Genome-wide analyses of direct target genes of an ERF11 transcription factor involved in plant defense against bacterial pathogens.

Ethylene responsive factor ERF11 containing the ERF-associated amphiphilic repression (EAR) motif enhances plant resistance to bacterial pathogens. However, the underlying molecular mechanisms regulated by transcription factor ERF11 are poorly understood, in tobacco or other model plants. Here, we revealed the genome-wide binding landscape of BrERF11b in Nicotiana benthamian by conducting chromatin immunoprecipitation experiments followed by high-throughput sequencing (ChIP-seq) and bioinformatic analyses. Our results also revealed a GCCbox-like consensus BrERF11b-binding DNA motif: VCGCCGCC. By further integrative analysis of ChIP-seq and RNA-seq data, and the confirmation of electrophoretic mobility shift assay (EMSA), we screened three direct target genes NbNIMIN2, NbTAF15b and NbERF4. These results suggest that ERF11 may be involved in NPR1-mediated systemic acquired resistance (SAR), nucleotide-binding leucine-rich repeat immune receptors (NLR) -mediated autoimmunity, and H2O2 generation, by direct transcriptional repression of NIM1-INTERACTING2 (NIMIN2), and transcriptional activation of TATA-binding protein-associated factor 15b (TAF15b) and ERF4. Our findings provide insightful information and valuable gene resource in unraveling the regulatory networks of plant defense responses to bacterial pathogens.

Sequence analysis of double-strand RNA6 and RNA9 from the fungus Sclerotium hydrophilum.

We have recently reported the identification of 10 double-strand RNA segments from Sclerotium hydrophilum [HZ11] mycelia and of virus-like particles isolated from the mycelia, as well as the sequences of dsRNA2 and dsRNA7. Phylogenetic analysis revealed that dsRNA2 and dsRNA7 belong to a group of unclassified viruses. In this report, we cloned and sequenced dsRNA6 and dsRNA9 from the 10 dsRNAs. We tentatively named the putative virus "Sclerotium hydrophilum virus 1", with isolates being abbreviated to ShV1, with dsRNA6 and dsRNA9 corresponding to dsRNA1 and dsRNA2, respectively, of ShV1. dsRNA1 was 1975 bp in length and encoded a putative RNA-dependent RNA polymerase (RdRp). dsRNA2 was 1728 bp and encoded a putative coat protein (CP). Phylogenetic analysis showed that the proteins encoded by dsRNA1 and dsRNA2 were highly related to known viral RdRps and CP, respectively, of viruses classified within the genus Alphapartitivirus of the family Partitiviridae. These members include Rhizoctonia solani dsRNA virus 2, Diuris pendunculata cryptic virus, and Heterobasidion partitivirus. The 5'- and 3'-untranslated regions (UTRs) of the two dsRNAs showed a high sequence identity. The 5'-UTR contained conserved sequences 5'-GAAGCAUCACUU(/G) G(/U)AGU(/A)UCGC(/U)CCA(/G) CAAUAACGAA-3' and 5'-AAAUUGAUCUUACCUCUCAC-3'. The 3'-UTR contained the conserved sequence 5'-UUGUUUU-3' and 5'-UUUA(/U)A(/C) UUAU-3'. These results indicate that dsRNA1 and dsRNA2 are phylogenetically related to members of the genus Alphapartitivirus of family Partitiviridae. We therefore propose that dsRNA1 and dsRNA2 are the genome sequences of a new partitivirus, ShV1.

Complete nucleotide sequences of dsRNA2 and dsRNA7 detected in the phytopathogenic fungus Sclerotium hydrophilum and their close phylogenetic relationship to a group of unclassified viruses.

Ten dsRNA segments were extracted from Sclerotium hydrophilum isolate (HZ11). The isolation of virus-like particles contained 10 dsRNA segments with the same number and migration as those extracted directly from the fungal mycelia. Two of these dsRNA segments, dsRNA2 and dsRNA7, were cloned and sequenced. They were 2121 and 1953 bp, respectively. The dsRNA2 encodes a RNA-dependent RNA polymerase. The dsRNA7 contains two open reading frames that encode putative proteins of unknown functions. Phylogenetic analysis of the putative proteins indicated that they are closely related to protein encoded by unclassified viruses, such as Cryphonectria parasitica bipartite mycovirus 1, Lactarius rufus RNA virus 1, Penicillium aurantiogriseum bipartite virus 1, and Curvularia thermal tolerance virus. The 5'- and 3'-untranslated regions of the two dsRNAs share significant sequence identity and contain conserved sequence stretches. It suggested that dsRNA2 and dsRNA7 have a common origin and a close phylogenetic relationship to a group of unclassified viruses.

Seed-borne viral dsRNA elements in three cultivated Raphanus and Brassica plants suggest three cryptoviruses.

Since the 1970s, several dsRNA viruses, including Radish yellow edge virus, Raphanus sativus virus 1, Raphanus sativus virus 2, and Raphanus sativus virus 3, have been identified and reported as infecting radish. In the present study, in conjunction with a survey of seed-borne viruses in cultivated Brassica and Raphanus using the dsRNA diagnostic method, we discovered 3 novel cryptoviruses that infect Brassica and Raphanus: Raphanus sativus partitivirus 1, which infects radish (Raphanus sativus); Sinapis alba cryptic virus 1, which infects Sinapis alba; and Brassica rapa cryptic virus 1 (BrCV1), which infects Brassica rapa. The genomic organization of these cryptoviruses was analyzed and characterized. BrCV1 might represent the first plant partitivirus found in Gammapartitivirus. Additionally, the evolutionary relationships among all of the partitiviruses reported in Raphanus and Brassica were analyzed.

Molecular characterization of a trisegmented chrysovirus isolated from the radish Raphanus sativus.

Radish (Raphanus sativus L.) is cultivated worldwide and is of agronomic importance. dsRNAs associated with partitiviruses were previously found in many R. sativus varieties. In this study, three large dsRNAs from radish were cloned using a modified single primer amplification technique. These three dsRNAs-of lengths 3638, 3517 and 3299 bp-shared conserved untranslated terminal regions, and each contained a major open reading frame putatively encoding the chrysoviral replicase, capsid protein and protease respectively. Isometric virus-like particles (VLP), approximately 45nm in diameter, were isolated from the infected radish plants. Northern blotting indicated that these dsRNAs were encapsidated in the VLP. The virus containing these dsRNA genome segments was named Raphanus sativus chrysovirus 1 (RasCV1). Phylogenetic analysis revealed that RasCV1 is a new species of the Chrysoviridae family and forms a plant taxon with another putative plant chrysovirus, Anthurium mosaic-associated virus (AmaCV). Furthermore, no fungal mycelia were observed in radish leaf tissues stained with trypan blue. These results indicated that RasCV1 is most likely a plant chrysovirus rather than a chrysovirus in symbiotic fungi. An exhaustive BLAST analysis of RasCV1 and AmaCV revealed that chrysovirus-like viruses might widely exist in eudicot and monocot plants and that endogenization of chrysovirus segments into plant genome might have ever happened.

Genomic characterization of a novel dsRNA virus detected in the phytopathogenic fungus Verticillium dahliae Kleb.

Four novel double-stranded RNA segments were detected in a Verticillium dahliae Kleb. strain (V. dahliae isolate 0-21), a causal fungal agent of Verticillium wilt disease of cotton. Each dsRNA genome segment contains a single large open reading frame (ORF) that encodes a distinctive protein with modest levels of sequence similarities to the corresponding putative proteins in the genus Chrysovirus. These include an RNA-dependent RNA polymerase (RdRp), a coat protein, an undefined replication-related protein and an ovarian tumor domain peptidase. Phylogenetic analysis of the four putative proteins unanimously indicated that they are evolutionarily related to viruses in Chrysovirus. The 5'- and 3'-untranslated regions of the four dsRNAs share highly similar internal sequence and contain conserved sequence stretches of UGAUAAAAAA(/U)UG(/U)AAAAA- (in the 5'-UTR) and -UUUACUACU (in the 3'-UTR), indicating that they have a common virus origin. Indeed, isometric virus-like particles (VLPs) with a diameter of approximately 34nm were extracted from the fungal mycelia, and the four dsRNA segments were also detected in the virus-like particle (VLP) fraction. These results suggest that the mycovirus with four different dsRNA genome segments from the fungal isolate 0-21 is a new member of the genus Chrysovirus. We named the virus Verticillium dahliae chrysovirus 1 (VdCV1).