The Rapid Methylation of T-DNAs Upon Agrobacterium Inoculation in Plant Leaves

Agrobacterium tumefaciens has been foundational in the development of transgenic plants for both agricultural biotechnology and plant molecular research. However, the transformation efficiency and level of transgene expression obtained for any given construct can be highly variable. These inefficiencies often require screening of many lines to find one with consistent and heritable transgene expression. Transcriptional gene silencing is known to affect transgene expression, and is associated with DNA methylation, especially of cytosines in symmetric CG and CHG contexts. While the specificity, heritability and silencing-associated effects of DNA methylation of transgene sequences have been analyzed in many stably transformed plants, the methylation status of transgene sequences in the T-DNA during the transformation process has not been well-studied. Here we used agro-infiltration of the eGFP reporter gene in Nicotiana benthamiana leaves driven by either an AtEF1α-A4 or a CaMV-35S promoter to study early T-DNA methylation patterns of these promoter sequences. The T-DNA was examined by amplicon sequencing following sodium bisulfite treatment using three different sequencing platforms: Sanger sequencing, Ion Torrent PGM, and the Illumina MiSeq. Rapid DNA methylation was detectable in each promoter region just 2–3 days post-infiltration and the levels continued to rapidly accumulate over the first week, then steadily up to 21 days later. Cytosines in an asymmetric context (CHH) were the most heavily and rapidly methylated. This suggests that early T-DNA methylation may be important in determining the epigenetic and transcriptional fate of integrated transgenes. The Illumina MiSeq platform was the most sensitive and robust way of detecting and following the methylation profiles of the T-DNA promoters. The utility of the methods was then used to show a subtle but significant difference in promoter methylation during intron-mediated enhancement. In addition, the method was able to detect an increase in promoter methylation when the eGFP reporter gene was targeted by siRNAs generated by co-infiltration of a hairpin RNAi construct

Vibrio cholerae Proteome-Wide Screen for Immunostimulatory Proteins Identifies Phosphatidylserine Decarboxylase as a Novel Toll-Like Receptor 4 Agonist

Recognition of conserved bacterial components provides immediate and efficient immune responses and plays a critical role in triggering antigen-specific adaptive immunity. To date, most microbial components that are detected by host innate immune system are non-proteinaceous structural components. In order to identify novel bacterial immunostimulatory proteins, we developed a new high-throughput approach called “EPSIA”, Expressed Protein Screen for Immune Activators. Out of 3,882 Vibrio cholerae proteins, we identified phosphatidylserine decarboxylase (PSD) as a conserved bacterial protein capable of activating host innate immunity. PSD in concentrations as low as 100 ng/ml stimulated RAW264.7 murine macrophage cells and primary peritoneal macrophage cells to secrete TNFα and IL-6, respectively. PSD-induced proinflammatory response was dependent on the presence of MyD88, a known adaptor molecule for innate immune response. An enzymatically inactive PSD mutant and heat-inactivated PSD induced ∼40% and ∼15% of IL-6 production compared to that by native PSD, respectively. This suggests that PSD induces the production of IL-6, in part, via its enzymatic activity. Subsequent receptor screening determined TLR4 as a receptor mediating the PSD-induced proinflammatory response. Moreover, no detectable IL-6 was produced in TLR4-deficient mouse macrophages by PSD. PSD also exhibited a strong adjuvant activity against a co-administered antigen, BSA. Anti-BSA response was decreased in TLR4-deficient mice immunized with BSA in combination with PSD, further proving the role of TLR4 in PSD signaling in vivo. Taken together, these results provide evidence for the identification of V. cholerae PSD as a novel TLR4 agonist and further demonstrate the potential application of PSD as a vaccine adjuvant

The widely used Nicotiana benthamiana 16c line has an unusual T-DNA integration pattern including a transposon sequence.

Nicotiana benthamiana is employed around the world for many types of research and one transgenic line has been used more extensively than any other. This line, 16c, expresses the Aequorea victoria green fluorescent protein (GFP), highly and constitutively, and has been a major resource for visualising the mobility and actions of small RNAs. Insights into the mechanisms studied at a molecular level in N. benthamiana 16c are likely to be deeper and more accurate with a greater knowledge of the GFP gene integration site. Therefore, using next generation sequencing, genome mapping and local alignment, we identified the location and characteristics of the integrated T-DNA. As suggested from previous molecular hybridisation and inheritance data, the transgenic line contains a single GFP-expressing locus. However, the GFP coding sequence differs from that originally reported. Furthermore, a 3.2 kb portion of a transposon, appears to have co-integrated with the T-DNA. The location of the integration mapped to a region of the genome represented by Nbv0.5scaffold4905 in the www.benthgenome.com assembly, and with less integrity to Niben101Scf03641 in the www.solgenomics.net assembly. The transposon is not endogenous to laboratory strains of N. benthamiana or Agrobacterium tumefaciens strain GV3101 (MP90), which was reportedly used in the generation of line 16c. However, it is present in the popular LBA4404 strain. The integrated transposon sequence includes its 5' terminal repeat and a transposase gene, and is immediately adjacent to the GFP gene. This unexpected genetic arrangement may contribute to the characteristics that have made the 16c line such a popular research tool and alerts researchers, taking transgenic plants to commercial release, to be aware of this genomic hitchhiker

The impact of emergency department crowding measures on time to antibiotics for patients with community-acquired pneumonia

STUDY OBJECTIVE: We seek to determine the impact of emergency department (ED) crowding on delays in antibiotic administration for patients with community-acquired pneumonia. METHODS: We performed a retrospective cohort study of adult patients admitted with community-acquired pneumonia from January 1, 2003, to April 31, 2005, at a single, urban academic ED. The main outcome was a delay (\u3e4 hours from arrival) or nonreceipt of antibiotics in the ED. Eight ED crowding measures were assigned at triage. Multivariable regression and bootstrapping were used to test the adjusted impact of ED crowding measures of delayed (or no) antibiotics. Predicted probabilities were then calculated to assess the magnitude of the impact of ED crowding on the probability of delayed (or no) antibiotics. RESULTS: In 694 patients, 44% (95% confidence interval [CI] 40% to 48%) received antibiotics within 4 hours and 92% (95% CI 90% to 94%) received antibiotics in the ED. Increasing levels of ED crowding were associated with delayed (or no) antibiotics, including waiting room number (odds ratio [OR] 1.05 for each additional waiting room patient [95% CI 1.01 to 1.10]) and recent ED length of stay for admitted patients (OR 1.14 for each additional hour [95% CI 1.04 to 1.25]). When the waiting room and recent length of stay were both at the lowest quartiles (ie, not crowded), the predicted probability of delayed (or no) antibiotics within 4 hours was 31% (95% CI 21% to 42%); when both were at the highest quartiles, the predicted probability was 72% (95% CI 61% to 81%). CONCLUSION: ED crowding is associated with delayed and nonreceipt of antibiotics in the ED for patients admitted with community-acquired pneumonia

Schematic of pBIN19-mGFP-ER and Tn5393 and their integration site in 16c.

<p><b>(A–B)</b> Schematic of genes within the T-DNA and transposon integrated in 16c genome. The sequence junctions between <i>N</i>. <i>benthamiana</i> genome-T-DNA-transposon-<i>N</i>. <i>benthamiana</i> genome are highlighted, GenBank accession number KY464890. <b>(C)</b> Log scale density plot of sequence reads aligning to T-DNA, transposon and flanking <i>N</i>. <i>benthamiana</i> genomic regions. <b>(D)</b> Location of primers used for amplification of various regions to confirm site of integration. <b>(E)</b> Log scale density plot of RNAseq reads aligning to T-DNA and transposon. <b>(F)</b> Gel image showing the endpoint (35 cycles) PCR amplicons of the T-DNA insert in 16c with LAB <i>N</i>. <i>benthamiana</i> used as a control. Expected sizes with primer pair B+E is 4174 nt and D+F is 4167 nt. NTC, no template control, 1 kb DNA Ladder (GeneRuler^™). <b>(G)</b> Leaves of <i>N</i>. <i>benthamiana</i> 16c photographed under UV light, showing mobile silencing, 14 days after local induction of RNAi at a lower leaf. Abbreviations are: RB: Right Border, pNOS: nopaline synthase promoter, <i>NPTII</i>: neomycin phosphotransferase II encoding gene, tNOS: nopaline synthase terminator, p35S: Cauliflower mosaic virus 35S promoter. IR: inverted terminal repeat, <i>tnpA</i> transposase gene, res: recombination region, <i>tnpR</i> resolvase gene, IS1133: an insertion element, <i>strA</i> and <i>strB</i>: streptomycin resistance genes, Nb Genome: Flanking DNA of <i>N</i>. <i>benthamiana</i>.</p

Schematic showing the alignment of Sanger sequences to scaffolds in benthgenome.com and solgenomics.net.

<p>Schematic showing the alignment of Sanger sequences to scaffolds in benthgenome.com and solgenomics.net.</p

List of primers used in this study.

<p>List of primers used in this study.</p