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Plants recognize microbe-associated molecular patterns (MAMPs) to activate immune responses and defense priming to defend against pathogen infections. Transcriptional regulation of gene expression is crucial for plant immunity and is mediated by multiple factors, including DNA methylation. However, it remains unknown whether and how DNA demethylation contributes to immune responses in MAMP-triggered immunity. Here, we report that active DNA demethylation is required for MAMP-triggered immunity to bacterial pathogens. The rdd-2 triple mutant carrying mutations in ROS1, DML2, and DML3 that encode DNA glycosylases, which are key DNA demethylation enzymes, exhibits compromised immune responses triggered by the MAMPs flg22 and elf18. Genome-wide methylome analysis reveals that flg22 induces rapid and specific DNA demethylation in an RDD-dependent manner. The expression levels of salicylic acid signaling-related and phytoalexin biosynthesis-related genes are tightly associated with the flg22-induced promoter demethylation. The compromised accumulation of priming compounds and antimicrobial metabolites ultimately leads to a defense priming defect in the rdd-2 mutant. Our results reveal the critical role of active DNA demethylation in the MAMP-triggered immune response and provide unique insight into the molecular mechanism of flg22-modulated DNA demethylation.

Fungal pathogens are seriously threatening food security and natural ecosystems; efficient and environmentally friendly control methods are essential to increase world crop production. S. sclerotiorum , a widespread pathogen of dicotyledons, can grow endophytically in wheat, providing protection against Fusarium head blight and stripe rust and enhancing wheat yield. In this study, we discovered that S. sclerotiorum treatment increased the diversity of the soil fungal and bacterial community in rhizosphere soil, while the diversity of the fungal community was obviously decreased in the wheat root.

Sclerotinia sclerotiorum is an economically important necrotrophic plant pathogen and mainly applies cell wall-degrading enzymes and oxalic acid to kill plant cells before colonization. In this research, we characterized a glycosylphosphatidylinositol (GPI)-anchored cell wall protein named SsGsr1, which is critical for the cell wall architecture and the pathogenicity of S. sclerotiorum .

Long noncoding RNAs (lncRNAs) are crucial regulators of gene expression in many biological processes, but their biological functions remain largely unknown, especially in fungi. Fusarium graminearum is an important pathogen that causes the destructive disease Fusarium head blight (FHB) or head scab disease on wheat and barley. In our previous RNA sequencing (RNA‐Seq) study, we discovered that lncRsp1 is an lncRNA that is located +99 bp upstream of a putative sugar transporter gene, Fgsp1, with the same transcription direction. Functional studies revealed that ΔlncRsp1 and ΔFgsp1 were normal in growth and conidiation but had defects in ascospore discharge and virulence on wheat coleoptiles. Moreover, lncRsp1 and Fgsp1 were shown to negatively regulate the expression of several deoxynivalenol (DON) biosynthesis genes, TRI4, TRI5, TRI6, and TRI13, as well as DON production. Further analysis showed that the overexpression of lncRsp1 enhanced the ability of ascospore release and increased the mRNA expression level of the Fgsp1 gene, while lncRsp1‐silenced strains reduced ascospore discharge and inhibited Fgsp1 expression during the sexual reproduction stage. In addition, the lncRsp1 complementary strains lncRsp1‐LC‐1 and lncRsp1‐LC‐2 restored ascospore discharge to the level of the wild‐type strain PH‐1. Taken together, our results reveal the distinct and specific functions of lncRsp1 and Fgsp1 in F. graminearum and principally demonstrate that lncRsp1 can affect the release of ascospores by regulating the expression of Fgsp1., lncRsp1, a long noncoding RNA located +99 bp upstream of a putative sugar transporter gene Fgsp1, can affect the release of ascospores by regulating the expression of Fgsp1 in Fusarium graminearum.

Sclerotinia sclerotiorum ourmiavirus 17 (SsOV17) was isolated from the hypovirulent strain GF3 of Sclerotinia sclerotiorum. The genome of SsOV17 is 2,802 nt in length and contains a single long open reading frame (ORF) flanked by a short structured 5'-untranslated region (5'-UTR) (28 nt) and a long 3'-UTR (788 nt), respectively. The ORF encodes a protein with 663 amino acids and a predicted molecular mass of 75.0 kDa. A BLASTp search indicated that the protein encoded by SsOV17 is closely related to the putative RNA-dependent RNA polymerase (RdRp) of Sclerotinia sclerotiorum ourmiavirus 13 (71% identity). A multiple sequence alignment indicated that eight conserved amino acid motifs were present in the RdRp conserved region of SsOV17. Phylogenetic analysis demonstrated that SsOV17 clustered with members of the genus Botoulivirus.

Sclerotinia sclerotiorum and Botrytis cinerea are typical necrotrophic pathogens that can attack more than 700 and 3000 plant species, respectively, and cause huge economic losses across numerous crops. In particular, the absence of resistant cultivars makes the stem rot because of S. sclerotiorum the major threat of rapeseed (Brassica napus) worldwide along with Botrytis. Previously, we identified an effector‐like protein (SsSSVP1) from S. sclerotiorum and a homologue of SsSSVP1 on B. cinerea genome and found that SsSSVP1 could interact with BnQCR8 of rapeseed, a subunit of the cytochrome b‐c1 complex. In this study, we found that BnQCR8 has eight homologous copies in rapeseed cultivar Westar and reduced the copy number of BnQCR8 using CRISPR/Cas9 to improve rapeseed resistance against S. sclerotiorum. Mutants with one or more copies of BnQCR8 edited showed strong resistance against S. sclerotiorum and B. cinerea. BnQCR8‐edited mutants did not show significant difference from Westar in terms of respiration and agronomic traits tested, including the plant shape, flowering time, silique size, seed number, thousand seed weight and seed oil content. These traits make it possible to use these mutants directly for commercial production. Our study highlights a common gene for breeding of rapeseed to unravel the key hindrance of rapeseed production caused by S. sclerotiorum and B. cinerea. In contrast to previously established methodologies, our findings provide a novel strategy to develop crops with high resistance against multiple pathogens by editing only a single gene that encodes the common target of pathogen effectors.

Over 110 million tons of nitrogen fertilizer every year is used for crop production. Scientists have dreamed of enabling rhizobial nitrogen fixation in non-leguminous crops to mitigate the increasing demand for nitrogen fertilizer. However, despite decades of research, rhizobial nitrogen fixation in non-host plants has not been demonstrated. Here, we reported that an N-fixing rhizobium and a clubroot pathogen Plasmodiophora brassicae exhibited a synergistic effect on fixing nitrogen in cruciferous plants. Rhizobia were found to invade P. brassicae-infected rapeseed (Brassica napus) roots in the field. The colonization of rhizobium on rapeseed roots was confirmed by co-inoculating Mesorhizobium huakuii with P. brassicae under controlled laboratory conditions. M. huakuii infection could alleviate clubroot symptoms and promote the growth of diseased rapeseeds. M. huakuii could fix nitrogen in P. brassicae-infected plants based on the results of 15N isotope dilution tests. The expression of homologs of legume genes required for symbiosis and early-nodulin genes was significantly upregulated in Arabidopsis during early infection by P. brassicae. More importantly, M. huakuii could even fix nitrogen in P. brassicae-resistant rapeseed cultivar and promote plant growth when co-inoculated with P. brassicae. Our findings provide a new avenue to understand the interaction of rhizobia with non-host plants, stimulate the exploration of fixing nitrogen in non-leguminous plants by nitrogen-fixing rhizobia, and develop a strategy for both disease control and nitrogen fixation on non-host crops.

Sclerotial development is a vital stage in the life cycles of many fungal plant pathogens. In this study, the protein Ss-CAD, which contains three conserved domains of cinnamyl alcohol dehydrogenase (CAD), was found to be required for sclerotial development in Sclerotinia sclerotiorum. Ss-CAD was significantly upregulated during early stage of sclerotial development. In Ss-CAD-silenced strains, sclerotial development was abnormal. In these silenced strains, formation of sclerotia was delayed or sclerotia yield was reduced, whereas hyphal growth and virulence were normal. Nox1, Nox2, and NoxR, which encode reactive oxygen species (ROS)-generating NADPH oxidases, were downregulated in Ss-CAD-silenced strains. NoxR-silenced strains displayed similar defects during sclerotial formation as Ss-CAD-silenced strains. Treatment of Ss-CAD-silenced strains with exogenous oxidants or NADPH restored normal sclerotial development. Sclerogenesis in Ss-CAD-silenced strains could also be recovered through Nox1 overexpression. The results suggest that Ss-CAD is linked to the NADPH oxidase pathways to affect sclerotial development in S. sclerotiorum.

The symbiosis of endophytes and plants is universal in nature. However, how endophytes grow in plants is not entirely clear. Previously, we reported that a virus-infected fungal pathogen could grow in plants as an endophyte. In this study, we utilized Sclerotinia sclerotiorum strain DT-8, a virus-mediated endophyte, to investigate the mechanism of symbiosis with rapeseed by dual unique molecular identifier-RNA sequencing (dual-UMI RNA-seq). We found that the expressions of genes encoding S. sclerotiorum amylase/glucoamylase, glucose transporters, and rapeseed s ugars w ill e ventually be e xported t ransporter 11 (SWEET11) were upregulated. It suggested that strain DT-8 might utilize plant starch as a nutrient. The defense systems of rapeseed were also activated, such as production of reactive oxygen species, phenylpropanoids, and brassinin, to control the growth of strain DT-8, while strain DT-8 counteracted host suppression by producing effector-like proteins, detoxification enzymes, and antioxidant components. Moreover, rapeseed also upregulated pectate lyase and pectinesterase genes to facilitate the colonization by strain DT-8. Our findings provide novel insights into the interaction of virus-mediated endophytes and their hosts that warrant further study. IMPORTANCE Although endophytes are widespread in nature, the interactions between endophytes and their hosts are still not fully understood. Members of a unique class of endophytes, the virus-mediated endophytic fungi, are continuously being discovered and have received wide attention. In this study, we investigated the interaction between a mycovirus-mediated endophytic fungus and its host rapeseed by using dual-UMI RNA-seq. According to the dual-UMI RNA-seq results, an aerial view of symbiotic mechanism under balanced regulation was suggested. This research expands our understanding of the symbiotic mechanisms of virus-fungus-plant interactions and could establish a foundation for the further development of practical application with virus-mediated hypovirulent fungi.

A novel positive single-stranded RNA virus, Sclerotinia sclerotiorum hypovirus 9 (SsHV9), was identified in the plant-pathogenic Sclerotinia sclerotiorum strain GB375, which was associated with a garden bean plant in the United States. The complete genome of SsHV9 is 14,067 nucleotides in length, excluding the poly(A) tail. It has a single large open reading frame encoding a putative polyprotein (4,196 amino acids), which is predicted to contain a papain-like protease, a protein of unknown function, an RNA-dependent RNA polymerase, and an RNA helicase. Phylogenetic analysis based on a multiple alignment of amino acid sequences of polyproteins that suggested SsHV9 belongs to the proposed genus "Alphahypovirus" in the family Hypoviridae.

Mycoviruses are an important component of the virosphere, but our current knowledge of their genome organization diversity and evolution remains rudimentary. In this study, the mycovirus composition in a hypovirulent strain of Sclerotinia sclerotiorum was molecularly characterized. Nine mycoviruses were identified and assigned into eight potential families. Of them, six were close relatives of known mycoviruses, while the other three had unique genome organizations and evolutionary positions. A deltaflexivirus with a tripartite genome has evolved via arrangement and horizontal gene transfer events, which could be an evolutionary connection from unsegmented to segmented RNA viruses. Two mycoviruses had acquired a second helicase gene by two different evolutionary mechanisms. A rhabdovirus representing an independent viral evolutionary branch was the first to be confirmed to occur naturally in fungi. The major hypovirulence-associated factor, an endornavirus, was finally corroborated. Our study expands the diversity of mycoviruses and potential virocontrol agents, and also provides new insights into virus evolutionary modes including virus genome segmentation., Author summary Identification of mycoviruses in phytopathogenic fungi is necessary for understanding the origin of viruses and developing virocontrol strategies to protect plants. Nine mycoviruses with RNA genomes were identified in a hypovirulent strain of Sclerotinia sclerotiorum and were classified into eight potential viral families, suggesting that the composition of mycoviral communities was complex in this single fungal strain. They included four previously characterized mycoviruses and three distant relatives of known mycoviruses, as well as the first reports of a deltaflexivirus with a tripartite genome, and a fungal rhabdovirus. In addition, we found an endornavirus associated with hypovirulence in a phytopathogenic fungus. Our study makes a significant contribution because it not only expands the diversity-related knowledge of mycoviruses and potential virocontrol agents, but also provides new insights into mycovirus evolution.

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.

Plasmodiophora brassicae is an important biotrophic eukaryotic plant pathogen and a member of the rhizarian protists. This biotrophic pathogen causes clubroot in cruciferous plants via novel intracellular mechanisms that are markedly different from those of other biotrophic organisms. To date, genomes from six single spore isolates of P. brassicae have been sequenced. An accurate description of the evolutionary status of this biotrophic protist, however, remains lacking. Here, we determined the draft genome of the P. brassicae ZJ-1 strain. A total of 10,951 protein-coding genes were identified from a 24.1 Mb genome sequence. We applied a comparative genomics approach to prove the Rhizaria supergroup is an independent branch in the eukaryotic evolutionary tree. We also found that the GPCR signaling pathway, the versatile signal transduction to multiple intracellular signaling cascades in response to extracellular signals in eukaryotes, is significantly enriched in P. brassicae-expanded and P. brassicae-specific gene sets. Additionally, treatment with a GPCR inhibitor relieved the symptoms of clubroot and significantly suppressed the development of plasmodia. Our findings suggest that GPCR signal transduction pathways play important roles in the growth, development, and pathogenicity of P. brassicae.

Mycovirus infection is a universal phenomenon in the major fungus groups. So far, however, mycoviruses have not been described in the phytopathogenic fungus Leptosphaeria biglobosa, the causal agent of phoma stem canker (blackleg). Here, we report the complete genome sequence of a novel mitovirus, Leptosphaeria biglobosa mitovirus 1 (LbMV1), isolated from the strain H3-38 of L. biglobosa. The LbMV1 genome comprises 2568 nucleotides with a low G+C content of 30%. Using the mitochondrial genetic code, the genome of LbMV1 was found to contain a single large open reading frame that encodes a predicted protein of 756 amino acids. Multiple alignment of the predicted protein sequence showed the highest similarity (51% identity) to the putative RNA-dependent RNA polymerase of Ophiostoma mitovirus 4. In addition, phylogenetic analysis grouped LbMV1 within the genus Mitovirus of the family Narnaviridae. Our results suggest that the double-stranded RNA found in strain H3-38 is the replicating genome of the mitovirus LbMV1, its deletion represents the first evidence of a mycovirus infecting L. biglobosa.

Background The eukaryotic unicellular protist Plasmodiophora brassicae is an endocellular parasite of cruciferous plants. In host cortical cells, this protist develops a unicellular structure that is termed the plasmodium. The plasmodium is actually a multinucleated cell, which subsequently splits and forms resting spores. The mechanism for the growth of this endocellular parasite in host cell is unclear. Results Here, combining de novo genome sequence and transcriptome analysis of strain ZJ-1, we identified top five significant enriched KEGG pathways of differentially expressed genes (DEGs), namely translation, cell growth and death, cell communication, cell motility and cancers. We detected 171 proto-oncogenes from the genome of P. brassicae that were implicated in cancer-related pathways, of which 46 were differential expression genes. Three predicted proto-oncogenes (Pb-Raf1, Pb-Raf2, and Pb-MYB), which showed homology to the human proto-oncogenes Raf and MYB, were specifically activated during the plasmodial growth in host cortical cells, demonstrating their involvement in the multinucleate development stage of the unicellular protist organism. Gene networks involved in the tumorigenic-related signaling transduction pathways and the activation of 12 core genes were identified. Inhibition of phosphoinositol-3-kinase relieved the clubroot symptom and significantly suppressed the development process of plasmodia. Conclusions Proto-oncogene-related regulatory mechanisms play an important role in the plasmodial growth of P. brassicae.

Nidovirales, which accommodates viruses with the largest RNA genomes, includes the notorious coronaviruses; however, the evolutionary route for nidoviruses is not well understood. We have characterized a positive-sense (+) single-stranded (ss) RNA mycovirus, Rhizoctonia solani hypovirus 2 (RsHV2), from the phytopathogenic fungus Rhizoctonia solani. RsHV2 has the largest RNA genome size of 22,219 nucleotides, excluding the poly(A) tail, in all known mycoviruses, and contains two open reading frames (ORF1 and ORF2). ORF1 encodes a protein of 2,009 amino acid (aa) that includes a conserved helicase domain belonging to helicase superfamily I (SFI). In contrast, ORF2 encodes a polyprotein of 4459 aa containing the hallmark genes of hypoviruses. The latter includes a helicase belonging to SFII. Following phylogenetic analysis, the ORF1-encoded helicase (Hel1) unexpectedly clustered in an independent evolutionary branch together with nidovirus helicases, including coronaviruses, and bacteria helicases. Thus, Hel1 presence indicates the occurrence of horizontal gene transfer between viruses and bacteria. These findings also suggest that RsHV2 is most likely a recombinant arising between hypoviruses and nidoviruses.

We previously identified Sclerotinia sclerotiorum negative-stranded virus 1 (SsNSRV-1), the first (&minus, ) ssRNA mycovirus, associated with hypovirulence of its fungal host Sclerotinia sclerotiorum. In this study, functional analysis of Open Reading Frame &Iota, (ORF &Iota, ) of SsNSRV-1 was performed. The integration and expression of ORF &Iota, led to defects in hyphal tips, vegetative growth, and virulence of the mutant strains of S. sclerotiorum. Further, differentially expressed genes (DEGs) responding to the expression of ORF &Iota, were identified by transcriptome analysis. In all, 686 DEGs consisted of 267 up-regulated genes and 419 down-regulated genes. DEGs reprogramed by ORF &Iota, were relevant to secretory proteins, pathogenicity, transcription, transmembrane transport, protein biosynthesis, modification, and metabolism. Alternative splicing was also detected in all mutant strains, but not in hypovirulent strain AH98, which was co-infected by SsNSRV-1 and Sclerotinia sclerotiorum hypovirus 1 (SsHV-1). Thus, the integrity of SsNSRV-1 genome may be necessary to protect viral mRNA from splicing and inactivation by the host. Taken together, the results suggested that protein ORF &Iota, could regulate the transcription, translation, and modification of host genes in order to facilitate viral proliferation and reduce the virulence of the host. Therefore, ORF &Iota, may be a potential gene used for the prevention of S. sclerotiorum.

Coniothyrium minitans is a mycoparasite of the notorious plant pathogen Sclerotinia sclerotiorum. To further understand the parasitism of C. minitans, we assembled and analysed its genome and performed transcriptome analyses. The genome of C. minitans strain ZS-1 was assembled into 350 scaffolds and had a size of 39.8 Mb. A total of 11 437 predicted genes and proteins were annotated, and 30.8 % of the blast hits matched proteins encoded by another member of the Pleosporales, Paraphaeosphaeria sporulosa, a worldwide soilborne fungus with biocontrol ability. The transcriptome of strain ZS-1 during the early interaction with S. sclerotiorum at 0, 4 and 12 h was analysed. The detected expressed genes were involved in responses to host defenses, including cell-wall-degrading enzymes, transporters, secretory proteins and secondary metabolite productions. Seventeen differentially expressed genes (DEGs) of fungal cell-wall-degrading enzymes (FCWDs) were up-regulated during parasitism, with only one down-regulated. Most of the monocarboxylate transporter genes of the major facilitator superfamily and all the detected ABC transporters, especially the heavy metal transporters, were significantly up-regulated. Approximately 8 % of the 11 437 proteins in C. minitans were predicted to be secretory proteins with catalytic activity. In the molecular function category, hydrolase activity, peptidase activity and serine hydrolase activity were enriched. Most genes involved in serine hydrolase activity were significantly up-regulated. This genomic analysis and genome-wide expression study demonstrates that the mycoparasitism process of C. minitans is complex and a broad range of proteins are deployed by C. minitans to successfully invade its host. Our study provides insights into the mechanisms of the mycoparasitism between C. minitans and S. sclerotiorum and identifies potential secondary metabolites from C. minitans for application as a biocontrol agent.

Mycoparasite Coniothyrium minitans parasitizes specifically the mycelia or sclerotia of Sclerotinia sclerotiorum, a worldwidely spread plant fungal pathogen causing serious diseases on crops. The interaction of C. minitans with S. sclerotiorum remains reciprocal and complex and little is known, especially on the side of the host (S. sclerotiorum). In this study, the early transcriptional response of S. sclerotiorum to the mycoparasitism by C. minitans was explored and the differentially expressed genes (DEGs) were analyzed. Based on GO ontology, KEGG pathway and fungal categories database, 887 up-regulated DEGs were enriched in the growth related function (i.e., rRNA processing, ribosome biogenesis, binding and transport), while the 546 down-regulated DEGs were enriched in the stress-related functions (i.e., oxidoreductase, response to stress and heat and the chorismate biosynthetic process). The expression of shikimate pathway and the biosynthesis of phenylalanine involving genes was significantly suppressed. Furthermore, 581 unenriched DEGs were explored in the parasitizing process and were mapped on the Pfam domains of redox enzymes, Alpha/Beta hydrolase, haloacid dehalogenase, and other universal conserved domain containing proteins. Thirty-two DEGs encoding candidate effectors, with 16 up-regulated and 16 down-regulated, were observed with diverse function. SS1G_11912 (encoding SsNEP2) was significantly up-regulated and may function in the parasitism. The involving of the shikimate pathway of phenylalanine biosynthesis and effector candidates were discussed. The results provide a basal understand on the interaction of S. sclerotiorum and C. minitans.

Background The eukaryotic unicellular protist Plasmodiophora brassicae is an endocellular parasite of cruciferous plants. In host cortical cells, this protist develops a unicellular structure that is termed the plasmodium. The plasmodium is actually a multinucleated cell, which subsequently splits and forms resting spores. The mechanism for the growth of this endocellular parasite in host cell is unclear. Results Here, combining de novo genome sequence and transcriptome analysis of strain ZJ-1, we identified top five significant enriched KEGG pathways of differentially expressed genes (DEGs), namely translation, cell growth and death, cell communication, cell motility and cancers. We detected 171 proto-oncogenes from the genome of P. brassicae that were implicated in cancer-related pathways, of which 46 were differential expression genes. Three predicted proto-oncogenes (Pb-Raf1, Pb-Raf2, and Pb-MYB), which showed homology to the human proto-oncogenes Raf and MYB, were specifically activated during the plasmodial growth in host cortical cells, demonstrating their involvement in the multinucleate development stage of the unicellular protist organism. Gene networks involved in the tumorigenic-related signaling transduction pathways and the activation of 12 core genes were identified. Inhibition of phosphoinositol-3-kinase relieved the clubroot symptom and significantly suppressed the development process of plasmodia. Conclusions Proto-oncogene-related regulatory mechanisms play an important role in the plasmodial growth of P. brassicae. Electronic supplementary material The online version of this article (10.1186/s12864-018-5307-4) contains supplementary material, which is available to authorized users.

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.

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.

Via virome sequencing, six viruses were detected from Magnaporthe oryzae strains YC81-2, including one virus in the family Tombusviridae, one virus in the family Narnaviridae and four viruses in the family Botourmiaviridae. Since the RNA-dependent RNA polymerase (RdRp) of one botourmiavirus show the highest identity (79%) with Magnaporthe oryzae ourmia-like virus 1 (MOLV1), the virus that was grouped into the genus Magoulivirus was designated as Magnaporthe oryzae botourmiavirus 2 (MOBV2). The three other novel botourmiaviruses were selected for further study. The complete nucleotide sequences of the three botourmiaviruses were determined. Sequence analysis showed that virus 1, virus 2, and virus 3 were 2598, 2385, and 2326 nts in length, respectively. The variable 3&prime, untranslated region (3&prime, UTR) and 5&prime, UTR of each virus could be folded into a stable stem-loop secondary structure. Each virus consisted of a unique ORF encoding a putative RdRp. The putative proteins with a conserved GDD motif of RdRp showed the highest sequence similarity to RdRps of viruses in the family Botourmiaviridae. Phylogenetic analysis demonstrated that these viruses were three distinct novel botourmiaviruses, clustered into the Botourmiaviridae family but not belonging to any known genera of this family. Thus, virus 1, virus 2, and virus 3 were designated as Magnaporthe oryzae botourmiavirus 5, 6, and 7 (MOBV5, MOBV6, and MOBV7), respectively. Our results suggest that four distinct botourmiaviruses, MOBV2, MOBV5, MOBV6, and MOBV7, co-infect a single strain of Magnaporthe oryzae, and MOBV5, MOBV6, and MOBV7 are members of three unclassified genera in the family Botourmiaviridae.

Ascospores act as the primary inoculum of Fusarium graminearum, which causes the destructive disease Fusarium head blight (FHB), or scab. MicroRNAs (miRNAs) have been reported in the F. graminearum vegetative stage, and Fgdcl2 is involved in microRNA-like RNA (milRNA) biogenesis but has no major impact on vegetative growth, abiotic stress or pathogenesis. In the present study, we found that ascospore discharge was decreased in the Fgdcl1 deletion mutant, and completely blocked in the double-deletion mutant of Fgdcl1 and Fgdcl2. Besides, more immature asci were observed in the double-deletion mutant. Interestingly, the up-regulated differentially expressed genes (DEGs) common to ΔFgdcl1 and ΔFgdcl1/2 were related to ion transmembrane transporter and membrane components. The combination of small RNA and transcriptome sequencing with bioinformatics analysis predicted 143 novel milRNAs in wild-type perithecia, and 138 of these milRNAs partly or absolutely depended on Fgdcl1, while only 5 novel milRNAs were still obtained in the Fgdcl1 and Fgdcl2 double-deletion mutant. Furthermore, 117 potential target genes were predicted. Overall, Fgdcl1 and Fgdcl2 genes were partly functionally redundant in ascospore discharge and perithecium-specific milRNA generation in F. graminearum, and these perithecium-specific milRNAs play potential roles in sexual development.

Sclerotinia sclerotiorum is a devastating plant pathogen that attacks numerous economically important broad acre and vegetable crops worldwide. Mycoviruses are widespread viruses that infect fungi, including S. sclerotiorum. As there were no previous reports of the presence of mycoviruses in this pathogen in Australia, studies were undertaken using RNA_Seq analysis to determine the diversity of mycoviruses in 84 Australian S. sclerotiorum isolates collected from various hosts. After RNA sequences were subjected to BLASTp analysis using NCBI database, 285 contigs representing partial or complete genomes of 57 mycoviruses were obtained, and 34 of these (59.6%) were novel viruses. These 57 viruses were grouped into 10 distinct lineages, namely Endornaviridae (four novel mycoviruses), Genomoviridae (isolate of SsHADV-1), Hypoviridae (two novel mycoviruses), Mononegavirales (four novel mycovirusess), Narnaviridae (10 novel mycoviruses), Partitiviridae (two novel mycoviruses), Ourmiavirus (two novel mycovirus), Tombusviridae (two novel mycoviruses), Totiviridae (one novel mycovirus), Tymovirales (five novel mycoviruses), and two non-classified mycoviruses lineages (one Botrytis porri RNA virus 1, one distantly related to Aspergillus fumigatus tetramycovirus-1). Twenty-five mitoviruses were determined and mitoviruses were dominant in the isolates tested. This is not only the first study to show existence of mycoviruses in S. sclerotiorum in Australia, but highlights how they are widespread and that many novel mycoviruses occur there. Further characterization of these mycoviruses is warranted, both in terms of exploring these novel mycoviruses for innovative biocontrol of Sclerotinia diseases and in enhancing our overall knowledge on viral diversity, taxonomy, ecology, and evolution.

Clubroot caused by Plasmodiophora brassicae, is a severe disease of cruciferous crops that causes large hypertrophic galls in the roots. The plant microbiome is important for growth promotion and disease suppression. In this study, using 16S rRNA and internal transcribed spacer (ITS) sequencing techniques, we compared the endosphere microbiome of symptomatic and asymptomatic B. napus roots infected with P. brassicae collected from the same natural clubroot field. The results showed that the microbial population and its relative abundance in the asymptomatic roots was far higher than that in the symptomatic roots, and that many microorganisms in asymptomatic roots have biological control and plant growth promotion functions that may be related to clubroot symptoms. These results suggest the importance of the endosphere microbiome in clubroot disease and provide potential bio-control resources for its prevention.

Two double-stranded RNA (dsRNA) mycoviruses were found in isolate QSP5 of the rice blast fungus Magnaporthe oryzae. Sequence analysis of the two dsRNA mycoviruses revealed that one is closely related to Magnaporthe oryzae virus 2 (MoV2), and the other one is related to Magnaporthe oryzae chrysovirus 1-A (MoCV1-A). Therefore, they were named Magnaporthe oryzae virus 3 (MoV3) and Magnaporthe oryzae chrysovirus 1-C (MoCV1-C), respectively. In this paper, the molecular and structural characteristics of MoV3 were analyzed in detail. The full genome sequence (5181 bp) of MoV3 was obtained by cDNA cloning. Sequence analysis indicated that MoV3 has two overlapping open reading frames (ORF1 and ORF2). The 5'-proximal ORF1 encodes a putative coat protein (CP) with a molecular weight of 80,939 Da; the 3'-proximal ORF2 encodes a putative RNA-dependent RNA polymerase (RdRp) with a molecular weight of 90,506 Da. The stop codon of ORF1 overlaps the start codon of ORF2, with the tetranucleotide sequence AUGA, which is characteristic of members of the genus Victorivirus of the family Totiviridae. Phylogenetic analysis of RdRp and CP further supported the view that MoV3, a novel mycovirus, belongs to the genus Victorivirus of the family Totiviridae.

A double-stranded RNA (dsRNA) segment was isolated from a hypovirulent strain, HC025, of Sclerotinia sclerotiorum. The complete nucleotide sequence of the dsRNA was determined to be 2530 bp in length. Using the fungal mitochondrial genetic code, the positive strand of the dsRNA was found to contain a single large open reading frame (ORF) with the characteristic conserved motifs of the RNA-dependent RNA polymerase (RdRp). BLAST analysis revealed that RdRp shares 74% sequence identity with Sclerotinia sclerotiorum mitovirus 1 (SsMV1/KL-1). The positive strand of the dsRNA could be folded into potentially stable stem-loop structures at both the 5' and 3' terminal sequences. Moreover, the 5' and 3' terminal sequences were inverted complementary sequences and formed a panhandle structure. These results reveal that this dsRNA segment represents the replicative form of a mitovirus that is a strain of SsMV1 from the genus Mitovirus in the family Narnaviridae and was tentatively designated as Sclerotinia sclerotiorum mitovirus 1 (SsMV1/HC025). Sequence comparison and phylogenetic analysis suggest that mitovirus RdRp gene was evolutionarily related to plant mitochondrial genome. Our results demonstrate that SsMV1/HC025 infection exerted obvious effects on host biological properties. Hypovirulence feature and SsMV1/HC025 could be co-transmitted from hypovirulent strains to other virulent strains via hyphal contact. Thus, SsMV1/HC025 related to plant mitochondrial gene confers hypovirulence on S. sclerotiorum.

Seven dsRNA segments were detected from a single Rhizoctonia solani strain HG81. From the full-length cDNA sequences of four smaller dsRNA segments, the genomes of two related partitiviruses, designated as Rhizoctonia solani partitivirus 3 (RsPV3) and RsPV4, were determined. The genomes of RsPV3 and RsPV4 are both composed of two separate dsRNA segments, with each segment possessing a single open reading frame (ORF). ORF1 from RsPV3 and RsPV4 encodes a putative RNA-dependent RNA polymerase, while ORF2 of RsPV3 and RsPV4 encodes a putative capsid protein. RsPV3 and RsPV4 share high sequence identity with viruses classified within the genus Alphapartitivirus, family Partitiviridae.

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 1903 and 1359 DEGs at 24 and 48 hours 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.

Non-self recognition is a common phenomenon among organisms; it often leads to innate immunity to prevent the invasion of parasites and maintain the genetic polymorphism of organisms. Fungal vegetative incompatibility is a type of non-self recognition which often induces programmed cell death (PCD) and restricts the spread of molecular parasites. It is not clearly known whether virus infection could attenuate non-self recognition among host individuals to facilitate its spread. Here, we report that a hypovirulence-associated mycoreovirus, named Sclerotinia sclerotiorum mycoreovirus 4 (SsMYRV4), could suppress host non-self recognition and facilitate horizontal transmission of heterologous viruses. We found that cell death in intermingled colony regions between SsMYRV4-infected Sclerotinia sclerotiorum strain and other tested vegetatively incompatible strains was markedly reduced and inhibition barrage lines were not clearly observed. Vegetative incompatibility, which involves Heterotrimeric guanine nucleotide-binding proteins (G proteins) signaling pathway, is controlled by specific loci termed het (heterokaryon incompatibility) loci. Reactive oxygen species (ROS) plays a key role in vegetative incompatibility-mediated PCD. The expression of G protein subunit genes, het genes, and ROS-related genes were significantly down-regulated, and cellular production of ROS was suppressed in the presence of SsMYRV4. Furthermore, SsMYRV4-infected strain could easily accept other viruses through hyphal contact and these viruses could be efficiently transmitted from SsMYRV4-infected strain to other vegetatively incompatible individuals. Thus, we concluded that SsMYRV4 is capable of suppressing host non-self recognition and facilitating heterologous viruses transmission among host individuals. These findings may enhance our understanding of virus ecology, and provide a potential strategy to utilize hypovirulence-associated mycoviruses to control fungal diseases., Author summary Self/non-self recognition, as the most fundamental immune system, is an essential part in response to nonself host defense in fungi, plants and animals against pathogens or molecular parasites. Vegetative incompatibility as a conspecific nonself system in filamentous fungi results in PCD when cells of incompatible strains contact each other, which also restricts the spread of molecular parasites. The hypovirulence-associated Sclerotinia sclerotiorum mycoreovirus 4 (SsMYRV4), was found to function as a potent inhibitor of G protein signaling pathway, ROS production and vegetative incompatibility-mediated PCD. This SsMYRV4-mediated unique feature enables horizontal transmission of heterologous mycoviruses among vegetatively incompatible S. sclerotiorum individuals in an interaction system (SsMYRV4/S. sclerotiorum) to create a bridge donor strain for mycovirus spread under natural conditions. Furthermore, this virus-mediated suppression of host non-self recognition provides a potential strategy for virus spread that can enhance our understanding of virus ecology.

Infection by diverse mycoviruses is a common phenomenon in Sclerotinia sclerotiorum. In this study, the full genome of a single-stranded RNA mycovirus, tentatively named Hubei sclerotinia RNA virus 1 (HuSRV1), was determined in the hypovirulent strain 277 of S. sclerotiorum. The HuSRV1 genome is 4492 nucleotides (nt) long and lacks a poly (A) tail at the 3ˊ- terminus. Sequence analyses showed that the HuSRV1 genome contains four putative open reading frames (ORFs). ORF1a was presumed to encode a protein with a conserved protease domain and a transmembrane domain. This protein is 27% identical to the P2a protein encoded by the subterranean clover mottle virus. ORF1b encodes a protein containing a conserved RNA-dependent RNA polymerase (RdRp) domain, which may be translated into a fusion protein by a -1 ribosome frameshift. This protein is 45.9% identical to P2b encoded by the sowbane mosaic virus. ORF2 was found to encode a putative coat protein, which shares 23% identical to the coat protein encoded by the olive mild mosaic virus. ORF3 was presumed to encode a putative protein with an unknown function. Evolutionary relation analyses indicated that HuSRV1 is related to members within Sobemovirus, but forms a unique phylogenetic branch, suggesting that HuSRV1 represents a new member within Solemoviridae. HuSRV1 virions, approximately 30 nm in diameter, were purified from strain 277. The purified virions were successfully introduced into virulent strain Ep-1PNA367, resulting in a new hypovirulent strain, which confirmed that HuSRV1 confers hypovirulence on S. sclerotiorum.

A double-stranded RNA (dsRNA) segment was isolated from the filamentous phytopathogenic fungus Rhizoctonia oryzae-sativae and its full-length cDNA sequence (3038 nucleotides) was determined. Sequence analysis revealed that a large open reading frame (ORF) is present on the positive strand of this dsRNA segment when the mitochondrial genetic code was applied. The ORF encodes a putative RNA-dependent RNA polymerase, which shares the closest similarity with Rhizoctonia mitovirus 1 and Rhizophagus sp. RF1 mitovirus, with 43% and 29% identity, respectively. This dsRNA segment represents the replication form of a novel mitovirus that was temporarily designated Rhizoctonia oryzae-sativae mitovirus 1 (RoMV1). Phylogenetic analysis further suggested that RoMV1 belongs to the family Narnaviridae. This is the first study to report a mitovirus genome sequence in the phytopathogenic fungus R. oryzae-sativae.

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.

Small, secreted proteins have been found to play crucial roles in interactions between biotrophic/hemi-biotrophic pathogens and plants. However, little is known about the roles of these proteins produced by broad host-range necrotrophic phytopathogens during infection. Here, we report that a cysteine-rich, small protein SsSSVP1 in the necrotrophic phytopathogen Sclerotinia sclerotiorum was experimentally confirmed to be a secreted protein, and the secretion of SsSSVP1 from hyphae was followed by internalization and cell-to-cell movement independent of a pathogen in host cells. SsSSVP1∆SP could induce significant plant cell death and targeted silencing of SsSSVP1 resulted in a significant reduction in virulence. Through yeast two-hybrid (Y2H), coimmunoprecipitation (co-IP) and bimolecular fluorescence complementation (BiFC) assays, we demonstrated that SsSSVP1∆SP interacted with QCR8, a subunit of the cytochrome b-c1 complex of mitochondrial respiratory chain in plants. Double site-directed mutagenesis of two cysteine residues (C38 and C44) in SsSSVP1∆SP had significant effects on its homo-dimer formation, SsSSVP1∆SP-QCR8 interaction and plant cell death induction, indicating that partial cysteine residues surely play crucial roles in maintaining the structure and function of SsSSVP1. Co-localization and BiFC assays showed that SsSSVP1∆SP might hijack QCR8 to cytoplasm before QCR8 targeting into mitochondria, thereby disturbing its subcellular localization in plant cells. Furthermore, virus induced gene silencing (VIGS) of QCR8 in tobacco caused plant abnormal development and cell death, indicating the cell death induced by SsSSVP1∆SP might be caused by the SsSSVP1∆SP-QCR8 interaction, which had disturbed the QCR8 subcellular localization and hence disabled its biological functions. These results suggest that SsSSVP1 is a potential effector which may manipulate plant energy metabolism to facilitate the infection of S. sclerotiorum. Our findings indicate novel roles of small secreted proteins in the interactions between host-non-specific necrotrophic fungi and plants, and highlight the significance to illuminate the pathogenic mechanisms of this type of interaction., Author Summary To resist biotrophic and hemibiotrophic phytopathogens, plants utilize an innate immune system, mediated through nucleotide binding (NB)-leucine rich repeat (LRR) proteins, to respond to effectors, most of which are small secreted proteins. Hypersensitive responses (HRs) resulting from this type of interaction can effectively restrain the expansion of biotrophic or hemibiotrophic phytopathogens in plant tissues. However, it is not effective against typical necrotrophs with remarkably broad host range, such as S. sclerotiorum, because these necrotrophs have long been thought to just simply kill hosts and complete their life cycles using nutrients derived mostly from dead plant tissues. This type of phytopathogen-plant interaction obviously does not comply with the gene-for-gene or inversed gene-for-gene relationship. The results in present study show that SsSSVP1 of S. sclerotiorum functions as an effector in pathogen-plant interactions. SsSSVP1 is dramatically induced during infection, and required for the full virulence of S. sclerotiorum. SsSSVP1 can be internalized by plant cells after being secreted from fungal cells in the absence of a pathogen during infection. Furthermore, SsSSVP1∆SP interacts with QCR8, a subunit of cytochrome b-c1 complex, and disturbs the localization of QCR8 in mitochondria, which may disable its biological function. The nonfunctionalization of QCR8 caused significant plant cell death. Hence, SsSSVP1 acts as an effector to manipulate the host cell physiology to facilitate the colonization of S. sclerotiorum. Obviously, this is a completely different interaction model from the gene-for-gene or inversed gene-for-gene paradigm. These findings suggest that the pathogenesis of S. sclerotiorum is more subtle and complex than previously appreciated and highlight the significance to investigate the interaction models between the host-non-specific necrotrophs and their hosts.

Coniothyrium minitans is an important biocontrol agent against Sclerotinia diseases. Previously, a conidiation-deficient mutant ZS-1T1000 was screened out from a T-DNA insertional library of C. minitans. CmBCK1, encoding MAP kinase kinase kinase and homologous to BCK1 of Saccharomyces cerevisiae, was disrupted by T-DNA insertion in this mutant. Targeted disruption of CmBCK1 led to the mutants undergoing autolysis and displaying hypersensitivity to the cell wall-degrading enzymes. The △CmBCK1 mutants lost the ability to produce pycnidia and conidia compared to the wild-type strain ZS-1. △CmBCK1 mutants could grow on the surface of sclerotia of Sclerotinia sclerotiorum but not form conidia, which resulted in much lower ability to reduce the viability of sclerotia of S. sclerotiorum. Furthermore, CmSlt2, a homolog of Slt2 encoding cell wall integrity-related MAP kinase and up-regulated by BCK1 in S. cerevisiae, was identified and targeted disrupted. The △CmSlt2 mutants had a similar phenotype to the △CmBCK1 mutants. The △CmSlt2 mutants also had autolytic aerial hyphae, hypersensitivity to cell wall-degrading enzymes, lack of conidiation and reduction of sclerotial mycoparasitism. Taken together, our results suggest that CmBCK1 and CmSlt2 are involved in conidiation and the hyperparasitic activities of C. minitans.

MicroRNAs are small non-coding RNAs of approximately 22 nt with important regulatory roles in post-transcriptional regulation of gene expression. To date, no miRNAs have been reported in plant pathogenic fungi. Here we used Solexa sequencing to find new miRNAs in a plant pathogenic fungus Sclerotinia sclerotiorum. 13,229,401 high-quality reads were obtained, a total of 5,645,718 reads were mapped to the S. sclerotiorum genome corresponding to 348,108 unique sRNAs. Among the sRNAs, 2 microRNA-like RNAs (milRNAs) and 42 mil-RNA candidates were identified by sequence analysis. Northern blot and RT-PCR results showed that some milRNAs and candidates are differentially expressed in sclerotial development suggesting the role of these milRNAs and candidates in this biological process. Our results provide new insights into the functional roles of small RNAs and add new resources for the study of plant pathogenic fungi.

In this study, we showed that curcumin treatment resulted in activation of Chk1-mediated G2 checkpoint, which was associated with the induction of G2/M arrest and the resistance of cancer cells to curcumin-induced apoptosis. Further investigation revealed that inhibition of Chk1 significantly abrogated G2/M arrest and sensitized curcumin-resistant cells to apoptosis via upregulation of Bad and in turn the loss of mitochondrial membrane potential. These results indicate that Chk1-mediated G2/M arrest may serve as a mechanism for curcumin resistance and Chk1 represents a potential target for the reversal of this resistance. Our findings should be helpful for clinical application of curcumin.

Recent studies have shown that Sclerotinia sclerotiorum, an important plant pathogen fungus, harbors diverse mycoviruses. A new mycovirus, tentatively named as Sclerotinia sclerotiorum deltaflexivirus 1 (SsDFV1), was isolated from a S. sclerotiorum strain AX19 containing multiple dsRNA elements. The complete genome of SsDFV1 was shown to be 8178 nucleotides long excluding the poly (A) tail. SsDFV1 has a large putative open reading frame (ORF1) and three smaller ORFs (2-4). ORF1 encodes a putative methyltransferase-helicase-RdRp polyprotein of 2075 amino acids. ORFs (2-4) encode three putative small hypothetical proteins (

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, S clerotinia s clerotiorum m ega b irna v irus 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 single-stranded 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. IMPORTANCE Mycoviruses are widespread in all major fungal groups, and they possess diverse genomes of mostly ssRNA and dsRNA and, recently, circular ssDNA. Here, we have characterized a novel dsRNA virus ( Sclerotinia sclerotiorum megabirnavirus 1 [SsMBV1]) that was isolated from an apparently hypovirulent strain, SX466, of Sclerotinia sclerotiorum . Although SsMBV1 is phylogenetically related to RnMBV1, SsMBV1 is markedly distinct from other reported megabirnaviruses with two features of VLPs and conserved domains. Our results convincingly showed that SsMBV1 is viable in the absence of L2-dsRNA/SsMBV1 (a potential large satellite-like RNA or genuine genomic virus component). More interestingly, we detected a conserved papain-like protease domain that commonly exists in ssRNA viruses, including members of the families Potyviridae and Hypoviridae . Phylogenetic analysis based on the protease domain suggests that horizontal gene transfer might have occurred from an ssRNA virus to a dsRNA virus, which may provide new insights into the evolutionary history of dsRNA and ssRNA viruses.

Coniothyrium minitans is a fungal biocontrol agent of the plant pathogen Sclerotinia sclerotiorum. Growth and sporulation of 21 strains of C. minitans were examined on potato dextrose agar (PDA) and compared with that in potato dextrose broth (PDB) in shaken culture after 12 days at 20 degrees C, to identify strains with potential for inoculum production in liquid culture. Four strains that produced high numbers of pycnidia in PDA also formed pycnidia on mycelial strands in PDB and 10(7) conidia ml(-1) broth were produced. The other strains formed pellets during shaking, resulting in production of less than 10(5) conidia ml(-1). Conidia from shaken PDB culture had the same ability to infect and rot sclerotia of S. sclerotiorum as conidia produced routinely on PDA, and survived well in dry kaolin dust for 6 months at temperatures less than 8 degrees C with less than 1 log(10) colony forming units mg(-1) loss. These results suggest that it might be possible to identify useful strains of C. minitans for future commercial conidial production in liquid fermentation systems based on morphological characteristics on agar.

The complete nucleotide sequence, 4975 bp, of the double-stranded RNA (dsRNA) mycovirus infecting the sclerotial parasite Coniothyrium minitans (CmRV) was determined. Sequence analysis revealed the occurrence of two overlapping open reading frames (ORFs): the 5'-proximal large ORF (ORF1; nucleotide positions 62-2389) encodes a putative coat protein (CP) with a predicted molecular mass of 80344 Da, and the 3'-proximal ORF (ORF2, nucleotide positions 2386-4875) encodes a putative RNA dependent RNA Polymerase (RdRp) with a predicted molecular mass of 82551 Da. The tetranucleotide AUGA at nucleotide positions 2386-2389 includes the predicted start codon of ORF2, which overlaps with the stop codon for ORF1. Based on genome organization and sequence analysis encoded proteins, the virus infecting C. minitans strain Chy-1, designated C. minitans RNA virus (CmRV), belongs to the family Totiviridae. Pairwise sequence comparisons of the deduced amino acid sequences encoded by CmRV as well as phylogentic analysis indicated that it is more closely related to the totiviruses that infect filamentous fungi than to those infecting protozoa, yeast and smut fungi. The role of CmRV in the abnormal phenotype associated with a variant of C. minitans is discussed.

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.

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. GAPDH-knockdown 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.

Background In addition to vertical transmission, organisms can also acquire genes from other distantly related species or from their extra-chromosomal elements (plasmids and viruses) via horizontal gene transfer (HGT). It has been suggested that phages represent substantial forces in prokaryotic evolution. In eukaryotes, retroviruses, which can integrate into host genome as an obligate step in their replication strategy, comprise approximately 8% of the human genome. Unlike retroviruses, few members of other virus families are known to transfer genes to host genomes. Results Here we performed a systematic search for sequences related to circular single-stranded DNA (ssDNA) viruses in publicly available eukaryotic genome databases followed by comprehensive phylogenetic analysis. We conclude that the replication initiation protein (Rep)-related sequences of geminiviruses, nanoviruses and circoviruses have been frequently transferred to a broad range of eukaryotic species, including plants, fungi, animals and protists. Some of the transferred viral genes were conserved and expressed, suggesting that these genes have been coopted to assume cellular functions in the host genomes. We also identified geminivirus-like and parvovirus-like transposable elements in genomes of fungi and lower animals, respectively, and thereby provide direct evidence that eukaryotic transposons could derive from ssDNA viruses. Conclusions Our discovery extends the host range of circular ssDNA viruses and sheds light on the origin and evolution of these viruses. It also suggests that ssDNA viruses act as an unforeseen source of genetic innovation in their hosts.

Parvoviruses infect humans and a broad range of animals, from mammals to crustaceans, and generally are associated with a variety of acute and chronic diseases. However, many others cause persistent infections and are not known to be associated with any disease. Viral persistence is likely related to the ability to integrate into the chromosomal DNA and to establish a latent infection. However, there is little evidence for genome integration of parvoviral DNA except for Adeno-associated virus (AAV). Here we performed a systematic search for homologs of parvoviral proteins in publicly available eukaryotic genome databases followed by experimental verification and phylogenetic analysis. We conclude that parvoviruses have frequently invaded the germ lines of diverse animal species, including mammals, fishes, birds, tunicates, arthropods, and flatworms. The identification of orthologous endogenous parvovirus sequences in the genomes of humans and other mammals suggests that parvoviruses have coexisted with mammals for at least 98 million years. Furthermore, some of the endogenized parvoviral genes were expressed in eukaryotic organisms, suggesting that these viral genes are also functional in the host genomes. Our findings may provide novel insights into parvovirus biology, host interactions, and evolution.

Cdc6 is essential for DNA replication and its deregulation is involved in carcinogenesis. To date, the biologi- cal significance of the polymorphism in Cdc6 promoter is still unknown. In this study, we aimed to evaluate the influence of the Cdc6 −515A>G polymorphism (rs4134994) on the individual's susceptibility to can- cer and on the function of Cdc6. The Cdc6 −515A>G polymorphism was genotyped in 387 hepatocellular carcinoma (HCC) and 389 age- and sex-matched healthy subjects. The association between the genotypes and the risk for HCC was then estimated by unconditional logistic regression analysis with adjustment for age, sex and HBV status. Compared with the AA homozygotes, the homozygous GG genotype (adjusted OR = 0.36, 95% confidence interval (CI) = 0.18-0.72, P = 0.004) or the combined AG/GG genotypes (adjusted OR = 0.56, 95% CI = 0.36-0.86, P = 0.008) were statistically significantly associated with the reduced risk for HCC. Moreover, the analysis using luciferase reporter system showed that the G-allelic Cdc6 promoter displayed a decreased transcriptional activity compared with the A-allelic one. These results indicate that the individuals with G allele may have reduced Cdc6 expression and are therefore in reduced risk for HCC. Further investigation using electrophoretic mobility shift assay (EMSA) revealed that the G allele had a stronger binding strength to nuclear protein(s) which might function as negative regulator(s) for Cdc6 transcription. Our findings suggest that the −515A>G polymorphism may affect the Cdc6 promoter binding affinity with nuclear protein(s) and in turn the Cdc6 expression, which consequently modulates the individual's susceptibility to HCC. © 2008 Elsevier B.V. All rights reserved.

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.