Genetic dissection of a TIR‐NB‐LRR locus from the wild North American grapevine species Muscadinia rotundifolia identifies paralogous genes conferring resistance to major fungal and oomycete pathogens in cultivated grapevine

The most economically important diseases of grapevine cultivation worldwide are caused by the fungal pathogen powdery mildew (Erysiphe necator syn. Uncinula necator) and the oomycete pathogen downy mildew (Plasmopara viticola). Currently, grapegrowers rely heavily on the use of agrochemicals to minimize the potentially devastating impact of these pathogens on grape yield and quality. The wild North American grapevine species Muscadinia rotundifolia was recognized as early as 1889 to be resistant to both powdery and downy mildew. We have now mapped resistance to these two mildew pathogens in M. rotundifolia to a single locus on chromosome 12 that contains a family of seven TIR‐NB‐LRR genes. We further demonstrate that two highly homologous (86% amino acid identity) members of this gene family confer strong resistance to these unrelated pathogens following genetic transformation into susceptible Vitis vinifera winegrape cultivars. These two genes, designated r esistance to P lasmopara v iticola (MrRPV1) are the first resistance genes to be cloned from a grapevine species. Both MrRUN1 and MrRPV1 were found to confer resistance to multiple powdery and downy mildew isolates from France, North America and Australia; however, a single powdery mildew isolate collected from the south‐eastern region of North America, to which M. rotundifolia is native, was capable of breaking MrRUN1‐mediated resistance. Comparisons of gene organization and coding sequences between M. rotundifolia and the cultivated grapevine V. vinifera at the MrRUN1/MrRPV1 locus revealed a high level of synteny, suggesting that the TIR‐NB‐LRR genes at this locus share a common ancestor.     

Cytidine derivatives as IspF inhibitors of Burkolderia pseudomallei

Published biological data suggest that the methyl erythritol phosphate (MEP) pathway, a non-mevalonate isoprenoid biosynthetic pathway, is essential for certain bacteria and other infectious disease organisms. One highly conserved enzyme in the MEP pathway is 2C-methyl-d-erythritol 2,4-cyclodiphosphate synthase (IspF). Fragment-bound complexes of IspF from Burkholderia pseudomallei were used to design and synthesize a series of molecules linking the cytidine moiety to different zinc pocket fragment binders. Testing by surface plasmon resonance (SPR) found one molecule in the series to possess binding affinity equal to that of cytidine diphosphate, despite lacking any metal-coordinating phosphate groups. Close inspection of the SPR data suggest different binding stoichiometries between IspF and test compounds. Crystallographic analysis shows important variations between the binding mode of one synthesized compound and the pose of the bound fragment from which it was designed. The binding modes of these molecules add to our structural knowledge base for IspF and suggest future refinements in this compound series.     

Bayesian parentage analysis reliably controls the number of false assignments in natural populations

Parentage analysis in natural populations is a powerful tool for addressing a wide range of ecological and evolutionary questions. However, identifying parent–offspring pairs in samples collected from natural populations is often more challenging than simply resolving the Mendelian pattern of shared alleles. For example, large numbers of pairwise comparisons and limited numbers of genetic markers can contribute to incorrect assignments, whereby unrelated individuals are falsely identified as parent–offspring pairs. Determining which parentage methods are the least susceptible to making false assignments is an important challenge facing molecular ecologists. In a recent paper, Harrison et al. (2013a) address this challenge by comparing three commonly used parentage methods, including a Bayesian approach, in order to explore the effects of varied proportions of sampled parents on the accuracy of parentage assignments. Unfortunately, Harrison et al. made a simple error in using the Bayesian approach, which led them to incorrectly conclude that this method could not control the rate of false assignment. Here, I briefly outline the basic principles behind the Bayesian approach, identify the error made by Harrison et al., and provide detailed guidelines as to how the method should be correctly applied. Furthermore, using the exact data from Harrison et al., I show that the Bayesian approach actually provides greater control over the number of false assignments than either of the other tested methods. Lastly, I conclude with a brief introduction to solomon , a recently updated version of the Bayesian approach that can account for genotyping error, missing data and false matching.     

Structural and dynamic properties of bacterial Type IV secretion systems (Review)

The type IV secretion systems (T4SS) are widely distributed among the Gram-negative and –positive bacteria. These systems mediate the transfer of DNA and protein substrates across the cell envelope to bacterial or eukaryotic cells generally through a process requiring direct cell-to-cell contact. Bacteria have evolved T4SS for survival during establishment of pathogenic or symbiotic relationships with eukaryotic hosts. The Agrobacterium tumefaciens VirB/D4 T4SS and related conjugation machines serve as models for detailed mechanistic studies aimed at elucidating the nature of translocation signals, machine assembly pathways and architectures, and the dynamics of substrate translocation. The A. tumefaciens VirB/D4 T4SS are polar-localized organelles composed of a secretion channel and an extracellular T pilus. These T4SS are assembled from 11 or more subunits. whose membrane topologies, intersubunit contacts and, in some cases, 3-dimensional structures are known. Recently, powerful in vivo assays have identified C-terminal translocation signals, defined for the first time the translocation route for a DNA substrate through a type IV secretion channel, and supplied evidence that ATP energy consumption contributes to a late stage of machine morphogenesis. Together, these recent findings describe the mechanics of type IV secretion in unprecedented detail.     

Fish oil disrupts MHC class II lateral organization on the B-cell side of the immunological synapse independent of B-T cell adhesion

Fish oil-enriched long chain n-3 polyunsaturated fatty acids disrupt the molecular organization of T-cell proteins in the immunological synapse. The impact of fish oil derived n-3 fatty acids on antigen-presenting cells, particularly at the animal level, is unknown. We previously demonstrated B-cells isolated from mice fed with fish oil-suppressed naïve CD4⁺ T-cell activation. Therefore, here we determined the mechanistic effects of fish oil on murine B-cell major histocompatibility complex (MHC) class II molecular distribution using a combination of total internal reflection fluorescence, Förster resonance energy transfer and confocal imaging. Fish oil had no impact on presynaptic B-cell MHC II clustering. Upon conjugation with transgenic T-cells, fish-oil suppressed MHC II accumulation at the immunological synapse. As a consequence, T-cell protein kinase C theta (PKCθ) recruitment to the synapse was also diminished. The effects were independent of changes in B-T cell adhesion, as measured with microscopy, flow cytometry and static cell adhesion assays with select immune ligands. Given that fish oil can reorganize the membrane by lowering membrane cholesterol levels, we then compared the results with fish oil to cholesterol depletion using methyl-B-cyclodextrin (MβCD). MβCD treatment of B-cells suppressed MHC II and T-cell PKCθ recruitment to the immunological synapse, similar to fish oil. Overall, the results reveal commonality in the mechanism by which fish oil manipulates protein lateral organization of B-cells compared to T-cells. Furthermore, the data establish MHC class II lateral organization on the B-cell side of the immunological synapse as a novel molecular target of fish oil.     

Circadian Locomotor Rhythms in Mice with Targeted Disruption of the Gene for the Carbon Monoxide Synthesizing Enzyme,Heme Oxygenase-2

Carbon monoxide (CO), generated in neurons by the enzyme heme oxygenase-2 (HO2), is postulated to be a gaseous signaling molecule in the mammalian brain. Because of the recent evidence suggesting an important role of another endogenously produced gas, nitric oxide (NO), in entrainment of circadian rhythms in mammals, we hypothesized that CO may also be involved in regulating these rhythms. Consistent with this idea, others have found a circadian rhythm of heme turnover and CO synthesis can be induced by bright light. Furthermore, HO2 is co-localized with guanylyl cyclase, the putative target of CO, throughout the brain, with high amounts of staining in the suprachiasmatic nucleus (SCN) of the hypothalamus. The goal of the present study was to evaluate the role of CO in photic entrainment in wild-type and HO2 deficient mice. HO2–/– mice did not display any abnormalities in circadian rhythmicity. Entrainment to a light–dark cycle, the ability to phase delay locomotor activity after a four hour phase shift in photoperiod, and the period of the free running rhythm (t) were similar between HO2–/– and wild-type mice. Taken together, these data suggest that CO does not play a major role in regulating circadian activity rhythms in mice.     

A Conserved Aspartic Acid Is Important for Agonist (VUAA1) and Odorant/Tuning Receptor-Dependent Activation of the Insect Odorant Co-Receptor (Orco)

Insect odorant receptors function as heteromeric odorant-gated cation channels comprising a conventional odorant-sensitive tuning receptor, and a conserved co-receptor (Orco). An Orco agonist, VUAA1, is able to activate both heteromeric and homomeric Orco-containing channels. Very little is known about specific residues in Orco that contribute to cation permeability and gating. We investigated the importance of two conserved Asp residues, one in each of transmembrane domains 5 and 7, for channel function by mutagenesis. Drosophila melanogaster Orco and its substitution mutants were expressed in HEK cells and VUAA1-stimulated channel activity was determined by Ca²⁺ influx and whole-cell patch clamp electrophysiology. Substitution of D466 in transmembrane 7 with amino acids other than glutamic acid resulted in a substantial reduction in channel activity. The D466E Orco substitution mutant was ∼2 times more sensitive to VUAA1. The permeability of the D466E Orco mutant to cations was unchanged relative to wild-type Orco. When D466E Orco is co-expressed with a conventional tuning odorant receptor, the heteromeric complex also shows increased sensitivity to an odorant. Thus, the effect of the D466E mutation is not specific to VUAA1 agonism or dependent on homomeric Orco assembly. We suggest the gain-of-activation characteristic of the D466E mutant identifies an amino acid that is likely to be important for activation of both heteromeric and homomeric insect odorant receptor channels.     

The Advocacy for Pedestrian Safety Study: Cluster Randomised Trial Evaluating a Political Advocacy Approach to Reduce Pedestrian Injuries in Deprived Communities

Objective

To determine whether advocacy targeted at local politicians leads to action to reduce the risk of pedestrian injury in deprived areas.

Design

Cluster randomised controlled trial.

Setting

239 electoral wards in 57 local authorities in England and Wales.

Participants

617 elected local politicians.

Interventions

Intervention group politicians were provided with tailored information packs, including maps of casualty sites, numbers injured and a synopsis of effective interventions.

Main outcome measures

25–30 months post intervention, primary outcomes included: electoral ward level: percentage of road traffic calmed; proportion with new interventions; school level: percentage with 20 mph zones, Safe Routes to School, pedestrian training or road safety education; politician level: percentage lobbying for safety measures. Secondary outcomes included politicians’ interest and involvement in injury prevention, and facilitators and barriers to implementation.

Results

Primary outcomes did not significantly differ: % difference in traffic calming (0.07, 95%CI: −0.07 to 0.20); proportion of schools with 20 mph zones (RR 1.47, 95%CI: 0.93 to 2.32), Safe Routes to School (RR 1.34, 95%CI: 0.83 to 2.17), pedestrian training (RR 1.23, 95%CI: 0.95 to 1.61) or other safety education (RR 1.16, 95%CI: 0.97 to 1.39). Intervention group politicians reported greater interest in child injury prevention (RR 1.09, 95%CI 1.03 to 1.16), belief in potential to help prevent injuries (RR 1.36, 95%CI 1.16 to 1.61), particularly pedestrian safety (RR 1.55, 95%CI 1.19 to 2.03). 63% of intervention politicians reported supporting new pedestrian safety schemes. The majority found the advocacy information surprising, interesting, effectively presented, and could identify suitable local interventions.

Conclusions

This study demonstrates the feasibility of an innovative approach to translational public health by targeting local politicians in a randomised controlled trial. The intervention package was positively viewed and raised interest but changes in interventions were not statistically significance. Longer term supported advocacy may be needed.

Trial Registration

Current Controlled Trials ISRCTN91381117     

Decoupled genomic elements and the evolution of partner quality in nitrogen‐fixing rhizobia

Understanding how mutualisms evolve in response to a changing environment will be critical for predicting the long‐term impacts of global changes, such as increased N (nitrogen) deposition. Bacterial mutualists in particular might evolve quickly, thanks to short generation times and the potential for independent evolution of plasmids through recombination and/or HGT (horizontal gene transfer). In a previous work using the legume/rhizobia mutualism, we demonstrated that long‐term nitrogen fertilization caused the evolution of less‐mutualistic rhizobia. Here, we use our 63 previously isolated rhizobium strains in comparative phylogenetic and quantitative genetic analyses to determine the degree to which variation in partner quality is attributable to phylogenetic relationships among strains versus recent genetic changes in response to N fertilization. We find evidence of distinct evolutionary relationships between chromosomal and pSym genes, and broad similarity between pSym genes. We also find that nifD has a unique evolutionary history that explains much of the variation in partner quality, and suggest MoFe subunit interaction sites in the evolution of less‐mutualistic rhizobia. These results provide insight into the mechanisms behind the evolutionary response of rhizobia to long‐term N fertilization, and we discuss the implications of our results for the evolution of the mutualism.