The Association between Broiler Litter Microbiota and the Supplementation of Bacillus Probiotics in a Leaky Gut Model

Probiotics provided from hatch have a major influence on microbiota development, and together with environmental and bedding microbiota, shape the microbial community of the litter. We investigated the influence of probiotic supplementation and a leaky gut challenge induced using dexamethasone (DEX) on the litter microbial community and litter parameters. The probiotic product was a mix of three Bacillus amyloliquefaciens strains. The litter microbiota were compared to the microbial communities from other gut sections. The litter samples had higher microbial diversity compared to the caecum, gizzard, jejunum, and jejunal mucosa. The high similarity between the litter phylum-level microbiota and gizzard microbiota detected in our study could be a consequence of ingested feed and litter passing through the gizzard. Moreover, the litter microbial community is fundamentally distinct from the intestinal microbiota, as evidenced by the number of genera present in the litter but absent from all the intestinal sections and vice versa. Furthermore, LEfSe analysis identified distinct microbial taxa across different groups, with specific genera associated with different treatments. In terms of litter quality, the birds in the DEX groups had a significantly higher moisture content, indicating successful leaky gut challenge, while probiotic supplementation did not significantly affect the moisture levels. These findings provide comprehensive insights into the distinct microbiota characteristics of litter

The role of CDC48 in the retro-translocation of non-ubiquitinated toxin substrates in plant cells

When the catalytic A subunits of the castor bean toxins ricin and Ricinus communis agglutinin (denoted as RTA and RCA A, respectively) are delivered into the endoplasmic reticulum (ER) of tobacco protoplasts, they become substrates for ER-associated protein degradation (ERAD). As such, these orphan polypeptides are retro-translocated to the cytosol, where a significant proportion of each protein is degraded by proteasomes. Here we begin to characterise the ERAD pathway in plant cells, showing that retro-translocation of these lysine-deficient glycoproteins requires the ATPase activity of cytosolic CDC48. Lysine polyubiquitination is not obligatory for this step. We also show that while RCA A is found in a mannose-untrimmed form prior to its retro-translocation, a significant proportion of newly synthesised RTA cycles via the Golgi and becomes modified by downstream glycosylation enzymes. Despite these differences, both proteins are similarly retro-translocated

Design and Construction of Multigenic Constructs for Plant Biotechnology Using the GoldenBraid Cloning Strategy

GoldenBraid (GB) is an iterative and standardized DNA assembling system specially designed for Multigene Engineering in Plant Synthetic Biology. GB is based on restriction–ligation reactions using type IIS restriction enzymes. GB comprises a collection of standard DNA pieces named “GB parts” and a set of destination plasmids (pDGBs) that incorporate the multipartite assembly of standardized DNA parts. GB reactions are extremely efficient: two transcriptional units (TUs) can be assembled from several basic GBparts in one T-DNA less than 24 h. Moreover, larger assemblies comprising 4–5 TUs are routinely built in less than 2 working weeks. Here we provide a detailed view of the GB methodology. As a practical example, a Bimolecular Fluorescence Complementation construct comprising four TUs in a 12 kb DNA fragment is presented.Sarrion-Perdigones, A.; Palací, J.; Granell Richart, A.; Orzáez Calatayud, DV. (2014). Design and Construction of Multigenic Constructs for Plant Biotechnology Using the GoldenBraid Cloning Strategy. Methods in Molecular Biology. 1116:133-151. doi:10.1007/978-1-62703-764-8_10S1331511116Haseloff J, Ajioka J (2009) Synthetic biology, history, challenges and prospects. J R Soc Interface 6(Suppl 4):S389–S391Check E (2005) Synthetic biology, designs on life. Nature 438:417–418Kosuri S, Eroshenko N, LeProust EM et al (2010) Scalable gene synthesis by selective amplification of DNA pools from high-fidelity microchips. Nat Biotechnol 28:1295–1299Ellis T, Adie T, Baldwin GS (2011) DNA assembly for synthetic biology, from parts to pathways and beyond. Integr Biol 3:109–118Gibson DG, Young L, Chuang R-Y et al (2009) Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat Methods 6: 343–345Gibson DG, Glass JI, Lartigue C et al (2010) Creation of a bacterial cell controlled by a chemically synthesized genome. Science 329:52–56Sarrion-Perdigones A, Falconi EE, Zandalinas SI et al (2011) GoldenBraid, an iterative cloning system for standardized assembly of reusable genetic modules. PLoS One 6:e21622Sarrion-Perdigones A, Vilar-Vazquez M et al (2013) GoldenBraid2.0, A comprehensive DNA assembly framework for plant synthetic biology. Plant Physiol 162:1618–1631Engler C, Gruetzner R, Kandzia R (2009) Golden gate shuffling, a one-pot DNA shuffling method based on type IIs restriction enzymes. PLoS One 4:e5553Engler C, Kandzia R, Marillonnet S (2008) A one pot, one step, precision cloning method with high throughput capability. PLoS One 3:e3647Bracha-Drori K, Shichrur K, Katz A et al (2004) Detection of protein-protein interactions in plants using bimolecular fluorescence complementation. Plant J 40:419–427Smaczniak C, Immink RG, Muino JM et al (2012) Characterization of MADS-domain transcription factor complexes in Arabidopsis flower development. Proc Natl Acad Sci U S A 109:1560–1565de Folter S, Immink RG, Kieffer M et al (2005) Comprehensive interaction map of the Arabidopsis MADS Box transcription factors. Plant Cell 17:1424–1433Lorenz WW, McCann RO, Longiaru M et al (1991) Isolation and expression of a cDNA encoding Renilla reniformis luciferase. Proc Natl Acad Sci U S A 88:4438–4442Voinnet O, Pinto YM, Baulcombe DC (1999) Suppression of gene silencing: a general strategy used by diverse DNA and RNA viruses of plants. Proc Natl Acad Sci U S A 96: 14147–14152Hellens RP, Edwards EA, Leyland NR et al (2000) pGreen: a versatile and flexible binary Ti vector for Agrobacterium-mediated plant transformation. Plant Mol Biol 42:819–832Butelli E, Titta L, Giorgio M et al (2008) Enrichment of tomato fruit with health-promoting anthocyanins by expression of select transcription factors. Nat Biotechnol 26: 1301–1308Kapila J, DeRycke R, VanMontagu M et al (1997) An Agrobacterium-mediated transient gene expression system for intact leaves. Plant Sci 122:101–10

Bacillus amyloliquefaciens probiotics mix supplementation in a broiler leaky gut model

The supplementation of antimicrobial growth promoters (AGPs) has been banned in many countries because of the emergence of antimicrobial-resistant pathogens in poultry products and the environment. Probiotics have been broadly studied and demonstrated as a promising AGP substitute. Our study is centred on the effects of a multi-strain Bacillus-based probiotic product on broiler production performance and gut microbial profile in a dexamethasone-induced leaky gut challenge. Two hundred and fifty-six broiler chicks were hatched and randomly assigned into four groups (wheat-soybean meal basal diet (BD) = non-supplemented control (C), BD supplemented with dexamethasone in week 4 (CD), BD containing a probiotic from day one (P), and BD containing a probiotic from day one and supplemented with dexamethasone during challenge week 4 (PD)). The production performance and caecal, gizzard, jejunal lumen and jejunal mucosa swab microbiota were studied by 16S rRNA gene sequencing. The Bacillus probiotic product significantly improved production performance and altered caecal gut microbiota (p ≤ 0.05), but no significant impact on microbiota was observed in other gut sections

The Agrobacterium-mediated transformation of common wheat (Triticum aestivum L.) and triticale (x Triticosecale Wittmack): role of the binary vector system and selection cassettes

The influence of two binary vector systems, pGreen and pCAMBIA, on the Agrobacterium-mediated transformation ability of wheat and triticale was studied. Both vectors carried selection cassettes with bar or nptII driven by different promoters. Two cultivars of wheat, Kontesa and Torka, and one cultivar of triticale, Wanad, were tested. The transformation rates for the wheat cultivars ranged from 0.00 to 3.58% and from 0.00 to 6.79% for triticale. The best values for wheat were 3.58% for Kontesa and 3.14% for Torka, and these were obtained after transformation with the pGreen vector carrying the nptII selection gene under the control of 35S promoter. In the case of the bar selection system, the best transformation rates were, respectively, 1.46 and 1.79%. Such rates were obtained when the 35S::bar cassette was carried by the pCAMBIA vector; they were significantly lower with the pGreen vector. The triticale cultivar Wanad had its highest transformation rate after transformation with nptII driven by 35S in pCAMBIA. The bar selection system for the same triticale cultivar was better when the gene was driven by nos and the selection cassette was carried by pGreen. The integration of the transgenes was confirmed with at least three pairs of specific starters amplifying the fragments of nptII, bar, or gus. The expression of selection genes, measured by reverse transcriptase polymerase chain reaction (RT-PCR) in relation to the actin gene, was low, ranging from 0.00 to 0.63 for nptII and from 0.00 to 0.33 for bar. The highest relative transcript accumulation was observed for nptII driven by 35S and expressed in Kontesa that had been transformed with pGreen

GoldenBraid: An Iterative Cloning System for Standardized Assembly of Reusable Genetic Modules

Synthetic Biology requires efficient and versatile DNA assembly systems to facilitate the building of new genetic modules/pathways from basic DNA parts in a standardized way. Here we present GoldenBraid (GB), a standardized assembly system based on type IIS restriction enzymes that allows the indefinite growth of reusable gene modules made of standardized DNA pieces. The GB system consists of a set of four destination plasmids (pDGBs) designed to incorporate multipartite assemblies made of standard DNA parts and to combine them binarily to build increasingly complex multigene constructs. The relative position of type IIS restriction sites inside pDGB vectors introduces a double loop (“braid”) topology in the cloning strategy that allows the indefinite growth of composite parts through the succession of iterative assembling steps, while the overall simplicity of the system is maintained. We propose the use of GoldenBraid as an assembly standard for Plant Synthetic Biology. For this purpose we have GB-adapted a set of binary plasmids for A. tumefaciens-mediated plant transformation. Fast GB-engineering of several multigene T-DNAs, including two alternative modules made of five reusable devices each, and comprising a total of 19 basic parts are also described

Molecular characterization of seven genes encoding ethylene-responsive transcriptional factors during plum fruit development and ripening

Seven ERF cDNAs were cloned from two Japanese plum (Prunus salicina L.) cultivars, ‘Early Golden’ (EG) and ‘Shiro’ (SH). Based on the sequence characterization, these Ps-ERFs could be classified into three of the four known ERF families. Their predicted amino acid sequences exhibited similarities to ERFs from other plant species. Functional nuclear localization signal analyses of two Ps-ERF proteins (Ps-ERF1a and -1b) were carried out using confocal microscopy. Expression analyses of Ps-ERF mRNAs were studied in the two plum cultivars in order to determine the role of this gene family in fruit development and ripening. The seven Ps-ERFs displayed differential expression pattern and levels throughout the various stages of flower and fruit development. The diversity in Ps-ERFs accumulation was largely due to the differences in their responses to the levels of ethylene production. However, other plant hormones such as cytokinin and auxin, which accumulate strongly throughout the various developmental stages, also influence the Ps-ERFs expression. The effect of the plant hormones, gibberellin, cytokinin, auxin, and ethylene in regulating the different Ps-ERF transcripts was investigated. A model was proposed in which the role played by the plant hormone auxin is as important as that of ethylene in initiating and determining the date and rate of ripening in Japanese plums

AGO1 and AGO2 Act Redundantly in miR408-Mediated Plantacyanin Regulation

Background: In Arabidopsis, AGO1 and AGO2 associate with small RNAs that exhibit a Uridine and an Adenosine at their 59 end, respectively. Because most plant miRNAs have a 59U, AGO1 plays many essential roles in miRNA-mediated regulation of development and stress responses. In contrast, AGO2 has only been implicated in antibacterial defense in association with miR393*, which has a 59A. AGO2 also participates in antiviral defense in association with viral siRNAs. Principal Findings: This study reveals that miR408, which has a 59A, regulates its target Plantacyanin through either AGO1 or AGO2. Indeed, neither ago1 nor ago2 single mutations abolish miR408-mediated regulation of Plantacyanin. Only an ago1 ago2 double mutant appears compromised in miR408-mediated regulation of Plantacyanin, suggesting that AGO1 and AGO2 have redundant roles in this regulation. Moreover, the nature of the 59 nucleotide of miR408 does not appear essential for its regulatory role because both a wildtype 59A-MIR408 and a mutant 59U-MIR408 gene complement a mir408 mutant. Conclusions/Significance: These results suggest that miR408 associates with both AGO1 and AGO2 based on criteria that differ from the 59 end rule, reminiscent of miR390-AGO7 and miR165/166-AGO10 associations, which are not based on the nature of the 59 nucleotide

Agrobacterium-mediated transformation systems of Primula vulgaris

Background: Genetic transformation is a valuable tool and an important procedure in plant functional genomics contributing to gene discovery, allowing powerful insights into gene function and genetically controlled characteristics. Primulaceae species provide one of the best-known examples of heteromorphic flower development, a breeding system which has attracted considerable attention, including that of Charles Darwin. Molecular approaches, including plant transformation give the best opportunity to define and understand the role of genes involved in floral heteromorphy in the common primrose, Primula vulgaris, along with other Primula species. Results: Two transformation systems have been developed in P. vulgaris. The first system, Agrobacterium-mediated vacuum infiltration of seedlings, enables the rapid testing of transgenes, transiently in planta. GUS expression was observed in the cotyledons, true leaves, and roots of Primula seedlings. The second system is based on Agrobacterium tumefaciens infection of pedicel explants with an average transformation efficiency of 4.6%. This transformation system, based on regeneration and selection of transformants within in vitro culture, demonstrates stable transgene integration and transmission to the next generation. Conclusion: The two transformation systems reported here will aid fundamental research into important traits in Primula. Although, stable integration of transgenes is the ultimate goal for such analyses, transient gene expression via Agrobacterium-mediated DNA transfer, offers a simple and fast method to analyse transgene functions. The second system describes, for the first time, stable Agrobacterium-mediated transformation of Primula vulgaris, which will be key to characterising the genes responsible for the control of floral heteromorphy