From genes to ecosystems: a synthesis of the effects of plant genetic factors across levels of organization

Using two genetic approaches and seven different plant systems, we present findings from a meta-analysis examining the strength of the effects of plant genetic introgression and genotypic diversity across individual, community and ecosystem levels with the goal of synthesizing the patterns to date. We found that (i) the strength of plant genetic effects can be quite high; however, the overall strength of genetic effects on most response variables declined as the levels of organization increased. (ii) Plant genetic effects varied such that introgression had a greater impact on individual phenotypes than extended effects on arthropods or microbes/fungi. By contrast, the greatest effects of genotypic diversity were on arthropods. (iii) Plant genetic effects were greater on above-ground versus below-ground processes, but there was no difference between terrestrial and aquatic environments. (iv) The strength of the effects of intraspecific genotypic diversity tended to be weaker than interspecific genetic introgression. (v) Although genetic effects generally decline across levels of organization, in some cases they do not, suggesting that specific organisms and/or processes may respond more than others to underlying genetic variation. Because patterns in the overall impacts of introgression and genotypic diversity were generally consistent across diverse study systems and consistent with theoretical expectations, these results provide generality for understanding the extended consequences of plant genetic variation across levels of organization, with evolutionary implications.     

A Rapid High-throughput Method for Mapping Ribonucleoproteins (RNPs) on Human pre-mRNA

Sequencing RNAs that co-immunoprecipitate (co-IP) with RNA binding proteins has increased our understanding of splicing by demonstrating that binding location often influences function of a splicing factor. However, as with any sampling strategy the chance of identifying an RNA bound to a splicing factor is proportional to its cellular abundance. We have developed a novel in vitro approach for surveying binding specificity on otherwise transient pre-mRNA. This approach utilizes a specifically designed oligonucleotide pool that tiles across introns, exons, splice junctions, or other pre-mRNA. The pool is subjected to some kind of molecular selection. Here, we demonstrate the method by separating the oligonucleotide into a bound and unbound fraction and utilize a two color array strategy to record the enrichment of each oligonucleotide in the bound fraction. The array data generates high-resolution maps with the ability to identify sequence-specific and structural determinates of ribonucleoprotein (RNP) binding on pre-mRNA. A unique advantage to this method is its ability to avoid the sampling bias towards mRNA associated with current IP and SELEX techniques, as the pool is specifically designed and synthesized from pre-mRNA sequence. The flexibility of the oligonucleotide pool is another advantage since the experimenter chooses which regions to study and tile across, tailoring the pool to their individual needs. Using this technique, one can assay the effects of polymorphisms or mutations on binding on a large scale or clone the library into a functional splicing reporter and identify oligonucleotides that are enriched in the included fraction. This novel in vitro high-resolution mapping scheme provides a unique way to study RNP interactions with transient pre-mRNA species, whose low abundance makes them difficult to study with current in vivo techniques. Download video file.^{(148M, mp4)}     

Comparative <Emphasis Type="Italic">in vivo</Emphasis> infection models yield insights on early host immune response to Campylobacter in chickens

Salmonella typhimurium and Campylobacter jejuni pose significant risks to human health and poultry are a major vector for infection. Comparative in vivo infection models were performed to compare the avian host immune response to both bacterial species. Forty-five commercial broiler chickens were orally challenged with either C. jejuni or S. typhimurium whilst 60 similar control birds were mock challenged in parallel. Birds were sacrificed at 0, 6, 20 and 48 h post-infection and cloacal swabs, blood and tissue samples taken. Peripheral blood leukocytes were isolated for flow cytometric analyses and RNA was extracted for gene expression profiling. Colonisation patterns were markedly different between the two bacterial species, with systemic colonisation of Campylobacter outside the gastrointestinal tract. Salmonella infection induced significant changes in circulating heterophil and monocyte/macrophage populations, whilst Campylobacter infection had no effect on the heterophil numbers but caused a significant early increase in circulating monocytes/macrophages. Toll-like receptor 1 (TLR1) gene expression was decreased, and avian β-defensin (AvBD) gene expression (AvBD3, AvBD10 and AvBD12) was significantly increased in response to Salmonella infection (P < 0.05). In contrast, Campylobacter infection induced increased TLR21 gene expression but significantly reduced expression of seven antimicrobial peptide (AMP) genes (AvBD3, AvBD4, AvBD8, AvBD13, AvBD14, CTHL2 and CTHL3; P < 0.05). Considered together, microbiological, cellular and gene expression profiles indicate that the innate immune system responds differently to Salmonella and to Campylobacter infection. Furthermore, reduction in the expression of AMPs may play a role in the persistence of high level colonisation of the host by Campylobacter. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

The cin and rai Quorum-Sensing Regulatory Systems in Rhizobium leguminosarum Are Coordinated by ExpR and CinS,a Small Regulatory Protein Coexpressed with CinI

To understand how the Rhizobium leguminosarum raiI-raiR quorum-sensing system is regulated, we identified mutants with decreased levels of RaiI-made N-acyl homoserine lactones (AHLs). A LuxR-type regulator, ExpR, is required for raiR expression, and RaiR is required to induce raiI. Since raiR (and raiI) expression is also reduced in cinI and cinR quorum-sensing mutants, we thought CinI-made AHLs may activate ExpR to induce raiR. However, added CinI-made AHLs did not induce raiR expression in a cinI mutant. The reduced raiR expression in cinI and cinR mutants was due to lack of expression of cinS immediately downstream of cinI. cinS encodes a 67-residue protein, translationally coupled to CinI, and cinS acts downstream of expR for raiR induction. Cloned cinS in R. leguminosarum caused an unusual collapse of colony structure, and this was delayed by mutation of expR. The phenotype looked like a loss of exopolysaccharide (EPS) integrity; mutations in cinI, cinR, cinS, and expR all reduced expression of plyB, encoding an EPS glycanase, and mutation of plyB abolished the effect of cloned cinS on colony morphology. We conclude that CinS and ExpR act to increase PlyB levels, thereby influencing the bacterial surface. CinS is conserved in other rhizobia, including Rhizobium etli; the previously observed effect of cinI and cinR mutations decreasing swarming in that strain is primarily due to a lack of CinS rather than a lack of CinI-made AHL. We conclude that CinS mediates quorum-sensing regulation because it is coregulated with an AHL synthase and demonstrate that its regulatory effects can occur in the absence of AHLs.Production of N-acyl homoserine lactones (AHLs) is common to many plant-associated bacteria (7), in which it is usually associated with population density-dependent regulation of genes affecting adaptive responses (49). Within the family Rhizobiaceae, population density-regulated gene expression (quorum sensing) mediated via AHLs has been identified in several agrobacteria and rhizobia (13, 51). In Agrobacterium spp., quorum-sensing regulation was initially identified as a mechanism of regulating plasmid transfer. As the bacterial population density increases, plasmid transfer genes are induced by TraR in response to AHLs made by TraI (55). In several rhizobia, traI-like AHL synthase genes are also in an operon along with plasmid transfer genes (13).There are other quorum-sensing loci in different strains of rhizobia. In Sinorhizobium meliloti strain Rm1021, AHLs produced by SinI activate SinR and ExpR, LuxR-type regulators, to induce several genes, including those determining the production of an exopolysaccharide, exopolysaccharide II (EPS-II) (17, 23, 24, 35), that plays an important role in the symbiosis. In S. meliloti, two LuxR-type regulators, VisN and VisR, are involved in chemotaxis and motility (24, 44). Rhizobium etli has multiple AHL synthase genes (9, 39), but the functions of many of the regulated genes remain to be established. The cinR and cinI genes are required for normal symbiotic nitrogen fixation and swarming in R. etli (5, 9, 11) and for normal levels of expression of raiI, which encodes another AHL synthase. The expression of raiI in R. etli is regulated by RaiR (39).Analysis of AHLs produced by strain A34 of Rhizobium leguminosarum bv. viciae led to the characterization of four LuxI-type AHL synthases (RhiI, CinI, RaiI, and TraI) and five LuxR-type regulators (RhiR, CinR, RaiR, TraR, and BisR) (8, 31, 50, 53). In this strain, the cinI and cinR genes are chromosomally located; CinI produces N-(3-hydroxy-7-cis-tetradecenoyl)-l-homoserine lactone (3-OH-C_14:1-HSL) (20, 31), CinR induces cinI expression in response to this AHL (31), and this appears to be associated with adaptation to starvation and salt stress (47). Mutation of cinI or cinR affects the expression of the other three AHL synthase genes in R. leguminosarum bv. viciae strain A34. Thus, in a cinI mutant, the expression of raiI is reduced, resulting in very low levels of 3-OH-C₈-HSL, the major AHL made by RaiI (53). Similarly, the expression levels of the traI and rhiI genes on the symbiotic plasmid pRL1JI are reduced in cinI and cinR mutants (31). RhiI-made AHLs activate RhiR to induce the expression of the rhiABC operon in R. leguminosarum bv. viciae (38), enhancing the interaction with the legume host (8).The cinI and cinR quorum-sensing genes control induction of the traI and traR quorum-sensing regulons via CinI-made 3-OH-C_14:1-HSL, which activates BisR (another LuxR-type regulator) to induce traR and hence traI (12). However, the mechanism by which cinI and/or cinR control raiI and raiR expression has not been established. In this work we demonstrate that raiI and raiR expression requires both expR and a small gene (cinS) cotranscribed with cinI. CinS also regulates the expression of plyB encoding an extracellular glycanase and is required for swarming of R. etli.     

The pore domain outer helix contributes to both activation and inactivation of the HERG K+ channel

Ion flow in many voltage-gated K(+) channels (VGK), including the (human ether-a-go-go-related gene) hERG channel, is regulated by reversible collapse of the selectivity filter. hERG channels, however, exhibit low sequence homology to other VGKs, particularly in the outer pore helix (S5) domain, and we hypothesize that this contributes to the unique activation and inactivation kinetics in hERG K(+) channels that are so important for cardiac electrical activity. The S5 domain in hERG identified by NMR spectroscopy closely corresponded to the segment predicted by bioinformatics analysis of 676 members of the VGK superfamily. Mutations to approximately every third residue, from Phe(551) to Trp(563), affected steady state activation, whereas mutations to approximately every third residue on an adjacent face and spanning the entire S5 segment perturbed inactivation, suggesting that the whole span of S5 experiences a rearrangement associated with inactivation. We refined a homology model of the hERG pore domain using constraints from the mutagenesis data with residues affecting inactivation pointing in toward S6. In this model the three residues with maximum impact on activation (W563A, F559A, and F551A) face out toward the voltage sensor. In addition, the residues that when mutated to alanine, or from alanine to valine, that did not express (Ala(561), His(562), Ala(565), Trp(568), and Ile(571)), all point toward the pore helix and contribute to close hydrophobic packing in this region of the channel.     

GeneComps and ChemComps: a new CTD metric to identify genes and chemicals with shared toxicogenomic profiles

The Comparative Toxicogenomics Database is a public resource that promotes understanding about the effects of environmental chemicals on human health. Currently, CTD describes over 184,000 molecular interactions for more than 5,100 chemicals and 16,300 genes/proteins. We have leveraged this dataset of chemical-gene relationships to compute similarity indices following the statistical method of the Jaccard index. These scores are used to produce lists of comparable genes (“GeneComps”) or chemicals (“ChemComps”) based on shared toxicogenomic profiles. GeneComps and ChemComps are now provided for every curated gene and chemical in CTD. ChemComps are particularly significant because they provide a way to group chemicals based upon their biological effects, instead of their physical or structural properties. These metrics provide a novel way to view and classify genes and chemicals and will help advance testable hypotheses about environmental chemical-genedisease networks.

Availability

CTD is freely available at http://ctd.mdibl.org/     

The evolution of floral variation without pollinator shifts in Gorteria diffusa (Asteraceae)

One of the most widely accepted explanations for floral diversification in angiosperms is the pollinator-shift model developed by Verne Grant and Ledyard Stebbins. According to this model, the most profound changes in floral traits (such as morphology, color, patterning and scent) occur when plants undergo adaptive shifts between pollinator classes. We tested this model through investigations of geographical variation in floral form and pollinator assemblages in the South African annual daisy Gorteria diffusa. This species has elaborate insect-like ornaments on the capitulum, which attract bee flies belonging to the genus Megapalpus. We found unprecedented levels of geographically structured intraspecific variation and identified 14 discrete forms that vary in the morphology and ornamentation of the capitulum. This variation is not due to phenotypic plasticity because differences among forms were maintained in plants grown from seed in a common garden experiment. Contrary to predictions from the pollinator-shift model, all populations, regardless of floral phenotype, were pollinated primarily by a single species of Megapalpus bee fly. Much of the extensive variation in floral form in G. diffusa therefore appears to have arisen without evolutionary shifts between pollinator types.