Gene regulation involves the orchestrated action of multiple regulators to fine-tune the expression of genes. Hierarchical interactions and co-regulation among regulators are commonly observed in biological systems, leading to complex regulatory networks. Small RNA (sRNAs) have been shown to be important regulators of gene expression due to their involvement in multiple cellular processes. In plants, microRNA (miRNAs) and phased small interfering RNAs (phasiRNAs) correspond to two well-characterized types of sRNAs involved in the regulation of posttranscriptional gene expression, although information about their targets and interactions with other gene expression regulators is limited. We describe an extended sRNA-mediated regulatory network in Arabidopsis thaliana that provides a reference frame to understand sRNA biogenesis and activity at the genomewide level. This regulatory network combines a comprehensive evaluation of phasiRNA production and sRNA targets supported by degradome data. The network includes 17% of genes in the A. thaliana genome, representing~50% annotated gene ontology (GO) functional categories. Approximately 14% of genes with GO annotations corresponding to regulation of gene expression were found to be under sRNA control. The unbiased bioinformatic approach used to produce the network was able to detect 107 PHAS loci (regions of phasiRNA production), 5,047 active phasiRNAs (~70% of which were non-canonical), and reconstruct 17 regulatory modules resulting from complex regulatory interactions between different sRNA-regulatory pathways. Known regulatory modules like miR173-TAS-PPR/TPR and miR390-TAS3-ARF/F-box were faithfully reconstructed and expanded, illustrating the accuracy and sensitivity of the methods and providing confidence for the validity of findings of previously unrecognized modules. The network presented here includes a 2X increase in the number of identified PHAS loci, a large complement (~70%) of non-canonical phasiRNAs, and the most comprehensive
Viruses and viroids prevalent in a population of 42 wild grapevines (i.e., free-living, uncultivated grapevines; Vitis spp.) were compared to those in a population of 85 cultivated grapevines collected in Tennessee, USA by RNA-seq analysis of pools of ribosomal RNA-depleted total RNA. The sequences of 10 viruses (grapevine fleck virus, grapevine leafroll-associated virus 2, grapevine rupestris stem pitting-associated virus, grapevine Syrah virus 1, grapevine vein-clearing virus, grapevine virus B, grapevine virus E, tobacco ringspot virus, tomato ringspot virus and a novel nano-like virus) and two viroids (hop stunt viroid and grapevine yellow speckle viroid 1) were detected in both grapevine populations. Sequences of four viruses (grapevine associated tymo-like virus, grapevine leafroll-associated virus 3, grapevine red blotch virus and grapevine virus H) were identified only from cultivated grapevines. High, moderate and low numbers of sequence reads were identified only from wild grapevines for a novel caulimovirus, an enamovirus, and alfalfa mosaic virus, respectively. The presence of most virus sequences and both viroids was verified independently in the original samples by reverse transcription-polymerase chain reaction followed by Sanger sequencing. Comparison of viral sequences shared by both populations showed that cultivated and wild grapevines harbored distinct sequence variants, which suggests that there was limited virus movement between the two populations. Collectively, this study represents the first unbiased survey of viruses and viroids in both cultivated and wild grapevines within a defined geographic region.
Plants in a single field of commercial tomato (Solanum lycopersicum) of unidentified cultivars in Virginia in July, 2012, were observed showing stunting, leaf distortion, twisting and thickening, discoloration, and color streaking and ringspots on fruits. Serological tests were negative for Cucumber mosaic virus, Groundnut ringspot virus, Tomato spotted wilt virus, Tomato chlorotic spot virus, Impatiens necrotic spot virus, Tobacco mosaic virus, and Tomato bushy stunt virus (Agdia, Inc., Elkhart, IN). Using a membrane-based macroarray (3), hybridization was observed to 8 of 9 70-mer oligonucleotide probes of Spinach latent virus (SpLV; genus Ilarvirus, family Bromoviridae). To confirm the hybridization results, complementary DNA (cDNA) was synthesized using random hexamers and MMLV reverse transcriptase (Promega, Madison, WI), followed by PCR amplification using ilarvirus degenerate primers (4). Fragments of approximately 380 bp were amplified and directly sequenced (GenBank Accession KC_466090); a BLAST search showed a 99% identity to the SpLV RNA 2 reference genome (NC_003809). Primers for SpLV RNA1 (SpLVRNA1f-GGTGTCACCATGCAAACTGG, SpLVRNA1r-AGCTCTTCGTAATAGGCCTGC) and SpLV RNA3 (SpLVCPf-GAAGTCTTTCCCAGGTGAGCA, SpLVCPr-AGGTGGGCATATGGACTTGG) were designed and cDNA was amplified using the IQ supermix (Biorad, Hercules, CA) with thermocycling of 94°C for 4 min, 35× (94°C 45 s, 55°C 45 s, 72°C 45 s), and 72°C for 10 min. The resulting fragments of 538 bp for RNA1 (KC_466088) and 661 bp for RNA3 (KC_466089) showed 100% identity to reference genome sequences for SpLV (NC_003808 and NC_003810, respectively). To demonstrate virus transmissibility, Chenopodium quinoa plants were mechanically inoculated using tomato leaf material (same source described above) ground in 30 mM Na2HPO4 buffer, pH 7.0. Necrotic spots developed on the inoculated leaves 10 dpi. Younger, non-inoculated leaves showed yellow mottling and tested positive for SpLV by RT-PCR (two of two plants tested). The detection of SpLV is rarely reported, with only one record from the United States (2). Although SpLV is described as a latent virus, it has been found associated with tomato fruit symptoms in New Zealand (1). It is not known if the fruit ringspot and other symptoms on the Virginia samples were due to virus infection. Since SpLV is seed-transmissible and seed production takes place in different parts of the world, it has the potential to spread with germplasm and become more widespread in North America. References: (1) B. S. M. Lebas et al. Plant Dis. 91:228, 2007. (2) H. Y. Liu and J. E. Duffus. Phytopathology 76:1087, 1986. (3) K. L. Perry and X. Lu. Phytopathology 100:S100, 2010. (4) M. Untiveros, et al. J. Virol. Methods 165:97, 2010.
A survey for the presence of Grapevine virus E (GVE, genus Vitivirus, family Betaflexiviridae) in vineyards in New York and California was conducted using macroarray hybridization or reverse-transcription polymerase chain reaction (RT-PCR) assays. In New York, GVE was detected in 10 of 46 vines of Vitis labrusca, one V. riparia, and one Vitis hybrid. All GVE-infected New York vines were coinfected with Grapevine leafroll-associated virus-3. In California, GVE was detected in 8 of 417 vines of V. vinifera. All GVE-infected California vines were also coinfected by one of the leafroll-associated viruses and other vitiviruses. In order to assess the genetic diversity among GVE isolates, a viral cDNA was amplified by RT-PCR, and a 675-nucleotide region that included the 3′ terminus of the coat protein gene, a short intergenic region, and the 5′ terminus of the putative nucleic acid binding protein gene was sequenced. All 20 GVE isolates sequenced in this study were very closely related, with >98% nucleotide identity to the SA94 isolate from South Africa. These findings confirm the presence of GVE in major grape-growing regions of the United States and indicate a very low level of genetic diversity.
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