article: Even though the fungal kingdom contains more than 3 million species, little is known about the biological roles of RNA silencing in fungi. The Colletotrichum genus comprises fungal species that are pathogenic for a wide range of crop species worldwide. To investigate the role of RNA silencing in the ascomycete fungus Colletotrichum higginsianum, knock-out mutants affecting genes for three RNA-dependent RNA polymerase (RDR), two Dicer-like (DCL), and two Argonaute (AGO) proteins were generated by targeted gene replacement. No effects were observed on vegetative growth for any mutant strain when grown on complex or minimal media. However, Δdcl1, Δdcl1Δdcl2 double mutant, and Δago1 strains showed severe defects in conidiation and conidia morphology. Total RNA transcripts and small RNA populations were analyzed in parental and mutant strains. The greatest effects on both RNA populations was observed in the Δdcl1, Δdcl1Δdcl2, and Δago1 strains, in which a previously uncharacterized dsRNA mycovirus [termed Colletotrichum higginsianum non-segmented dsRNA virus 1 (ChNRV1) ] was derepressed. Phylogenetic analyses clearly showed a close relationship between ChNRV1 and members of the segmented Partitiviridae family, despite the non-segmented nature of the genome. Immunoprecipitation of small RNAs associated with AGO1 showed abundant loading of 5’U-containing viral siRNA. C. higginsianum parental and Δdcl1 mutant strains cured of ChNRV1 revealed that the conidiation and spore morphology defects were primarily caused by ChNRV1. Based on these results, RNA silencing involving ChDCL1 and ChAGO1 in C. higginsianum is proposed to function as an antiviral mechanism.
  RNA-mediated silencing mechanisms regulate gene expression at the transcriptional and post-transcriptional level [1]. Although pathways have proliferated and specialized in various lineages, a core RNA silencing mechanism is conserved among plants, animals, fungi and other eukaryotes [2]. Canonical RNA silencing involves highly base-paired or double-stranded RNA (dsRNA) that is processed into 21–30-nucleotide small RNAs by the activity of one or more ribonucleaseIII-like enzymes called Dicer or Dicer-like (DCL) [3]. Small RNAs are incorporated into an RNA-induced silencing complex (RISC) that contains a member of the Argonaute (AGO) protein family [4]. The small RNA programs the complex to recognize target RNA (s) through base pair complementarity, while the AGO protein functions as an effector to modulate the abundance or activity of the target [5,6]. The fungal kingdom comprises an enormous, diverse group of organisms. Two major fungal RNA silencing pathways have been described: the quelling and the meiotic silencing by unpaired DNA (MSUD) pathways [7,8]. Both appear to be effective genome defense mechanisms that operate during asexual (quelling) [9,10] and sexual development (MSUD) [11]. Fungal RNA silencing pathways function in genome protection and have been proposed to be involved in pathogenicity [12], development [13] and antiviral defense [14]. Fungal viruses, or mycoviruses, are widespread. Most characterized mycoviruses have dsRNA genomes packaged in spherical particles (Totiviridae, Partititiviridae, Chrysoviridae) or a (+) single stranded (ssRNA) genome without the ability to form particles (Hypoviridae, Endornaviridae) [15]. Both (-) ssRNA [16] and ssDNA mycoviruses [17] have also been described. Mycoviruses do not have an extracellular phase, but rather are transmitted vertically by spores or horizontally by hyphal anastomosis [15]. Although mycoviral infections are generally associated with cryptic (non-symptomatic) and latent (expressed under specific conditions) infections, some mycoviruses cause debilitating phenotypes in their host, making mycoviruses a potential tool for the control of fungal plant pathogens [18–20]. In other cases, mycoviruses have coevolved mutualistically with their hosts [21,22]. Cryphonectria parasitica is a model filamentous fungus for the study of virus-host interactions [23,24], and provided the first example that RNA silencing functions as an antiviral defence mechanism in fungi [14]. C. parasitica has four RNA-dependent RNA Polymerases, two Dicer, and four Argonaute genes, but only dcl-2 and agl-2 have been shown to have roles in antiviral defense [14,25,26]. Genetic studies have revealed that C. parasitica antiviral defense is active against members from the Hypoviridae, Reoviridae [14,25], Partitiviridae [27], Totiviridae [28] and Megabirnaviridae [29] families. Indirect evidence of an RNA silencing-mycovirus interaction has also been described in fungi with killer viruses. In Saccharomyces cerevisiae the M satellites of the dsRNA mycovirus L-A from the Totiviridae family produce a toxin that kills uninfected neighbour cells but renders the host immune to the toxin [30]. Strains with active RNA silencing suppress the virus and lose the advantage provided by the virus [31]. The incompatibility between the killer virus and the RNA silencing machinery might explain the existence of several RNA silencing-deficient fungi [31,32]. The genus Colletotrichum is considered one of the most economically important groups of plant pathogens, causing anthracnose disease in over 3,200 species of monocot and dicot plants [33], with some infections leading to post-harvest losses of up to 100%. Colletotrichum higginsianum infects plants of the Brassicaceae family, including Arabidopsis thaliana, and is emerging as a model for the study of plant-pathogen interactions in dicotyledonous species [34]. C. higginsianum is a hemibiotrophic fungus that forms an intracellular hyphae during the initial symptomless biotrophic stage before entering a destructive necrotrophic colonization phase. C. higginsianum has a relatively small haploid genome that was recently sequenced, the ability to be cultured axenically, and stable transformation methods that allow for the analysis of gene function by targeted disruption [35]. The primary goal of this study was to identify and analyze the role of the RNA silencing machinery in the fungal pathogen C. higginsianum. Knock-out mutants and high-throughput sequencing was used to functionally characterize transcriptomes and small RNA populations in C. higginsianum mycelia. ChAGO1 and ChDCL1 were determined to be critical for maintaining low levels of accumulation of a novel dsRNA virus, designated as ChNRV1. Production of viral small RNAs was ChDCL1-dependent and loading into ChAGO1 demonstrated a strong preference for 21-nt, 5’U sequences.
  Genes encoding three homologs of RNA-dependent RNA Polymerase (RDR1, RDR2 and RDR3), two homologs of Dicer (DCL1 and DCL2) and two homologs of Argonaute (AGO1 and AGO2) were identified in the Colletotrichum higginsianum genome (Fig 1A). All three RDRs contained an RNA-dependent RNA Polymerase domain (RdRP). Both DCLs contain an RNA helicase domain, double-stranded RNA-binding domain (dsRBD) and two RNaseIII catalytic domains. Neither DCL contained a PAZ (Piwi/Argonaute/Zwille) domain, which binds the two-nucleotide, 3’ overhangs on canonical DCL substrates [36,37]. Dicer-like proteins in some eukaryotes lack the PAZ domain [38–40] but retain the capacity to generate small RNAs, suggesting the possibility of other substrate recognition mechanisms (S1A Fig). Both C. higginsianum AGO proteins possess conserved PAZ and PIWI domains, and a conserved MID domain required for 5’ anchoring of the guide RNA. The catalytic triad residues (Aspartic-Aspartic-Aspartic), required for slicer activity [41], are present in the PIWI domain of both proteins (S1B Fig). Additionally, AGO1 contains an amino-terminal RGG box, with 12 copies of the Arginine-Glycine-Glycine motif (Fig 1A). The RGG domain functions in nucleic-acid binding and protein-protein interactions, and is present in some other eukaryotic AGO proteins [39,42,43]. The evolutionary relationships of C. higginsianum RDRs, DCLs and AGOs with those of other fungal species from the Ascomycota clade was assessed by phylogenetic analysis of protein sequences for each family. Members of C. higginsianum RDR, DCL, and AGO families grouped clearly with known members that function in quelling or MSUD pathways (Fig 1B and 1D). RDR3, DCL1, and AGO1 grouped with quelling factors, while RDR1, DCL2, and AGO2 grouped with proteins from the MSUD pathway. RDR2 was identified in a separate group with RRP3 from Neurospora crassa and RDR4 from C. parasitica (Fig 1B), the function of which are not clear [44]. Expression of RRP3 and RDR4 is elevated by the introduction of dsRNA [45] or in response to viral infection [26], respectively. Expression levels of C. higginsianum silencing genes were determined using quantitative real time PCR (qRT-PCR). Based on the phylogenetic analysis, we hypothesized that genes in the quelling and MSUD pathways would be expressed during asexual and sexual stages of fungal development, respectively. Colletotrichum is generally recognized as an asexual genus, although some species are able to adopt a sexual form, which are classified under the genus name Glomerella [33]. Colletotrichum asexual morphs are generally associated with disease symptoms, while sexual stages tend to develop in moribund or dead host tissues [33]. The C. higginsianum strain IMI349063A used in this study has not been observed to reproduce sexually [35], thus, gene expression analysis was limited to mycelia, conidia, and germinated conidia. RDR3, DCL1 and AGO1 had the highest levels of expression in mycelia (Fig 1E, left panel), conidia, and germinated conidia (S2 Fig, left panels), which is consistent with their predicted role in the quelling pathway during vegetative growth and asexual reproduction. Conversely, transcripts for RDR1, DCL2 and AGO2 (MSUD pathway) were expressed at detectable but relatively low levels in mycelia, conidia, and germinated conidia (Figs 1E and S2, middle panels). Thus, while a role for RDR1, DCL2 and AGO2 during sexual reproduction is anticipated, it is possible that these genes have roles in asexual developmental phases. Transcripts for RDR2 were expressed at intermediate levels in mycelia, but relatively high in conidia and germinated conidia (Fig 1E and S2 Fig, right panels). To identify functions of the RNA silencing components in C. higginsianum, single deletion mutants with loss of RDR (S3 Fig), DCL (S4 Fig) and AGO (S5 Fig) genes were generated by targeted gene replacement. A double DCL1/DCL2 deletion mutant was also generated (S4 Fig). Single-site gene disruptions were confirmed by Southern blot and RT-PCR analysis, and four independent deletion mutants were selected for further analysis (S3 and S5 Figs). Previous work in Mucor circinelloides [46], Magnaporthe oryzae [47], Candida albicans [48], Botrytis cinerea [12], and Trichoderma atroviride [13] demonstrated that disruptions of DCL1 orthologs were sufficient to slow vegetative growth. However, in other fungi, such as C. parasitica [14] and Saccharomyces castellii [38], comparable mutations did not affect growth. To evaluate C. higginsianum silencing mutants, mycelial growth and morphology on conidiation (Mathur’s), complete (PDA), and minimal (CDA) media were measured. For all single and double mutants, colony morphology and growth were indistinguishable from that of the wild-type strain (Figs 2A and S6). The Δdcl1, Δdcl2, and Δdcl1Δdcl2 mutants were also exposed to oxidative stress (H2O2 and Methyl Viologen), cell wall stress (Calcofluor white), salt-related stress (NaCl, and LiCl), osmotic stress (sorbitol, and sucrose) and nutrient deprivation (carbon and nitrogen). Growth of the Δdcl1, Δdcl2, and Δdcl1Δdcl2 mutant strains was indistinguishable from that of the wild-type on all media (S7 Fig), suggesting that small RNA-mediated processes are not necessary for vegetative growth under several abiotic stress conditions. These results are in agreement with those reported for silencing mutants of the yeasts S. castellii [38] and C. neoformans [49]. Conidiation was analyzed for all mutant strains after growth in Mathur’s media for seven days. Compared to the control strains, conidiation was significantly reduced in the Δdcl1, Δdcl1Δdcl2, and Δago1 strains by 8. 8-fold, 11. 6-fold, and 4-fold, respectively (Fig 2B). There was no significant difference between the Δdcl1 and Δdcl1Δdcl2 strains, indicating that conidiation effect was due to DCL1 with little or no redundancy with DCL2. DCL genes have been implicated in conidiation in other fungi [13,50]. In B. cinerea, at least partial redundancy of the two DCL proteins was observed [12] where the double mutant dcl1dcl2 had a stronger, negative effect on sporulation than did the single mutants dcl1 and dcl2. Argonautes were also shown to affect spore production in M. circinelloides [51], in which an ago-1 mutant strain produced ~50% fewer spores than the control strain. Conidia produced by the Δdcl1, Δdcl1Δdcl2, and Δago1 mutants were shorter and wider than those produced by the controls strain (Fig 2C). Most conidia from the Δdcl1 and Δdcl1Δdcl2 strains exhibited an abnormal, slightly rounded morphology (Fig 2D), and were more variable in length and width than those control strains. Deletion mutants lacking RDR1, RDR2, or RDR3 genes had no measurable growth or conidiation phenotypes (Fig 2B). However, it is not clear whether RDR1, RDR2 and RDR3 are not functional during growth and conidiation, or are functional but redundant, as combinatorial mutants were not analyzed. The deletion mutant analysis revealed that DCL1 and AGO1 both affect conidia production and asexual spore morphology in C. higginsianum. To identify loci that are affected by RNA silencing factors, in particular DCL1 and AGO1, transcripts and small RNA from all mutant strains (Fig 3) were analyzed. In addition, small RNAs were analyzed after immunoprecipitation (IP) of 6His-3FLAG (6H3F) epitope-tagged versions of AGO1 (6H3F-AGO1) and AGO2 (6H3F-AGO2), which were expressed in Δago1 and Δago2 strains, respectively (S8 Fig). Transformants containing and expressing 6H3F-AGO1 had significantly increased conidia production relative to the original Δago1 background, indicating the tagged version of AGO1 complemented the loss of AGO1 (S8B and S8D Fig). Transcript RNA reads were de-multiplexed and aligned to the C. higginsianum genome using Bowtie2 (S2 Table) [52]. Strikingly, 40±3% of the RNA-seq reads in Δago1,54±5% of the reads in Δdcl1, and 46±6% of the reads in Δdcl1Δdcl2 did not map to the C. higginsianum genome (Fig 3A and S2 Table). Small RNA libraries were also de-multiplexed, trimmed of the 3’adaptor sequence, and aligned to the C. higginsianum genome using the aligner Bowtie (S3 Table) [53]. Here, 63±3% of the small RNA reads in Δago1, and 46±8% of AGO1-bound small RNAs did not map to the C. higginsianum genome (Fig 3B and 3C, S3 Table). High-throughput sequencing usually yields a proportion of reads that do not map to the target genome such as sequencing errors, sequence variants, and chimeric sequences [54]. Draft genome sequences present further difficulties; the available C. higginsianum IMI349063A genome sequence contains approximately 48. 2 Mbp of a predicted 53. 4 Mbp nuclear genome across 10,235 contigs, where 5. 2% of genes are split across multiple contigs, 4% of genes are truncated and the assembly does not include the mitochondrial genome [35]. Further, exogenous species (viral or bacterial) may be present in the target sample. Several infectious agents have been identified from high-throughput sequencing reads that did not map reference sequences [55–57]. Since the increase in unmapped transcript and small reads was specific to the Δdcl1, Δdcl1Δdcl2, Δago1 strains, as well as the small RNA reads from the 6F3H-AGO1 IP, we hypothesized that the C. higginsianum silencing machinery is important for the regulation of one or more loci of an undetermined origin. To determine the relationship between ChNRV1 and other mycoviruses we used a maximum-likelihood phylogenetic analysis on the amino acid sequence of the RdRP of ChNRV1 and other mycoviruses from the Totiviridae, Partitiviridae, and Unclassified dsRNA viruses (S6 Table). We initially hypothesized that ChNRV1 is a Totiviridae based on its non-segmented genome organization and the predicated structural similarity between ORF1 and the capsid protein of ScV-L-A. However ChNRV1 was not placed into either the monophyletic Totiviridae or the Partitiviridae groups (Fig 6A). Instead, ChNRV1 grouped in as a sister clade to the Partitiviridae family RdRPs with a number of recently described members of the family Amalgaviridae that primarily have monopartite genomes (Fig 6A). A set of RdRP sequences from dsRNA viruses currently unclassified is also part of this sister clade, but separate from the Amalgaviridae members. Further supporting a relationship with Partitiviridae RdRPs, an analysis within the eight conserved domains of RdRP proteins of dsRNA viruses determined that all residues specific to the Partitiviridae family RdRPs are present in ChNRV1, while only 4 of 29 Totiviridae-specific residues are observed [78] (Fig 6B). While the evolutionary relationship between ChNRV1 and existing mycovirus families is unclear, it seems likely that ChNRV1 belongs to a distinct group of dsRNA viruses with some similarities to both totiviruses and partitiviruses. The recent discovery of both fungal and plant dsRNA viruses with high levels of shared sequence homology indicates these viruses are either new members of the Amalgamaviridae family or belong to a yet to be determined family [74–76,79–84] (Fig 6A). After determining that DCL1 and AGO1 were involved in virus regulation, we hypothesized that the defects in conidiation observed in these strains might be due to the activity of ChNRV1 rather than endogenous regulatory activity of RNA silencing. Therefore, we used a cyclohexamide treatment to obtain a C. higginsianum wild-type strain IMI 349063A cured of ChNRV1. DCL1 deletion mutants were generated by targeted gene replacement using the C. higginsianum wild-type cured strain as the recipient background. Gene disruptions were confirmed by RT-PCR analysis of DCL1, identifying two independent deletion mutants for conidia quantification (Fig 9B). Virus-free Δdcl1 strains produced an average of 10. 7x105 conidia/ml more than ChNRV1-infected Δdcl1 (95% confidence interval: 9. 6x105–11. 9x105 conidia/ml), demonstrating an 8. 8X increase in conidia production (Fig 9A). Further, conidiation levels in the Δdcl1 strain were restored to nearly 80% of the levels observed in the cured wild-type strain. These results indicate that virus proliferation in the Δdcl1 strain is the major contributing factor to the severe conidiation defect observed in the ChNRV1-infected Δdcl1 strains. Further work to generate and characterize of virus-free strains will be used to elucidate the contribution of RNA silencing during conidia production, particularly as work with T. atroviride strains lacking the ChDCL1 ortholog demonstrated severe deficiencies in conidia production [13]. As well, the virus-free strain can be used as a system to query the effects of viral RNA versus viral proteins in an RNA-silencing deficient strain to better understand the relative contribution of each element to the conidiation phenotype observed. Here we describe the antiviral role of Colletotrichum higginsianum silencing machinery against ChNRV1, a novel dsRNA virus present in the wild-type strain (IMI 349063A). When the fungal silencing machinery is functional, ChNRV1 is maintained at low levels. However, loss of either DCL1 or AGO1 leads to de-repression of ChNRV1, which consequently accumulates at very high levels. Increase in viral accumulation is responsible for the severe conidiation defect observed in Δdcl1, Δdcl1Δdcl2 strains, as virus-free Δdcl1 strains show strong recovery of the phenotype. The question remains why and how is ChNRV1 able to avoid the full impact of the antiviral defense. Possibly ChNRV1 confers some advantage to C. higginsianum in a yet to be studied environment, or it may act as vector a contributing to fungal genomic plasticity, enhancing the emergence of new virulence traits through evolution.
  The Colletotrichum higginsianum (IMI 349063A) wild-type strain used in this study corresponds to the sequenced strain isolated from Brassica rapa (Trinidad Tobago), kindly provided by Dr. Richard O’Connell [35]. The fungal strains deficient in the RNA silencing machinery (Δago1, Δago2, Δdcl1, Δdcl2, Δdcl1Δdcl2 (Δdcl1/2), Δrdr1, Δrdr2, and Δrdr3) were obtained in this study by gene replacement. The fungal strains expressing the 6His-3FLAG-tagged AGO1 and AGO2 proteins were generated in this study by transformation of the previously generated Δago1 and Δago2 strains, respectively. Specific details related to the generation and confirmation of knock-out and tagged strains can be found in S2 Text. Growth tests were performed by culturing the fungus in Potato Dextrose Agar (PDA), Czapek dox agar (CDA), or Mathur’s Agar (Conidiation media) [94] at 25°C in dark conditions. Growth tests under nutrient limitations were performed using CDA depleted in carbon (CDA-C) or nitrogen (CDA-N) at 25°C in dark conditions. For growth tests under osmotic stress conditions, fungal strains were grown for 10 days in Mathur’s agar supplemented with 0. 4 M NaCl, 0. 2 M LiCl, 0. 5 M Sorbitol, or 0. 6 M Sucrose. Oxidative stress responses were analysed in Mathur’s media supplemented with 2 mM H2O2. Fungal conidiation was induced by growing the fungus on Mathur’s agar for 7 days at 25°C and dark conditions. Conidia were collected from fungal mycelium by adding sterile water to the surface of the mycelium. After filtration and microscopic observation, conidia were counted with a Neubauer counting chamber. The search for putative homologs of the RDR, DCL and AGO proteins in Ascomycete fungi analyzed in this work was done with BLASTP, using N. crassa protein domain sequences as input. Identification of homologs was as follows: RDR proteins were required to have an RNA-dependent Polymerase Domain, DCL proteins were required to have two RNAseIII-domains, and AGO proteins were required to have a PAZ and a PIWI domain. C. higginsianum gene names were assigned based to the silencing pathway to which they belong: AGO1, DCL1 and RDR3 for the Quelling pathway; AGO2, DCL2 and RDR1 for the MSUD pathway; and RDR2 for the Unknown pathway. The deduced amino acid sequence for each RNA silencing protein was aligned with MAFFT [95]. Aligned sequences were imported into Molecular Evolutionary Genetics Analysis (MEGA v6. 06) [96], and trimmed to exclude positions with gaps. The best model of protein evolution was determined using Prottest [97], based on the Akaike Information Criterion (AIC). The LG+G+F model was determined by Prottest to be the best-fit for both fungal RNA silencing protein and the viral RDR alignments. A maximum likelihood (ML) phylogenetic tree was constructed with RAXML [98] using the appropriate model of amino acid substitution. The tree was drawn using Dendroscope v3. 2. 10 [99]. For conidia enumeration, a 10 μl drop of conidia suspension was placed in a Neubauer chamber and covered with a cover slip. Three biological replicates from four individual transformants were counted for each genotype. Three different mm2 were analyzed from each biological replicate. For conidia measurements, pictures were taken using a Planapo x20 water immersion objective with a Confocal Laser Scanning microscope Leica TCS SP8, and image analysis was done with open-source ImageJ/Fiji v1. 48 software (http: //fiji. sc/Fiji). At least 200 conidia were measured for each genotype. Viral particles were partially isolated from the Δdcl1, Δdcl1Δdcl2 and Δago1 strains following the methods described by Dunn and colleagues [69] with minor modifications. Fungal mycelia were grown for one month with constant agitation at (150 rpm, 25°C, dark). Approximately 70 mg of mycelia were homogenized with 6 ml of Tris buffer (0. 1 M Tris-HCl, 0. 15 M NaCl, 5 mM EDTA (pH 7. 8). Hyphal debris was removed by centrifugation (10,000 rcf for 20 min at 4°C). The supernatant was collected and filtered through four layers of Miracloth (Calbiochem). To concentrate the virus particles, 0. 15 M NaCl and 6% PEG6000 was added to the solution, incubated 1 h on ice, and precipitated by low-speed centrifugation (3,600 rcf for 20 min at 4°C). The supernatant was removed and the pellet was resuspended in 100 μl 0. 1 M Tris-buffer (pH 7. 8). Samples were analyzed immediately. The coat protein from viral particles was observed by running a 5 μl aliquot of resuspended viral particles on a NUPAGE-Novex 4–12% Bis-Tris protein gel (Invitrogen) and stained with Commassie blue G-250. dsRNA from viral particles was extracted from a 5 μl aliquot by phenol-chloroform-isoamyl alcohol, followed by ethanol precipitation and visualized on a 1% (wt/vol) agarose gel. The gel bands were cut, de-stained and in-gel digested as previously described [100]. Five microliters of the digested peptides were injected to the LC-MS/MS system. The LC-MS/MS was carried out on a LTQ-Orbitrap Velos Pro (ThermoFisher Scientific, Waltham, MA) coupled with a U3000 RSLCnano HPLC (ThermoFisher Scientific, Waltham, MA). The protein digests were first loaded onto a C18 trap column (PepMap100,300 μm ID × 5 mm, 5 μm particle size, 100 Å; ThermoFisher Scientific) at a flow rate of 5 μL/min for 4 min equilibrated with 2% acetonitrile, 0. 1% formic acid. Peptide separation was carried out on a C18 column (Acclaim PepMap RSLC, 15 cm × 75 μm nanoViper, C18,2 μm, 100 Å, ThermoFisher Scientific) at a flow rate of 0. 3 μL/min and the following gradient: 0–3 min, 2% B isocratic; 3–41 min, 2–45% B; 45–47. 8 min, 45–98% B. Mobile phase A, 0. 1% formic acid; mobile phase B, 0. 1% formic acid in 80: 20 acetonitrile: water. The Orbitrap mass analyzer was operated in positive ionization mode using collision induced dissociation (CID) to fragment the HPLC separated peptides. The mass range for the MS survey scan done using the FTMS was 300 to 2000 m/z with resolution set to 60,000 @ 400 m/z and the automatic gain control (AGC) target set to 1,000,000 ions with a maximum fill time of 10 ms and 1 μscan. The 20 most intense signals in the survey scans were selected and fragmented in the ion trap using an isolation window of 1. 5 m/z, an AGC target value of 10,000 ions, a maximum fill time of 100 ms, normalized collision energy of 35 and activation time of 30 ms. Dynamic exclusion was performed with a repeat count of 1, exclusion duration of 30 s, and a minimum MS ion count for triggering MS/MS set to 5,000 counts. All MS/MS samples were analyzed using Mascot (Matrix Science, London, UK; version 2. 5. 0). Mascot was set up to search the Colletotrichum higginsianum database from http: //www. broadinstitute. org/annotation/genome/colletotrichum_group/ToolsIndex. html, (16,263 entries) including the sequence from CP, RdRP and CP-RdRP assuming the digestion enzyme trypsin. Mascot was searched with a fragment ion mass tolerance of 0. 80 Da and a parent ion tolerance of 15 PPM. Deamidated of asparagine and glutamine, oxidation of methionine and carbamidomethyl of cysteine were specified in Mascot as variable modifications. Scaffold (version Scaffold_4. 3. 4, Proteome Software Inc. , Portland, OR) was used to validate MS/MS based peptide and protein identifications. Peptide identifications were accepted if they could be established at greater than 95. 0% probability by the Peptide Prophet algorithm [101] with Scaffold delta-mass correction. Protein identifications were accepted if they could be established at greater than 99. 0% probability and contained at least 2 identified peptides. Protein probabilities were assigned by the Protein Prophet algorithm [102]. Proteins that contained similar peptides and could not be differentiated based on MS/MS analysis alone were grouped to satisfy the principles of parsimony. Proteins sharing significant peptide evidence were grouped into clusters. Cells were packed in specimen carriers in 75 mM PIPES buffer (pH 6. 8) containing 50 mM sucrose, and ultra-rapidly frozen in a Bal-Tec high-pressure freezer (HPM 010, Technotrade International). Frozen samples were substituted in 2% osmium tetroxide plus 0. 1% uranyl acetate in acetone for 5 days at -85°C, slowly thawed to 20°C and embedded in Spurr’s resin. Thin sections were cut using a LeicaUCT ultramicrotome, stained in uranyl and lead salts, and observed using a LEO 912 AB energy filter TEM (Zeiss). The viral particles were stained with 2% (wt/vol) uranyl-acetate solution (pH 7. 4) and observed using a transmission electron microscope. Cycloheximide treatment was used to remove ChNRV1 from the wild-type C. higginsianum strain. In order to increase the chances of obtaining single conidia free of virus, mycelial plugs were inoculated in Mathur’s Agar supplemented with Cycloheximide at 10 μg/ml (Crescent Chemical Company) and allowed to conidiate for 15 days at 25°C in dark conditions. Conidia were collected by adding sterile water, then counted with a Neubauer counting chamber. About 100 conidia were spread onto 1% agar plates containing 0. 5 ug/ml cycloheximide, and allowed to germinate at 25°C. After 3–4 days the margin of the colonies were collected and transferred to fresh Mathur’s agar. Total nucleic acid from C. higginsianum was extracted using previously described methods [103], but with a mixed alkyltri-methylammonium bromide solution (MATAB) [0. 1 M Tris HCl, pH 8. 0,1. 4 M NaCl, 20 mM EDTA, 2% MATAB, 1% PEG 6000,0. 5% sodium sulfite] as the extraction buffer. DNA was purified by chloroform-isoamyl alcohol, followed by RNase treatment, chloroform-isoamyl alcohol, and ethanol precipitation. Ten μg of DNA was digested to completion with the indicated restriction enzymes (S3, S4 and S5 Figs) (New England Biolabs), separated on a 1% (wt/vol) agarose gel, and desired fragments were visualized after hybridized with the corresponding digoxigenin (DIG) -dUTP-labelled probe. Oligonucleotides used for probe preparation are listed in S7 Table. Probes were labeled with digoxigenin (DIG) -dUTP using the Random Primed DNA Labeling Kit (Roche). To determine the terminal sequence of the dsRNA ChNSV1 genome, a RNA ligase mediated RACE (RLM-RACE) protocol was followed [59]. or dsRNA enrichment, total RNA was extracted using Trizol reagent (Life Technologies), followed by precipitation with 2M LiCl to remove single stranded (ss) RNA. Supernatant was collected and dsRNAs were precipitated with 4 M LiCl. dsRNAs were fractionated by 1% (wt/vol) agarose gel electrophoresis, gel-purified (PureLink Quick Gel Extraction Kit—Life Technologies) and denatured at 95°C for 5 min. The miRNA Cloning Linker 1 (IDT) (5' App/CTGTAGGCACCATCAAT/3' ddC/) was ligated to the 3’end of each strand of the denatured RNA using AIR Ligase (Bioscientific) in the presence of RNAseOUT (Invitrogen). The ligated products were used as templates for cDNA synthesis using Superscript III Reverse Transcriptase (Invitrogen) following the manufacturer’s instructions but with a denaturation step of 98°C for 10 min. The resulting cDNAs were amplified with the primers ORF1-reverse/P7-modban and ORF2-forward/P7-Modban to amplify the 3’ end from each strand. A total of 12 PCR reaction products for both strands were cloned into pCR-BluntII (Invitrogen) for sequence analysis. Total RNA from C. higginsianum tissue was extracted using TRIzol reagent (Life Technologies). One microgram was treated with TURBO DNase I DNA-free (Ambion) to remove genomic DNA contamination. cDNA synthesis was performed using the Superscript III system (Life Technologies) following the manufacturer’s instructions. RT-PCR analysis was performed using 10% of the first-strand reaction and 40 cycles of amplification to guarantee the detection of the amplicon. RT-qPCR analysis used 3% of the first strand reaction as previously described [104,105]. Oligonucleotides used for RT-PCR and RT-qPCR analysis are listed in S7 Table. For Northern blot analysis of viral siRNAs, 2. 5 μg of Total RNA was subjected to 17% polyacrylamide (containing 7M Urea) electrophoresis, and transferred to a positively charged, nylon membrane (Roche) using a semi-dry electroblotting apparatus (Biostep). Blots were pre-hybridized in PerfectHyb Hybridization buffer (Sigma), followed by hybridization with a DIG-labeled DNA probe corresponding to the CP (ORF1) sequence. The DIG-labeled DNA probe was created by amplifying the coat protein sequence using oligonucleotides listed in S7 Table, cloning the resulting amplicon into pCR4 (Life Technologies), digestion with EcoRI (NEB) and gel purification (Life Technologies), then labeled following the manufacturer’s instructions for the DIG DNA Labeling Kit (Roche). One gram of mycelia tissue from wild-type, Δago1/6H3F-AGO1 and Δago2/6H3F-AGO2 C. higginsianum strains was used as the starting material for AGO-immunoprecipitation described in Carbonell et al. [104], but with the following modifications. Clarified lysates were incubated with 4 μg/ml of Monoclonal ANTI-FLAG M2 Antibody (Sigma) for 3 h at 4°C, followed by 100 μl of Protein-G agarose (Roche) per milliliter for 30 min at 4°C. RNAs recovered from input (before immunoprecipitation) and IP fraction were used for preparation of small RNA libraries as described below. Small RNA libraries were prepared following the detailed protocol previously described [105,106]. Specific modifications include using 40 μg of total RNA of C. higginsianum 4-days old mycelia and a 2 hr incubation for the 3’ ligation reaction. Libraries were sequenced using the Illumina HiSeq 2000, v3 chemistry at the Genome Technology Access Center (GTAC) (Washington University, Saint Louis, Missouri). Scripts used for initial processing of the small RNA data are available on GitHub (https: //github. com/carringtonlab/srtools). LibParse. pl was used to remove the 3’ adaptor and de-multiplex the sequences in the raw (FASTQ) file. For all lanes of data, the following common settings were used: -t fastq -r 50 -m 18 -e CTGTAG–E (comma-separated list of the 10–12 barcodes used to index the individual samples during library preparation) -l (log_file. txt) -a (failed_parsing_file. txt). Reads less than 18 nt in length and reads containing one or more N nucleotides were discarded. Remaining small RNA reads were aligned to the C. higginsianum genome (version 1 assembly; Colletotrichum Sequencing Project, Broad Institute of Harvard and MIT (http: //www. broadinstitute. org/) ) using the Bowtie algorithm [53] (version 0. 12. 8) allowing only perfect matches and reporting all mappings for reads that map multiple times to the genome. Reads that mapped and reads that did not map to the genome were quantified using the Bowtie output files and get_parsed_mapped_unmapped_stats. pl. RepeatMasker (version open-3. 3. 0 [107]) and IRF (Inverted Repeats Finder, version 3. 05) [108] were used to identify repetitive elements within the C. higginsianum genome. These features were added to the features table in a custom SQLite3 database along with the C. higginsianum version 1 gene annotation available from the Colletotrichum Sequencing Project (Broad Institute of Harvard and MIT, http: //www. broadinstitute. org/). Strand-specific RNAseq libraries were produced using the same RNA samples used for preparation of small RNA libraries. Ten μg of total RNA was treated with TURBO DNase I DNA-free (Ambion). For depletion of ribosomal RNAs, 1. 5μg of DNase-treated RNA was treated with the Yeast RiboZero Magnetic Gold Kit (Epicentre) according to manufacturer’s instructions. cDNA synthesis was performed using 70 ng of RiboZero-treated RNA as previously described [104,109] with the following modifications. RNAs were fragmented at 94°C for 7 min, and 12 cycles were used in the linear PCR reaction. DNA adaptors 1 and 2 were annealed to generate the Y-shape adaptors, and oligonucleotides PE-Primer-F and PE-Primer-R (series N701-N712) were used for linear PCR and multiplexing of transcript libraries (S7 Table). DNA amplicons were analyzed with a Bioanalyzer (DNA HS kit, Agilent) to ensure a library size of ~250 bp, quantified using the Qubit HS Assay Kit (Invitrogen) and sequenced on a HiSeq 2000 sequencer (Illumina) at GTAC (Washington University). FASTQ files of RNA-seq data were de-multiplexed using the script parseFastq. pl and then Bowtie2 (version 2. 1. 0) [52] was used to align the reads to the reference genome. The indexed bam output from bowtie2 and the script get_RNAseq_mapped_unmapped_stats. pl were used to identify the total number of reads that mapped and that did not map for each genotype. The program Trinity (version Trinityrnaseq_r20131110, http: //trinityrnaseq. github. io/) was used for de novo assembly of RNA-seq reads. The FASTQ files for the four Δdcl1 replicates were combined into one FASTQ file and used as the input for Trinity. Separately the unmapped reads from the bowtie2 alignment for Δdcl1 (replicate 1) were converted into FASTQ format (bam_2_fastq. noHit. pl), and aligned to the Trinity contigs using the utility program alignReads. pl provided with the r20131110 version of Trinity. As the C. higginsianum genome does not contain the mitochondrial genome, a custom BLAST database was created from the mitochondrial sequences of C. graminicola (available at http: //www. broadinstitute. org/) and C. lindemuthianum (http: //www. ncbi. nlm. nih. gov/nuccore/KF953885. 1). Trinity contigs were aligned to this mitochondrial database using BLASTN. A non-redundant set of 26 contigs was concatenated together to create the C. higginsianum mitochondrial RNA feature. To identify high-coverage contigs, the samtools idxstats command was used on the output from alignReads. pl: coordSorted. bam. Putative gene identities were determined using BLASTX and the nr database. In total, three additional features were added to the features table of the SQLite3 database and the C. higginsianum genome sequence: (1) viral sequence (2,923 bp), (2) a large subunit rRNA sequence (4,077 bp), and (3) mitochondrial RNA sequence (30,330 bp). A new reference genome sequence was created with the addition of the three contigs to the C. higginsianum genome sequence. RNA-seq reads were aligned to the modified genome using Bowtie2 (version 2. 1. 0) [52]. Mapped and unmapped reads were determined using get_RNAseq_mapped_unmapped_stats. pl. To determine the distribution of the newly mapped reads, the script reads_mapped_2_chromNum. RNAseq. pl was used. The distribution of RNA-seq by strand mapped to the virus sequence was determined using readsPerFeature. strand. byRep. RNAseq. pl. Small RNAs were aligned to the new reference sequence using bowtie, with perfect matches only allowed. An SQLite3 database was created from the parsed and aligned reads using the scripts PopulateDB. pl and Merge. pl (https: //github. com/carringtonlab/srtools). Mapped and unmapped read counts were determined using the script get_parsed_mapped_unmapped_stats. pl. The distribution of newly mapped reads was determined using reads_mapped_2_chromNum. FinalGenome. pl. To determine the distribution of mapped reads by genomic feature category sizeNT_byCatID. FinalGenome. pl was used. This script also created the 5’ nucleotide profile data. Small RNAs mapped by strand to the virus were identified using readsPerFeature. strand. byRep. smRNA. pl. Size distribution of viral small RNAs by strand was determined using the script sizeNT_byCatID. byRep. byStrand. FinalGenome. pl. Small RNA and RNA-seq reads mapped to the virus genome were scaled using the total number of reads in each respective library. Additionally, the scaled abundance of reads mapped to the sense strand and antisense strand were also determined. Data were analyzed in R (version 3. 0. 3) with a one-way ANOVA with Tukey post hoc analysis. Visualization of scaled and ratio data were also performed in R. For plotting small RNAs along the virus genome, first the scaled read count per nucleotide, by strand, was determined using hitsPerNT. smRNA. strand. pl. The per-nucleotide values were then each divided by the average number of scaled RNA-seq reads mapped to that strand of the virus. A density map of reads along the sense and antisense strands of the virus was created in R. The hits per nucleotide per strand from the small RNA data of the AGO1 IP similarly plotted, but was not normalized by RNA-seq data. 3D structure prediction of ORF1 of the viral genome was performed with I-Tasser (version 3) on-line server [65]. The known structure of the Saccharomyces cerevisiae L-A virus major coat protein (PDBID: 1m1c) [64] was used as a template. The resulting protein structures were visualized using Chimera (http: //www. cgl. ucsf. edu/chimera) [110]. Accession numbers for the fungal protein sequences analyzed in this study are listed in S1 Table. Accession numbers for Colletotrichum destructivum/higginsianum species are listed in S4 Table. Accession numbers for viral proteins are listed in S6 Table. The complete nucleotide sequence of the dsRNA virus ChNRV1 was deposited at NCBI in GenBank under the accession number KM923925. De-multiplexed fastq files of the small RNA, IP, and RNA-seq libraries, along with a gff3 file of counts and alignments to the viral genome, are available at the Gene Expression Omnibus (GEO, http: //www. ncbi. nlm. nih. gov/geo/) under the SuperSeries accession GSE62708; subseries GSE62705, GSE62706, and GSE62707.
summary: Colletotrichum sp. comprises a diverse group of fungal pathogens that attack over 3000 plant species worldwide. Understanding the underlying mechanisms that govern fungal development and pathogenicity may enable more effective and sustainable approaches to crop disease management and control. In most organisms, RNA silencing is an important mechanism to control endogenous and exogenous RNA. RNA silencing utilizes small regulatory molecules (small RNAs) produced by proteins called Dicer (DCL), and exercise their function though effector proteins named Argonaute (AGO). Here, we investigated the role of RNA silencing machinery in the fungus Colletotrichum higginsianum, by generating deletions in genes encoding RNA silencing components. Severe defects were observed in both conidiation and conidia morphology in the Δdcl1, Δdcl1Δdcl2, and Δago1 strains. Analysis of transcripts and small RNAs revealed an uncharacterized dsRNA virus persistently infecting C. higginsianum. The virus was shown (1) to be de-repressed in the Δdcl1, Δdcl1Δdcl2 and Δago1 strains, and (2) to cause the conidiation and spore mutant phenotypes. Our results indicate that C. higginsianum employs RNA silencing as an antiviral mechanism to suppress viruses and their debilitating effects.
keywords: sequencing techniques
  rna interference
  viruses
  mutation
  rna viruses
  genome analysis
  epigenetics
  molecular biology techniques
  rna sequencing
  research and analysis methods
  sequence analysis
  genomic libraries
  mutant strains
  rna sequence analysis
  antisense rna
  genetic interference
  sequence alignment
  gene expression
  molecular biology
  biochemistry
  rna
  nucleic acids
  genetics
  biology and life sciences
  dsrna viruses
  genomics
  computational biology
  organisms
title: Small RNA-Based Antiviral Defense in the Phytopathogenic Fungus Colletotrichum higginsianum