Mycoviruses in Fusarium Species: An Update

Fusarium is an important genus of plant pathogenic fungi, and is widely distributed in soil and associated with plants worldwide. The diversity of mycoviruses in Fusarium is increasing continuously due to the development and extensive use of state-of-the-art RNA deep sequencing techniques. To date, fully-sequenced mycoviruses have been reported in 13 Fusarium species: Fusarium asiaticum, F. boothii, F. circinatum, F. coeruleum, F. globosum, F. graminearum, F. incarnatum, F. langsethiae, F. oxysporum, F. poae, F. pseudograminearum, F. solani, and F. virguliforme. Most Fusarium mycoviruses establish latent infections, but some mycoviruses such as Fusarium graminearum virus 1 (FgV1), Fusarium graminearum virus-ch9 (FgV-ch9), Fusarium graminearum hypovirus 2 (FgHV2), and Fusarium oxysporum f. sp. dianthi mycovirus 1 (FodV1) cause hypovirulence. Rapid advances in various omics technologies used to elucidate genes or biological processes can facilitate an improved understanding of mycovirus-host interactions. The review aims to illuminate the recent advances in studies of mycoviruses in Fusarium, including those related to diversity, molecular mechanisms of virus-host interaction. We also discuss the induction and suppression of RNA silencing including the role of RNAi components as an antiviral defense response.

Mycoviruses are generally transmitted vertically via spores and horizontally via anastomosis, with one exception, Sclerotinia gemycircularvirus 1 (formerly named Sclerotinia sclerotiorum hypovirulence-associated DNA virus 1), which can extracellularly infect fungi . Although many mycoviruses show latent infections, some cause phenotypic alterations that reduce or increase the virulence of the host (hypoor hypervirulence) (Nuss, 2005;Pearson et al., 2010;Jiang et al., 2013;Ghabrial et al., 2015). Fungal viruses causing hypovirulence attract much attention as potential candidates for the biological control of phytopathogenic fungal diseases and provide an opportunity to understand the interactions between mycoviruses and plant-pathogenic fungal hosts (Nuss, 2005;Xie and Jiang, 2014;Ghabrial et al., 2015). Because most viruses lack an extracellular replication phase, researchers have developed many transformation and transfection techniques using cDNA infectious clones (Choi and Nuss, 1992;Chen et al., 1993;Zhang et al., 2016), dsRNA (Stanway and Buck, 1984;Kanhayuwa et al., 2015), in vitro-transcribed RNA transcripts (Chen et al., 1994;Chen and Nuss, 1999;Moleleki et al., 2003;Lin et al., 2007;Marzano et al., 2015), and purified virions (Hillman et al., 2004;Sasaki et al., 2007). These techniques have accelerated the identification of host and/or viral factor(s) involved in the interactions between mycoviruses and their hosts . These methods can also be used to extend the experimental host ranges of some mycoviruses, without the restriction of fungal vegetative incompatibility .
Fusarium is a widely dispersed filamentous phytopathogenic fungus genus of the phylum Ascomycota, which causes serious damage to many field, ornamental, forest, and horticultural crops (Sharma et al., 2018). Many Fusarium species also produce mycotoxins such as trichothecenes and fumonisins that cause health problems in humans and livestock, and some of the greatest economic impacts are associated with deoxynivalenol (DON) and its derivatives (Munkvold, 2017). Many species of Fusarium also act as hosts for mycoviruses. Although many mycoviruses have been reported from different species of Fusarium, only a few isolates have a hypovirulent effect on their host, which is one of the major challenges to using mycoviruses efficiently as a biocontrol agent against Fusarium species. This review discusses recent advances in research related to mycoviruses infecting Fusarium species.

DIVERSE VIRUSES IN FUSARIUM
Due to the advancement and widespread use of next generation sequencing (NGS) techniques, knowledge of the diversity of known mycoviruses has rapidly increased in the past few years. To date, complete genome sequences are available for 29 mycoviruses, which have been identified from different species of Fusarium (Table 1), including 17 dsRNA viruses from six established or provisionally designated families (Crysoviridae, Megabirnaviridae, Partitiviridae, Totiviridae, Alternaviridae, Fusagraviridae) and one unassigned dsRNA group, 11 (+)ssRNA viruses from five established or provisionally designated families (Deltaflexiviridae, Hypoviridae, Narnaviridae, Fusariviridae, and Tymoviridae) and one unassigned (+)ssRNA group, and one (-)ssRNA virus from the family Mymonaviridae.

Family Totiviridae
A member of the genus Victorivirus, family Totiviridae, has been isolated from Fusarium asiaticum strain F16176 and is designated Fusarium asiaticum victorivirus 1 (FaVV1) (Li et al., 2019). The genome of FaVV1 consists of a single, linear dsRNA of 5,281 bp that contains two ORFs. ORF1 is predicted to encode a CP with a molecular mass of 79 kDa. ORF2 is predicted to encode an RdRp with a molecular mass of 92 kDa. Interestingly, the CP and RdRp of FaVV1 are most similar (77 and 75% identical, respectively) to those of Rosellinia necatrix victorivirus 1 (RnVV1), which infects the white root rot pathogen fungus Rosellinia necatrix. Both ORFs overlap with each other at a pentanucleotide "UAAUG, " and the overlapping pentanucleotide is one of the features of the genus Victorivirus (Li et al., 2019).

Family Fusagraviridae
Five members of the newly proposed family Fusagraviridae (Wang et al., 2016a) have been isolated from Fusarium, Fusarium poae dsRNA virus 2 (FpV2), and Fusarium poae dsRNA virus 3 (FpV3) isolated from the same Fusarium poae strain SX63 (Wang et al., 2016a), Fusarium virguliforme dsRNA mycovirus 1 (FvV1) and Fusarium virguliforme dsRNA mycovirus 2 (FvV2) isolated from Fusarium virguliforme (Marvelli et al., 2014), and Fusarium graminearum virus 3 (FgV3) isolated from F. graminearum strain DK3 . The respective genomes of FpV2 and FpV3 consist of a single dsRNA, 9,518 and 9,419 bp, respectively. Each contains two ORFs (ORF1 and ORF2), and encode a protein of unknown function and an RdRp, respectively (Wang et al., 2016a). A phytoreo_S7 domain is found downstream of the RdRp domain of the ORF2-coded proteins of both FpV2 and FpV3. The same shifty heptamer motif (GGAAAAC) is found immediately before the stop codon UAG of ORF1 in both FpV2 and FpV3, which could mediate programmed −1 ribosomal frameshifting (−1 PRF). The respective genomes of both FvV1 and FvV2 consist of a dsRNA, 9,402 and 9,327 bp, respectively, and contain two ORFs (ORF1 and ORF2) that overlap by 25 nt. The 25-nt overlap between ORFs 1 and 2 contain a slippery sequence (AAAAAAC) that is a−1 frameshift signal. ORF2 encodes an RdRp, while the function of ORF1 is unclear (Marvelli et al., 2014). The FgV3 genome also has a dsRNA (9,098 bp), and possesses two ORFs. ORF1 encodes a protein of unknown function, while ORF2 encodes an RdRp. A phytoreovirus S7 protein domain is also found downstream of the RdRp domain of ORF2 ).

Unassigned (+)ssRNA Mycoviruses in Fusarium
Fusarium boothii large flexivirus 1 (FbLFV1) has been isolated from Fusarium boothii strain Ep-BL13 (Mizutani et al., 2018). The FbLFV1 genome consists of a linear, positive-sense ssRNA of 12,579 nt, and encompasses a single large ORF encoding a replicase of 444 kDa, containing three conserved domains, Mtr, Hel, and RdRp. There are also two regions of unknown function in the polypeptide with similarity to a PHA03247 domain found in herpesviruses (large dsDNA viruses). Sequence and phylogenetic analyses show that FbLFV1 belongs to a novel virus species that may form an independent genus, or even a novel family, in the order Tymovirales (Mizutani et al., 2018).

Negative-Sense, Single-Stranded RNA Mycoviruses
Family Mymonaviridae A member of the family Mymonaviridae has been isolated from Fusarium graminearum strain HN1 and is designated Fusarium graminearum negative-stranded RNA virus 1 (FgNSRV-1) (Wang et al., 2018). The genome of FgNSRV-1 harbors a linear, negative-sense ssRNA of 9,072 nt, and contains five discontinuous but linear ORFs (ORF I-V), encoding five proteins (termed P I-P V) with molecular masses ranging from 6 to 221 kDa. Among the five FgNSRV-1 proteins, P II and P IV are related to the putative nucleoprotein N and large protein L of Sclerotinia sclerotiorum negative-stranded RNA virus 1 (SsNSRV-1) and SsNSRV-3 Lee Marzano et al., 2016). The 3 ′ and 5 ′ -ends of FgNSRV-1 RNA have perfect complementarity of the first six residues (3 ′ -UCCUGC-GCAGGA-5 ′ ), which is a common feature among mononegaviruses (Chen et al., 2016).

EFFECT OF VIRUSES ON FUSARIUM
Due to recent technological advances, a number of virus/fungal host systems can be established by curing viruses along with artificial introduction Suzuki, 2017). The vegetative growth, development, and physiological properties of host fungi are highly influenced by the harmful, beneficial, or neutral (little or no) effects of fungal viruses . In this review, we mainly focus on the interplay between mycoviruses and Fusarium spp. Mitigation of fungal virulence or "hypovirulence" is a good example of the harmful effect of several viruses on their host fungi. Therefore, fungal viruses could act as a potential candidate for the biological control of fungi (Hillman et al., 2018). The most studied example of reduced fungal virulence is CHV1/C. parasitica (Dawe and Nuss, 2013). In the case of Fusarium spp., four mycoviruses have harmful effects on fungal phenotypes. FgHV2, a novel hypovirus of the newly proposed genus Alphahypovirus, is associated with hypovirulence phenotypes, including reduced mycelial growth rate, conidia production, and DON concentration. In the virulence assay, the virusfree strain spreads more quickly from the inoculation sites to nearby spikelets than the virus-carrying strain JS16 (Li et al., 2015). FgV1, a member of the novel family Fusariviridae, has been reported to reduce the virulence of F. graminearum, delay mycelial growth, increase pigmentation, and reduce the production of mycotoxin (Chu et al., 2002). FgV-ch9, a member of the family Crysoviridae, is associated with hypovirulence of F. graminearum at high and medium viral dsRNA levels, including reduced mycelial growth rate and conidiation capacity, abnormal colony morphology, disorganized cytoplasm, and reduced virulence in wheat and maize plants (Darissa et al., 2012). FodV1, a member of the family Chrysoviridae, causes significant phenotypic alterations in the vegetative growth and virulence of the fungal host (Lemus-Minor et al., 2018).
In most cases, mycovirus infection causes little or no symptoms in the host (Ghabrial and Suzuki, 2009). For example, FgV3 and FgV4 infections do not cause any phenotypic change in the host F. graminearum (Lee et al., 2014). FgHV1 infection has mild or no effects on the fungal phenotypes of F. graminearum including morphology, mycelial growth, conidiation, and virulence or toxin production (Wang et al., 2013). FsV1 and FpV1 are also not associated with phenotypic changes in their hosts F. solani and F. poae (Nogawa et al., 1996;Compel et al., 1999). Another two mycoviruses, FpV2 and FpV3, isolated from the same strain of Fusarium poae, do not show any deleterious effect on their host fungus (Wang et al., 2016a).

GENE REGULATIONS ASSOCIATED WITH FUSARIUM VIRUS INFECTION
The proteome and transcriptome analysis of virus-infected fungi will help to elucidate the proteins and genes regulated by mycovirus infection that are involved in growth, development, and stress responses and direct further studies on the interactions between pathogenic fungi and viruses. The differential expression of F. graminearum proteins caused by FgV1 infection was investigated by two-dimensional electrophoresis with mass spectrometry . Seven proteins, including sporulation-specific gene SPS2, triose phosphate isomerase, nucleoside diphosphate kinase, and woronin body major protein precursor, were induced or significantly upregulated by FgV1 infection. The significant decrease or down regulation of 16 proteins, including enolase, saccharopine dehydrogenase, flavohemoglobin, mannitol dehydrogenase, and malate dehydrogenase, caused by FgV1 infection was also identified. However, the number of proteins identified was insufficient to obtain a comprehensive understanding of the host response Cho et al., 2013). Therefore, genome-wide expression analyses were carried out using a 3 ′ -tiling microarray in FgV1-infected F. graminearum. Genes associated with protein synthesis (such as ribosome assembly, nucleolus, and ribosomal RNA processing) and genes required for transcription and signal transduction (such as fungal-specific transcription factors and cAMP signaling), were significantly up-regulated in fungal host cells, which seems to be related to virus replication. In contrast, significant down-regulation of genes required for metabolism (such as carboxylic acids, aromatic amino acids, nitrogen compounds, and polyamines) and transporting systems in a fungal host containing the virus appears to be related to the host defense mechanism and fungal virulence (Cho et al., 2012(Cho et al., , 2013. Furthermore, a comprehensive genome-wide gene expression analysis elucidated completely distinct expression patterns of F. graminearum transcriptomes in response to infections by two hypovirulent mycoviruses (FgV1 and FgV2) and two non hypovirulent mycoviruses (FgV3 and FgV4), respectively. Interestingly, changes in the host transcriptome caused by different mycoviruses are not always correlated with observed host phenotypes. The fungal host transcriptome was more affected by FgV1 and FgV4 infections than by FgV2 and FgV3 infections. In addition, 12 differentially expressed genes were identified in response to all four mycovirus infections, but functions of most of these genes are still unknown (Lee et al., 2014). In the FgHV1-infected F. graminearum strain, enriched genes related to redox regulation are differentially expressed. It has been demonstrated that FgHV1-encoded p20 could induce H 2 O 2 accumulation and a hypersensitive response in Nicotiana benthamiana leaves (Wang et al., 2016c).
Subsequently, the hexagonal peroxisome protein FgHex1 was screened using transcriptional and proteomic analyses. By generating FgHEX1 gene deletion and overexpression mutants, it was indicated that the FgHEX1 gene plays a direct role in the asexual reproduction and virulence of F. graminearum and facilitates viral RNA accumulation in the FgV1-infected host fungus . To investigate the molecular mechanism underlying the production of FgHEX1 and the replication of FgV1 viral RNA, electrophoretic mobility shift assays (EMSA) was conducted with recombinant FgHex1 protein and RNA sequences derived from various regions of FgV1 genomic RNA. These analyses demonstrated that the FgHex1 functions in the synthesis of both strands of FgV1 RNA and therefore in FgV1 replication probably by specifically binding the FgV1 genomic RNA. This is the first report about the regulation of viral RNA replication by a fungal cellular protein that can directly bind to viral genomic RNA (Son et al., 2016). Otherwise, another host gene, the putative 3 ′ (2 ′ ),5 ′ -bisphosphate nucleotidase gene, FgHal2, in F. graminearum is down-regulated following FgV1 infection. The possible function(s) of FgHal2 was investigated in F. graminearum using gene deletion and gene over-expression mutants. It was found that deletion of FgHal2 reduced conidiation, mycelial growth, and the production of secondary metabolites. Moreover, deletion of FgHal2 decreased viral RNA accumulation and the vertical transmission of FgV1 via conidia. Together, these results indicate that F. graminearum can down-regulate one of its major multifunctional genes, FgHal2, in response to FgV1 infection . Recently, an mRNA binding protein (named virus response 1, vr1) was identified to be involved in symptom expression in F. graminearum. Downregulation of vr1 in the virus FgV-ch9 infected fungus and vr1 deletion evoke virus-infection like symptoms while constitutive expression overrules the cytopathic effects of the virus infection. Intriguingly, the presence of a specific viral structural protein P3 is sufficient to trigger the fungal response, i.e., vr1 downregulation, and symptom development similar to virus infection. Hence, vr1 represents a fundamental host factor for the expression of virus-related symptoms and helps to understand the underlying mechanism of hypovirulence. The advancements in understanding fungal infection and response may aid biological pest control approaches using mycoviruses or viral proteins to prevent future Fusarium epidemics (Bormann et al., 2018).

RNAI IN FUSARIUM RESPONDING TO VIRAL INFECTION
RNA silencing or RNA interference (RNAi) functions as an antiviral defense mechanism in eukaryotic organisms. The core elements of the cross-kingdom RNA silencing defense response consists of conserved ribonucleases: members of the Dicerlike (DCL) and Argonaute-like (AGO) protein families. Dicer nucleases recognize viral double-stranded and structured RNAs and use the associated RNase III-type activity to process these RNAs into small RNAs of 21-24 nts in length, termed virusderived small (vs.) RNAs. The vsRNAs are then incorporated into an effector complex termed the RNA-induced silencing complex (RISC) with the aid of an Argonaute family protein.
One strand of the vsRNA is degraded and the remaining guide strand targets the effector complex to the cognate viral RNA, which is then cleaved by the Argonaute-associated RNAse Hlike activity. Subsequently, the antiviral RNA silencing response is further amplified by host RNA-dependent RNA polymerases (RdRPs) (Ding, 2010;Nuss, 2011). To combat RNA silencingbased antiviral defense, viruses of plants, insects, mammals, and fungi encode proteins, designated viral suppressors of RNA silencing (VSR), that employ a variety of mechanisms to suppress RNA silencing pathways. RNA silencing and viruses against host RNA silencing have been well-characterized in the Cryphonectria parasitica-hypovirus system (Ding, 2010;Nuss, 2011). More encouragingly, the roles of core components of the RNAi pathway have been also been gradually revealed in F. graminearum.
F. graminearum contains genes encoding two dicer proteins (FgDicer1 and FgDicer2), two Argonaute proteins (FgAgo1 and FgAgo2), and five RdRp proteins (FgRdRp1 to 5). Chen et al. showed that RNAi machinery was not involved in growth, abiotic stress and pathogenesis in F. graminearum. In addition, it was demonstrated that the hairpin RNA (hpRNA) can efficiently silence the expression level of target gene and the argonaute protein FgAgo1 and dicer protein FgDicer2 played a critical role in the hpRNA mediated gene silencing process . FgDicer2 was also involved in small RNA (sRNA, 17-40 nt) transcription and microlike-RNA (milRNA) generation in this fungus . Strikingly, a recent study showed that FgDicer2 is the primary DCL component for defense against viroids in F. graminearum (Wei et al., 2019). Furthermore, a combined analysis of functional genetics and deep sequencing of small non-coding RNA (sRNA), mRNA, and the degradome indicate that the sex-specifically induced exonic small interference RNA (exsiRNA)-mediated RNA interference (RNAi) mechanism has an important role in fine-tuning the transcriptome during ascospore formation in F. graminearum. It is FgDicer1 and FgAgo2 that primarily mediate the sex-specific RNAi pathway. Each fungal species appears to have evolved RNAi-based gene regulation for specific developmental stages or stress responses. This study provides new insights into the regulatory role of sRNAs in fungi and other lower eukaryotes (Son et al., 2017).
To preliminarily elucidate the RNA silencing mechanism of the F. graminearum/hypovirus system, the properties of sRNAs in FgHV1 and FgHV2 were analyzed by using HiSeq deep sequencing in F. graminearum. The length distributions of F. graminearum sRNAs were altered by hypoviral infection. Potential milRNA candidates were differentially expressed between the hypovirus-free and hypovirus-infected library types. The 1,831,081 and 3,254,758 total reads generated from the FgHV1 and FgHV2 genomes in F. graminearum yielded the first high-resolution sRNA maps of fungal viruses. In particular, FgDicer1 and FgRdRp5 were predicted targets of FgHV1-and FgHV2-derived siRNAs, possibly revealing a novel anti-RNA silencing strategy employed by mycoviruses (Wang et al., 2016b). To investigate the contributions of RNAi components to the antiviral response against Fusarium graminearum viruses (FgV1, 2, and 3), reverse genetics and virus-derived small RNA profiling were used. FgV1-3 infection differentially induces the gene expression of RNAi components in F. graminearum. FgDICER2 and FgAGO1 transcripts accumulated at lower levels following FgV1 infection than following FgV2 or FgV3 infection. FgAGO1 can efficiently robust RNAi response against FgV1 infection, but the functions of two dicer genes, i.e., FgDICER1 and FgDICER2, might be partially redundant in response to FgV 1-3 infections. These results show that F. graminearum developed more complex and robust RNA silencing system against mycoviruses (Yu et al., 2018).

CONCLUSION AND PROSPECTS
The characterization of the viruses isolated from Fusarium mentioned above has enhanced our understanding of the great diversity of mycoviruses. Most Fusarium mycoviruses have double-stranded or single-stranded, positive-or negativesense RNA genomes. No DNA viruses have been isolated from Fusarium species. Additionally, purified virions of Fusarium mycoviruses can be obtained in large quantities and of high quality and can serve as good material for structural analysis, which will promote a better understanding of mycovirus assembly, function, evolution, and its uses in nanotechnological applications (Ghabrial et al., 2015;De Ruiter et al., 2019).
Fungal viruses causing hypovirulence can be used as potential biocontrol agents of phytopathogenic fungal diseases, which are always restricted by fungal vegetative incompatibility . Recent studies have demonstrated that some mycoviruses can be transmitted between vegetatively incompatible strains, particularly SsHADV-1, Sclerotinia sclerotiorum partitivirus 1 (SsPV1), Sclerotinia sclerotiorum mycoreovirus 4 (SsMYRV4), and Sclerotinia sclerotiorum deltaflexivirus 2 (SsDFV2) Xiao et al., 2014;Wu et al., 2017;Hamid et al., 2018). In addition, these mycoviruses can cause hypovirulence. Their hypovirulent properties suggest that SsHADV-1, SsPV1, SsMYRV4, and SsDFV2, have potential for controlling plant disease in the field. Therefore, further research is required to clarify whether the transmission of Fusarium mycoviruses can occur and their corresponding transmission efficiency between vegetative-incompatible individuals or interspecific fungi.
Fusarium graminearum has emerged as a good model system for studying mycovirus-host interactions, similar to three other host-virus interaction systems: C. parasitica-mycovirus, S. sclerotiorum-mycovirus, and R. necatrix-mycovirus. There are some advantages of using F. graminearum-mycovirus for such studies. For example, the genome of the F. graminearum strain PH-1 is sequenced and is available publicly, allowing RNAseq-based, genome-wide expression analysis and modification of targeted gene disruptions. Additionally, protoplasts of F. graminearum are easy to prepare and store and are competent in genetics transformation systems with DNA. Infectious cDNA clones have been successfully used in a few fungal species, including C. parasitica, Diaporthe ambigua, and Sclerotinia sclerotiorum (Choi and Nuss, 1992;Chen et al., 1993Chen et al., , 1994Chen and Nuss, 1999;Moleleki et al., 2003;Lin et al., 2007;Marzano et al., 2015;Zhang et al., 2016), but not so far in F. graminearum. The construction of infectious cDNA clones can be used to explore the properties of mycoviruses and employ mycoviruses as vectors to introduce genes deleterious to the fungal host (Pearson and Bailey, 2013).
Future studies should focus on systematically identifying viral and host factors important for the interactions between mycoviruses and their hosts, especially crucial determinants responsible for the phenotypic changes (including hypovirulence) and reduction of Fusarium mycotoxin production caused by mycovirus infection. SW, LZ, and IA performed the literature search and prepared the  manuscript table. LG drafted and revised the manuscript. PL and PB contributed equally to writing the manuscript. All authors revised and agree with the final manuscript version.

FUNDING
This work was supported by the National Key R&D Program of China (2018YFD0200500) to LG.