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In eukaryotes, antisense transcription can regulate sense transcription by induction of epigenetic modifications. We showed previously that antisense transcription triggers Dicer-independent siRNA (disiRNA) production and disiRNA locus DNA methylation (DLDM) in Neurospora crassa. Here we show that the conserved exonuclease ERI-1 (enhanced RNAi-1) is a critical component in this process. Antisense transcription and ERI-1 binding to target RNAs are necessary and sufficient to trigger DLDM. Convergent transcription causes stalling of RNA polymerase II during transcription, which permits ERI-1 to bind nascent RNAs in the nucleus and recruit a histone methyltransferase complex that catalyzes chromatin modifications. Furthermore, we show that, in the cytoplasm, ERI-1 targets hundreds of transcripts from loci without antisense transcription to regulate RNA stability. Together, our results demonstrate a critical role for transcription kinetics in long noncoding RNA-mediated epigenetic modifications and identify ERI-1 as an important regulator of cotranscriptional gene silencing and post-transcriptional RNA metabolism. [ABSTRACT FROM AUTHOR]

Recent studies have demonstrated that a diverse array of mycoviruses infect the plant pathogenic fungus Sclerotinia sclerotiorum. Here, we report the molecular characterization of a newly identified mycovirus, Sclerotinia sclerotiorum fusarivirus 1 (SsFV1), which was isolated from a sclerotia-defective strain JMTJ14 of S. sclerotiorum. Excluding a poly (A) tail, the genome of SsFV1 comprises 7754 nucleotides (nts) in length with 83 and 418 nts for 5'- and 3'-untranslated regions, respectively. SsFV1 has four non-overlapping open reading frames (ORFs): ORF1 encodes a 191 kDa polyprotein (1664 amino acid residues in length) containing conserved RNA-dependent RNA polymerase (RdRp) and helicase domains; the other three ORFs encode three putative hypothetical proteins of unknown function. Phylogenetic analysis, based on RdRp and Helicase domains, indicated that SsFV1 is phylogenetically related to Rosellinia necatrix fusarivirus 1 (RnFV1), Fusarium graminearum virus-DK21 (FgV1), and Penicillium roqueforti RNA mycovirus 1 (PrRV1), a cluster of an independent group belonging to a newly proposed family Fusarividae. However, SsFV1 is markedly different from FgV1 and RnFV1 in genome organization and nucleotide sequence. SsFV1 was transmitted successfully to two vegetatively incompatible virus-free strains. SsFV1 is not responsible for the abnormal phenotype of strain JMTJ14. [ABSTRACT FROM AUTHOR]

Members of the family Partitiviridae have bisegmented double-stranded RNA (dsRNA) genomes and are not generally known to cause obvious symptoms in their natural hosts. An unusual partitivirus, Sclerotinia sclerotiorum partitivirus 1 (SsPV1/WF-1), conferred hypovirulence on its natural plant-pathogenic fungal host, Sclerotinia sclerotiorum strain WF-1. Cellular organelles, including mitochondria, were severely damaged. Hypovirulence and associated traits of strain WF-1 and SsPV1/WF-1 were readily cotransmitted horizontally via hyphal contact to different vegetative compatibility groups of S. sclerotiorum and interspecifically to Sclerotinia nivalis and Sclerotinia minor. S. sclerotiorum strain 1980 transfected with purified SsPV1/WF-1 virions also exhibited hypovirulence and associated traits similar to those of strain WF-1. Moreover, introduction of purified SsPV1/WF-1 virions into strain KY-1 of Botrytis cinerea also resulted in reductions in virulence and mycelial growth and, unexpectedly, enhanced conidial production. However, virus infection suppressed hyphal growth of most germinating conidia of B. cinerea and was eventually lethal to infected hyphae, since very few new colonies could develop following germ tube formation. Taken together, our results support the conclusion that SsPV1/WF-1 causes hypovirulence in Sclerotinia spp. and B. cinerea. Cryo-EM (cryo-electron microscopy) reconstruction of the SsPV1 particle shows that it has a distinct structure with similarity to the closely related partitiviruses Fusarium poae virus 1 and Penicillium stoloniferum virus F. These findings provide new insights into partitivirus biological activities and clues about molecular interactions between partitiviruses and their hosts. [ABSTRACT FROM AUTHOR]

Mycoviruses are usually transmitted horizontally via hyphal anastomosis and vertically via sexual/asexual spores. Previously, we reported that a gemycircularvirus, Sclerotinia sclerotiorum hypovirulenceassociated DNA virus 1 (SsHADV-1), could infect its fungal host extracellularly. Here, we discovered that SsHADV-1 could infect a mycophagous insect, Lycoriella ingenua, and use it as a transmission vector. Virus acquired by larvae feeding on colonies of a virus-infected strain of S. sclerotiorum was replicated and retained in larvae, pupae, adults, and eggs. Virus could be transmitted to insect offspring when larvae were injected with virus particles and allowed to feed on a non-host fungus. Virus replication in insect cells was further confirmed by inoculating Spodoptera frugiperda cells with virus particles and analyzing with RT-PCR, Northern blot, immunofluorescence, and flow cytometry assays. Larvae could transmit virus once they acquired virus by feeding on virus-infected fungal colony. Offspring larvae hatched from viruliferous eggs were virus carriers and could also successfully transmit virus. Virus transmission between insect and fungus also occurred on rapeseed plants. Virus-infected isolates produced less repellent volatile substances to attract adults of L. ingenua. Furthermore, L. ingenua was easily observed on Sclerotinia lesions in rapeseed fields, and viruliferous adults were captured from fields either sprayed with a virus-infected fungal strain or nonsprayed. Our findings may facilitate the exploration of mycoviruses for control of fungal diseases and enhance our understanding of the ecology of SsHADV-1 and other newly emerging SsHADV-1-like viruses, which were recently found to be widespread in various niches including human HIV-infected blood, human and animal feces, insects, plants, and even sewage. [ABSTRACT FROM AUTHOR]

Although segmented negative-sense RNA viruses (SNSRVs) have been frequently discovered in various fungi, most SNSRVs reported only the large segments. In this study, we investigated the diversity of the mycoviruses in the phytopathogenic fungus Fusarium asiaticum using the metatranscriptomic technique. We identified 17 fungal single-stranded RNA (ssRNA) viruses including nine viruses within Mitoviridae, one each in Narnaviridae, Botourmiaviridae, Hypoviridae, Fusariviridae, and Narliviridae, two in Mymonaviridae, and one trisegmented virus temporarily named Fusarium asiaticum mycobunyavirus 1 (FaMBV1). The FaMBV1 genome comprises three RNA segments, large (L), medium (M), and small (S) with 6,468, 2,639, and 1,420 nucleotides, respectively. These L, M, and S segments putatively encode the L protein, glycoprotein, and nucleocapsid, respectively. Phylogenetic analysis based on the L protein showed that FaMBV1 is phylogenetically clustered with Alternaria tenuissima negative-stranded RNA virus 2 (AtNSRV2) and Sclerotinia sclerotiorum negative-stranded RNA virus 5 (SsNSRV5) but distantly related to the members of the family Phenuiviridae. FaMBV1 could be vertically transmitted by asexual spores with lower efficiency (16.7%, 2/42). Comparison between FaMBV1-free and -infected fungal strains revealed that FaMBV1 has little effect on hyphal growth, pathogenicity, and conidium production, and its M segment is dispensable for viral replication and lost during subculture and asexual conidiation. The M and S segments of AtNSRV2 and SsNSRV5 were found using bioinformatics methods, indicating that the two fungal NSRVs harbor trisegmented genomes. Our results provide a new example of the existence and evolution of the segmented negative-sense RNA viruses in fungi. IMPORTANCE Fungal segmented negative-sense RNA viruses (SNSRVs) have been frequently found. Only the large segment encoding RNA-dependent RNA polymerase (RdRp) has been reported in most fungal SNSRVs, except for a few fungal SNSRVs reported to encode nucleocapsids, nonstructural proteins, or movement proteins. Virome analysis of the Fusarium spp. that cause Fusarium head blight discovered a novel virus, Fusarium asiaticum mycobunyavirus 1 (FaMBV1), representing a novel lineage of the family Phenuiviridae. FaMBV1 harbors a trisegmented genome that putatively encodes RdRp, glycoproteins, and nucleocapsids. The putative glycoprotein was first described in fungal SNSRVs and shared homology with glycoprotein of animal phenuivirus but was dispensable for its replication in F. asiaticum. Two other trisegmented fungal SNSRVs that also encode glycoproteins were discovered, implying that three-segment bunyavirus infections may be common in fungi. These findings provide new insights into the ecology and evolution of SNSRVs, particularly those infecting fungi. [ABSTRACT FROM AUTHOR]

Sclerotinia sclerotiorum is a well-known phytopathogenic fungus with a wide host range. Identifying novel mycoviruses in phytopathogenic fungi is necessary to develop novel strategies for plant health protection and contribute to understanding the origin of viruses. Six new mycoviruses with positive single-stranded RNA genomes co-infecting the hypovirulent strain SCH733 of S. sclerotiorum were identified using a metatranscriptomic approach, and their complete genome sequences were molecularly determined. These mycoviruses belong to the following five families: Narnaviridae, Mitoviridae, Deltaflexviridae, Botourmiaviridae, and Ambiguiviridae. Three of these mycoviruses belong toexisting International Committee on Taxonomy of Viruses (ICTV)-recognized species. Two of these newly identified mycoviruses have unique genomic features that are significantly different from those of all known mycoviruses. Phylogenetic analysis revealed that these six mycoviruses included close as well as distant relatives of known mycoviruses, thereby providing new insight into virus evolution and classification. Mycovirus horizontal transmission and elimination experiments revealed that Sclerotinia sclerotiorum narnavirus 5 is associated with hypovirulence of S. sclerotiorum, although we have not shown that it is independently responsible for the hypovirulence phenotype. This study broadens the diversity of known mycoviruses infecting 5. sclerotiorum and provides a clue toward limiting hypovirulence in 5. sclerotiorum. [ABSTRACT FROM AUTHOR]

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 (1ssRNA) 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. [ABSTRACT FROM AUTHOR]

Rhabdoviruses are ubiquitous and diverse viruses that propagate owing to bidirectional interactions with their vertebrate, arthropod, and plant hosts, and some of them could pose global health or agricultural threats. However, rhabdoviruses have rarely been reported in fungi. Here, two newly identified fungal rhabdoviruses, Rhizoctonia solani rhabdovirus 1 (RsRhV1) and RsRhV2, were discovered and molecularly characterized from the phytopathogenic fungus Rhizoctonia solani. The genomic organizations of RsRhV1 and RsRhV2 are 11,716 and 11,496 nucleotides (nt) in length, respectively, and consist of five open reading frames (ORFs) (ORFs I to V). ORF I, ORF IV, and ORF V encode the viral nucleocapsid (N), glycoprotein (G), and RNA polymerase (L), respectively. The putative protein encoded by ORF III has a lower level of identity with the matrix protein of rhabdoviruses. ORF II encodes a hypothetical protein with unknown function. Phylogenetic trees based on multiple alignments of N, L, and G proteins revealed that RsRhV1 and RsRhV2 are new members of the family Rhabdoviridae, but they form an independent evolutionary branch significantly distinct from other known nonfungal rhabdoviruses, suggesting that they represent a novel viral evolutionary lineage within Rhabdoviridae. Compared to strains lacking rhabdoviruses, strains harboring RsRhV2 and RsRhV1 showed hypervirulence, suggesting that RsRhV1 and RsRhV2 might be associated with the virulence of R. solani. Taken together, this study enriches our understanding of the diversity and host range of rhabdoviruses. [ABSTRACT FROM AUTHOR]

Mycoviruses are widespread in nature and often occur with dsRNA and positive-stranded RNA genomes. Recently, strong evidence from RNA sequencing analysis suggested that negative-stranded (-)ssRNA viruses could infect fungi. Here we describe a (-)ssRNA virus, Sclerotinia sclerotiorum negative-stranded RNA virus 1 (SsNSRV-1), isolated from a hypovirulent strain of Sclerotinia sclerotiorum. The complete genome of SsNSRV-1 is 10,002 nt with six ORFs that are nonoverlapping and linearly arranged. Conserved gene-junction sequences that occur widely in mononegaviruses, (A/U)(U/A/C)UAUU (U/A)AA(U/G)AAAACUUAGG(A/U)(G/U), were identified between these ORFs. The analyses 5' and 3' rapid amplification of cDNA ends showed that all genes can be transcribed independently. ORF V encodes the largest protein that contains a conserved mononegaviral RNA-dependent RNA polymerase (RdRp) domain. Putative enveloped virion-like structures with filamentous morphology similar to members of Filoviridae were observed both in virion preparation samples and in ultrathin hyphal sections. The nudleocapsids are long, flexible, and helical; and are 22 nm in diameter and 200-2,000 nm in length. SDS/PAGE showed that the nucleocapsid possibly contains two nucleoproteins with different molecular masses, ~43 kDa (p43) and ~41 kDa (p41), and both are translated from ORF II. Purified SsNSRV-1 virions successfully transfected a virus-free strain of S. sclerotiorum and conferred hypovirulence. Phylogenetic analysis based on RdRp showed that SsNSRV-1 is clustered with viruses of Nyamiviridae and Bornaviridae. Moreover, SsNSRV-1 is widely distributed, as it has been detected in different regions of China. Our findings demonstrate that a (-)ssRNA virus can occur naturally in fungi and enhance our understanding of the ecology and evolution of (-)ssRNA viruses. [ABSTRACT FROM AUTHOR]

Coniothyrium minitans is a sclerotial parasite of the plant-pathogenic fungusSclerotinia sclerotiorum, and conidial production and parasitism are two important aspects for commercialization of this biological control agent. To understand the mechanism of conidiation and parasitism at the molecular level, we constructed a transfer DNA (tDNA) insertional library with the wild-type strain ZS-1. A conidiation-deficient mutant, ZS-1TN22803, was uncovered through screening of this library. This mutant could produce pycnidia on potato dextrose agar (PDA), but most were immature and did not bear conidia. Moreover, this mutant lost the ability to parasitize or rot the sclerotia of S. sclerotiorum. Analysis of the tDNA flanking sequences revealed that a peroxisome biogenesis factor 6 (PEX6) homolog of Saccharomyces cerevisiae, named CmPEX6, was disrupted by the tDNA insertion in this mutant. Targeted gene replacement and gene complementation tests confirmed that a null mutation of CmPEX6 was responsible for the phenotype of ZS-1TN22803. Further analysis showed that both ZS-1TN22803 and the targeted replacement mutants could not grow on PDA medium containing oleic acid, and they produced much less nitric oxide (NO) and hydrogen peroxide (H2O2) than wild-type strain ZS-1. The conidiation of ZS-1TN22803 was partially restored by adding acetyl-CoA or glyoxylic acid to the growth media. Our results suggest that fatty acid β-oxidation, reactive oxygen and nitrogen species, and possibly other unknown pathways in peroxisomes are involved in conidiation and parasitism by C. minitans. [ABSTRACT FROM AUTHOR]

The infection by a single-stranded DNA virus, Sclerotinia sclerotiorum hypovirulence- associated DNA virus 1 (SsHADV-1), causes hypovirulence, a reduced growth rate, and other colony morphological changes in its host Sclerotinia sclerotiorum strain DT-8. However, the mechanisms of the decline are still unclear. Using digital RNA sequencing, a transcriptome analysis was conducted to elucidate the phenotype-related genes with expression changes in response to SsHADV-1 infection. A total of 3110 S. sclerotiorum differentially expressed genes (DEGs) were detected during SsHADV-1 infection, 1741 of which were up-regulated, and 1369 were down-regulated. The identified DEGs were involved in several important pathways. DNA replication, DNA damage response, carbohydrate and lipid metabolism, ribosomal assembly, and translation were the affected categories in S. sclerotiorum upon SsHADV-1 infection. Moreover, the infection of SsHADV-1 also suppressed the expression of antiviral RNA silencing and virulence factor genes. These results provide further detailed insights into the effects of SsHADV-1 infection on the whole genome transcription in S. sclerotiorum. [ABSTRACT FROM AUTHOR]

This document is an erratum for an article titled "Discovery and Characterization of Putative Glycoprotein-Encoding Mycoviruses in the Bunyavirales" published in the Journal of Virology. The erratum corrects errors in the original article, including incorrect accession numbers for certain viruses and a typo in the description of a conserved domain. The document also provides the ORCID of one of the authors and includes a link to the original article. [Extracted from the article]

Coniothyrium minitans is an important mycoparasite of the notorious phytopathogenic fungus Sclerotinia sclerotiorum. The mycoparasitism system of C. minitans/S. sclerotiorum is unique and important to probe fungi and fungal interaction. Here, we report a conidiation-deficient mutant ZS-1TN1961 which was screened from a T-DNA insertional library of C. minitans. A single copy gene, coding a protein with high sequence similarity to an altered inheritance of mitochondria protein 24 (Aim24) in yeast, was disrupted by T-DNA insertion in this mutant. Gene replacement and complementation experiments confirmed that mutants lacking CmAim24 exhibited significantly reduced conidial production and germination as well as sclerotial mycoparasitic ability. Furthermore, cellular localization assays showed that CmAim24 localized to mitochondria and abnormal mitochondria were observed in ΔCmAim24. ΔCmAim24 exhibited significant accumulation of reactive oxygen species (ROS) and a reduced ATP content in mycelia. In summary, our results suggest that CmAim24 plays a key role in mitochondrial architecture and function, conidiogenesis and mycoparasitism in C. minitans. [ABSTRACT FROM AUTHOR]

Mitogen-activated protein kinase (MAPK) cascades play a central role in cellular growth, proliferation, and survival. MAPK cascade genes have been extensively investigated in model plants, mammals, yeast, and fungi but are not characterized in Plasmodiophora brassicae, which causes clubroot disease in cruciferous plants. Here, we identified 7 PbMAPK, 3 PbMAPKK, and 9 PbMAPKKK genes in the P. brassicae genome. Transcriptional profiling analysis demonstrated that several MAPK, MAPK kinase (MAPKK), and MAPK kinase kinase (MAPKKK) genes were preferentially expressed in three different zoosporic stages. Based on yeast two-hybrid assays, PbMAKKK7 interacted with PbMAKK3 and PbMAKK3 interacted with PbMAKlI PbMAK3. The PbMAKKK7-PbMAKK3-PbMAKl/PbMAK3 cascade may be present in P. brassicae. U0126, a potent and specific inhibitor of MAPKK, could inhibit the germination of P. brassicae resting spores. U0126 was used to treat the resting spores of P. brassicae and coinoculate rapeseed, and was proven to significantly relieve the severity of clubroot symptoms in the host plant and delay the life cycle of P. brassicae. These results suggest that MAPK signaling pathways may play important roles in P. brassicae growth, development, and pathogenicity. [ABSTRACT FROM AUTHOR]

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: Abisegmented 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. [ABSTRACT FROM AUTHOR]

Mycoviruses have been detected in all major groups of filamentous fungi, and their study represents an important branch of virology. Here, we characterized a novel double-stranded RNA (dsRNA) mycovirus, Sclerotinia sclerotiorum megabirnavirus 1 (SsMBV1), in an apparently hypovirulent strain (SX466) of Sclerotinia sclerotiorum. Two similarly sized dsRNA segments (L1- and L2-dsRNA), the genome of SsMBV1, are packaged in rigid spherical particles purified from strain SX466. The full-length cDNA sequence of L1-dsRNA/SsMBV1 comprises two large open reading frames (ORF1 and ORF2), which encode a putative coat protein and an RNA-dependent RNA polymerase (RdRp), respectively. Phylogenetic analysis of the RdRp domain clearly indicates that SsMBV1 is related to Rosellinia necatrix megabirnavirus 1 (RnMBV1). L2-dsRNA/SsMBV1 comprises two nonoverlapping ORFs (ORFA and ORFB) encoding two hypothetical proteins with unknown functions. The 5'-terminal regions of L1- and L2-dsRNA/SsMBV1 share strictly conserved sequences and form stable stem-loop structures. Although L2-dsRNA/SsMBV1 is dispensable for replication, genome packaging, and pathogenicity of SsMBV1, it enhances transcript accumulation of L1- dsRNA/SsMBV1 and stability of virus-like particles (VLPs). Interestingly, a conserved papain-like protease domain similar to a multifunctional protein (p29) of Cryphonectria hypovirus 1 was detected in the ORFA-encoded protein of L2-dsRNA/SsMBV1. Phylogenetic analysis based on the protease domain suggests that horizontal gene transfer may have occurred from a singlestranded RNA (ssRNA) virus (hypovirus) to a dsRNA virus, SsMBV1. Our results reveal that SsMBV1 has a slight impact on the fundamental biological characteristics of its host regardless of the presence or absence of L2-dsRNA/SsMBV1. [ABSTRACT FROM AUTHOR]

Mycoviruses are thought not to be infectious as free particles and to lack an extracellular phase in their life cycles, limiting the broad use of hypovirulence-associated mycoviruses in controlling fungal disease. Here, we demonstrate that purified particles of a DNA mycovirus, Sclerotinia sclerotiorum hypovirulence-associated DNA virus 1 (SsHADV-1), are infectious when applied extracellularly to its host Sclerotinia sclerotiorum. Virus particles isolated from an infected host can infect the hyphae of virus-free S. sclerotiorum directly when applied to hyphae grown on potato dextrose agar or sprayed on leaves of Arabidopsis thaliana and Brassica napus, regardless of vegetative compatibility affiliation. When applied to leaves, the virus can suppress the development of lesions. SsHADV-1 can also reduce disease severity and enhance rapeseed yield significantly under field conditions. SsHADV-1 has a narrow host range; it can infect Sclerotinia minor and Sclerotinia nivalis, sister species of S. sclerotiorum, and cause debilitation of these two fungi, but cannot infect or transfect other tested fungi, such as Botrytis cinerea, which shares the same family with S. sclerotiorum. Virus particles are likely to be very stable on the leaves of A. thaliana plants because viral DNA could be detected at 15 d postinoculation on unwounded leaves and at 10 d postinoculation on wounded leaves, respectively; however, this virus could not infect and move in plant cells. Our findings may prompt a reconsideration of the generalization that mycoviruses lack an extracellular phase in their life cycles and stimulate the search for other DNA mycoviruses with potential use as natural fungicides. [ABSTRACT FROM AUTHOR]

SSITL (SS1G_14133) of Sclerotinia sclerotiorum encodes a protein with 302 amino acid residues including a signal peptide, its secretion property was confirmed with immunolocalization and immunofluorescence techniques. SSITL was classified in the integrin alpha N-terminal domain superfamily, and its 3D structure is similar to those of human integrin α4-subunit and a fungal integrin-like protein. When S. sclerotiorum was inoculated to its host, high expression of SSITL was detected during the initial stages of infection (1.5-3.0 hpi). Targeted silencing of SSITL resulted in a significant reduction in virulence; on the other hand, inoculation of SSITL silenced transformant A10 initiated strong and rapid defense response in Arabidopsis, the highest expressions of defense genes PDF1.2 and PR-1 appeared at 3 hpi which was 9 hr earlier than that time when plants were inoculated with the wild-type strain of S. sclerotiorum. Systemic resistance induced by A10 was detected by analysis of the expression of PDF1.2 and PR-1, and confirmed following inoculation with Botrytis cinerea. A10 induced much larger lesions on Arabidopsis mutant ein2 and jar1, and slightly larger lesions on mutant pad4 and NahG in comparison with the wild-type plants. Furthermore, both transient and constitutive expression of SSITL in Arabidopsis suppressed the expression of PDF1.2 and led to be more susceptible to A10 and the wild-type strain of S. sclerotiorum and B. cinerea. Our results suggested that SSITL is an effector possibly and plays significant role in the suppression of jasmonic/ethylene (JA/ET) signal pathway mediated resistance at the early stage of infection. [ABSTRACT FROM AUTHOR]

Genome sequence of viruses can contribute greatly to the study of viral evolution, diversity and the interaction between viruses and hosts. Traditional molecular cloning methods for obtaining RNA viral genomes are time-consuming and often difficult because many viruses occur in extremely low titers. DsRNA viruses in the families, Partitiviridae, Totiviridae, Endornaviridae, Chrysoviridae, and other related unclassified dsRNA viruses are generally associated with symptomless or persistent infections of their hosts. These characteristics indicate that samples or materials derived from eukaryotic organisms used to construct cDNA libraries and EST sequencing might carry these viruses, which were not easily detected by the researchers. Therefore, the EST databases may include numerous unknown viral sequences. In this study, we performed in silico cloning, a procedure for obtaining full or partial cDNA sequence of a gene by bioinformatics analysis, using known dsRNA viral sequences as queries to search against NCBI Expressed Sequence Tag (EST) database. From this analysis, we obtained 119 novel virus-like sequences related to members of the families, Endornaviridae, Chrysoviridae, Partitiviridae, and Totiviridae. Many of them were identified in cDNA libraries of eukaryotic lineages, which were not known to be hosts for these viruses. Furthermore, comprehensive phylogenetic analysis of these newly discovered virus-like sequences with known dsRNA viruses revealed that these dsRNA viruses may have co-evolved with respective host supergroups over a long evolutionary time while potential horizontal transmissions of viruses between different host supergroups also is possible. We also found that some of the plant partitiviruses may have originated from fungal viruses by horizontal transmissions. These findings extend our knowledge of the diversity and possible host range of dsRNA viruses and offer insight into the origin and evolution of relevant viruses with their hosts. [ABSTRACT FROM AUTHOR]

The sclerotium is an important dormant body for many plant fungal pathogens. Here, we reported that a protein, named Ss- Sl2, is involved in sclerotial development of Sclerotinia sclerotiorum. Ss-Sl2 does not show significant homology with any protein of known function. Ss-Sl2 contains two putative PAN modules which were found in other proteins with diverse adhesion functions. Ss-Sl2 is a secreted protein, during the initial stage of sclerotial development, copious amounts of Ss-Sl2 are secreted and accumulated on the cell walls. The ability to maintain the cellular integrity of RNAi-mediated Ss-Sl2 silenced strains was reduced, but the hyphal growth and virulence of Ss-Sl2 silenced strains were not significantly different from the wild strain. Ss-Sl2 silenced strains could form interwoven hyphal masses at the initial stage of sclerotial development, but the interwoven hyphae could not consolidate and melanize. Hyphae in these interwoven bodies were thin-walled, and arranged loosely. Co-immunoprecipitation and yeast two-hybrid experiments showed that glyceraldehyde-3-phosphate dehydrogenase (GAPDH), Woronin body major protein (Hex1) and elongation factor 1-alpha interact with Ss-Sl2. GAPDHknockdown strains showed a similar phenotype in sclerotial development as Ss-Sl2 silenced strains. Hex1-knockdown strains showed similar impairment in maintenance of hyphal integrity as Ss-Sl2 silenced strains. The results suggested that Ss-Sl2 functions in both sclerotial development and cellular integrity of S. sclerotiorum. [ABSTRACT FROM AUTHOR]

Background: Double-stranded (ds) RNA fungal viruses are typically isometric single-shelled particles that are classified into three families, Totiviridae, Partitiviridae and Chrysoviridae, the members of which possess monopartite, bipartite and quadripartite genomes, respectively. Recent findings revealed that mycovirus-related dsRNA viruses are more diverse than previously recognized. Although an increasing number of viral complete genomic sequences have become available, the evolution of these diverse dsRNA viruses remains to be clarified. This is particularly so since there is little evidence for horizontal gene transfer (HGT) among dsRNA viruses. Results: In this study, we report the molecular properties of two novel dsRNA mycoviruses that were isolated from a field strain of Sclerotinia sclerotiorum, Sunf-M: one is a large monopartite virus representing a distinct evolutionary lineage of dsRNA viruses; the other is a new member of the family Partitiviridae. Comprehensive phylogenetic analysis and genome comparison revealed that there are at least ten monopartite, three bipartite, one tripartite and three quadripartite lineages in the known dsRNA mycoviruses and that the multipartite lineages have possibly evolved from different monopartite dsRNA viruses. Moreover, we found that homologs of the S7 Domain, characteristic of members of the genus phytoreovirus in family Reoviridae are widely distributed in diverse dsRNA viral lineages, including chrysoviruses, endornaviruses and some unclassified dsRNA mycoviruses. We further provided evidence that multiple HGT events may have occurred among these dsRNA viruses from different families. Conclusions: Our study provides an insight into the phylogeny and evolution of mycovirus-related dsRNA viruses and reveals that the occurrence of HGT between different virus species and the development of multipartite genomes during evolution are important macroevolutionary mechanisms in dsRNA viruses. [ABSTRACT FROM AUTHOR]

Horizontal gene transfer commonly occurs from cells to viruses but rarely occurs from viruses to their host cells, with the exception of retroviruses and some DNA viruses. However, extensive sequence similarity searches in public genome databases for various organisms showed that the capsid protein and RNA-dependent RNA polymerase genes from totiviruses and partitiviruses have widespread homologs in the nuclear genomes of eukaryotic organisms, including plants, arthropods, fungi, nematodes, and protozoa. PCR amplification and sequencing as well as comparative evidence of junction coverage between virus and host sequences support the conclusion that these viral homologs are real and occur in eukaryotic genomes. Sequence comparison and phylogenetic analysis suggest that these genes were likely transferred horizontally from viruses to eukaryotic genomes. Furthermore, we present evidence showing that some of the transferred genes are conserved and expressed in eukaryotic organisms and suggesting that these viral genes are also functional in the recipient genomes. Our findings imply that horizontal transfer of double-stranded RNA viral genes is widespread among eukaryotes and may give rise to functionally important new genes, thus entailing that RNA viruses may play significant roles in the evolution of eukaryotes. [ABSTRACT FROM AUTHOR]

Mycoviruses are viruses that infect fungi and have the potential to control fungal diseases of crops when associated with hypovirulence. Typically, mycoviruses have double-stranded (ds) or single-stranded (ss) RNA genomes. No mycoviruses with DNA genomes have previously been reported. Here, we describe a hypovirulence- associated circular ssDNA mycovirus from the plant pathogenic fungus Scierotinia sclerotiorum. The genome of this ssDNA virus, named Sclerotinia sclerotiorum hypovirulence-associated DNA virus 1 (SsHADV-1), is 2166 nt, coding for a replication initiation protein (Rep) and a coat protein (CP). Although phylogenetic analysis of Rep showed that SsHADV-1 is related to geminiviruses, it is notably distinct from geminiviruses both in genome organization and particle morphology. Polyethylene glycol-mediated transfection of fungal protoplasts was successful with either purified SsHADV-1 particles or viral DNA isolated directly from infected mycelium. The discovery of an ssDNA mycovirus enhances the potential of exploring fungal viruses as valuable tools for molecular manipulation of fungi and for plant disease control and expands our knowledge of global virus ecology and evolution. [ABSTRACT FROM AUTHOR]

Understanding signaling pathways that modulate conidiation of mitosporic fungi is of both practical and theoretical importance. The enzymatic origin of nitric oxide (NO) and its roles in conidiation by the sclerotial parasite Coniothyrium minitans were investigated. The activity of a nitric oxide synthase-like (NOS-like) enzyme was detected in C. minitans as evidenced by the conversion of L-arginine to L-citrulline. Guanylate cyclase (GC) activity was also detected indirectly in C. minitans with the GC-specific inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), which significantly reduced production of cyclic GMP (cGMP). The dynamics of NOS activity were closely mirrored by the cGMP levels during pycnidial development, with the highest levels of both occurring at the pycnidial initiation stage of C. minitans. Furthermore, the NO donor, sodium nitroprusside (SNP), stimulated the accumulation of cGMP almost instantly in mycelium during the hyphal growth stage. When the activity of NOS or GC was inhibited with Nω-nitro-L-arginine or ODQ, conidial production of C. minitans was suppressed or completely eliminated; however, the suppression of conidiation by ODQ could be reversed by exogenous cGMP. The results also showed that conidiation of an L-arginine auxotroph could be restored by the NO donor SNP, but not by cGMP. Thus, NO-mediated conidiation has more than one signal pathway, including the cGMP signal pathway and another yet-unknown pathway, and both are essential for conidiation in C. minitans. [ABSTRACT FROM AUTHOR]