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. 2016 Oct 12;18(8):1127–1137. doi: 10.1111/mpp.12466

Deep sequencing of mycovirus‐derived small RNAs from Botrytis species

Livia Donaire 1, María A Ayllón 2,3,✉
PMCID: PMC6638239  PMID: 27578449

Summary

RNA silencing is an ancient regulatory mechanism operating in all eukaryotic cells. In fungi, it was first discovered in Neurospora crassa, although its potential as a defence mechanism against mycoviruses was first reported in Cryphonectria parasitica and, later, in several fungal species. There is little evidence of the antiviral potential of RNA silencing in the phytopathogenic species of the fungal genus Botrytis. Moreover, little is known about the RNA silencing components in these fungi, although the analysis of public genome databases identified two Dicer‐like genes in B. cinerea, as in most of the ascomycetes sequenced to date. In this work, we used deep sequencing to study the virus‐derived small RNA (vsiRNA) populations from different mycoviruses infecting field isolates of Botrytis spp. The mycoviruses under study belong to different genera and species, and have different types of genome [double‐stranded RNA (dsRNA), (+)single‐stranded RNA (ssRNA) and (–)ssRNA]. In general, vsiRNAs derived from mycoviruses are mostly of 21, 20 and 22 nucleotides in length, possess sense or antisense orientation, either in a similar ratio or with a predominance of sense polarity depending on the virus species, have predominantly U at their 5′ end, and are unevenly distributed along the viral genome, showing conspicuous hotspots of vsiRNA accumulation. These characteristics reveal striking similarities with vsiRNAs produced by plant viruses, suggesting similar pathways of viral targeting in plants and fungi. We have shown that the fungal RNA silencing machinery acts against the mycoviruses used in this work in a similar manner independent of their viral or fungal origin.

Keywords: antiviral RNA silencing, Botrytis; mycoviruses; quelling; small RNAs

Introduction

RNA silencing is an evolutionarily conserved regulatory mechanism which, in invertebrates, was first described in Caenorhabditis elegans as a phenomenon called RNA interference (Fire et al., 1998). Related phenomena have been described in plants (referred to as post‐transcriptional gene silencing) and fungi (known as quelling) (Napoli et al., 1990; Romano and Macino, 1992). RNA silencing groups several specialized pathways involved in a number of apparently distant biological processes, e.g. defence against invasive nucleic acids, such as viruses, transposons and transgenes, or regulation of several developmental and physiological processes (Chapman and Carrington, 2007; Ding and Voinnet, 2007). The role of RNA silencing as a defence mechanism against viruses has been broadly studied in plants and invertebrates (Ding and Voinnet, 2007). Replicating RNA viruses initiate an RNA silencing response that involves the formation of viral double‐stranded RNAs (dsRNAs), followed by Dicer‐mediated production of 21‐ to 24‐nucleotide virus‐derived small RNAs (vsiRNAs). In some organisms, host RNA‐dependent RNA polymerases (RDRs) amplify the antiviral RNA silencing response by producing new dsRNAs from viral single‐stranded RNA (ssRNA) templates, which, in turn, promote the biosynthesis of secondary vsiRNAs (Baulcombe, 2007; Qu, 2010). vsiRNAs are known to bind to specialized ARGONAUTE (AGO) proteins likely to promote the silencing of their complementary viral RNA genomes (Ding and Voinnet, 2007; Llave, 2010; Ruiz‐Ferrer and Voinnet, 2009). Some viruses encode suppressor proteins (VSRs) that act at any step of the silencing pathway to attenuate or repress the antiviral effect of RNA silencing (Csorba et al., 2015).

Many fungal species are susceptible to infection by mycoviruses (Donaire et al., 2016; Howitt et al., 2001; Rodríguez‐García et al., 2014; Wu et al., 2010), and some may have a significant effect on the virulence of the fungus on its plant host (Ghabrial et al., 2015). In fungi, the antiviral role of RNA silencing was first described in the chestnut blight Cryphonectria parasitica infected by the mycovirus Cryphonectria hypovirus 1 (CHV1) (family Hypoviridae) (Nuss, 2011). The papain‐like protease p29 of CHV1, which shares sequence and functional similarities with the potyviral VSR HCPro, suppresses virus‐induced RNA silencing in the fungal host (Segers et al., 2006). Furthermore, loss of function of dcl‐2 (dcl‐2 single mutant or dcl‐1 dcl‐2 double mutant) in C. parasitica increases susceptibility to CHV1 compared with the wild‐type fungus or a dcl‐1 single mutant (Segers et al., 2007). It was also shown that the antiviral defence response in C. parasitica requires the Argonaute‐like protein agl‐2, and that the suppression activity of p29 is a result of the transcriptional repression of this effector protein (Sun et al., 2009). Interestingly, none of the four rdr genes in C. parasitica is active in silencing the CHV1 RNA genome (Zhang et al., 2014). More recently, a dcl‐2 mutant of C. parasitica has been described with enhanced susceptibility to Rosellinia necatrix partitivirus 2 (RnPV2) in single or mixed infections with a defective‐interfering dsRNA (DI‐dsRNA) (Chiba et al., 2013). Moreover, the disruption of dcl‐2 abolishes the interference of the replication and lateral transmission of Rosellinia necatrix victovirus 1 (RnVV1) in C. parasitica (Chiba and Suzuki, 2015). It was also shown that a dsRNA mycovirus suppresses RNA silencing in Aspergillus nidulans by altering the levels of endogenous small RNAs (sRNAs) (Hammond et al., 2008).

Although vsiRNA populations have been extensively profiled in multiple plant and animal host species (reviewed by Ding and Lu, 2012; Llave, 2010), little is known about the structure and composition of the vsiRNA populations in fungal cells infected by mycoviruses. During the infection of Magnaporthe oryzae with Magnaporthe oryzae virus 2 (MoV2), vsiRNAs of 21 nucleotides from both positive and negative strands are produced in infected mycelia (Himeno et al., 2010). DCL2‐dependent vsiRNAs of both polarities are also produced during CHV1 infection in C. parasitica (Zhang et al., 2008). The characterization of vsiRNA populations from two hypoviruses infecting Fusarium graminearum, and from different encapsidated dsRNA mycoviruses in Rosellinia necatrix, using next‐generation sequencing (NGS), has been published recently (Wang et al., 2016; Yaegashi et al., 2016). Furthermore, NGS of vsiRNAs has been used to assemble whole fungal virus genomes, revealing a characteristic overlapping configuration of mycovirus‐derived sRNAs, similar to that observed in animal and plant viruses (Al Rwahnih et al., 2011; Bi et al., 2012; Donaire et al., 2009; Kreuze et al., 2009; Lin et al., 2012; Vainio et al., 2015; Wu et al., 2010).

Botrytis cinerea is an important plant‐pathogenic fungus that causes severe economic losses in over 200 crop hosts worldwide (Williamson et al., 2007). Botrytis paeoniae, together with B. cinerea, causes grey mould disease in peonies (Paeonia lactiflora Pall.) (Muñoz et al., 2016). In B. cinerea, RNA silencing controls the expression of host endogenous genes (Patel et al., 2008, 2010), and at least two Dicer genes have been identified by homology‐based search in its genome, as in most of the ascomycetes sequenced to date (Weiberg et al., 2013). Transfection of the wild‐type or a dcl‐2 mutant of B. cinerea with Botrytis virus F (BVF) had no effects on the fungal growth rate or virulence in the plant, which could mean that dcl‐2 is not essential for antiviral silencing, at least for BVF (Tauati et al., 2014). In this article, we use NGS to profile and characterize the vsiRNA populations in field isolates of B. cinerea and B. paeoniae infected with 15 viral genomes corresponding to 10 different mycoviral species. Mycovirus‐derived sRNA populations have similar features in terms of size, polarity and distribution to virus‐derived sRNAs in plant and animal hosts. This work represents the first exhaustive study of the vsiRNA composition of different mycoviruses infecting Botrytis spp.

Results

Deep sequencing of mycovirus‐derived sRNAs

Total RNA from the Pi258.8 and V448 field isolates of B. cinerea and the V446 isolate of B. paeoniae were used for deep sequencing of sRNAs. Previous reports by our group have documented the presence of replicating mycoviruses in these fungal isolates (Donaire et al., 2016; Rodríguez‐García et al., 2014; M. A. Ayllón, unpublished data). The Pi258.8 isolate was infected with a mixture of four (+)ssRNA mycoviruses, all belonging to family Narnaviridae: Botrytis cinerea mitovirus 1 (BcMV‐1), BcMV‐2, BcMV‐3 and Grapevine‐associated narnavirus 1 (GaNV‐1) (Al Rwahnih et al., 2011). The isolate V446 was infected with a mixture of a dsRNA mycovirus, Sclerotinia sclerotiorum nonsegmented virus L (SsNsV‐L) (Liu et al., 2012), and a (+)ssRNA mycovirus, Botrytis ourmia‐like virus (BOLV) (Donaire et al., 2016). Finally, the V448 isolate was infected with a mixture of BcMV‐1, BcMV‐2, BcMV‐3, GaNV‐1 detected in Pi258.8 and SsNsV‐L detected in V448, BcMV‐4, Sclerotinia sclerotiorum mitovirus 3 (SsMV‐3) (Khalifa and Pearson, 2013), BVF, a well‐characterized Mycoflexivirus (family Gammaflexiviridae) (Howitt et al., 2001), and a (–)ssRNA mycovirus, Botrytis cinerea negative‐stranded RNA virus 1 (BcNSRV‐1).

The three sRNA libraries were sequenced using the IIlumina platform in a multiplex format to yield 24.5 million insert reads for Pi258.8, 21.4 million insert reads for V446 and 34.4 million insert reads for V448 (Table 1). After filtering by length (18–30 nucleotides) and removing reads that matched with the Rfam database (rRNA and tRNA sequences), the remaining sRNA reads were mapped against the corresponding viral genomes in each sample (Table 1) and only perfect matches were allowed; 9.8% of total sRNA reads matched to viral genomes in V448, whereas 6.3% and 3.9% matches were found in Pi258.8 and V446, respectively (Table 1). The calculation of the number of reads per million (RPM) allowed the comparison of total vsiRNAs among the three Botrytis isolates, showing that the normalized number of vsiRNAs was almost equal in Pi258.8 and V448, but only one‐half the amount in V446 (Table 1).

Table 1.

Summary of small RNA sequencing (sRNAseq) results.

Pi258.8 V446 V448
# of reads # of reads # of reads
Insert reads 24 539 828 21 383 147 34 370 233
sRNA sequences (18–30 nucleotides) 11 503 418 8 619 036 10 952 493
Match with Rfam 1 936 254 1 110 848 2 016 980
Match with viral genomes 601 010 292 498 875 155
RPM of vsiRNAs 24 491.21 13 678.90 25 462.59
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RPM, reads per million; vsiRNA, virus‐derived small RNA.

The number of total vsiRNAs varied not only among fungal isolates, but also among viruses infecting each isolate (Table 2). The highest number of total vsiRNAs was derived from BcMV‐1 (214 010 reads) in the Pi258.8 isolate, whereas the lowest number of vsiRNAs reads was reported in BcNSRV‐1 in the V448 isolate (23 780 reads) (Table 2). Interestingly, we found that the number of vsiRNAs of each virus was significantly lower in the fungal isolate V448, which supports the largest number of mixed‐infecting viruses (Table 2).

Table 2.

Sequenced mycovirus‐derived small RNAs.

Fungal isolate Virus acronym Family* Genus* Genome type Genome length (kb) Total vsiRNAs Unique vsiRNAs
Pi258.8 BcMV‐1 Narnaviridae Mitovirus (+)ssRNA 2.9 214 010 18 094
BcMV‐2 Narnaviridae Mitovirus (+)ssRNA 2.5 82 390 12 149
BcMV‐3 Narnaviridae Mitovirus (+)ssRNA 2.9 163 801 16 420
GaNV‐1 Narnaviridae Narnavirus (+)ssRNA 2.7 140 809 13 897
V446 BOLV Unclassified Unclassified (+)ssRNA 2.9 76 693 7082
SsNsV‐L Unclassified Unclassified dsRNA 9 131 524 17 443
V448 BcMV‐1 Narnaviridae Mitovirus (+)ssRNA 2.8 160 065 15 773
BcMV‐2 Narnaviridae Mitovirus (+)ssRNA 2.5 72 011 12 485
BcMV‐3 Narnaviridae Mitovirus (+)ssRNA 2.9 143 646 14 449
BcMV‐4 Narnaviridae Mitovirus (+)ssRNA 2.8 173 765 16 931
GaNV‐1 Narnaviridae Narnavirus (+)ssRNA 2.7 79 111 11 182
SsMV‐3 Narnaviridae Mitovirus (+)ssRNA 2.6 48 549 9510
BVF Gammaflexiviridae Mycoflexivirus (+)ssRNA 6.8 38 860 10 150
SsNsV‐L Unclassified Unclassified dsRNA 9 111 936 23 736
BcNSRV‐1 Unclassified Unclassified (–)ssRNA 8.5 23 780 5723
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dsRNA, double‐stranded RNA; ssRNA, single‐stranded RNA; vsiRNA, virus‐derived small RNA. Virus acronyms are defined in the text.

*Proposed classification by King et al. (2012).

The number of unique vsiRNAs was then calculated to show the sequence diversity produced during mycoviral infections. The highest number of unique sequences was found for SsNsV‐L (17 443 in V446 and 23 736 in V448), as could be anticipated because of the longer size of its viral genome (9 kb) (Table 2). Interestingly, BcNSRV‐1 (8.5 kb) produced not only a lower number of total reads compared with other mycoviruses, but also a lower number of unique vsiRNA sequences (5723) (Table 2). Although the total number of vsiRNAs differed significantly between isolates of the same species, differences in the number of unique sequences were, in general, subtle. For instance, the number of total GaNV‐1‐derived reads was two‐fold higher in Pi258.8 than in V448, whereas the number of unique sequences was quite similar in both isolates (13 897 and 11 182, respectively) (Table 2).

Size distribution of mycovirus‐derived sRNAs

We profiled the size distribution of sequenced mycovirus‐derived sRNAs. The most abundant size class was 21 nucleotides when total vsiRNAs were counted, ranging from 53.7% of BcNSRV‐1‐derived vsiRNAs to 19.9% of BcMV‐3‐derived vsiRNAs in V448 (Fig. 1, top). Exceptionally, SsNsV‐L‐derived sRNAs of 22 nucleotides in V446 were slightly more abundant than 21‐nucleotide vsiRNAs: 40% and 37.3%, respectively (Fig. 1, top). vsiRNAs of 20 nucleotides were more abundant than vsiRNAs of 22 nucleotides for most of the mycoviruses tested, ranging from 21.2% in BVF‐derived vsiRNAs to 13.7% and 13.9% in both BcMV‐3 isolates (Fig. 1, top). The percentage of total vsiRNAs of 22 nucleotides ranged from 11.90% and 12.38% in both BcMV‐1 isolates to 23.17% in BOLV (Fig. 1, top). Likewise, the size distribution of unique vsiRNA sequences showed that the greatest sequence diversity was found for vsiRNAs of 21 nucleotides in length (values ranging from 38.5% of BVF vsiRNAs to 12.8% in both BcMV‐3 isolates), compared with vsiRNAs of 20 nucleotides (ranging from 20.2% of BVF vsiRNAs to 11.3% of vsiRNAs derived from BcMV‐3 in Pi258.8), and followed by vsiRNAs of 22 nucleotides (from approximately 18% in BVF‐derived vsiRNAs to around 10% in all mitochondria‐infecting viruses) (Fig. 1, bottom). Therefore, it is likely that the over‐representation of one or a few unique vsiRNAs of 22 nucleotides in the sequenced pool accounted for the predominance of the SsNsV‐L‐derived 22‐nucleotide class over the 21‐nucleotide class in the isolate V446. In general, the size distribution of vsiRNAs was very similar for any given virus irrespective of the fungal isolate (Fig. 1). Our data clearly demonstrated that 21‐nucleotide vsiRNAs were the most abundant and diverse size class of vsiRNAs in mycoviruses infecting B. cinerea and B. paeoniae. This finding is in agreement with previous reports in fungi and recalls the size distribution patterns produced by plant viruses on infection (Donaire et al., 2009; Hammond et al., 2008; Himeno et al., 2010; Wang et al., 2016).

Figure 1.

Figure 1

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Size distribution of mycovirus‐derived small RNAs (vsiRNAs). Histograms show the percentage of each size length of total reads (top) and unique sequences (bottom) from mycovirus‐derived sRNAs sequenced in the three fungal isolates Pi258.8, V446 and V448. nts, nucleotides.

Polarity distribution of mycovirus‐derived sRNAs

We next studied the sense and antisense strand distribution using our pools of sequenced vsiRNAs. Data using total or unique sequences are shown in Fig. 2. With the exception of total vsiRNA reads from SsNsV‐L, all virus species tested that were found in two different fungal isolates shared the same pattern of vsiRNA orientation (Fig. 2). vsiRNAs from BcMV‐2, BcMV‐4, BVF, SsNsV‐L, BOLV and BcNSRV‐1 were almost equally derived from positive and negative viral strands (Fig. 2). In contrast, vsiRNAs from BcMV‐1, BcMV‐3, GaNV‐1 and SsMV‐3 exhibited an asymmetrical distribution in strand polarity, with a dominance of vsiRNAs of sense orientation (nearly 70%–80%) (Fig. 2). Likewise, SsNsV‐L‐derived vsiRNAs in V446 were preferentially of sense polarity when total reads were counted (Fig. 2). However, unique sequences revealed an equimolecular distribution of sense and antisense strand polarity, suggesting that the predominance of sense vsiRNAs could be caused by one or few unique vsiRNA sequences of sense polarity that were sequenced many times. Indeed, as discussed later, there were two predominant vsiRNA species of 21 and 22 nucleotides, respectively, which explains the bias towards the sense polarity of the pool of vsiRNAs found in SsNsV‐L infecting V446.

Figure 2.

Figure 2

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Orientation of mycovirus‐derived small RNAs (vsiRNAs). Histograms show the percentage of sense and antisense total reads (top) and unique sequences (bottom) from mycovirus‐derived sRNAs sequenced in the three fungal isolates Pi258.8, V446 and V448.

5′ end nucleotide composition of mycovirus‐derived sRNAs

We next analysed whether there was any preference in the composition of the 5′ nucleotides of mycovirus‐derived sRNAs. There was a clear preference for U residues at the 5′ end of total vsiRNAs (all sizes), which varied from 93% in BcNSRV‐1 vsiRNAs to 49% in BcMV‐3 vsiRNAs in V448 (data not shown). The individual analysis of 20‐, 21‐ and 22‐nucleotide vsiRNAs gave similar results (Fig. 3 and data not shown). However, vsiRNAs of 24 nucleotides contained a predominant A residue at the 5′ end for some viruses (BcMV‐1, BcMV‐2 in Pi258.8, BcMV‐3, GaNV‐1 and SsMV‐L) (Fig. 3). These results were in accordance with the highest abundance of A and U residues in the viral genomes for most mycoviruses tested (Fig. 3). However, the observed frequency of U residues at the 5′ end of vsiRNAs of 21, 20 and 22 nucleotides was much higher than the expected frequency considering the composition of the viral genomes (χ 2 3 > 3315.97, P = 0).

Figure 3.

Figure 3

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Nucleotide composition of the 5′ end of mycovirus‐derived small RNAs (vsiRNAs) and full length of the viral genomes. Histograms show the percentage of appearance of each nucleotide at the 5′ end position of mycovirus‐derived sRNAs of 21, 22 and 24 nucleotides (nts), and the percentage of each nucleotide in the total length of the viral genomes.

Viral genome distribution of mycovirus‐derived sRNAs

To study the viral origin of mycovirus‐derived sRNAs, total reads of 18–30 nucleotides from both the sense and antisense polarities were plotted along the corresponding viral genomes (Figs 4 and S1, see Supporting Information). vsiRNAs were distributed along the entire viral genomes, but with a heterogeneous distribution (Figs 4 and S1). The existence of peaks of vsiRNA abundance of both polarities revealed the occurrence of discrete genomic regions that were particularly productive in the formation of vsiRNAs (Figs 4 and S1). Interestingly, these vsiRNA formation hotspots were located on both strands of the viral RNAs (Figs 4 and S1).

Figure 4.

Figure 4

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Distribution of total mycovirus‐derived small RNAs (vsiRNAs) along the viral genomes. Histograms show the number of sense (blue) and antisense (red) vsiRNAs of 18–30 nucleotides (nts) along the corresponding viral genomes.

To test whether the vsiRNA hotspots proceed from highly structured genomic regions with the potential to serve as dsRNA templates for Dicer activities, secondary structures of regions surrounding vsiRNA hotspots at the indicated genomic coordinates were analysed for the following viruses. In the Pi258.8 isolate: BcMV‐1 (position 892, 2894 reads) and BcMV‐3 (position 2900, 3629 reads). In the V446 isolate: SsNsV‐L (position 465, 72 904 reads). In the V448 isolate: BcMV‐3 (position 2774, 6143 reads, and position 2890, 2641 reads) and BcMV‐4 (position 417, 2671 reads). Fold‐back structures were calculated in windows of 70–120 nucleotides for peaks that contained more than 2500 reads using Mfold (Zuker, 2003). We only found a vsiRNA hotspot at the coordinates 460–528 in the SsNsV‐L genome infecting the V446 isolate, which located within a near‐perfect, self‐complementary region similar to miRNA precursors in plants and animals (Fig. 5) (Jones‐Rhoades and Bartel, 2004). Interestingly, this hotspot was mostly composed of two highly abundant vsiRNA sequences of 21 and 22 nucleotides that were sequenced 29 126 and 40 994 times, respectively. Their 5′ end mapped at the same nucleotide position (nucleotide 465) in the near‐perfect complementary arm of the predicted fold‐back structure. Furthermore, the opposite sequence generated from the other arm of the hairpin structure, which would correspond to the passenger strand, was sequenced to a much lesser extent, 11 times, as described for the miRNA:miRNA* duplex (Fig. 5). Neither the hairpin structure nor the vsiRNA sequence were found in SsNsV‐L infecting V448, which only shares 76.3% identity with SsNsV‐L infecting V446. This result suggests that mycoviruses could produce miRNA‐like sequences, as described for animal viruses (Pfeffer, 2004), although their existence has not been proven for any plant or fungal RNA virus.

Figure 5.

Figure 5

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Hairpin structure at position 465 of V446 Sclerotinia sclerotiorum nonsegmented virus L (SsNsV‐L). Schematic representation of the region surrounding the hotspot at position 465 of the viral genome using Mfold. The location of the high‐abundant small RNA (sRNA) sequences of 21 and 22 nucleotides is highlighted in dark grey and the location of the other sequence in the arm is highlighted in light grey. The free energy of the folding structure is given in kcal/mol.

Discussion

In this work, we profiled, for the first time, the vsiRNA populations produced in the plant‐pathogenic fungi B. cinerea and B. paeoniae on infection with a large variety of mycoviruses. With the exception of two recent publications (Wang et al., 2016; Yaegashi et al., 2016), all previous published studies applied NGS technology only for mycovirus screening and detection (Nerva et al., 2016; Vainio et al., 2015). Although NGS of mycoviral sRNAs has also been used to discover novel endornaviruses (Sela et al., 2012), in this study, we failed to obtain the complete sequence of the endornavirus infecting the V446 isolate, by either sRNAseq or RNAseq technologies, perhaps because of the low accumulation of this mycovirus in the RNA sample used for sequencing.

In general, all mycoviruses tested in this study exhibited similar patterns of vsiRNA length and orientation, suggesting that they were all targeted by identical RNA silencing pathways. The mycovirus‐derived sRNAs obtained shared striking similarities with the vsiRNAs produced in other fungi, but also in virus‐infected plants and animals (Donaire et al., 2009; Parameswaran et al., 2010; Wang et al., 2016; Yaegashi et al., 2016): they were mostly of 21, 20 and 22 nucleotides in length, derived from both polarities in a similar ratio or predominantly from sense polarity, had predominantly U at their 5′ end, and were distributed along the complete viral genomes with discrete hotspots of vsiRNA accumulation. These findings reveal strong commonalities in the manner in which viral genomes are targeted by the RNA silencing machinery in different kingdoms.

The number of total vsiRNA reads was similar among fungal isolates infected with different mycoviruses. Despite the high similarity between vsiRNA populations found in our datasets, there were some discrepancies in terms of total amount of vsiRNAs among fungal samples and between viruses of the same species in different fungal hosts. Differences in the total amount of vsiRNAs were higher in V446 compared with the two other fungal isolates. This observation could be a result of the intrinsic characteristics of the fungal isolate, as a phylogenetic analysis showed that Pi258.8 and V448 were close to the reference genome of B. cinerea and V446 was probably an isolate of B. paeoniae (Dr Ernesto P. Benito, CIALE, Salamanca, Spain, personal communication).

We found that any given virus produced, in general, different amounts of vsiRNAs in different hosts. Particularly, we documented a dramatic reduction in the content of species‐specific vsiRNAs in fungal isolates that supported mixed infections by multiple viruses. We reasoned that multiple infections might compromise the host RNA silencing machinery by saturating core components in the sRNA biogenesis pathway. In addition, differences in the viral replication strategies of each virus and/or the activity of potential virus‐encoded VSRs may also explain different levels of vsiRNAs in the infected fungal samples. Interestingly, mycoviruses with the simplest genomes (2.5–2.9 kb), including members of the Narnaviridae family and their relative BOLV, produced, in our study, comparatively greater amounts of vsiRNAs than viruses with longer genomes, such as BVF, SsNsV‐L or BcNSRV‐1 (6.8, 9 and 8.5 kb, respectively). The fact that narnaviruses of the genus Narnavirus replicate in the cytoplasm may favour the accessibility of Dicer proteins to the viral dsRNA templates and explain, at least in part, the greater amount of vsiRNAs produced in GaNV‐1. Interestingly, a large number of vsiRNAs were also sequenced in fungal isolates infected with narnaviruses of the genus Mitovirus which replicate inside the mitochondria. This finding suggests that RNA silencing in fungi may be fully operative inside the mitochondria, as described for human mitochondria (Bandiera et al., 2011). Alternatively, we cannot rule out the possibility that mitovirus dsRNA intermediates exit outside the mitochondria and could be targeted by Dicer proteins.

In our study, the only virus that encodes a coat protein (CP) and can form viral particles is BVF. BVF consistently produced small amounts of vsiRNAs in infected tissues, which may reflect ineffective targeting against the BVF genome. This finding agrees with the idea that the recruitment of viral genomes inside viral particles could preserve genomes from being targeted by host RNA silencing proteins, thus limiting the production of vsiRNAs (Hammond et al., 2008; Himeno et al., 2010). However, a recent study has suggested that, in encapsidated dsRNA mycoviruses, vsiRNA production may be affected by their replication and/or counter‐defence strategies and not by the recruitment of their genomes in a viral particle or viral RNA accumulation (Yaegashi et al., 2016). Alternatively, the levels of vsiRNAs in BVF‐infected fungi may be largely influenced by the ability of BVF to alter the normal expression of components of the RNA silencing pathway in B. cinerea (Tauati et al., 2014). The small amount of vsiRNAs derived from the negative‐stranded BcNRSV‐1 in our sequenced pool may be related to its replication strategy, which yields low viral accumulation levels in the infected tissue, and/or the existence of a VSR in the BcNRSV‐1 viral genome (M. A. Ayllón, unpublished results). For instance, Tomato spotted wilt tospovirus (TSWV) possesses a negative‐sense RNA genome and produces vsiRNAs at low relative frequencies in infected plants (less than 10% of total sRNA reads). However, the number of TSWV‐derived vsiRNAs increased three‐fold when the expression of VSR was abolished (Margaria et al., 2015).

Although the hierarchical activities of Dicer genes have been well characterized in plants and animals, the knowledge of the contribution is limited of the two Dicer proteins in the biogenesis of each size class of mycovirus‐derived sRNAs is limited. For example, CHV‐1‐derived vsiRNAs of 21 and 22 nucleotides in length are dependent on the activity of dcl‐2, but not dcl‐1, suggesting that these two proteins act in a non‐redundant manner (Nuss, 2011; Zhang et al., 2008). Furthermore, dcl‐2 has increased antiviral activity in comparison with dcl‐1 in several fungi (Chiba et al., 2013; Segers et al., 2007). In our sRNA sequencing datasets, reads that mapped with viral genomes were mostly of 18–24 nucleotides in length, with a predominance of vsiRNAs of 21, 20 and 22 nucleotides. Previous studies have demonstrated that dcl‐2 is dispensable for effective antiviral silencing against BVF in B. cinerea (Tauati et al., 2014), although the contribution of each Dicer protein of B. cinerea to each sRNA size class remains unknown. As we have shown in this work that all mycoviruses tested seem to be targeted by the same RNA silencing pathways, we can suppose that, unlike in other fungal infections, dcl‐1 is the major contributor to all size classes of vsiRNAs in Botrytis spp. However, further investigation using silencing mutants of two Dicer proteins in Botrytis spp. infected with different mycoviruses is needed to clarify the role of dcl‐1 and dcl‐2 in vsiRNA biogenesis.

We have shown that mycoviral vsiRNAs originate from both sense and antisense RNA strands, irrespective of the nature of the RNA genome. For viruses with a clear predominance of vsiRNA species of sense polarity (BcMV‐1, BcMV‐3, GaNV‐1 and SsMV‐3), it is possible that RNA genomic regions with local base pairing could provide major hotspots of vsiRNA production (Molnár et al., 2005). However, with the exception of SsNsV‐L in V446, the bias towards sense species could not be explained on the basis of local or extended secondary structures in the viral genome. For viruses producing comparable amounts of sense and antisense vsiRNAs (BcMV‐2, BcMV‐4, BVF, SsNsV‐L in V448, BOLV and BcNRSV‐1), it is tempting to propose that they are processed from long dsRNAs generated by viral RNA polymerases as an intermediate during viral replication and transcription, or by the activity of host RDR proteins (Donaire et al., 2009). However, the contribution of each mechanism of dsRNA formation to the final pool of vsiRNAs has not been experimentally validated for any plant, animal or fungal virus analysed. As mycoviruses with similar genome organization display different polarity distribution of their vsiRNAs, it is reasonable to speculate that other specific viral or host factors are involved in the biosynthesis of viral dsRNAs.

Our dataset reveals that vsiRNAs of 20–22 nucleotides display a clear tendency to begin with U or A, as reported for other mycoviruses and for plant viruses (Donaire et al., 2009; Himeno et al., 2010; Margaria et al., 2015; Wang et al., 2016). It is not clear, however, whether vsiRNAs starting with a U or an A have preferential affinities for AGO proteins in Botrytis. In addition, whether sorting of vsiRNAs into AGO proteins in this species is determined by the identity of the 5′ end nucleotide, the sRNA size or the thermodynamic stability of the sRNA duplex remains unknown. In the ascomycete Neurospora crassa, Argonaute‐like proteins interact with a class of sRNAs called qiRNAs, which generally have a 5′ end U (Lee et al., 2009). In the zygomycete Mucor circinelloides, Ago‐1 binds endogenous siRNAs with a strong preference for a U at the 5′ end that map to exon regions and regulate the expression of the protein coding genes from which they derive (Cervantes et al., 2013). Although these findings suggest a contribution of the 5′ nucleotide identity in determining vsiRNA loading into AGO proteins, further research is needed to elucidate the nature of the silencing effector complex in Botrytis species.

Despite the striking similarities in the spatial distribution profile of unique vsiRNAs produced by the same virus in different fungal isolates, quantitative differences could be observed. This is probably a consequence of the existence of conspicuous peaks of vsiRNA accumulation or hotspots distributed along the viral genomes. A vsiRNA hotspot in SsNsV‐L infecting V446 could be explained by the presence of a highly structured secondary structure at that position in the SsNsV‐L genome (Fig. 5). As a result of the sequence diversity between isolates, neither the hairpin structure nor the hotspot were found in the V448 isolate. The biological relevance of this highly abundant vsiRNA produced by SsNsV‐L in V446 is unknown, although it is tempting to assign it a regulatory role. Moreover, future work is needed to demonstrate the possible role of this vsiRNA and its biological relevance in the final outcome of mycovirus infection.

Experimental Procedures

Fungal isolates and culture conditions

The isolates Pi258.8 and V448 of B. cinerea were obtained from pepper and grapevine, respectively, and the isolate V446 of B. paeoniae was obtained from grapevine. All three have been described previously (Rodríguez‐García et al., 2014). Stock cultures were stored in 20% glycerol at −80 ºC and on potato dextrose agar (PDA) plates at 4 ºC.

RNA extraction and high‐throughput sequencing

Fresh mycelia were dried by pressing with sterile filter paper, and total RNA was purified from 1 g of dry mycelia using TRIZOL reagent (Invitrogen, Carlsbad, CA, USA), as described previously (Rodríguez‐García et al., 2014). For virus detection by RNAseq, 5–10 μg of total RNA were used for library preparation, as described previously (Al Rwahnih et al., 2011), and subjected to deep sequencing using the Illumina platform (HiSeq2000, 2 × 100 bp length, IGA Technology Services, Udine, Italy; http://www.igatechnology.com) (Donaire et al., 2016). For mycovirus‐derived sRNA sequencing, 5–10 µg of total RNA were used for library preparation, applying the Small TruSeq protocol (Illumina), and subjected to high‐throughput sequencing with the Illumina platform in a multiplex format (HiSeq2000, 1 × 50 bp length, Fasteris S.A., Plan‐les‐Ouates, Switzerland http://www.fasteris.com).

NGS data mining

To search for viral sequences, reads obtained in the RNAseq experiment were de novo assembled using CLC Genomic Workbench software with a minimum contig length of 61 nucleotides and other parameters as default. Large contigs with no match with Botrytis databases were subjected to BLASTN and BLASTX alignments against the National Center for Biotechnology Information (NCBI) database. For mycovirus‐derived sRNA analysis, sequence adapters were trimmed from sRNAseq raw reads, and clean reads between 18 and 30 nucleotides in length were filtered using custom Perl scripts. Reads were subsequently mapped against the Rfam database to remove rRNA and tRNA sequences, and against known infecting mycoviral genomes, previously detected by RNAseq, using Bowtie, allowing zero mismatches with the reference genomes (Langmead et al., 2009). Table files containing counts of sRNA reads at each position of the reference sequence from forward and reverse strands were performed using MISIS (Mapped Short Interfering RNA Spot Identification Software) (Seguin et al., 2014). Compositions at the 5′ nucleotide of vsiRNAs and unique sequences were obtained using custom Perl scripts. The accession numbers of the mycoviruses described are: Pi258.8‐BcMV‐1 (LN827940), Pi258.8‐BcMV‐2 (LN827941), Pi258.8‐BcMV‐3 (LN827942), Pi258.8‐GaNV‐1 (LN827943), V448‐BcMV‐1 (LN827944), V448‐BcMV‐2 (LN827945), V448‐BcMV‐3 (LN827946), V448‐BcMV‐4 (LN827947), V448‐GaNV‐1 (LN827948), SsMV‐3 (LN827949), V446‐SsNsV‐L (LN827951), V448‐SsNsV‐L (LN827952), BVF (LN827953), BOLV (LN827955) and BcNSRV‐1 (LN827956). The partial sequence of the endornavirus found in V446 is available under the accession number LN827950. Raw reads as well as vsiRNA reads are available at the GEO database under the accession number GSE83282.

Statistical analysis

Differences in nucleotide composition at the 5′ end nucleotide were analysed by chi‐squared goodness‐of‐fit test using the online tool www.quantpsy.org/chisq/chisq.htm. Expected frequencies were calculated according to the nucleotide composition of the corresponding viral genomes.

Supporting information

Additional Supporting Information may be found in the online version of this article at the publisher‘s website:

Fig. S1 Distribution of total mycovirus‐derived small RNAs (vsiRNAs) along the viral genomes. Histograms show the number of sense (blue) and antisense (red) vsiRNAs of 18–30 nucleotides along the corresponding viral genomes.

Click here for additional data file. (390.9KB, pptx)

Acknowledgements

We thank Dr Ernesto P. Benito for Botrytis cinerea grapevine isolates (CIALE, Centro Hispanoluso de Investigaciones Agrarias, Universidad de Salamanca, Salamanca, Spain) and Dra. Paloma Melgarejo for the Botrytis cinerea pepper isolate (Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria, INIA, Madrid, Spain). We thank Dr César Llave (Centro de Investigaciones Biológicas, CSIC, Madrid, Spain) for critical reading of the manuscript. This research was financed by project AGL2009‐11778 from the Spanish Ministry of Science and Innovation.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Additional Supporting Information may be found in the online version of this article at the publisher‘s website:

Fig. S1 Distribution of total mycovirus‐derived small RNAs (vsiRNAs) along the viral genomes. Histograms show the number of sense (blue) and antisense (red) vsiRNAs of 18–30 nucleotides along the corresponding viral genomes.

Click here for additional data file. (390.9KB, pptx)

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