elf3_pif4_project_data_code

data and code pertinent to submitted manuscript

<i>elf3</i> and <i>pif4</i> null mutant phenotypes are independent under LD treatments and robust to conditions.

<p>(A), (B), and (C): 22°: constant 22° LD growth; 27° 14d: transfer from 22° to 27° at 14 days post-germination; 27° 1d: transfer from 22° to 27° at 1 day post-germination. (A): Col (WT), <i>elf3-200</i>, and <i>pif4-2</i> plants grown under long days with three different temperature regimes were photographed at 20 days post germination. Experiment was repeated with similar results. (B and C): Petiole elongation responses of the indicated genotypes, measured by ratio of petiole to whole leaf length at 25 days post germination. Regression analysis of data in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161791#pone.0161791.s007" target="_blank">S3 Table</a>. In each case, **: Bonferroni-corrected p < 0.01, *: Bonferroni-corrected p < 0.05, in testing whether the genotype x environment interaction term (difference of 22°-27 response from the Col 22°-27° response) differs from zero. Outliers (defined as >1.5 interquartile ranges away from the median) of each distribution are indicated as points.</p

Current and suggested GWAS approaches.

<p>(A) Current approach. GWAS identify variants that are overrepresented in cases. Rare variants of large effect (red square, blue star) may escape detection, thereby contributing to missing heritability. Common variants that are overrepresented in cases (small yellow bar, 6 versus 2) do not contribute strongly to disease risk. A cryptic disease-related variant does not show significant overrepresentation in cases (open circle). (B) Suggested approach. Individuals are first analyzed for phenotypic robustness (bold box) and then for variants associated with disease. Rare variants of large effect will be enriched in robust cases, although they may also be present in nonrobust cases. Variants that are overrepresented in all cases (robust, nonrobust) will show higher penetrance in nonrobust individuals (large yellow bars). The formerly cryptic, disease-related variant (open circle) is significantly enriched in nonrobust cases versus nonrobust controls (and robust cases) and can therefore be identified. Together, heritability significantly increases. The formerly cryptic genetic variant and higher penetrance variant can be thought of as “disease-specifiers” as they determine the specific disease phenotype of individuals carrying them. Note symbols represent highly simplified frequencies of specific variant in indicated groups and not individuals carrying certain variants.</p

Summary of combined statistical and functional support for loci underlying root length.

<p>Four epistatic pairs, involving seven unique loci (connected boxes), were identified at a genome-wide significance-threshold in a two-dimensional genome-wide GWA scan. Green, yellow, and red lines connect pairs of loci with very low (p = 0.003), intermediate (0.28 < p < 0.37), and high (p = 0.95) risk for the interaction being a false-positive when accounting for population size. The risk of the statistical epistatic association resulting from high-order LD to an unobserved functional variant in the genome (i.e. “apparent epistasis”) is illustrated by arrow color, in which yellow indicates an intermediate risk (0.18 < p < 0.42) and green a very low risk (p = 0.0021). Green boxes indicate loci for which the T-DNA insertion line analyses suggest the named genes to be involved in root development. When considering the joint statistical and functional results, two pairs emerge as highly likely true positive two-locus associations: 3_66596/3_9273674 due to very strong statistical support and one identified functional candidate gene, and 3_10891195/5_1027939 where the identification of functional candidate genes at both loci suggest that the two-locus association in the original genome-wide scan is true despite the lower statistical support in after the conservative statistical correction for sample-size. For the other two pairs, the results are inconclusive. There is strong support for one of the two associated loci (3_66596 from its statistical interaction with 3_9273674 and 1_17257526 by the detection of the a functional candidate gene in the T-DNA analysis), but weaker support for the second locus. Further work is thus needed to conclude whether these pairs represent true positive two-locus associations, or whether they are false-positives due to the small population-size or high-order LD (“apparent epistasis”) to unknown functional variants.</p

Function and expression patterns of genes in LD with leading SNP from epistatic GWAS analysis.

<p>¹ Locations of high expression were obtained from the BAR eFP <i>Arabidopsis</i> Browser [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005541#pgen.1005541.ref044" target="_blank">44</a>].</p><p>The gene names or proposed function is listed for all genes harboring polymorphisms in LD with the leading epistatic SNPs in the whole genome interaction analysis.</p

Estimated narrow (h²) and broad sense (H²) heritabilities of root length mean and variance in a population of 93 natural <i>A</i>. <i>thaliana</i> accessions.

<p>¹Estimated using ANOVA on accession,</p><p>²Estimated using the R/hglm package (hglm) by fitting a linear mixed model including both additive and epistatic kinship matrices as random effects,</p><p>³p-values from Wald tests for the heritability being larger than zero, and s.e. are the standard errors estimated via jackknife resampling.</p><p>The broad-sense heritability estimates obtained using the phenotypic variances within and between accessions (H² = V_G/V_P; ANOVA) and the genomic relationships of the accessions (H² = (V_A + V_AA)/V_P; hglm) were similar and intermediate. The narrow-sense heritability (h² = V_A/V_P), estimated based on the genomic relationships between the accessions, was negligible.</p

Estimation of the risk that epistatic pairs identified in the GWA analysis are due to high-order LD to unobserved functional variants (“apparent epistasis”).

<p>¹Locus detected as part of epistatic pair named as Chromosome_PositionInBp; r²_mean: mean high-order LD between epistatic pseudo-marker and genome-wide sequence variants ± standard error; r²_max: maximum high-order LD between epistatic pseudo-marker and _genome-wide sequence variants ± standard error; P(r²_max > 0.8): probability of observing a high-order LD larger then 0.8 in a random sample of 93 accessions with MAF_PM for the epistatic pseudo-marker; MAF_PM: minor-allele frequency for the epistatic pseudo-marker, i.e. the frequency of the minor-allele double-homozygote for the epistatic pair.</p><p>Using the whole-genome re-sequencing data from the reference 1001 Genomes <i>A</i>. <i>thaliana</i> collection, we estimated the high-order LD (r²) between the four pseudo-markers representing our epistatic pairs and all sequencing variants that were not genotyped in our GWA analysis. A bootstrap approach was used to estimate the mean and max r² between the epistatic pseudo-markers and all the genome-wide sequencing-variants. The risk that an association might be due to “apparent epistasis” was calculated as the risk of observing an r²_max > 0.8 in this analysis.</p

Four statistical epistatic interactions associated with mean root length.

<p>(A) The x- and y-axes represent the five <i>A</i>. <i>thaliana</i> chromosomes. The positions of the seven SNPs that are part of the four significant interacting pairs (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005541#pgen.1005541.t002" target="_blank">Table 2</a>) are indicated by a black dot. Solid lines indicate support for an interaction by more than one linked SNP and dotted lines indicate support by a single SNP. (B) Genotype-phenotype (G-P) map of the root means for the four genotype combinations constituting the interaction between the SNPs on chromosome 1 (17,257,526 bp) and chromosome 5 (15,862,026 bp). The major allele is indicated by -1, and the minor allele is indicated by 1. This G-P-map is a representative example for the other pairs in which the accessions with a minor and major allele have lower phenotypic values than those with either both major or both minor alleles (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005541#pgen.1005541.s003" target="_blank">S2 Fig</a>). (C) G-P map for the most significant epistatic pair illustrates how this type of epistasis cancels the additive genetic variance at the allele-frequencies observed for the two loci in the analyzed population of wild-collected <i>A</i>. <i>thaliana</i> accessions.</p

Genome-scale Co-evolutionary Inference Identifies Functions and Clients of Bacterial Hsp90

<div><p>The molecular chaperone Hsp90 is essential in eukaryotes, in which it facilitates the folding of developmental regulators and signal transduction proteins known as Hsp90 clients. In contrast, Hsp90 is not essential in bacteria, and a broad characterization of its molecular and organismal function is lacking. To enable such characterization, we used a genome-scale phylogenetic analysis to identify genes that co-evolve with bacterial Hsp90. We find that genes whose gain and loss were coordinated with Hsp90 throughout bacterial evolution tended to function in flagellar assembly, chemotaxis, and bacterial secretion, suggesting that Hsp90 may aid assembly of protein complexes. To add to the limited set of known bacterial Hsp90 clients, we further developed a statistical method to predict putative clients. We validated our predictions by demonstrating that the flagellar protein FliN and the chemotaxis kinase CheA behaved as Hsp90 clients in <i>Escherichia coli</i>, confirming the predicted role of Hsp90 in chemotaxis and flagellar assembly. Furthermore, normal Hsp90 function is important for wild-type motility and/or chemotaxis in <i>E. coli</i>. This novel function of bacterial Hsp90 agreed with our subsequent finding that Hsp90 is associated with a preference for multiple habitats and may therefore face a complex selection regime. Taken together, our results reveal previously unknown functions of bacterial Hsp90 and open avenues for future experimental exploration by implicating Hsp90 in the assembly of membrane protein complexes and adaptation to novel environments.</p></div