Using cross-linking coupled to matrix-assisted laser desorption/ionization mass spectrometry and CLIP-Seq sequencing, we determined the peptide and oligonucleotide sequences at the interfaces between the capsid proteins and the genomic RNA of bacteriophage MS2. The results suggest that the same coat protein (CP)–RNA and maturation protein (MP)–RNA interfaces are used in every viral particle. The portions of the viral RNA in contact with CP subunits span the genome, consistent with a large number of discrete and similar contacts within each particle. Many of these sites match previous predictions of the locations of multiple, dispersed and degenerate RNA sites with cognate CP affinity termed packaging signals (PSs). Chemical RNA footprinting was used to compare the secondary structures of protein-free genomic fragments and the RNA in the virion. Some PSs are partially present in protein-free RNA but others would need to refold from their dominant solution conformations to form the contacts identified in the virion. The RNA-binding peptides within the MP map to two sections of the N-terminal half of the protein. Comparison of MP sequences from related phages suggests a similar arrangement of RNA-binding sites, although these N-terminal regions have only limited sequence conservation. In contrast, the sequences of the C-termini are highly conserved, consistent with them encompassing pilin-binding domains required for initial contact with host cells. These results provide independent and unambiguous support for the assembly of MS2 virions via a PS-mediated mechanism involving a series of induced-fit viral protein interactions with RNA.

Permafrost thaw is leading to rapid shifts in boreal ecosystem function. Permafrost thaw affects soil carbon turnover through changes in soil hydrology; however, the biotic mechanisms regulating plant community response remain elusive. Here, we measured the response of fungal community composition and soil nutrient content in an intact permafrost plateau forest soil and an adjacent thermokarst bog and evaluated their potential to mediate shifts in plant composition. We used barcoded amplicon targeting ITS2 and 28S rRNA genes to determine fungal community composition. Next, we used the soils from the permafrost plateau and the thermokarst bog as soil inoculum in a greenhouse experiment to measure whether shifts in fungal community and soil water level regulate plant productivity and composition. Overall, we found that fungal community composition differed significantly between the thawed and intact permafrost sites, but soil nutrient content did not. Relative abundance of mycorrhizal fungal taxa decreased while relative abundance of putative fungal pathogens increased with permafrost thaw. In the greenhouse, we found that ecto‐ and arbuscular‐associated host plants had higher productivity in permafrost‐intact soils relative to thawed soils. However, productivity of non‐mycorrhizal tussock grass was more affected by soil water levels than soil communities. Synthesis. Our results suggest that fungal communities are crucial in mediating plant community response to permafrost thaws inducing hydrology changes.

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BackgroundBAC-based physical maps provide for sequencing across an entire genome or a selected sub-genomic region of biological interest. Such a region can be approached with next-generation whole-genome sequencing and assembly as if it were an independent small genome. Using the minimum tiling path as a guide, specific BAC clones representing the prioritized genomic interval are selected, pooled, and used to prepare a sequencing library.ResultsThis pooled BAC approach was taken to sequence and assemble a QTL-rich region, of ~3 Mbp and represented by twenty-seven BACs, on linkage group 5 of the Theobroma cacao cv. Matina 1-6 genome. Using various mixtures of read coverages from paired-end and linear 454 libraries, multiple assemblies of varied quality were generated. Quality was assessed by comparing the assembly of 454 reads with a subset of ten BACs individually sequenced and assembled using Sanger reads. A mixture of reads optimal for assembly was identified. We found, furthermore, that a quality assembly suitable for serving as a reference genome template could be obtained even with a reduced depth of sequencing coverage. Annotation of the resulting assembly revealed several genes potentially responsible for three T. cacao traits: black pod disease resistance, bean shape index, and pod weight.ConclusionsOur results, as with other pooled BAC sequencing reports, suggest that pooling portions of a minimum tiling path derived from a BAC-based physical map is an effective method to target sub-genomic regions for sequencing. While we focused on a single QTL region, other QTL regions of importance could be similarly sequenced allowing for biological discovery to take place before a high quality whole-genome assembly is completed.

Stranded whole transcriptome RNA-Seq described in this unit captures quantitative expression data for all types of RNA including, but not limited to miRNA (microRNA), piRNA (Piwi-interacting RNA), snoRNA (small nucleolar RNA), lincRNA (large non-coding intergenic RNA), SRP RNA (signal recognition particle RNA), tRNA (transfer RNA), mtRNA (mitochondrial RNA) and mRNA (messenger RNA). The size and nature of these types of RNA are irrelevant to the approach described here. Barcoded libraries for multiplexing on the Illumina platform are generated with this approach but it can be applied to other platforms with a few modifications.