Nucleotide bias of DCL and AGO in plant anti-virus gene silencing

Plant Dicer-like (DCL) and Argonaute (AGO) are the key enzymes involved in anti-virus post-transcriptional gene silencing (AV-PTGS). Here we show that AV-PTGS exhibited nucleotide preference by calculating a relative AV-PTGS efficiency on processing viral RNA substrates. In comparison with genome sequences of dicot-infecting Turnip mosaic virus (TuMV) and monocot-infecting Cocksfoot streak virus (CSV), viral-derived small interfering RNAs (vsiRNAs) displayed positive correlations between AV-PTGS efficiency and G+C content (GC%). Further investigations on nucleotide contents revealed that the vsiRNA populations had G-biases. This finding was further supported by our analyses of previously reported vsiRNA populations in diverse plant-virus associations, and AGO associated Arabidopsis endogenous siRNA populations, indicating that plant AGOs operated with G-preference. We further propose a hypothesis that AV-PTGS imposes selection pressure(s) on the evolution of plant viruses. This hypothesis was supported when potyvirus genomes were analysed for evidence of GC elimination, suggesting that plant virus evolution to have low GC% genomes would have a unique function, which is to reduce the host AV-PTGS attack during infections

Biogeochemical and climate drivers of wetland phosphorus and nitrogen release: implications for nutrient legacies and eutrophication risk

The dynamics and processes of nutrient cycling and release were examined for a lowland wetland‐pond system, draining woodland in southern England. Hydrochemical and meteorological data were analyzed from 1997 to 2017, along with high‐resolution in situ sensor measurements from 2016 to 2017. The results showed that even a relatively pristine wetland can become a source of highly bioavailable phosphorus (P), nitrogen (N), and silicon (Si) during low‐flow periods of high ecological sensitivity. The drivers of nutrient release were primary production and accumulation of biomass, which provided a carbon (C) source for microbial respiration and, via mineralization, a source of bioavailable nutrients for P and N co‐limited microorganisms. During high‐intensity nutrient release events, the dominant N‐cycling process switched from denitrification to nitrate ammonification, and a positive feedback cycle of P and N release was sustained over several months during summer and fall. Temperature controls on microbial activity were the primary drivers of short‐term (day‐to‐day) variability in P release, with subdaily (diurnal) fluctuations in P concentrations driven by water body metabolism. Interannual relationships between nutrient release and climate variables indicated “memory” effects of antecedent climate drivers through accumulated legacy organic matter from the previous year's biomass production. Natural flood management initiatives promote the use of wetlands as “nature‐based solutions” in climate change adaptation, flood management, and soil and water conservation. This study highlights potential water quality trade‐offs and shows how the convergence of climate and biogeochemical drivers of wetland nutrient release can amplify background nutrient signals by mobilizing legacy nutrients, causing water quality impairment and accelerating eutrophication risk

Detection of Cereal yellow dwarf virus using small interfering RNAs and enhanced infection rate with Cocksfoot streak virus in wild cocksfoot grass (Dactylis glomerata)

Small RNA sequences were obtained from leaf extracts of wild Dactylis glomerata (cocksfoot grass) using deep sequencing (454 Life Sciences, Roche Diagnostics), and were screened against virus sequences in GenBank using a local BLASTn search program (BioEdit). Putative small interfering (si)RNAs complementary in sequence to Cereal yellow dwarf virus (CYDV, genus Luteovirus) genomes were identified. Primer sequences were made against the “high scoring” siRNA sequences and RT-PCR was used to amplify a 438 bp CYDV fragment in total RNA extracts from D. glomerata leaves. Sequencing of the RT-PCR product confirmed the occurrence of a previously undescribed CYDV population with phylogenetic affinity to CYDV-RPS. In D. glomerata the CYDV infection rates were 42.3% (n = 78) in 2008 and 50.0% (n = 48) in 2009. Specific RT-PCR tests also showed that this D. glomerata population harboured Cocksfoot streak virus (CSV, genus Potyvirus). Dual infections by these viruses were observed in 20.5–22.9% of all plants tested in 2008–2009. Interestingly, infections of either CYDV or CSV enhanced the occurrence of the other virus in individual grasses. Opportunities are discussed for using siRNA sequencing approaches in virus survey and other ecology studies under field conditions

Proof of concept pilot study: prevalence of grass virus infection and the potential for effects on the allergenic potency of pollen

Background: Wild plants harbour a variety of viruses and these have the potential to alter the composition of pollen. The potential consequences of virus infection of grasses on pollen-induced allergic disease are not known. Methods: We have collected pollen from Dactylis glomerata (cocksfoot; a grass species implicated as a trigger of allergic rhino-conjunctivitis) from Wytham Wood, Oxfordshire UK. Extracts were prepared from pollen from uninfected grass, and from grass naturally infected by the Cocksfoot streak potyvirus (CSV). Preparations of pollen from virus-infected and non-infected grasses were employed in skin testing 15 grass pollen-allergic subjects with hayfever. Allergen profiles of extracts were investigated by Western blotting for IgE with sera from allergic subjects. Results: The prevalence of CSV infection in cocksfoot grasses sampled from the study site varied significantly over an eight-year period, but infection rates of up to 70% were detected. Virus infection was associated with small alterations in the quantities of pollen proteins detected by polyacrylamide gel electrophoresis, and in the patterns of allergens identified by Western blotting with IgE from grass pollen allergic subjects. For individual subjects there were differences in potencies of standardised extracts of pollen from virus-free and virus-infected plants as assessed by skin testing, though a consistent pattern was not established for the group of 15 subjects. Conclusion: Infection rates for CSV in cocksfoot grass can be high, though variable. Virus-induced alterations in components of grass pollen have the potential to alter the allergenic potency

Conserved short fragments (CSF) of the virus population.

<p>Between-species conserved regions (consecutively larger than 25 nt, without any SNP) were identified by using sequence alignment of the VDV-1/DWV/KV clade (Figure S5). Within-population SNP was calculated by using the sRNA deep sequencing dataset. Each CSF was labelled using DWV genome positions. Colour coded positions represented within-population mutation rate (Pi) as Pi = 0 (dark green), Pi < 5% (light green), 5% ≤ Pi < 10% (yellow) and Pi ≥ 10% (red). The mutation rate at each nucleotide position was reported in Table S3. Black dots label CSFs with 20 consecutive low Pi sites at the within-population level.</p

Virus derived small interfering (vsiRNA) mapping profiles.

<p>(A) Unique (non-redundant) vsiRNAs mapped to either VDV-1 or DWV or KV genomes. Shared reads were excluded. (B) Positions of vsiRNAs mapped to DWV and KV genomes. Shared reads with VDV-1 were excluded. (C) Shared vsiRNAs mapped to all of the three virus genomes. Note that the Y-axis represents both plus and minus strands and the scale is different among the three panels.</p

Comparison of assembled virus genomes.

<p>(A) A Neighbour Joint tree constructed with 1000 bootstraps. NCBI reference sequences were labelled with virus names followed by the GenBank accession numbers. The Maq assembled sequences were labelled accordingly to the reference sequences used. The <i>Sacbrood </i><i>virus</i> (SBV) reference genome was used as an outer sequence. (B) SNP profile using the 3 viral reference sequences. (C) SNP profile using the 3 Maq assembled virus genomes.</p

Sequence recombination and conservation of Varroa destructor virus-1 and deformed wing virus in field collected honey bees (Apis mellifera)

We sequenced small (s)RNAs from field collected honeybees (Apis mellifera) and bumblebees (Bombus pascuorum) using the Illumina technology. The sRNA reads were assembled and resulting contigs were used to search for virus homologues in GenBank. Matches with Varroa destructor virus-1 (VDV1) and Deformed wing virus (DWV) genomic sequences were obtained for A. mellifera but not B. pascuorum. Further analyses suggested that the prevalent virus population was composed of VDV-1 and a chimera of 5’-DWV-VDV1-DWV-3’. The recombination junctions in the chimera genomes were confirmed by using RT-PCR, cDNA cloning and Sanger sequencing. We then focused on conserved short fragments (CSF, size > 25 nt) in the virus genomes by using GenBank sequences and the deep sequencing data obtained in this study. The majority of CSF sites confirmed conservation at both between-species (GenBank sequences) and within-population (dataset of this study) levels. However, conserved nucleotide positions in the GenBank sequences might be variable at the within-population level. High mutation rates (Pi>10%) were observed at a number of sites using the deep sequencing data, suggesting that sequence conservation might not always be maintained at the population level. Virus-host interactions and strategies for developing RNAi treatments against VDV1/DWV infections are discussed