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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]

Coniothyrium minitans is an important mycoparasite of Sclerotinia sclerotiorum. In addition, it also produces small amounts of antifungal substances. ZS-1TN1812, an abnormal mutant, was originally screened from a T-DNA insertional library. This mutant showed abnormal growth phenotype and could significantly inhibit the growth of S. sclerotiorum when dual-cultured on a PDA plate. When spraying the filtrate of ZS-1TN1812 on the leaves of rapeseed, S. sclerotiorum infection was significantly inhibited, suggesting that the antifungal substances produced by this mutant were effective on rapeseed leaves. The thermo-tolerant antifungal substances could specifically suppress the growth of S. sclerotiorum, but could not significantly suppress the growth of another fungus, Colletotrichum higginsianum. However, C. higginsianum was more sensitive to proteinous antibiotics than S. sclerotiorum. The T-DNA insertion in ZS-1TN1812 activated the expression of CmSIT1, a gene involved in siderophore-mediated iron transport. It was also determined that mutant ZS-1TN1812 produced hypha with high iron levels. In the wild-type strain ZS-1, CmSIT1 was expressed only when in contact with S. sclerotiorum, and consistent overexpression of CmSIT1 showed similar phenotypes as ZS-1TN1812. Therefore, activated expression of CmSIT1 leads to the enhanced antifungal ability, and CmSIT1 is a potential gene for improving the control ability of C. minitans. [ABSTRACT FROM AUTHOR]

Clubroot disease is a serious threat to cruciferous plants worldwide, especially to oilseed rape. However, knowledge on pathogenic molecular mechanisms and host interaction is limited. We presume that the recognition between Arabidopsis thaliana and Plasmodiophora brassicae occurs at the early stage of infection and within a relatively short period. In this study, we demonstrated changes on gene expression and pathways in A. thaliana during early infection with P. brassicae using transcriptome analysis. We identified 1,903 and 1,359 DEGs at 24 and 48 h post-inoculation (hpi), respectively. Flavonoids and the lignin synthesis pathways were enhanced, glucosinolates, terpenoids, and proanthocyanidins accumulated and many hormonal- and receptor-kinase related genes were expressed, caused by P. brassicae infection during its early phase. Therefore, the early interaction between A. thaliana and P. brassicae plays an important role in the entire infection process. The results provide a new contribution to a better understanding of the interaction between host plants and P. brassicae, as well as the development of future measures for the prevention of clubroot. [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]

Botrytis-induced kinase1 (BIK1), a receptor-like cytoplasmic kinase, plays an important role in resistance against pathogens and insects in Arabidopsis thaliana. However, it remains unknown whether BIK1 functions against Plasmodiophora brassicae, an obligate biotrophic protist that attacks cruciferous plants and induces gall formation on roots. Here, we investigated the potential roles of receptors FLS2, BAK1, and BIK1 in the infection of P. brassicae cruciferous plants. Wild-type plants, fls2, and bak1 mutants showed typical symptom on roots, and the galls were filled with large quantities of resting spores, while bik1 mutant plants exhibited strong resistance to P. brassicae. Compared with that of the wild-type plants, the root hair and cortical infection rate of bik1 mutant were significantly reduced by about 40-50%. A considerable portion of bik1 roots failed to form typical galls. Even if some small galls were formed, they were filled with multinucleate secondary plasmodia. The bik1 plants accumulated less reactive oxygen species (ROS) at infected roots than other mutants and wild-type plants. Exogenous salicylic acid (SA) treatment alleviated the clubroot symptoms in wild-type plants, and the expression of the SA signaling marker gene PR1 was significantly increased in bik1. Both sid2 (salicylic acid induction-deficient 2) and npr1-1 [non-expresser of PR genes that regulate systemic acquired resistance (SAR)] mutants showed increased susceptibility to P. brassicae compared with wild-type plants. These results suggest that the resistance of bik1 to P. brassicae is possibly mediated by SA inducible mechanisms. [ABSTRACT FROM AUTHOR]

Histone methylation is widely present in animals, plants and fungi, and the methylation modification of histone H3 has important biological functions. Methylation of Lys9 of histone H3 (H3K9) has been proven to regulate chromatin structure, gene silencing, transcriptional activation, plant metabolism, and other processes. In this work, we investigated the functions of a H3K9 methyltransferase gene BcDIM5 in Botrytis cinerea, which contains a PreSET domain, a SET domain and a PostSET domain. Characterization of BcDIM5 knockout transformants showed that the hyphal growth rate and production of conidiophores and sclerotia were significantly reduced, while complementary transformation of BcDIM5 could restore the phenotypes to the levels of wild type. Pathogenicity assays revealed that BcDIM5 was essential for full virulence of B. cinerea. BcDIM5 knockout transformants exhibited decreased virulence, down-regulated expression of some pathogenic genes and drastically decreased H3K9 trimethylation level. However, knockout transformants of other two genes heterochromatin protein 1 (HP1) BcHP1 and DNA methyltransferase (DIM2) BcDIM2 did not exhibit significant change in the growth phenotype and virulence compared with the wild type. Our results indicate that H3K9 methyltransferase BcDIM5 is required for H3K9 trimethylation to regulate the development and virulence of B. cinerea. [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]

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]

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]

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]

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]

Background: Horizontal gene transfer, also called lateral gene transfer, frequently occurs among prokaryotic organisms, and is considered an important force in their evolution. However, there are relatively few reports of transfer to or from fungi, with some notable exceptions in the acquisition of prokaryotic genes. Some fungal species have been found to contain sequences resembling those of bacterial genes, and with such sequences absent in other fungal species, this has been interpreted as horizontal gene transfer. Similarly, a few fungi have been found to contain genes absent in close relatives but present in more distantly related taxa, and horizontal gene transfer has been invoked as a parsimonious explanation. There is a paucity of direct experimental evidence demonstrating the occurrence of horizontal gene transfer in fungi. Results: We found a fungal field isolate from rice (Oryzae sativa) that contains ribosomal DNA sequences from two species of fungal rice pathogens (Thanatephorus cucumeris and Ceratobasidium oryzae-sativae). This field isolate has four types of ribosomal DNA internal transcribed spacers (ITS), namely pure ITS of C. oryzae-sativae, which was dominant in this field isolate, pure ITS of T. cucumeris, and two chimeric ITS, with ITS1 derived from C. oryzae-sativae and ITS2 from T. cucumeris, or ITS1 from T. cucumeris and ITS2 from C. oryzae-sativae. The presence of chimeric forms indicates that the intergeneric hybrid was not merely composed of nuclei from the parental species, but that nuclear fusion and crossing over had taken place. Conclusion: Hyphae of T. cucumeris and C. oryzae-sativae are vegetatively incompatible, and do not successfully anastomose. However, they parasitize the same host, and perhaps under the influence of host enzymes targeted to weaken pathogen cells or in dying host plant tissue, the fungal hyphae lost their integrity, and normal vegetative incompatibility mechanisms were overcome, allowing the hyphae to fuse. Based on the presence of other similarly anomalous isolates from the field, we speculate that these types of intergeneric hybridization events and occurrences of horizontal gene transfer may not be so rare in the field. [ABSTRACT FROM AUTHOR]