A large-scale metagenomic survey dataset of the post-weaning piglet gut lumen

BackgroundEarly weaning and intensive farming practices predispose piglets to the development of infectious and often lethal diseases, against which antibiotics are used. Besides contributing to the build-up of antimicrobial resistance, antibiotics are known to modulate the gut microbial composition. As an alternative to antibiotic treatment, studies have previously investigated the potential of probiotics for the prevention of postweaning diarrhea. In order to describe the post-weaning gut microbiota, and to study the effects of two probiotics formulations and of intramuscular antibiotic treatment on the gut microbiota, we sampled and processed over 800 faecal time-series samples from 126 piglets and 42 sows.ResultsHere we report on the largest shotgun metagenomic dataset of the pig gut lumen microbiome to date, consisting of >8 Tbp of shotgun metagenomic sequencing data. The animal trial, the workflow from sample collection to sample processing, and the preparation of libraries for sequencing, are described in detail. We provide a preliminary analysis of the dataset, centered on a taxonomic profiling of the samples, and a 16S-based beta diversity analysis of the mothers and the piglets in the first 5 weeks after weaning.ConclusionsThis study was conducted to generate a publicly available databank of the faecal metagenome of weaner piglets aged between 3 and 9 weeks old, treated with different probiotic formulations and intramuscular antibiotic treatment. Besides investigating the effects of the probiotic and intramuscular antibiotic treatment, the dataset can be explored to assess a wide range of ecological questions with regards to antimicrobial resistance, host-associated microbial and phage communities, and their dynamics during the aging of the host

DRB2 Is Required for MicroRNA Biogenesis in Arabidopsis thaliana

Background The Arabidopsis thaliana (Arabidopsis) DOUBLE-STRANDED RNA BINDING (DRB) protein family consists of five members, DRB1 to DRB5. The biogenesis of two developmentally important small RNA (sRNA) species, the microRNAs (miRNAs) and trans-acting small interfering RNAs (tasiRNAs) by DICER-LIKE (DCL) endonucleases requires the assistance of DRB1 and DRB4 respectively. The importance of miRNA-directed target gene expression in plant development is exemplified by the phenotypic consequence of loss of DRB1 activity (drb1 plants). Principal Findings Here we report that the developmental phenotype of the drb235 triple mutant plant is the result of deregulated miRNA biogenesis in the shoot apical meristem (SAM) region. The expression of DRB2, DRB3 and DRB5 in wild-type seedlings is restricted to the SAM region. Small RNA sequencing of the corresponding tissue of drb235 plants revealed altered miRNA accumulation. Approximately half of the miRNAs detected remained at levels equivalent to those of wild-type plants. However, the accumulation of the remaining miRNAs was either elevated or reduced in the triple mutant. Examination of different single and multiple drb mutants revealed a clear association between the loss of DRB2 activity and altered accumulation for both the elevated and reduced miRNA classes. Furthermore, we show that the constitutive over-expression of DRB2 outside of its wild-type expression domain can compensate for the loss of DRB1 activity in drb1 plants. Conclusions/Significance Our results suggest that in the SAM region, DRB2 is both antagonistic and synergistic to the role of DRB1 in miRNA biogenesis, adding an additional layer of gene regulatory complexity in this developmentally important tissue

Proteomic identification of putative microRNA394 target genes in <em>Arabidopsis thaliana</em> identifies major latex protein family members critical for normal development

Expression of the F-Box protein Leaf Curling Responsiveness (LCR) is regulated by microRNA, miR394, and alterations to this interplay in <i>Arabidopsis thaliana</i> produce defects in leaf polarity and shoot apical meristem organization. Although the miR394-LCR node has been documented in Arabidopsis, the identification of proteins targeted by LCR F-box itself has proven problematic. Here, a proteomic analysis of shoot apices from plants with altered LCR levels identified a member of the Latex Protein (MLP) family gene as a potential LCR F-box target. Bioinformatic and molecular analyses also suggested that other MLP family members are likely to be targets for this post-translational regulation. Direct interaction between LCR F-Box and MLP423 was validated. Additional MLP members had reduction in protein accumulation, in varying degrees, mediated by LCR F-Box. Transgenic Arabidopsis lines, in which MLP28 expression was reduced through an artificial miRNA technology, displayed severe developmental defects, including changes in leaf patterning and morphology, shoot apex defects, and eventual premature death. These phenotypic characteristics resemble those of Arabidopsis plants modified to over-express LCR. Taken together, the results demonstrate that MLPs are driven to degradation by LCR, and indicate that MLP gene family is target of miR394-LCR regulatory node, representing potential targets for directly post-translational regulation mediated by LCR F-Box. In addition, MLP28 family member is associated with the LCR regulation that is critical for normal Arabidopsis development

Community composition and development of the post-weaning piglet gut microbiome

ABSTRACT We report on the largest metagenomic analysis of the pig gut microbiome to date. By processing over 800 faecal time-series samples from 126 piglets and 42 sows, we generated over 8Tbp of metagenomic shotgun sequence data. Here we describe the animal trial procedures, the generation of our metagenomic dataset and the analysis of the microbial community composition using a phylogenetic framework. We assess the effects of intramuscular antibiotic treatment and probiotic oral treatment on the diversity of gut microbial communities. We found differences between individual hosts such as breed, litter, and age, to be important contributors to variation in the community composition. Treatment effects of the antibiotic and probiotic treatments were found but were subtle, while host age was the dominant factor in shaping the gut microbiota of piglets after weaning. The post-weaning pig gut microbiome appears to follow a highly structured developmental program with characteristic post-weaning changes that can distinguish hosts that were born as little as two days apart in the second month of life

Erysipelothrix spp. past, present and future directions in vaccine research

Erysipelothrix spp. comprise a group of small Gram-positive bacteria that can infect a variety of hosts including mammals, fish, birds, reptiles and insects. Among the eight Erysipelothrix species that have been described to date, only E. rhusiopathiae plays a major role in farmed livestock where it is the causative agent of erysipelas. E. rhusiopathiae also has zoonotic potential and can cause erysipeloid in humans with a clear occupational link to meat and fish industries. While there are 28 known Erysipelothrix serovars, over 80% of identified isolates belong to serovars 1 or 2. Vaccines to protect pigs against E. rhusiopathiae first became available in 1883 as a response to an epizootic of swine erysipelas in southern France. The overall vaccine repertoire was notably enlarged between the 1940s and 1960s following major outbreaks of swine erysipelas in the Midwest USA and have changed little since. Traditionally, E. rhusiopathiae serovar 1a or 2 isolates were inactivated (bacterins) or attenuated and these types of vaccines are still used today on a global basis. E. rhusiopathiae vaccines are most commonly used in pigs, poultry and sheep where the bacterium can cause considerable economic losses. In addition, erysipelas vaccination is also utilized in selected vulnerable susceptible populations, such as marine mammals in aquariums, which are commonly vaccinated at regular intervals. While commercially produced erysipelas vaccines appear to provide good protection against clinical disease, in recent years there has been an increase in perceived vaccine failures in farmed animals, especially in organic outdoor operations. Moreover, clinical erysipelas outbreaks have been reported in animal populations not previously considered at risk. This has raised concerns over a possible lack of vaccine protection across various production species. This review focuses on summarizing the history and the present status of E. rhusiopathiae vaccines, the current knowledge on protection including surface antigens, and also provides an outlook into future directions for vaccine development

High-Throughput Construction of Intron-Containing Hairpin RNA Vectors for RNAi in Plants

With the wide use of double-stranded RNA interference (RNAi) for the analysis of gene function in plants, a high-throughput system for making hairpin RNA (hpRNA) constructs is in great demand. Here, we describe a novel restriction-ligation approach that provides a simple but efficient construction of intron-containing hpRNA (ihpRNA) vectors. The system takes advantage of the type IIs restriction enzyme BsaI and our new plant RNAi vector pRNAi-GG based on the Golden Gate (GG) cloning. This method requires only a single PCR product of the gene of interest flanked with BsaI recognition sequence, which can then be cloned into pRNAi-GG at both sense and antisense orientations simultaneously to form ihpRNA construct. The process, completed in one tube with one restriction-ligation step, produced a recombinant ihpRNA with high efficiency and zero background. We demonstrate the utility of the ihpRNA constructs generated with pRNAi-GG vector for the effective silencing of various individual endogenous and exogenous marker genes as well as two genes simultaneously. This method provides a novel and high-throughput platform for large-scale analysis of plant functional genomics

Viral Small Interfering RNAs Target Host Genes to Mediate Disease Symptoms in Plants

The Cucumber mosaic virus (CMV) Y-satellite RNA (Y-Sat) has a small non-protein-coding RNA genome that induces yellowing symptoms in infected Nicotiana tabacum (tobacco). How this RNA pathogen induces such symptoms has been a longstanding question. We show that the yellowing symptoms are a result of small interfering RNA (siRNA)-directed RNA silencing of the chlorophyll biosynthetic gene, CHLI. The CHLI mRNA contains a 22-nucleotide (nt) complementary sequence to the Y-Sat genome, and in Y-Sat-infected plants, CHLI expression is dramatically down-regulated. Small RNA sequencing and 5′ RACE analyses confirmed that this 22-nt sequence was targeted for mRNA cleavage by Y-Sat-derived siRNAs. Transformation of tobacco with a RNA interference (RNAi) vector targeting CHLI induced Y-Sat-like symptoms. In addition, the symptoms of Y-Sat infection can be completely prevented by transforming tobacco with a silencing-resistant variant of the CHLI gene. These results suggest that siRNA-directed silencing of CHLI is solely responsible for the Y-Sat-induced symptoms. Furthermore, we demonstrate that two Nicotiana species, which do not develop yellowing symptoms upon Y-Sat infection, contain a single nucleotide polymorphism within the siRNA-targeted CHLI sequence. This suggests that the previously observed species specificity of Y-Sat-induced symptoms is due to natural sequence variation in the CHLI gene, preventing CHLI silencing in species with a mismatch to the Y-Sat siRNA. Taken together, these findings provide the first demonstration of small RNA-mediated viral disease symptom production and offer an explanation of the species specificity of the viral disease

TRBP and eIF6 Homologue in Marsupenaeus japonicus Play Crucial Roles in Antiviral Response

Plants and invertebrates can suppress viral infection through RNA silencing, mediated by RNA-induced silencing complex (RISC). Trans-activation response RNA-binding protein (TRBP), consisting of three double-stranded RNA-binding domains, is a component of the RISC. In our previous paper, a TRBP homologue in Fenneropenaeus chinensis (Fc-TRBP) was reported to directly bind to eukaryotic initiation factor 6 (Fc-eIF6). In this study, we further characterized the function of TRBP and the involvement of TRBP and eIF6 in antiviral RNA interference (RNAi) pathway of shrimp. The double-stranded RNA binding domains (dsRBDs) B and C of the TRBP from Marsupenaeus japonicus (Mj-TRBP) were found to mediate the interaction of TRBP and eIF6. Gel-shift assays revealed that the N-terminal of Mj-TRBP dsRBD strongly binds to double-stranded RNA (dsRNA) and that the homodimer of the TRBP mediated by the C-terminal dsRBD increases the affinity to dsRNA. RNAi against either Mj-TRBP or Mj-eIF6 impairs the dsRNA-induced sequence-specific RNAi pathway and facilitates the proliferation of white spot syndrome virus (WSSV). These results further proved the important roles of TRBP and eIF6 in the antiviral response of shrimp