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

This document is the Accepted Manuscript version, made available in accordance to Springer Nature Terms of reuse of archived manuscripts.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.Peer reviewe

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.Fil: Liang, Shih Chieh. University of Edinburgh; Reino UnidoFil: Hartwig, Ben. Max Planck Institute for Plant Breeding Research; AlemaniaFil: Perera, Pumi. University of Edinburgh; Reino UnidoFil: Mora Garcia, Santiago. Fundación Instituto Leloir; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquimicas de Buenos Aires; ArgentinaFil: de Leau, Erica. University of Edinburgh; Reino UnidoFil: Thornton, Harry. University of Edinburgh; Reino UnidoFil: Lima de Alves, Flavia. University of Edinburgh; Reino UnidoFil: Rapsilber, Juri. University of Edinburgh; Reino UnidoFil: Yang, Suxin. University of Edinburgh; Reino UnidoFil: James, Geo Velikkakam. Max Planck Institute for Plant Breeding Research; AlemaniaFil: Schneeberger, Korbinian. Max Planck Institute for Plant Breeding Research; AlemaniaFil: Finnegan, E. Jean. University of Edinburgh; Reino UnidoFil: Turck, Franziska. Max Planck Institute for Plant Breeding Research; AlemaniaFil: Goodrich, Justin. Mc Gill University; Canad

Fast Isogenic Mapping-by-Sequencing of Ethyl Methanesulfonate-Induced Mutant Bulks

Mapping-by-sequencing (or SHOREmapping) has revitalized the powerful concept of forward genetic screens in plants. However, as in conventional genetic mapping approaches, mapping-by-sequencing requires phenotyping of mapping populations established from crosses between two diverged accessions. In addition to the segregation of the focal phenotype, this introduces natural phenotypic variation, which can interfere with the recognition of quantitative phenotypes. Here, we demonstrate how mapping-by-sequencing and candidate gene identification can be performed within the same genetic background using only mutagen-induced changes as segregating markers. Using a previously unknown suppressor of mutants of like heterochromatin protein1 (lhp1), which in its functional form is involved in chromatin-mediated gene repression, we identified three closely linked ethyl methanesulfonate-induced changes as putative candidates. In order to assess allele frequency differences between such closely linked mutations, we introduced deep candidate resequencing using the new Ion Torrent Personal Genome Machine sequencing platform to our mutant identification pipeline and thereby reduced the number of causal candidate mutations to only one. Genetic analysis of two independent additional alleles confirmed that this mutation was causal for the suppression of lhp1

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

Nuclear-enforced expression of EDS1 in transgenic <i>A</i>. <i>thaliana</i>.

<p>A. Schematic drawing of the EDS1-YFP^NLS/nls* expression cassette used for <i>A</i>. <i>thaliana</i> Col <i>eds1-2</i> transformation. gEDS1, EDS1 genomic sequence with exons shown as boxes and introns as lines; pEDS1, EDS1 promoter; T35S, CaMV35S terminator; attB, recombination sites; PKK/TKRKRVG*, SV40 NLS and non-functional nls* variant with stop fused to the last codon of YFP. B. Immunoblot analysis of total protein extracts from 3-week-old plants separated by SDS-PAGE and probed with α-EDS1 antibody. Positions of endogenous EDS1 and transgenic EDS1-YFP are marked. Transgenic lines were generated in the Col <i>eds1-2</i> background. Ponceau S staining of the membrane is shown as a loading control. C. Confocal live cell imaging of representative leaf epidermal cells from EDS1-YFP and EDS1-YFP^NLS transgenic plants. YFP channel, bright field image and an intensity projection of the YFP channel are shown. D. Nuclear accumulation of EDS1-YFP and EDS1-YFP^NLS analyzed by biochemical fractionation. 4-week-old plants before the onset of developmental defects were used for fractionation. Equal amounts of total protein extracts (T), and nuclear fractions (N) were separated by SDS-PAGE and used for immunodetection. A Coomassie-stained section of the membrane is shown as loading control (CBB). Black line indicates splicing of additional lanes from the same membrane. E. Development of macroscopic growth phenotypes in transgenic EDS1-YFP^NLS lines #A3, #A5 and #B2 compared to wild-type Col after 3 and 6 weeks cultivation at 22°C, respectively.</p