Geo Velikkakam James

The genomic landscape of meiotic crossovers and gene conversions in Arabidopsis thaliana

Knowledge of the exact distribution of meiotic crossovers (COs) and gene conversions (GCs) is essential for understanding many aspects of population genetics and evolution, from haplotype structure and long-distance genetic linkage to the generation of new allelic variants of genes. To this end, we resequenced the four products of 13 meiotic tetrads along with 10 doubled haploids derived from Arabidopsis thaliana hybrids. GC detection through short reads has previously been confounded by genomic rearrangements. Rigid filtering for misaligned reads allowed GC identification at high accuracy and revealed an ∼80-kb transposition, which undergoes copy-number changes mediated by meiotic recombination. Non-crossover associated GCs were extremely rare most likely due to their short average length of ∼25–50 bp, which is significantly shorter than the length of CO-associated GCs. Overall, recombination preferentially targeted non-methylated nucleosome-free regions at gene promoters, which showed significant enrichment of two sequence motifs. DOI: http://dx.doi.org/10.7554/eLife.01426.001

Mutation identification by direct comparison of whole-genome sequencing data from mutant and wild-type individuals using k -mers

Genes underlying mutant phenotypes can be isolated by combining marker discovery, genetic mapping and resequencing, but a more straightforward strategy for mapping mutations would be the direct comparison of mutant and wild-type genomes. Applying such an approach, however, is hampered by the need for reference sequences and by mutational loads that confound the unambiguous identification of causal mutations. Here we introduce NIKS (needle in the k-stack), a reference-free algorithm based on comparing k-mers in whole-genome sequencing data for precise discovery of homozygous mutations. We applied NIKS to eight mutants induced in nonreference rice cultivars and to two mutants of the nonmodel species Arabis alpina. In both species, comparing pooled F2 individuals selected for mutant phenotypes revealed small sets of mutations including the causal changes. Moreover, comparing M3 seedlings of two allelic mutants unambiguously identified the causal gene. Thus, for any species amenable to mutagenesis, NIKS enables forward genetics without requiring segregating populations, genetic maps and reference sequences.

Genome expansion of Arabis alpina linked with retrotransposition and reduced symmetric DNA methylation

Despite evolutionary conserved mechanisms to silence transposable element activity, there are drastic differences in the abundance of transposable elements even among closely related plant species. We conducted a de novo assembly for the 375 Mb genome of the perennial model plant, Arabis alpina. Analysing this genome revealed long-lasting and recent transposable element activity predominately driven by Gypsy long terminal repeat retrotransposons, which extended the low-recombining pericentromeres and transformed large formerly euchromatic regions into repeat-rich pericentromeric regions. This reduced capacity for long terminal repeat retrotransposon silencing and removal in A. alpina co-occurs with unexpectedly low levels of DNA methylation. Most remarkably, the striking reduction of symmetrical CG and CHG methylation suggests weakened DNA methylation maintenance in A. alpina compared with Arabidopsis thaliana. Phylogenetic analyses indicate a highly dynamic evolution of some components of methylation maintenance machinery that might be related to the unique methylation in A. alpina.

User guide for mapping-by-sequencing in Arabidopsis

Mapping-by-sequencing combines genetic mapping with whole-genome sequencing in order to accelerate mutant identification. However, application of mapping-by-sequencing requires decisions on various practical settings on the experimental design that are not intuitively answered. Following an experimentally determined recombination landscape of Arabidopsis and next generation sequencing-specific biases, we simulated more than 400,000 mapping-by-sequencing experiments. This allowed us to evaluate a broad range of different types of experiments and to develop general rules for mapping-by-sequencing in Arabidopsis. Most importantly, this informs about the properties of different crossing scenarios, the number of recombinants and sequencing depth needed for successful mapping experiments.

The DET1-COP1-HY5 Pathway Constitutes a Multipurpose Signaling Module Regulating Plant Photomorphogenesis and Thermomorphogenesis

Developmental plasticity enables plants to respond to elevated ambient temperatures by adapting their shoot architecture. On the cellular level, the basic-helix-loop-helix (bHLH) transcription factor phytochrome interacting factor 4 (PIF4) coordinates this response by activating hormonal modules that in turn regulate growth. In addition to an unknown temperature-sensing mechanism, it is currently not understood how temperature regulates PIF4 activity. Using a forward genetic approach in Arabidopsis thaliana, we present extensive genetic evidence demonstrating that the de-etiolated 1 (DET1)-constitutive photomorphogenic 1 (COP1)-elongated hypocotyl 5 (HY5)-dependent photomorphogenesis pathway transcriptionally regulates PIF4 to coordinate seedling growth in response to elevated temperature. Our findings demonstrate that two of the most prevalent environmental cues, light and temperature, share a much larger set of signaling components than previously assumed. Similar to the toolbox concept in animal embryonic patterning, multipurpose signaling modules might have evolved in plants to translate various environmental stimuli into adaptational growth processes.

Natural variation reveals that intracellular distribution of ELF3 protein is associated with function in the circadian clock

Natural selection of variants within the Arabidopsis thaliana circadian clock can be attributed to adaptation to varying environments. To define a basis for such variation, we examined clock speed in a reporter-modified Bay-0 x Shakdara recombinant inbred line and localized heritable variation. Extensive variation led us to identify EARLY FLOWERING3 (ELF3) as a major quantitative trait locus (QTL). The causal nucleotide polymorphism caused a short-period phenotype under light and severely dampened rhythm generation in darkness, and entrainment alterations resulted. We found that ELF3-Sha protein failed to properly localize to the nucleus, and its ability to accumulate in darkness was compromised. Evidence was provided that the ELF3-Sha allele originated in Central Asia. Collectively, we showed that ELF3 protein plays a vital role in defining its light-repressor action in the circadian clock and that its functional abilities are largely dependent on its cellular localization. DOI: http://dx.doi.org/10.7554/eLife.02206.001

Complementary Activities of TELOMERE REPEAT BINDING Proteins and Polycomb Group Complexes in Transcriptional Regulation of Target Genes.

TELOMERE REPEAT BINDING proteins sustain repression of target genes they share with Polycomb Group repressive complexes. In multicellular organisms, Polycomb Repressive Complex 1 (PRC1) and PRC2 repress target genes through histone modification and chromatin compaction. Arabidopsis thaliana mutants strongly compromised in the pathway cannot develop differentiated organs. LIKE HETEROCHROMATIN PROTEIN1 (LHP1) is so far the only known plant PRC1 component that directly binds to H3K27me3, the histone modification set by PRC2, and also associates genome-wide with trimethylation of lysine 27 of histone H3 (H3K27me3). Surprisingly, lhp1 mutants show relatively mild phenotypic alterations. To explain this paradox, we screened for genetic enhancers of lhp1 mutants to identify novel components repressing target genes together with, or in parallel to, LHP1. Two enhancing mutations were mapped to TELOMERE REPEAT BINDING PROTEIN1 (TRB1) and its paralog TRB3. We show that TRB1 binds to thousands of genomic sites containing telobox or related cis-elements with a significant increase of sites and strength of binding in the lhp1 background. Furthermore, in combination with lhp1, but not alone, trb1 mutants show increased transcription of LHP1 targets, such as floral meristem identity genes, which are more likely to be bound by TRB1 in the lhp1 background. By contrast, expression of a subset of LHP1-independent TRB1 target genes, many involved in primary metabolism, is decreased in the absence of TRB1 alone. Thus, TRB1 is a bivalent transcriptional modulator that maintains downregulation of Polycomb Group (PcG) target genes in lhp1 mutants, while it sustains high expression of targets that are regulated independently of PcG.

Kicking against the PRCs - A Domesticated Transposase Antagonises Silencing Mediated by Polycomb Group Proteins and Is an Accessory Component of Polycomb Repressive Complex 2.

The Polycomb group (PcG) and trithorax group (trxG) genes play crucial roles in development by regulating expression of homeotic and other genes controlling cell fate. Both groups catalyse modifications of chromatin, particularly histone methylation, leading to epigenetic changes that affect gene activity. The trxG antagonizes the function of PcG genes by activating PcG target genes, and consequently trxG mutants suppress PcG mutant phenotypes. We previously identified the ANTAGONIST OF LIKE HETEROCHROMATIN PROTEIN1 (ALP1) gene as a genetic suppressor of mutants in the Arabidopsis PcG gene LIKE HETEROCHROMATIN PROTEIN1 (LHP1). Here, we show that ALP1 interacts genetically with several other PcG and trxG components and that it antagonizes PcG silencing. Transcriptional profiling reveals that when PcG activity is compromised numerous target genes are hyper-activated in seedlings and that in most cases this requires ALP1. Furthermore, when PcG activity is present ALP1 is needed for full activation of several floral homeotic genes that are repressed by the PcG. Strikingly, ALP1 does not encode a known chromatin protein but rather a protein related to PIF/Harbinger class transposases. Phylogenetic analysis indicates that ALP1 is broadly conserved in land plants and likely lost transposase activity and acquired a novel function during angiosperm evolution. Consistent with this, immunoprecipitation and mass spectrometry (IP-MS) show that ALP1 associates, in vivo, with core components of POLYCOMB REPRESSIVE COMPLEX 2 (PRC2), a widely conserved PcG protein complex which functions as a H3K27me3 histone methyltransferase. Furthermore, in reciprocal pulldowns using the histone methyltransferase CURLY LEAF (CLF), we identify not only ALP1 and the core PRC2 components but also plant-specific accessory components including EMBRYONIC FLOWER 1 (EMF1), a transcriptional repressor previously associated with PRC1-like complexes. Taken together our data suggest that ALP1 inhibits PcG silencing by blocking the interaction of the core PRC2 with accessory components that promote its HMTase activity or its role in inhibiting transcription. ALP1 is the first example of a domesticated transposase acquiring a novel function as a PcG component. The antagonistic interaction of a modified transposase with the PcG machinery is novel and may have arisen as a means for the cognate transposon to evade host surveillance or for the host to exploit features of the transposition machinery beneficial for epigenetic regulation of gene activity.

Chromosome-level assembly of Arabidopsis thaliana Ler reveals the extent of translocation and inversion polymorphisms.

Significance Despite widespread reports on deciphering the sequences of all kinds of genomes, most of these reconstructed genomes rely on a comparison of short DNA sequencing reads to a reference sequence, rather than being independently reconstructed. This method limits the insights on genomic differences to local, mostly small-scale variation, because large rearrangements are likely overlooked by current methods. We have de novo assembled the genome of a common strain of Arabidopsis thaliana Landsberg erecta and revealed hundreds of rearranged regions. Some of these differences suppress meiotic recombination, impacting the haplotypes of a worldwide population of A. thaliana. In addition to sequence changes, this work, which, to our knowledge is the first comparison of an independent, chromosome-level assembled A. thaliana genome, revealed hundreds of unknown, accession-specific genes. Resequencing or reference-based assemblies reveal large parts of the small-scale sequence variation. However, they typically fail to separate such local variation into colinear and rearranged variation, because they usually do not recover the complement of large-scale rearrangements, including transpositions and inversions. Besides the availability of hundreds of genomes of diverse Arabidopsis thaliana accessions, there is so far only one full-length assembled genome: the reference sequence. We have assembled 117 Mb of the A. thaliana Landsberg erecta (Ler) genome into five chromosome-equivalent sequences using a combination of short Illumina reads, long PacBio reads, and linkage information. Whole-genome comparison against the reference sequence revealed 564 transpositions and 47 inversions comprising ∼3.6 Mb, in addition to 4.1 Mb of nonreference sequence, mostly originating from duplications. Although rearranged regions are not different in local divergence from colinear regions, they are drastically depleted for meiotic recombination in heterozygotes. Using a 1.2-Mb inversion as an example, we show that such rearrangement-mediated reduction of meiotic recombination can lead to genetically isolated haplotypes in the worldwide population of A. thaliana. Moreover, we found 105 single-copy genes, which were only present in the reference sequence or the Ler assembly, and 334 single-copy orthologs, which showed an additional copy in only one of the genomes. To our knowledge, this work gives first insights into the degree and type of variation, which will be revealed once complete assemblies will replace resequencing or other reference-dependent methods.

Arabidopsis thaliana DM2h (R8) within the Landsberg RPP1-like Resistance Locus Underlies Three Different Cases of EDS1-Conditioned Autoimmunity.

Plants have a large panel of nucleotide-binding/leucine rich repeat (NLR) immune receptors which monitor host interference by diverse pathogen molecules (effectors) and trigger disease resistance pathways. NLR receptor systems are necessarily under tight control to mitigate the trade-off between induced defenses and growth. Hence, mis-regulated NLRs often cause autoimmunity associated with stunting and, in severe cases, necrosis. Nucleocytoplasmic ENHANCED DISEASE SUSCEPTIBILITY1 (EDS1) is indispensable for effector-triggered and autoimmune responses governed by a family of Toll-Interleukin1-Receptor-related NLR receptors (TNLs). EDS1 operates coincidently or immediately downstream of TNL activation to transcriptionally reprogram cells for defense. We show here that low levels of nuclear-enforced EDS1 are sufficient for pathogen resistance in Arabidopsis thaliana, without causing negative effects. Plants expressing higher nuclear EDS1 amounts have the genetic, phenotypic and transcriptional hallmarks of TNL autoimmunity. In a screen for genetic suppressors of nuclear EDS1 autoimmunity, we map multiple, independent mutations to one gene, DM2h, lying within the polymorphic DANGEROUS MIX2 cluster of TNL RPP1-like genes from A. thaliana accession Landsberg erecta (Ler). The DM2 locus is a known hotspot for deleterious epistatic interactions leading to immune-related incompatibilities between A. thaliana natural accessions. We find that DM2hLer underlies two further genetic incompatibilities involving the RPP1-likeLer locus and EDS1. We conclude that the DM2hLer TNL protein and nuclear EDS1 cooperate, directly or indirectly, to drive cells into an immune response at the expense of growth. A further conclusion is that regulating the available EDS1 nuclear pool is fundamental for maintaining homeostatic control of TNL immune pathways.