Molecular characterization of a new botybirnavirus that infects Alternaria sp. from tobacco.

The genus Alternaria comprises many important fungal pathogens that infect a wide variety of organisms. In this report, we present the discovery of a new double-stranded RNA (dsRNA) mycovirus called Alternaria botybirnavirus 2 (ABRV2) from a phytopathogenic strain, XC21-21C, of Alternaria sp. isolated from diseased tobacco leaves in China. The ABRV2 genome consists of two dsRNA components, namely dsRNA1 and dsRNA2, with lengths of 6,162 and 5,865 base pairs (bp), respectively. Each of these genomic dsRNAs is monocistronic, encoding hypothetical proteins of 201.6 kDa (P1) and 2193.3 kDa (P2). ABRV2 P1 and P2 share 50.54% and 63.13% amino acid sequence identity with the corresponding proteins encoded by dsRNA1 of Alternaria botybirnavirus 1 (ABRV1). Analysis of its genome organization and phylogenetic analysis revealed that ABRV2 is a new member of the genus Botybirnavirus.

A novel botybirnavirus with a unique satellite dsRNA causes latent infection in Didymella theifolia isolated from tea plants.

IMPORTANCE: A novel botybirnavirus, infecting the tea plant pathogen Didymella theifolia and tentatively named Didymella theifolia botybirnavirus 1 (DtBRV1), together with an additional double-stranded RNA (dsRNA), was characterized. DtBRV1 comprises two dsRNAs (1 and 2) encapsidated in isometric virions, while dsRNA3 is a satellite. The satellite represents a unique specimen since it contains a duplicated region and has high similarity to the two botybirnavirus dsRNAs, supporting the notion that it most likely originated from a deficient genomic component. The biological characteristics of DtBRV1 were further determined. With their unique molecular traits, DtBRV1 and its related dsRNA expand our understanding of virus diversity, taxonomy, and evolution.

A Botybirnavirus Isolated from Alternaria tenuissima Confers Hypervirulence and Decreased Sensitivity of Its Host Fungus to Difenoconazole.

Alternaria alternata botybirnavirus 1 (AaBRV1) was isolated from a strain of Alternaria alternata, causing watermelon leaf blight in our previous research. The effect of AaBRV1 on the phenotype of its host fungus, however, was not determined. In the present study, a novel strain of AaBRV1 was identified in A. tenuissima strain TJ-NH-51S-4, the causal agent of cotton Alternaria leaf spot, and designated as AaBRV1-AT1. A mycovirus AaBRV1-AT1-free strain TJ-NH-51S-4-VF was obtained by protoplast regeneration, which eliminated AaBRV1-AT1 from the mycovirus AaBRV1-AT1-infected strain TJ-NH-51S-4. Colony growth rate, spore production, and virulence of strain TJ-NH-51S-4 were greater than they were in TJ-NH-51S-4-VF, while the sensitivity of strain TJ-NH-51S-4 to difenoconazole, as measured by the EC50, was lower. AaBRV1-AT1 was capable of vertical transmission via asexual spores and horizontal transmission from strain TJ-NH-51S-4 to strain XJ-BZ-5-1hyg (another strain of A. tenuissima) through hyphal contact in pairing cultures. A total of 613 differentially expressed genes (DEGs) were identified in a comparative transcriptome analysis between TJ-NH-51S-4 and TJ-NH-51S-4-VF. Relative to strain TJ-NH-51S-4-VF, the number of up-regulated and down-regulated DEGs in strain TJ-NH-51S-4 was 286 and 327, respectively. Notably, the expression level of one DEG-encoding cytochrome P450 sterol 14α-demethylase and four DEGs encoding siderophore iron transporters were significantly up-regulated. To our knowledge, this is the first documentation of hypervirulence and reduced sensitivity to difenoconazole induced by AaBRV1-AT1 infection in A. tenuissima.

A Capsidless Virus Is trans-Encapsidated by a Bisegmented Botybirnavirus.

RNA viruses usually have linear genomes and are encapsidated by their own capsids. Here, we newly identified four mycoviruses and two previously reported mycoviruses (a fungal reovirus and a botybirnavirus) in the hypovirulent strain SCH941 of Sclerotinia sclerotiorum. One of the newly discovered mycoviruses, Sclerotinia sclerotiorum yadokarivirus 1 (SsYkV1), with a nonsegmented positive-sense single-stranded RNA (+ssRNA) genome, was molecularly characterized. SsYkV1 is 5,256 nucleotides (nt) in length, excluding the poly(A) structure, and has a large open reading frame that putatively encodes a polyprotein with the RNA-dependent RNA polymerase (RdRp) domain and a 2A-like motif. SsYkV1 was phylogenetically positioned into the family Yadokariviridae and was most closely related to Rosellinia necatrix yadokarivirus 2 (RnYkV2), with 40.55% identity (78% coverage). Although SsYkV1 does not encode its own capsid protein, the RNA and RdRp of SsYkV1 are trans-encapsidated in virions of Sclerotinia sclerotiorum botybirnavirus 3 (SsBV3), a bisegmented double-stranded RNA (dsRNA) mycovirus within the genus Botybirnavirus. In this way, SsYkV1 likely replicates inside the heterocapsid comprised of the SsBV3 capsid protein, like a dsRNA virus. SsYkV1 has a limited impact on the biological features of S. sclerotiorum. This study represents an example of a yadokarivirus trans-encapsidated by an unrelated dsRNA virus, which greatly deepens our knowledge and understanding of the unique life cycles of RNA viruses. IMPORTANCE RNA viruses typically encase their linear genomes in their own capsids. However, a capsidless +ssRNA virus (RnYkV1) highjacks the capsid of a nonsegmented dsRNA virus for the trans-encapsidation of its own RNA and RdRp. RnYkV1 belongs to the family Yadokariviridae, which already contains more than a dozen mycoviruses. However, it is unknown whether other yadokariviruses except RnYkV1 are also hosted by a heterocapsid, although dsRNA viruses with capsid proteins were detected in fungi harboring yadokarivirus. It is noteworthy that almost all presumed partner dsRNA viruses of yadokariviruses belong to the order Ghabrivirales (most probably a totivirus or toti-like virus). Here, we found a capsidless +ssRNA mycovirus, SsYkV1, from hypovirulent strain SCH941 of S. sclerotiorum, and the RNA and RdRp of this mycovirus are trans-encapsidated in virions of a bisegmented dsRNA virus within the free-floating genus Botybirnavirus. Our results greatly expand our knowledge of the unique life cycles of RNA viruses.

Discovery of Two Mycoviruses by High-Throughput Sequencing and Assembly of Mycovirus-Derived Small Silencing RNAs From a Hypovirulent Strain of Sclerotinia sclerotiorum.

Sclerotinia sclerotiorum, an important phytopathogenic fungus, harbors rich diversity of mycoviruses. Lately, more mycoviruses can be successfully and accurately discovered by deep sequencing, especially those that could not be detected by traditional double-stranded RNA (dsRNA) extraction. Previously, we reported that the hypovirulent S. sclerotiorum strain SZ-150 is coinfected by Sclerotinia sclerotiorum hypovirus 1 (SsHV1) and its related satellite RNA. Here, aside from SsHV1, we detected two other mycoviruses, Sclerotinia sclerotiorum botybirnavirus 3 (SsBV3/SZ-150) and Sclerotinia sclerotiorum mycotymovirus 1 (SsMTV1/SZ-150), coinfecting strain SZ-150, by deep sequencing and assembly of mycovirus-derived small RNAs and determined their full-length genomes. The genome of SsBV3/SZ-150 was found to be composed of two linear dsRNA segments, 6,212, and 5,880 bp in size, respectively. Each dsRNA segment of SsBV3/SZ-150 contains a large open reading frame (ORF) encoding RNA-dependent RNA polymerase (RdRp) and a hypothetical protein. The whole genome of SsBV3/SZ-150 shares more than 95% sequence identity with Botrytis porri botybirnavirus 1 (BpBV1) at the nucleotide (nt) or amino acid level. Thus, SsBV3/SZ-150 was assumed to be a strain of BpBV1. The genome of SsMTV1/SZ-150 consists of 6,391 nt excluding the poly(A) tail. SsMTV1/SZ-150 was predicted to contain a large ORF that encodes a putative replication-associated polyprotein (RP) with three conserved domains of viral RNA methyltransferase, viral RNA helicase, and RdRp. Phylogenetic analyses suggest that SsMTV1/SZ-150 is related, albeit distantly, to members of the family Tymoviridae. Analysis of the small RNAs derived from SsBV3/SZ-150 and SsMTV1/SZ-150 revealed that small-RNA lengths mainly range from 20 to 24 nt, with a peak at 22 nt, and the most abundant 5'-terminal nucleotide is uridine, suggesting that the Dicer 2 and Argonaute 1, two key components in the RNA inference pathway, may play important roles in the resistance to mycoviral infection in S. sclerotiorum. Neither SsBV3/SZ-150 nor SsMTV1/SZ-150 is a causal agent of hypovirulence in strain SZ-150.

Characterization of a Botybirnavirus Conferring Hypovirulence in the Phytopathogenic Fungus Botryosphaeria dothidea.

A double-stranded RNA (dsRNA) virus was isolated and characterized from strain EW220 of the phytopathogenic fungus Botryosphaeria dothidea. The full-length cDNAs of the dsRNAs were 6434 bp and 5986 bp in size, respectively. The largest dsRNA encodes a cap-pol fusion protein that contains a coat protein gene and an RNA-dependent RNA polymerase (RdRp) domain, and the second dsRNA encodes a hypothetical protein. Genome sequence analysis revealed that the sequences of the dsRNA virus shared 99% identity with Bipolaris maydis botybirnavirus 1(BmBRV1) isolated from the causal agent of corn southern leaf blight, Bipolaris maydis. Hence, the dsRNA virus constitutes a new strain of BmBRV1 and was named Bipolaris maydis botybirnavirus 1 strain BdEW220 (BmBRV1-BdEW220). BmBRV1-BdEW220 contains spherical virions that are 37 nm in diameter and consist of two dsRNA segments. The structural proteins of the BmBRV1-BdEW220 virus particles were 110 kDa, 90 kDa, and 80 kDa and were encoded by dsRNA1 and 2-ORFs. Phylogenetic reconstruction indicated that BmBRV1 and BmBRV1-BdEW220 are phylogenetically related to the genus Botybirnavirus. Importantly, BmBRV1-BdEW220 influences the growth of B. dothidea and confers hypovirulence to the fungal host. To our knowledge, this is the first report of a botybirnavirus in B. dothidea.

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.

The complete genomic sequence of a novel botybirnavirus isolated from a phytopathogenic Bipolaris maydis.

Bipolaris maydis is the causal agent of corn southern leaf blight. Here, we report a novel double-stranded RNA (dsRNA) mycovirus designated Bipolaris maydis botybirnavirus 1 (BmBRV1) from B. maydis strain JZ11 in Jingzhou, Hubei province of China. BmBRV1 has a genome consisting of two dsRNAs (dsRNA1 and dsRNA2) with a size of 6435 and 5987 bp, respectively, each of which contains a single open reading frame (ORF). The two polyproteins encoded by dsRNA1 and dsRNA2 share the highest amino acid identities of 81.8 and 75.3%, respectively, with the RdRp and coat protein of Sclerotinia sclerotiorum botybirnavirus 1 (SsBRV1), a tentative species of the genus Botybirnavirus. Phylogenetic analysis based on the amino acid sequences of RdRp indicated that BmBRV1 belongs to a distinct species of the newly proposed family Botybirnaviridae.

Co-infection of a hypovirulent isolate of Sclerotinia sclerotiorum with a new botybirnavirus and a strain of a mitovirus.

BACKGROUND: Sclerotinia sclerotiorum, a notorious plant fungal pathogen, causes yield loss of many crops and vegetables, and is a natural host of a diverse viruses with positive-sense RNA (+ssRNA), negative-sense RNA (-ssRNA), double-stranded RNA (dsRNA), or DNA genomes. Mixed-infection with multiple related or unrelated mycoviruses is a common phenomenon in S. sclerotiorum. However, a single strain co-infected with dsRNA and + ssRNA viruses has not been reported in S. sclerotiorum. RESULTS: We report two unrelated viruses, Sclerotinia sclerotiorum botybirnavirus 2 (SsBRV2) with a bipartite dsRNA genome and Sclerotinia sclerotiorum mitovirus 4 (SsMV4/AH16) with a + ssRNA genome, which were originally detected in a single hypovirulent strain AH16 of S. sclerotiorum. SsMV4/AH16 has a typical genome of mitovirus and is a strain of mitovirus SsMV4. The genome of SsBRV2 consists of two separated dsRNA segments. The large dsRNA segment is 6159 bp in length and only has a single open reading frame (ORF) encoding a putative 1868-aa polyprotein with a conserved RNA dependent RNA polymerase (RdRp) domain. The small dsRNA segment is 5872 bp in length and encodes a putative 1778-aa protein. Phylogenetic analysis using RdRp conserved domain sequences revealed that SsBRV2 is phylogenetically related to the previously reported three bipartite viruses SsBRV1, Botrytis porri RNA virus 1 (BpRV1), and soybean leaf-associated botybirnavirus 1 (SlaBRV1). Electron microscopy demonstrated that SsBRV2 forms rigid spherical virions with a diameter of approximately 40 nm in infected mycelia. The virion of SsBRV2 was successfully introduced into a virus-free strain, which provides conclusive evidence that SsBRV2 confers hypovirulence on phytopathogenic fungus S. sclerotiorum. CONCLUSIONS: A bisegmented dsRNA virus (SsBRV2/AH16) and a nonsegmented + ssRNA virus (SsMV4/AH16) were characterized in a hypovirulent strain AH16 of S. sclerotiorum. SsMV4/AH16 is a strain of a reported mitovirus, whereas SsBRV2 is a new botybirnavirus. SsBRV2 is the causal agent of hypovirulence on S. sclerotiorum. Our findings supplied a first evidence that a single S. sclerotiorum strain is co-infected by dsRNA and + ssRNA mycoviruses.

Molecular characterization of a bipartite double-stranded RNA virus and its satellite-like RNA co-infecting the phytopathogenic fungus Sclerotinia sclerotiorum.

A variety of mycoviruses have been found in Sclerotinia sclerotiorum. In this study, we report a novel mycovirus S. sclerotiorum botybirnavirus 1 (SsBRV1) that was originally isolated from the hypovirulent strain SCH941 of S. sclerotiorum. SsBRV1 has rigid spherical virions that are ∼38 nm in diameter, and three double-stranded RNA (dsRNA) segments (dsRNA1, 2, and 3 with lengths of 6.4, 6.0, and 1.7 kbp, respectively) were packaged in the virions. dsRNA1 encodes a cap-pol fusion protein, and dsRNA2 encodes a polyprotein with unknown functions but contributes to the formation of virus particles. The dsRNA3 is dispensable and may be a satellite-like RNA of SsBRV1. Although phylogenetic analysis of the RdRp domain demonstrated that SsBRV1 is related to Botrytis porri RNA virus 1 (BpRV1) and Ustilago maydis dsRNA virus-H1, the structure proteins of SsBRV1 do not have any significant sequence similarities with other known viral proteins with the exception of those of BpRV1. SsBRV1 carrying dsRNA3 seems to have no obvious effects on the colony morphology, but can significantly reduce the growth rate and virulence of S. sclerotiorum. These findings provide new insights into the virus taxonomy, virus evolution and the interactions between SsBRV1 and the fungal hosts.