PIF4 and ELF3 Act Independently in <i>Arabidopsis thaliana</i> Thermoresponsive Flowering

<div><p>Plants have evolved elaborate mechanisms controlling developmental responses to environmental stimuli. A particularly important stimulus is temperature. Previous work has identified the interplay of PIF4 and ELF3 as a central circuit underlying thermal responses in <i>Arabidopsis thaliana</i>. However, thermal responses vary widely among strains, possibly offering mechanistic insights into the wiring of this circuit. ELF3 contains a polyglutamine (polyQ) tract that is crucial for ELF3 function and varies in length across strains. Here, we use transgenic analysis to test the hypothesis that natural polyQ variation in ELF3 is associated with the observed natural variation in thermomorphogenesis. We found little evidence that the polyQ tract plays a specific role in thermal responses beyond modulating general ELF3 function. Instead, we made the serendipitous discovery that ELF3 plays a crucial, PIF4-independent role in thermoresponsive flowering under conditions more likely to reflect field conditions. We present evidence that ELF3 acts through the photoperiodic pathway, pointing to a previously unknown symmetry between low and high ambient temperature responses. Moreover, in analyzing two strain backgrounds with different thermal responses, we demonstrate that responses may be shifted rather than fundamentally rewired across strains. Our findings tie together disparate observations into a coherent framework in which multiple pathways converge in accelerating flowering in response to temperature, with some such pathways modulated by photoperiod.</p></div

ELF3 and GI regulate thermoresponsive flowering.

<p>(A): Temperature-responsive expression of photoperiodic pathway components at ZT0. Expression of each gene is quantified relative to levels in Col at 22° (Col 22 = 1.0). Error bars represent SEM across three biological replicates. <i>elf3-4</i>: <i>elf3</i> null in Ws background; <i>elf3-200</i>: <i>elf3</i> null in Col background. (B): Thermoresponsive flowering in various flowering mutants. LD RLN = rosette leaf number at flowering under long days. *: 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; details of regression model in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161791#pone.0161791.s013" target="_blank">S9 Table</a>. (C) Thermoresponsive petiole elongation in various flowering mutants. For (B) and (C), n > = 8 plants of each genotype in each condition; white boxes indicate measurements at 22°, red boxes indicate measurements at 27°. <i>gi</i>: <i>gi-2</i>, <i>co</i>: <i>co-101</i>, <i>spy</i>: <i>spy-3</i>, <i>soc1</i>: <i>soc1</i> T-DNA insertion, <i>elf3</i>: <i>elf3-200</i>. Outliers (defined as >1.5 interquartile ranges away from the median) of each distribution are indicated as points. This experiment was repeated with similar results. (D): Models of thermoresponsive flowering under long and short photoperiods. Dashed edges indicate speculated temperature sensing mechanisms. Edges with increased weight indicate relative increases of influence between conditions. Pathways are indicated, along with other important actors reported elsewhere.</p

<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

Response to elevated temperature (27°, relative to 22°) among transgenic lines expressing ELF3-polyQ variants.

<p>Mean response and error were estimated by regression, based on two independently-generated transgenic lines for each genotype, with n > = 30 seedlings of each genotype in each condition (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161791#pone.0161791.s005" target="_blank">S1 Table</a>). WT = Ws, <i>elf3</i> = <i>elf3</i> mutant+vector control, 0Q = <i>elf3</i> mutant+<i>ELF3</i> transgene lacking polyQ, etc. Error bars indicate standard error of the mean. (A): Ws (Wassilewskija) strain background. Lines are generated in an <i>elf3-4</i> background. (B): Response in the Col (Columbia) strain background, lines were generated in an <i>elf3-200</i> background. In both (A) and (B), response is defined as the change in hypocotyl length in mm; **: Bonferroni-corrected p < 0.01, *: Bonferroni-corrected p < 0.05,.: Bonferroni-corrected p < 0.1 in testing the interaction term (different response from WT, Ws or Col). (C): Temperature response is a function of ELF3 functionality (repression of hypocotyl elongation at 22°). Simple means of 22° hypocotyl length, regression estimates of temperature response. PCC = Pearson correlation coefficient; p-value is from a Pearson correlation test.</p

MIPSTR: a method for multiplex genotyping of germline and somatic STR variation across many individuals

Short tandem repeats (STRs) are highly mutable genetic elements that often reside in regulatory and coding DNA. The cumulative evidence of genetic studies on individual STRs suggests that STR variation profoundly affects phenotype and contributes to trait heritability. Despite recent advances in sequencing technology, STR variation has remained largely inaccessible across many individuals compared to single nucleotide variation or copy number variation. STR genotyping with short-read sequence data is confounded by (1) the difficulty of uniquely mapping short, low-complexity reads; and (2) the high rate of STR amplification stutter. Here, we present MIPSTR, a robust, scalable, and affordable method that addresses these challenges. MIPSTR uses targeted capture of STR loci by single-molecule Molecular Inversion Probes (smMIPs) and a unique mapping strategy. Targeted capture and our mapping strategy resolve the first challenge; the use of single molecule information resolves the second challenge. Unlike previous methods, MIPSTR is capable of distinguishing technical error due to amplification stutter from somatic STR mutations. In proof-of-principle experiments, we use MIPSTR to determine germline STR genotypes for 102 STR loci with high accuracy across diverse populations of the plant A. thaliana. We show that putatively functional STRs may be identified by deviation from predicted STR variation and by association with quantitative phenotypes. Using DNA mixing experiments and a mutant deficient in DNA repair, we demonstrate that MIPSTR can detect low-frequency somatic STR variants. MIPSTR is applicable to any organism with a high-quality reference genome and is scalable to genotyping many thousands of STR loci in thousands of individuals

MIPSTR: a method for multiplex genotyping of germline and somatic STR variation across many individuals

Short tandem repeats (STRs) are highly mutable genetic elements that often reside in regulatory and coding DNA. The cumulative evidence of genetic studies on individual STRs suggests that STR variation profoundly affects phenotype and contributes to trait heritability. Despite recent advances in sequencing technology, STR variation has remained largely inaccessible across many individuals compared to single nucleotide variation or copy number variation. STR genotyping with short-read sequence data is confounded by (1) the difficulty of uniquely mapping short, low-complexity reads; and (2) the high rate of STR amplification stutter. Here, we present MIPSTR, a robust, scalable, and affordable method that addresses these challenges. MIPSTR uses targeted capture of STR loci by single-molecule Molecular Inversion Probes (smMIPs) and a unique mapping strategy. Targeted capture and our mapping strategy resolve the first challenge; the use of single molecule information resolves the second challenge. Unlike previous methods, MIPSTR is capable of distinguishing technical error due to amplification stutter from somatic STR mutations. In proof-of-principle experiments, we use MIPSTR to determine germline STR genotypes for 102 STR loci with high accuracy across diverse populations of the plant A. thaliana. We show that putatively functional STRs may be identified by deviation from predicted STR variation and by association with quantitative phenotypes. Using DNA mixing experiments and a mutant deficient in DNA repair, we demonstrate that MIPSTR can detect low-frequency somatic STR variants. MIPSTR is applicable to any organism with a high-quality reference genome and is scalable to genotyping many thousands of STR loci in thousands of individuals

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

Functional enrichments in the classes of <i>hsp90A</i>-associated genes.

*<p><b>The number of genes with this functional annotation in the </b><b><i>hsp90A</i></b><b>-associated set and in the background set.</b></p

<i>ΔhtpG E. coli</i> cells spread less efficiently on soft-agar plates.

<p>Upon equal mixing, WT and <i>ΔhtpG</i> cells were competed for 8 hours at 34° on the same soft-agar plates, where bacteria spread in a motility- and chemotaxis-dependent fashion. Samples from the outer edge of the plate are thus enriched in cells with optimal chemotaxis and motility, whereas cells from the center are less chemotactic and/or motile. (<b>A</b>) A representative image of assay plate. (<b>B</b>) Quantitation of different genotypes as determined by percentage of the YFP-labeled WT vs. CFP-labeled <i>ΔhtpG</i> cells at the indicated locations. YFP and CFP expression was induced by 1 µM IPTG. An essentially identical result was obtained for the CFP-labeled WT vs. YFP-labeled <i>ΔhtpG</i> cells (data not shown), confirming that it is label-independent. Error bars indicate standard errors from four replicates. <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003631#s2" target="_blank">Results</a> were similar at 42°C (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003631#pgen.1003631.s010" target="_blank">Table S3</a>).</p