Molecular and biological characterization of a novel partitivirus from Talaromyces pinophilus.

Talaromyces spp. have a worldwide distribution, are ecologically diverse and have been isolated from numerous different substrates. Talaromyces spp. are considered biotechnologically important due to their ability to produce a range of enzymes and pigments. Talaromyces pinophilus, belonging to genus Talaromyces and family Trichocomaceae, is known for producing several important bioactive metabolites. Here we report the isolation and characterisation of a partitivirus from T. pinophilus which we have nominated Talaromyces pinophilus partitivirus-1 (TpPV-1). TpPV-1 possesses a genome consisting of three double stranded (ds) RNA segments i.e., dsRNAs1-3, 1824 bp, 1638 bp and 1451 bp respectively, which are encapsidated in icosahedral particles 35 nm in diameter. Both dsRNA1 and dsRNA2 contain a single open reading frame (ORF) encoding respectively a 572 amino acid (aa) protein of 65 kDa and a 504 aa protein of 50 kDa. The third segment (dsRNA3) is potentially a satellite RNA. Phylogenetic analysis revealed that the TpPV-1 belongs to the family Partitiviridae in the proposed genus Zetapartitivirus. TpPV-1 infection decreases the mycelial growth rate of the host fungus and alters pigmentation as indicated by time course experiments performed on a range of different solid media comparing virus-infected and virus-free isogenic lines. This is the first report of mycovirus infection in T. pinophilus and may provide insights into understanding the effect of the mycovirus on the production of enzymes and pigments by the host fungus.

The complete mitochondrial genome of Penicillium expansum: Insights into the fungal evolution and phylogeny.

Penicillium expansum is an important phytopathogenic fungus that causes blue mold disease. In this study, the novel mitochondrial genome of P. expansum was sequenced, assembled, annotated, and compared with the previously published Penicillium mitogenomes. P. expansum mitogenome is composed of circular DNA molecules with a genome size of 25,496 bp. It encodes 16 protein-encoding genes (PCGs), two rRNA genes, and 25 tRNA genes. Comparative analysis with six other Penicillium species revealed that gene length, GC content, AT skew, and GC skew were variable among the core protein-coding genes. The Penicillium species' gene synteny analysis identified several gene rearrangements. Among the core 15 PCGs, atp8 had the lowest K2P genetic distance, which shows that this gene is highly conserved. The Ka/Ks value of most PCGs was less than 1, which shows that these genes have undergone purifying selection. Phylogenetic analysis based on 14 concatenated core mitochondrial genes revealed that P. expansum shares a close relationship with P. solitum. This study served as a first report on the complete mitochondrial genome of P. expansum and its comparative analysis that will contribute to population genetics and rapid evolutionary studies among Penicillium species.

Molecular and biological characterization of a partitivirus from Paecilomyces variotii.

Paeciliomyces variotii is a thermo-tolerant, ubiquitous fungus commonly found in food products, indoor environments, soil and clinical samples. It is a well-known biocontrol agent used against phytopathogenic fungi and its metabolites have many industrial applications. Rare reports of P. variotii-related human infections have been found in the medical literature. In this study, we report for the first time the infection of P. variotii isolated from a soil sample collected in a rice field with a double-stranded RNA virus, Paeciliomyces variotii partitivirus 1 (PvPV-1) in the family Partitiviridae. P. variotii harboured icosahedral virus particles 30 nm in diameter with two dsRNA segments 1758 and 1356 bp long. Both dsRNA1 and dsRNA2 have a single open reading frame encoding proteins of 63 and 40 kDa, respectively. These proteins have significant similarity to the RNA-dependent RNA polymerase and capsid protein encoded by the genomic segments of several viruses from the family Partitiviridae. Phylogenetic analysis revealed that PvPV-1 belongs to the family Partitiviridae but in an unclassified group/genus, tentatively nominated Zetapartitivirus. PvPV-1 was found to increase the growth rate of the host fungus, as indicated by time course experiments performed on a range of different media for virus-infected and virus-free isogenic lines. Further, dual-culture assays performed for both isogenic lines confirmed the antagonistic potential of P. variotii against other phytopathogenic fungi. The findings of this study assist us in understanding P. variotii as a potential biocontrol agent, together with plant-fungus-virus interactions.

Genome-wide characterization of the NLR gene family in tomato (Solanum lycopersicum) and their relatedness to disease resistance.

Nucleotide-binding leucine-rich-repeat receptors (NLR), the largest group of genes associated with plant disease resistance (R), have attracted attention due to their crucial role in protecting plants from pathogens. Genome-wide studies of NLRs have revealed conserved domains in the annotated tomato genome. The 321 NLR genes identified in the tomato genome have been randomly mapped to 12 chromosomes. Phylogenetic analysis and classification of NLRs have revealed that 211 genes share full-length domains categorized into three major clades (CNL, TNL, and RNL); the remaining 110 NLRs share partial domains and are classified in CN, TN, and N according to their motifs and gene structures. The cis-regulatory elements of NLRs exhibit the maximum number of these elements and are involved in response to biotic and abiotic stresses, pathogen recognition, and resistance. Analysis of the phylogenetic relationship between tomato NLRs and orthologs in other species has shown conservation among Solanaceae members and variation with A. thaliana. Synteny and Ka/Ks analyses of Solanum lycopersicum and Solanum tuberosum orthologs have underscored the importance of NLR conservation and diversification from ancestral species millions of years ago. RNA-seq data and qPCR analysis of early and late blight diseases in tomatoes revealed consistent NLR expression patterns, including upregulation in infected compared to control plants (with some exceptions), suggesting the role of NLRs as key regulators in early blight resistance. Moreover, the expression levels of NLRs associated with late blight resistance (Solyc04g007060 [NRC4] and Solyc10g008240 [RIB12]) suggested that they regulate S. lycopersicum resistance to P. infestans. These findings provide important fundamental knowledge for understanding NLR evolution and diversity and will empower the broader characterization of disease resistance genes for pyramiding through speed cloning to develop disease-tolerant varieties.

Aflatoxin Contamination, Its Impact and Management Strategies: An Updated Review.

Aflatoxin, a type of mycotoxin, is mostly produced by Aspergillus flavus and Aspergillus parasiticus. It is responsible for the loss of billions of dollars to the world economy, by contaminating different crops such as cotton, groundnut, maize, and chilies, and causing immense effects on the health of humans and animals. More than eighteen different types of aflatoxins have been reported to date, and among them, aflatoxins B1, B2, G1, and G2 are the most prevalent and lethal. Early detection of fungal infection plays a key role in the control of aflatoxin contamination. Therefore, different methods, including culture, chromatographic techniques, and molecular assays, are used to determine aflatoxin contamination in crops and food products. Many countries have set a maximum limit of aflatoxin contamination (2-20 ppb) in their food and agriculture commodities for human or animal consumption, and the use of different methods to combat this menace is essential. Fungal infection mostly takes place during the pre- and post-harvest stage of crops, and most of the methods to control aflatoxin are employed for the latter phase. Studies have shown that if correct measures are adopted during the crop development phase, aflatoxin contamination can be reduced by a significant level. Currently, the use of bio-pesticides is the intervention employed in many countries, whereby atoxigenic strains competitively reduce the burden of toxigenic strains in the field, thereby helping to mitigate this problem. This updated review on aflatoxins sheds light on the sources of contamination, and the on occurrence, impact, detection techniques, and management strategies, with a special emphasis on bio-pesticides to control aflatoxins.

Antimicrobial Activity of Pinus wallachiana Leaf Extracts against Fusarium oxysporum f. sp. cubense and Analysis of Its Fractions by HPLC.

Fusarium wilt has ruined banana production and poses a major threat to its industry because of highly virulent Fusarium oxysporum f. sp. cubense (Foc) race 4. The present study focused on the efficacy of Pinus wallachiana leaf extracts and its organic fractions against Foc in in vitro and greenhouse experiments. The presence of polyphenols in the fractions was also investigated using high performance liquid chromatography (HPLC). The in vitro tests carried out for the leaf extract of P. wallachiana showed its inhibitory effect on the mycelial growth and, based on this evidence, further characterization of fractions were done. Complete mycelial inhibition and the highest zone of inhibition against Foc was observed for the n-butanol fraction in vitro, while the n-hexane and dichloromethane fractions showed lower disease severity index (DSI) in greenhouse experiments. The fractions were further analysed by HPLC using nine polyphenolic standards, namely quercitin, myrecitin, kaempferol, rutin, gallic acid, trans-ferulic acid, coumeric acid, epicatechin and catechin. The highest content of polyphenols, based on standards used, was quantified in the n-butanol fraction followed by the ethyl acetate fraction of the leaf extract. This is the first report of antimicrobial activity of Pinus wallachiana extracts against Foc to the best of our knowledge.

A novel victorivirus from the phytopathogenic fungus Neofusicoccum parvum.

Neofusicoccum parvum is an important plant-pathogenic ascomycetous fungus that causes trunk diseases in a variety of plants. A limited number of reports on mycoviruses from this fungus are available. Here, we report the characterization of a novel victorivirus, Neofusicoccum parvum victorivirus 3 (NpVV3). An agarose gel dsRNA profile of a Pakistani strain of N. parvum, NFN, showed a band of ~5 kbp that was not detectable in Japanese strains of N. parvum. Taking a high-throughput and Sanger sequencing approach, the complete genome sequence of NpVV3 was determined to be 5226 bp in length with two open reading frames (ORF1 and ORF2) that encode a capsid protein (CP) and an RNA-dependent RNA polymerase (RdRP). The RdRP appears to be translated by a stop/restart mechanism facilitated by the junction sequence AUGucUGA, as is found in some other victoriviruses. BLASTp searches showed that NpVV3 CP and RdRP share the highest amino acid sequence identity (80.5% and 72.4%, respectively) with the corresponding proteins of NpVV1 isolated from a French strain of N. parvum. However, NpVV3 was found to be different from NpVV1 in its terminal sequences and the stop/restart facilitator sequence. NpVV3 particles ~35 nm in diameter were partially purified and used to infect an antiviral-RNA-silencing-deficient strain (∆dcl2) of an experimental ascomycetous fungal host, Cryphonectria parasitica. NpVV3 showed symptomless infection in the new host strain.

Assessment of mycoviral diversity in Pakistani fungal isolates revealed infection by 11 novel viruses of a single strain of Fusarium mangiferae isolate SP1.

An extensive screening survey was conducted on Pakistani filamentous fungal isolates for the identification of viral infections. A total of 396 fungal samples were screened, of which 36 isolates were found double-stranded (ds) RNA positive with an overall frequency of 9% when analysed by a classical dsRNA isolation method. One of 36 dsRNA-positive strains, strain SP1 of a plant pathogenic fungus Fusarium mangiferae, was subjected to virome analysis. Next-generation sequencing and subsequent completion of the entire genome sequencing by a classical Sanger sequencing method showed the SP1 strain to be co-infected by 11 distinct viruses, at least seven of which should be described as new taxa at the species level according to the ICTV (International Committee on Taxonomy of Viruses) species demarcation criteria. The newly identified F. mangiferae viruses (FmVs) include two partitivirids, one betapartitivirus (FmPV1) and one gammapartitivirus (FmPV2); six mitovirids, three unuamitovirus (FmMV2, FmMV4, FmMV6), one duamitovirus (FmMV5), and two unclassified mitovirids (FmMV1, FmMV3); and three botourmiavirids, two magoulivirus (FmBOV1, FmBOV3) and one scleroulivirus (FmBOV2). The number of coinfecting viruses is among the largest ones of fungal coinfections. Their molecular features are thoroughly described here. This represents the first large virus survey in the Indian sub-continent.

A second capsidless hadakavirus strain with 10 positive-sense single-stranded RNA genomic segments from Fusarium nygamai.

A unique capsidless virus with a positive-sense, single-stranded RNA genome (hadakavirus 1, HadV1), a member of the extended picorna-like supergroup, was isolated previously from the phytopathogenic fungus Fusarium oxysporum. Here, we describe the molecular and biological characterisation of a second hadakavirus strain from Fusarium nygamai, which has not been investigated in detail previously as a virus host. This virus, hadakavirus 1 strain 1NL (HadV1-1NL), has features similar to the first hadakavirus, HadV1-7n, despite having a different number of segments (10 for HadV1-1NL vs. 11 for HadV1-7n). The 10 genomic RNA segments of HadV1-1NL range in size from 0.9 kb to 2.5 kb. All HadV1-1NL segments show 67% to 86% local nucleotide sequence identity to their HadV1-7n counterparts, whereas HadV1-1NL has no homolog of HadV1-7n RNA8, which encodes a zinc-finger motif. Another interesting feature is the possible coding incapability of HadV1-1NL RNA10. HadV1-1NL was predicted to be capsidless based on the RNase A susceptibility of its replicative form dsRNA. Phenotypic comparison of multiple virus-infected and virus-free single-spore isolates indicated asymptomatic infection by HadV1-1NL. Less-efficient vertical transmission via spores was observed as the infected fungal colonies from which the spores were derived became older, as was observed for HadV1-7n. This study shows a second example of a hadakavirus that appears to have unusual features.

Molecular Characterization of a Novel Polymycovirus From Penicillium janthinellum With a Focus on Its Genome-Associated PASrp.

The genus Polymycovirus of the family Polymycoviridae accommodates fungal RNA viruses with different genomic segment numbers (four, five, or eight). It is suggested that four members form no true capsids and one forms filamentous virus particles enclosing double-stranded RNA (dsRNA). In both cases, viral dsRNA is associated with a viral protein termed "proline-alanine-serine-rich protein" (PASrp). These forms are assumed to be the infectious entity. However, the detailed molecular characteristics of PASrps remain unclear. Here, we identified a novel five-segmented polymycovirus, Penicillium janthinellum polymycovirus 1 (PjPmV1), and characterized its purified fraction form in detail. The PjPmV1 had five dsRNA segments associated with PASrp. Density gradient ultracentrifugation of the PASrp-associated PjPmV1 dsRNA revealed its uneven structure and a broad fractionation profile distinct from that of typical encapsidated viruses. Moreover, PjPmV1-PASrp interacted in vitro with various nucleic acids in a sequence-non-specific manner. These PjPmV1 features are discussed in view of the diversification of genomic segment numbers of the genus Polymycovirus.

Hadaka Virus 1: a Capsidless Eleven-Segmented Positive-Sense Single-Stranded RNA Virus from a Phytopathogenic Fungus, Fusarium oxysporum.

The search for viruses infecting fungi, or mycoviruses, has extended our knowledge about the diversity of RNA viruses, as exemplified by the discovery of polymycoviruses, a phylogenetic group of multisegmented RNA viruses with unusual forms. The genomic RNAs of known polymycoviruses, which show a phylogenetic affinity for animal positive-sense single-stranded RNA [(+)RNA] viruses such as caliciviruses, are comprised of four conserved segments with an additional zero to four segments. The double-stranded form of polymycovirus genomic RNA is assumed to be associated with a virally encoded protein (proline-alanine-serine-rich protein [PASrp]) in either of two manners: a capsidless colloidal form or a filamentous encapsidated form. Detailed molecular characterizations of polymycoviruses, however, have been conducted for only a few strains. Here, a novel polymyco-related virus named Hadaka virus 1 (HadV1), from the phytopathogenic fungus Fusarium oxysporum, was characterized. The genomic RNA of HadV1 consisted of an 11-segmented positive-sense RNA with highly conserved terminal nucleotide sequences. HadV1 shared the three conserved segments with known polymycoviruses but lacked the PASrp-encoding segment. Unlike the known polymycoviruses and encapsidated viruses, HadV1 was not pelleted by conventional ultracentrifugation, possibly due to the lack of PASrp. This result implied that HadV1 exists only as a soluble form with naked RNA. Nevertheless, the 11 genomic segments of HadV1 have been stably maintained through host subculturing and conidiation. Taken together, the results of this study revealed a virus with a potential novel virus lifestyle, carrying many genomic segments without typical capsids or PASrp-associated forms.IMPORTANCE Fungi collectively host various RNA viruses. Examples include encapsidated double-stranded RNA (dsRNA) viruses with diverse numbers of genomic segments (from 1 to 12) and capsidless viruses with nonsegmented (+)RNA genomes. Recently, viruses with unusual intermediate features of an infectious entity between encapsidated dsRNA viruses and capsidless (+)RNA viruses were found. They are called polymycoviruses, which typically have four to eight dsRNA genomic segments associated with one of the virus-encoded proteins and are phylogenetically distantly related to animal (+)RNA caliciviruses. Here, we identified a novel virus phylogenetically related to polymycoviruses, from the phytopathogenic fungus Fusarium oxysporum The virus, termed Hadaka virus 1 (HadV1), has 11 (+)RNA genomic segments, the largest number in known (+)RNA viruses. Nevertheless, HadV1 lacked a typical structural protein of polymycoviruses and was not pelleted by standard ultracentrifugation, implying an unusual capsidless nature of HadV1. This study reveals a potential novel lifestyle of multisegmented RNA viruses.

Novel Victorivirus from a Pakistani Isolate of Alternaria alternata Lacking a Typical Translational Stop/Restart Sequence Signature.

The family Totiviridae currently contains five genera Totivirus, Victorivirus, Leishmavirus, Trichomonasvirus, and Giardiavirus. Members in this family generally have a set of two-open reading frame (ORF) elements in their genome with the 5'-proximal ORF (ORF1) encoding a capsid protein (CP) and the 3'-proximal one (ORF2) for RNA-dependent RNA polymerase (RdRp). How the downstream open reading frames (ORFs) are expressed is genus-specific. All victoriviruses characterized thus far appear to use the stop/restart translation mechanism, allowing for the expression of two separate protein products from bicitronic genome-sized viral mRNA, while the totiviruses use a -1 ribosomal frame-shifting that leads to a fusion product of CP and RdRp. We report the biological and molecular characterization of a novel victorivirus termed Alternaria alternata victorivirus 1 (AalVV1) isolated from Alternaria alternata in Pakistan. The phylogenetic and molecular analyses showed AalVV1 to be distinct from previously reported victoriviruses. AalVV1 appears to have a sequence signature required for the -1 frame-shifting at the ORF1/2 junction region, rather than a stop/restart key mediator. By contrast, SDS-polyacrylamide gel electrophoresis and peptide mass fingerprinting analyses of purified virion preparations suggested the expression of two protein products, not a CP-RdRp fusion product. How these proteins are expressed is discussed in this study. Possible effects of infection by this virus were tested in two fungal species: A. alternata and RNA silencing proficient and deficient strains of Cryphonectria parasitica, a model filamentous fungus. AalVV1 showed symptomless infection in all of these fungal strains, even in the RNA silencing deficient C. parasitica strain.

Molecular and biological characterization of a novel botybirnavirus identified from a Pakistani isolate of Alternaria alternata.

Mycoviruses ubiquitously infect a wide range of fungal hosts in the world. The current study reports a novel double stranded RNA (dsRNA) virus, termed Alternaria alternata botybirnavirus 1 (AaBbV1), infecting a Pakistani strain, 4a, of a phytopathogenic ascomycetous fungus Alternaria alternata. A combined approach of next generation and conventional terminal end sequencing of the viral genome revealed that the virus is a distinct member of the genus Botybirnavirus. This virus comprised of two segments (dsRNA1 and dsRNA2) of sizes 6127 bp and 5860 bp respectively. The dsRNA1-encoded protein carrying the RNA-dependent RNA polymerase domain showed 61% identity to the counterpart of Botrytis porri botybirnavirus 1 and lower levels of amino acid similarity with those of other putative botybirnaviruses and the fungal dsRNA viruses such as members of the families Totiviridae, Chrysoviridae and Megabirnaviridae. The dsRNA2-encoded protein showed resemblance with corresponding proteins of botybirnaviruses. Electron microscopy showed AaBbV1 to form spherical particles of 40 nm in diameter. Biochemical analyses showed that two structural proteins encoded by dsRNA1 and dsRNA2 underwent processing to some extent during particle purification, resulting in the appearance of multiple smaller products. Phylogenetic analyses of structural proteins suggested that their coding region might have been duplicated once and maintained without recombination. Protoplast fusion technique allowed for the introduction of AaBbV1 into a virus free Japanese strain of A. alternata and demonstrated its symptomless infection by the virus. Interesting similarities and dissimilarities between AaBbV1 and other previously reported botybirnaviruses are also discussed.

First Evidence for Internal Ribosomal Entry Sites in Diverse Fungal Virus Genomes.

In contrast to well-established internal ribosomal entry site (IRES)-mediated translational initiation in animals and plants, no IRESs were established in fungal viral or cellular RNAs. To identify IRES elements in mycoviruses, we developed a luciferase-based dual-reporter detection system in Cryphonectria parasitica, a model filamentous fungus for virus-host interactions. A bicistronic construct entails a codon-optimized Renilla and firefly luciferase (ORluc and OFluc, respectively) gene, between which potential IRES sequences can be inserted. In this system, ORluc serves as an internal control, while OFluc represents IRES activity. Virus sequences in the 5' untranslated regions (UTRs) of the genomes of diverse positive-sense single-stranded RNA and double-stranded RNA (dsRNA) viruses were analyzed. The results show relatively high IRES activities for Cryphonectria hypovirus 1 (CHV1) and CHV2 and faint but measurable activity for CHV3. The weak IRES signal of CHV3 may be explained by its monocistronic nature, differing from the bicistronic nature of CHV1 and CHV2. This would allow these three hypoviruses to have similar rates of translation of replication-associated protein per viral mRNA molecule. The importance of 24 5'-proximal codons of CHV1 as well as the 5' UTR for IRES function was confirmed. Furthermore, victoriviruses and chrysoviruses tested IRES positive, whereas mycoreoviruses, partitiviruses, and quadriviruses showed similar Fluc activities as the negative controls. Overall, this study represents the first development of an IRES identification system in filamentous fungi based on the codon-optimized dual-luciferase assay and provides evidence for IRESs in filamentous fungi.IMPORTANCE Cap-independent, internal ribosomal entry site (IRES)-mediated translational initiation is often used by virus mRNAs and infrequently by cellular mRNAs in animals and plants. However, no IRESs have been established in fungal virus RNAs or cellular RNAs in filamentous fungi. Here, we report the development of a dual-luciferase assay system and measurement of the IRES activities of fungal RNA viruses in a model filamentous fungal host, Cryphonectria parasitica Viruses identified as IRES positive include hypoviruses (positive-sense RNA viruses, members of the expanded Picornavirus supergroup), totiviruses (nonsegmented dsRNA viruses), and chrysoviruses (tetrasegmented dsRNA viruses). No IRES activities were observed in the 5' untranslated regions of mycoreoviruses (11-segmented dsRNA viruses), quadriviruses (tetrasegmented dsRNA viruses), or partitiviruses (bisegmented dsRNA viruses). This study provides the first evidence for IRES activities in diverse RNA viruses in filamentous fungi and is a first step toward identifying trans-acting host factors and cis-regulatory viral RNA elements.

Novel, diverse RNA viruses from Mediterranean isolates of the phytopathogenic fungus, Rosellinia necatrix: insights into evolutionary biology of fungal viruses.

To reveal mycovirus diversity, we conducted a search of as-yet-unexplored Mediterranean isolates of the phytopathogenic ascomycete Rosellinia necatrix for virus infections. Of seventy-nine, eleven fungal isolates tested RNA virus-positive, with many showing coinfections, indicating a virus incidence of 14%, which is slightly lower than that (approximately 20%) previously reported for extensive surveys of over 1000 Japanese R. necatrix isolates. All viral sequences were fully or partially characterized by Sanger and next-generation sequencing. These sequences appear to represent isolates of various new species spanning at least 6 established or previously proposed families such as Partiti-, Hypo-, Megabirna-, Yado-kari-, Fusagra- and Fusarividae, as well as a newly proposed family, Megatotiviridae. This observation greatly expands the diversity of R. necatrix viruses, because no hypo-, fusagra- or megatotiviruses were previously reported from R. necatrix. The sequence analyses showed a rare horizontal gene transfer event of the 2A-like protease domain between a dsRNA (phlegivirus) and a positive-sense, single-stranded RNA virus (hypovirus). Moreover, many of the newly detected viruses showed the closest relation to viruses reported from fungi other than R. necatrix, such as Fusarium spp., which are sympatric to R. necatrix. These combined results imply horizontal virus transfer between these soil-inhabitant fungi.

SAGA complex mediates the transcriptional up-regulation of antiviral RNA silencing.

Pathogen recognition and transcriptional activation of defense-related genes are crucial steps in cellular defense responses. RNA silencing (RNAi) functions as an antiviral defense in eukaryotic organisms. Several RNAi-related genes are known to be transcriptionally up-regulated upon virus infection in some host organisms, but little is known about their induction mechanism. A phytopathogenic ascomycete, Cryphonectria parasitica (chestnut blight fungus), provides a particularly advantageous system to study RNAi activation, because its infection by certain RNA viruses induces the transcription of dicer-like 2 (dcl2) and argonaute-like 2 (agl2), two major RNAi players. To identify cellular factors governing activation of antiviral RNAi in C. parasitica, we developed a screening protocol entailing multiple transformations of the fungus with cDNA of a hypovirus mutant lacking the RNAi suppressor (CHV1-Δp69), a reporter construct with a GFP gene driven by the dcl2 promoter, and a random mutagenic construct. Screening for GFP-negative colonies allowed the identification of sgf73, a component of the SAGA (Spt-Ada-Gcn5 acetyltransferase) complex, a well-known transcriptional coactivator. Knockout of other SAGA components showed that the histone acetyltransferase module regulates transcriptional induction of dcl2 and agl2, whereas histone deubiquitinase mediates regulation of agl2 but not dcl2 Interestingly, full-scale induction of agl2 and dcl2 by CHV1-Δp69 required both DCL2 and AGL2, whereas that by another RNA virus, mycoreovirus 1, required only DCL2, uncovering additional roles for DCL2 and AGL2 in viral recognition and/or RNAi activation. Overall, these results provide insight into the mechanism of RNAi activation.

Sequence determination of a quadripartite dsRNA virus isolated from Aspergillus foetidus.

Virus infection of Aspergillus foetidus was documented over 40 years ago and was one of the first mycovirus infections described in a filamentous fungus. The virus, named Aspergillus foetidus virus (AfV), contains at least two types of icosahedral particles, called AfV-fast (-F) and AfV-slow (-S) virions, based on their relative electrophoretic mobilities. Here, we report the complete nucleotide sequence of the AfV-F genome isolated from virions purified from the prototype isolate of the fungus. The AfV-F double-stranded (ds) RNA genome is tetra-segmented, and the plus strands of each of the four segments, but not the minus strands, are polyadenylated. The organisation and sequences of the four AfV-F dsRNAs are similar to those described for Alternaria alternata virus 1, which we propose is a member of an emerging mycovirus genus ("Alternavirus") and family ("Alternaviridae"), which also includes AfV-F.

Incidence of dsRNA mycoviruses in a collection of Aspergillus fumigatus isolates.

A collection of clinical and environmental isolates of the opportunistic human pathogen, Aspergillus fumigatus, were screened for the presence of mycoviruses and 6.6 % of 366 isolates contained dsRNA segments ranging in size from ~1.0 to 4.0 kbp. The dsRNAs were categorised into three different groups comprising bipartite dsRNAs, quadripartite dsRNAs, representative isolates of which have both been sequenced, and an uncharacterised mycovirus, whose genome apparently consists of four dsRNAs 1-2.5 kbp in size. Here, we describe dsRNA incidence in the A. fumigatus isolates examined, their provenance and also note that on occasion individual isolates were infected with two groups of different dsRNAs.

The effects of dsRNA mycoviruses on growth and murine virulence of Aspergillus fumigatus.

Some isolates of the opportunistic human pathogenic fungus Aspergillus fumigatus are known to be infected with mycoviruses. The dsRNA genomes of two of these mycoviruses, which include a chrysovirus and a partitivirus, have been completely sequenced and an RT-PCR assay for the viruses has been developed. Through curing virus-infected A. fumigatus isolates by cycloheximide treatment and transfecting virus-free isolates with purified virus, as checked by RT-PCR, isogenic virus-free and virus-infected lines of the fungus were generated whose phenotypes and growth have been directly compared. Mycovirus infection of A. fumigatus with either the chrysovirus or the partitivirus resulted in significant aberrant phenotypic alterations and attenuation of growth of the fungus but had no effect on susceptibility to common antifungals. Chrysovirus infection of A. fumigatus caused no significant alterations to murine pathogenicity.

Incidence of Phlebiopsis gigantea large virus-1 in a collection of Phlebiopsis gigantea isolates.

Eighty six Phlebiopsis gigantea isolates from at least 9 different tree species from various locations in 12 different European countries and North America were screened for the presence of large molecular weight dsRNA >10 kbp in size. In 7 isolates, which contained large dsRNAs, the presence of Phlebiopsis gigantea large virus-1 (PgLV-1) was suggested following the sequencing of the RT-PCR amplicons generated with PgLV-1 specific oligonucleotide primers which also revealed little genetic diversity between the virus isolates.