as described above. RNA was quantified using the Qubit 4 Fluorometer (Thermo Fisher). Small RNA libraries were then prepared from 10 ng of small RNA using the NEBNext Multiplex Small RNA library preparation set for Illumina (New England Biolabs #E7300S) according to the manufacturer's recommendation using NEBNext Multiplex Oligos for Illumina (Index Primer Set 1, #E7335S) (Supplemental Table S1). Size selection was performed according to the manufacturer using AMPure XP beads (Beckman Coulter). Libraries were validated for the correct size by polyacrylamide gel electrophoresis. The barcoded libraries were pooled without further adjustment, followed by 50-cycle single-end sequencing using the Illumina HiSeq 4000 platform (Eurofins Genomics). In this case, the sRNA data produced are not strictly suitable for comparison of read abundance between time points but were deemed appropriate for sRNA discovery.

Adaptors were trimmed from sRNA sequencing data using BBDuk from BBTools v38.22 (https://sourceforge.net/projects/bbmap/) and quality profiles assessed using FastQC. Candidate sRNAs were identified from merged replicate data using Manatee v1.2 (Handzlik et al. 2020) along with the Ensembl A. fumigatus A1163 genome (ASM15014v1) sequence and annotated transfer RNAs as a reference. Sequence hits between 19 and 25 nt in length and with more than 200 counts were retained for further analysis. Hits were visually inspected using sRNA- and mRNA-seq data and the complete A. fumigatus gene annotation viewed in Integrative Genomics Viewer (IGV) to validate their existence and identify antisense overlap with gene transcription (Thorvaldsdottir et al. 2013). FASTA sequences for the final candidate sRNAs were extracted using Gffread from GFF utilities (Pertea and Pertea 2020).

Data availability

The mass spectrometry proteomics data were deposited in the ProteomeXchange Consortium via the PRIDE partner repository (Perez-Riverol et al. 2022) with the data set identifiers PXD033984 (48-h mycelium) and PXD039739 (conidia). Small RNA and mRNA sequencing data sets can be found on GEO under identifiers GSE223619 and GSE223618, respectively.

Statistical analysis

The proteomics data were analyzed as described. For the remaining analyses, P-values were determined by ANOVA or two-tailed Student's t-tests where appropriate using Prism 5.0.0 software (GraphPad Software). Differences between the groups were considered significant at a P-value of <0.05. Throughout the article, significance is denoted as follows: P ≤ 0.05 (*), P ≤ 0.01 (**), P ≤ 0.001 (***), and nonsignificant (ns).