Building pangenome graphs.

Pangenome graphs can represent all variation between multiple reference genomes, but current approaches to build them exclude complex sequences or are based upon a single reference. In response, we developed the PanGenome Graph Builder, a pipeline for constructing pangenome graphs without bias or exclusion. The PanGenome Graph Builder uses all-to-all alignments to build a variation graph in which we can identify variation, measure conservation, detect recombination events and infer phylogenetic relationships.

Atlas of telomeric repeat diversity in Arabidopsis thaliana.

BACKGROUND: Telomeric repeat arrays at the ends of chromosomes are highly dynamic in composition, but their repetitive nature and technological limitations have made it difficult to assess their true variation in genome diversity surveys. RESULTS: We have comprehensively characterized the sequence variation immediately adjacent to the canonical telomeric repeat arrays at the very ends of chromosomes in 74 genetically diverse Arabidopsis thaliana accessions. We first describe several types of distinct telomeric repeat units and then identify evolutionary processes such as local homogenization and higher-order repeat formation that shape diversity of chromosome ends. By comparing largely isogenic samples, we also determine repeat number variation of the degenerate and variant telomeric repeat array at both the germline and somatic levels. Finally, our analysis of haplotype structure uncovers chromosome end-specific patterns in the distribution of variant telomeric repeats, and their linkage to the more proximal non-coding region. CONCLUSIONS: Our findings illustrate the spectrum of telomeric repeat variation at multiple levels in A. thaliana-in germline and soma, across all chromosome ends, and across genetic groups-thereby expanding our knowledge of the evolution of chromosome ends.

miR472 deficiency enhances Arabidopsis thaliana defense without reducing seed production.

After having co-existed in plant genomes for at least 200 million years, the products of microRNA (miRNA) and Nucleotide-Binding Leucine Rich Repeat protein (NLR) genes formed a regulatory relationship in the common ancestor of modern gymnosperms and angiosperms. From then on, DNA polymorphisms occurring at miRNA target sequences within NLR transcripts must have been compensated by mutations in the corresponding mature miRNA sequence. The potential evolutionary advantage of such regulation remains largely unknown and might be related to two non-exclusive scenarios: miRNA-dependent regulation of NLR levels might prevent defense mis-activation with negative effects on plant growth and reproduction; or reduction of active miRNA levels in response to pathogen derived molecules (PAMPS and silencing suppressors) might rapidly release otherwise silent NLR transcripts for rapid translation and thereby enhance defense. Here, we used Arabidopsis thaliana plants deficient for miR472 function to study the impact of releasing its NLR targets on plant growth and reproduction and on defense against the fungal pathogen Plectospharaella cucumerina. We show that miR472 regulation has a dual role, participating both in the tight regulation of plant defense and growth. MIM472 lines, with reduced active miR472, are more resistant to pathogens and, correlatively, have reduced relative growth compared to wild-type plants although the end of their reproductive phase is delayed, exhibiting higher adult biomass and similar seed yield as the wild-type. Our study highlights how negative consequences of defense activation might be compensated by changes in phenology and that miR472 reduction is an integral part of plant defense responses.

Continental-scale associations of Arabidopsis thaliana phyllosphere members with host genotype and drought.

Plants are colonized by distinct pathogenic and commensal microbiomes across different regions of the globe, but the factors driving their geographic variation are largely unknown. Here, using 16S ribosomal DNA and shotgun sequencing, we characterized the associations of the Arabidopsis thaliana leaf microbiome with host genetics and climate variables from 267 populations in the species' native range across Europe. Comparing the distribution of the 575 major bacterial amplicon variants (phylotypes), we discovered that microbiome composition in A. thaliana segregates along a latitudinal gradient. The latitudinal clines in microbiome composition are predicted by metrics of drought, but also by the spatial genetics of the host. To validate the relative effects of drought and host genotype we conducted a common garden field study, finding 10% of the core bacteria to be affected directly by drought and 20% to be affected by host genetic associations with drought. These data provide a valuable resource for the plant microbiome field, with the identified associations suggesting that drought can directly and indirectly shape genetic variation in A. thaliana via the leaf microbiome.

Activation of an atypical plant NLR with an N-terminal deletion initiates cell death at the vacuole.

Plants evolve nucleotide-binding leucine-rich repeat receptors (NLRs) to induce immunity. Activated coiled-coil (CC) domain containing NLRs (CNLs) oligomerize and form apparent cation channels promoting calcium influx and cell death, with the alpha-1 helix of the individual CC domains penetrating the plasma membranes. Some CNLs are characterized by putative N-myristoylation and S-acylation sites in their CC domain, potentially mediating permanent membrane association. Whether activated Potentially Membrane Localized NLRs (PMLs) mediate cell death and calcium influx in a similar way is unknown. We uncovered the cell-death function at the vacuole of an atypical but conserved Arabidopsis PML, PML5, which has a significant deletion in its CCG10/GA domain. Active PML5 oligomers localize in Golgi membranes and the tonoplast, alter vacuolar morphology, and induce cell death, with the short N-terminus being sufficient. Mutant analysis supports a potential role of PMLs in plant immunity. PML5-like deletions are found in several Brassicales paralogs, pointing to the evolutionary importance of this innovation. PML5, with its minimal CC domain, represents the first identified CNL utilizing vacuolar-stored calcium for cell death induction.

Widespread position-dependent transcriptional regulatory sequences in plants.

Much of what we know about eukaryotic transcription stems from animals and yeast; however, plants evolved separately for over a billion years, leaving ample time for divergence in transcriptional regulation. Here we set out to elucidate fundamental properties of cis-regulatory sequences in plants. Using massively parallel reporter assays across four plant species, we demonstrate the central role of sequences downstream of the transcription start site (TSS) in transcriptional regulation. Unlike animal enhancers that are position independent, plant regulatory elements depend on their position, as altering their location relative to the TSS significantly affects transcription. We highlight the importance of the region downstream of the TSS in regulating transcription by identifying a DNA motif that is conserved across vascular plants and is sufficient to enhance gene expression in a dose-dependent manner. The identification of a large number of position-dependent enhancers points to fundamental differences in gene regulation between plants and animals.

The genetic architecture of repeated local adaptation to climate in distantly related plants.

Closely related species often use the same genes to adapt to similar environments. However, we know little about why such genes possess increased adaptive potential and whether this is conserved across deeper evolutionary lineages. Adaptation to climate presents a natural laboratory to test these ideas, as even distantly related species must contend with similar stresses. Here, we re-analyse genomic data from thousands of individuals from 25 plant species as diverged as lodgepole pine and Arabidopsis (~300 Myr). We test for genetic repeatability based on within-species associations between allele frequencies in genes and variation in 21 climate variables. Our results demonstrate significant statistical evidence for genetic repeatability across deep time that is not expected under randomness, identifying a suite of 108 gene families (orthogroups) and gene functions that repeatedly drive local adaptation to climate. This set includes many orthogroups with well-known functions in abiotic stress response. Using gene co-expression networks to quantify pleiotropy, we find that orthogroups with stronger evidence for repeatability exhibit greater network centrality and broader expression across tissues (higher pleiotropy), contrary to the 'cost of complexity' theory. These gene families may be important in helping wild and crop species cope with future climate change, representing important candidates for future study.

Diploid genome assembly of the Malbec grapevine cultivar enables haplotype-aware analysis of transcriptomic differences underlying clonal phenotypic variation.

To preserve their varietal attributes, established grapevine cultivars (Vitis vinifera L. ssp. vinifera) must be clonally propagated, due to their highly heterozygous genomes. Malbec is a France-originated cultivar appreciated for producing high-quality wines and is the offspring of cultivars Prunelard and Magdeleine Noire des Charentes. Here, we have built a diploid genome assembly of Malbec, after trio binning of PacBio long reads into the two haploid complements inherited from either parent. After haplotype-aware deduplication and corrections, complete assemblies for the two haplophases were obtained with a very low haplotype switch-error rate (<0.025). The haplophase alignment identified > 25% of polymorphic regions. Gene annotation including RNA-seq transcriptome assembly and ab initio prediction evidence resulted in similar gene model numbers for both haplophases. The annotated diploid assembly was exploited in the transcriptomic comparison of four clonal accessions of Malbec that exhibited variation in berry composition traits. Analysis of the ripening pericarp transcriptome using either haplophases as a reference yielded similar results, although some differences were observed. Particularly, among the differentially expressed genes identified only with the Magdeleine-inherited haplotype as reference, we observed an over-representation of hypothetically hemizygous genes. The higher berry anthocyanin content of clonal accession 595 was associated with increased abscisic acid responses, possibly leading to the observed overexpression of phenylpropanoid metabolism genes and deregulation of genes associated with abiotic stress response. Overall, the results highlight the importance of producing diploid assemblies to fully represent the genomic diversity of highly heterozygous woody crop cultivars and unveil the molecular bases of clonal phenotypic variation.

The first year of a new era.

What happened when eLife decided to eliminate accept/reject decisions after peer review?

Transposon dynamics in the emerging oilseed crop Thlaspi arvense.

Genome evolution is partly driven by the mobility of transposable elements (TEs) which often leads to deleterious effects, but their activity can also facilitate genetic novelty and catalyze local adaptation. We explored how the intraspecific diversity of TE polymorphisms might contribute to the broad geographic success and adaptive capacity of the emerging oil crop Thlaspi arvense (field pennycress). We classified the TE inventory based on a high-quality genome assembly, estimated the age of retrotransposon TE families and comprehensively assessed their mobilization potential. A survey of 280 accessions from 12 regions across the Northern hemisphere allowed us to quantify over 90,000 TE insertion polymorphisms (TIPs). Their distribution mirrored the genetic differentiation as measured by single nucleotide polymorphisms (SNPs). The number and types of mobile TE families vary substantially across populations, but there are also shared patterns common to all accessions. Ty3/Athila elements are the main drivers of TE diversity in T. arvense populations, while a single Ty1/Alesia lineage might be particularly important for transcriptome divergence. The number of retrotransposon TIPs is associated with variation at genes related to epigenetic regulation, including an apparent knockout mutation in BROMODOMAIN AND ATPase DOMAIN-CONTAINING PROTEIN 1 (BRAT1), while DNA transposons are associated with variation at the HSP19 heat shock protein gene. We propose that the high rate of mobilization activity can be harnessed for targeted gene expression diversification, which may ultimately present a toolbox for the potential use of transposition in breeding and domestication of T. arvense.

Structural variation and DNA methylation shape the centromere-proximal meiotic crossover landscape in Arabidopsis.

BACKGROUND: Centromeres load kinetochore complexes onto chromosomes, which mediate spindle attachment and allow segregation during cell division. Although centromeres perform a conserved cellular function, their underlying DNA sequences are highly divergent within and between species. Despite variability in DNA sequence, centromeres are also universally suppressed for meiotic crossover recombination, across eukaryotes. However, the genetic and epigenetic factors responsible for suppression of centromeric crossovers remain to be completely defined. RESULTS: To explore the centromere-proximal meiotic recombination landscape, we map 14,397 crossovers against fully assembled Arabidopsis thaliana (A. thaliana) genomes. A. thaliana centromeres comprise megabase satellite repeat arrays that load nucleosomes containing the CENH3 histone variant. Each chromosome contains a structurally polymorphic region of ~3-4 megabases, which lack crossovers and include the satellite arrays. This polymorphic region is flanked by ~1-2 megabase low-recombination zones. These recombination-suppressed regions are enriched for Gypsy/Ty3 retrotransposons, and additionally contain expressed genes with high genetic diversity that initiate meiotic recombination, yet do not crossover. We map crossovers at high-resolution in proximity to CEN3, which resolves punctate centromere-proximal hotspots that overlap gene islands embedded in heterochromatin. Centromeres are densely DNA methylated and the recombination landscape is remodelled in DNA methylation mutants. We observe that the centromeric low-recombining zones decrease and increase crossovers in CG (met1) and non-CG (cmt3) mutants, respectively, whereas the core non-recombining zones remain suppressed. CONCLUSION: Our work relates the genetic and epigenetic organization of A. thaliana centromeres and flanking pericentromeric heterochromatin to the zones of crossover suppression that surround the CENH3-occupied satellite repeat arrays.

Deleterious phenotypes in wild Arabidopsis arenosa populations are common and linked to runs of homozygosity.

In this study, we aimed to systematically assess the frequency at which potentially deleterious phenotypes appear in natural populations of the outcrossing model plant Arabidopsis arenosa, and to establish their underlying genetics. For this purpose, we collected seeds from wild A. arenosa populations and screened over 2,500 plants for unusual phenotypes in the greenhouse. We repeatedly found plants with obvious phenotypic defects, such as small stature and necrotic or chlorotic leaves, among first-generation progeny of wild A. arenosa plants. Such abnormal plants were present in about 10% of maternal sibships, with multiple plants with similar phenotypes in each of these sibships, pointing to a genetic basis of the observed defects. A combination of transcriptome profiling, linkage mapping and genome-wide runs of homozygosity patterns using a newly assembled reference genome indicated a range of underlying genetic architectures associated with phenotypic abnormalities. This included evidence for homozygosity of certain genomic regions, consistent with alleles that are identical by descent being responsible for these defects. Our observations suggest that deleterious alleles with different genetic architectures are segregating at appreciable frequencies in wild A. arenosa populations.

The plant immune receptor SNC1 monitors helper NLRs targeted by a bacterial effector.

Plants deploy intracellular receptors to counteract pathogen effectors that suppress cell-surface-receptor-mediated immunity. To what extent pathogens manipulate intracellular receptor-mediated immunity, and how plants tackle such manipulation, remains unknown. Arabidopsis thaliana encodes three similar ADR1 class helper nucleotide-binding domain leucine-rich repeat receptors (ADR1, ADR1-L1, and ADR1-L2), which are crucial in plant immunity initiated by intracellular receptors. Here, we report that Pseudomonas syringae effector AvrPtoB suppresses ADR1-L1- and ADR1-L2-mediated cell death. ADR1, however, evades such suppression by diversifying into two ubiquitination sites targeted by AvrPtoB. The intracellular sensor SNC1 interacts with and guards the CCR domains of ADR1-L1/L2. Removal of ADR1-L1/L2 or delivery of AvrPtoB activates SNC1, which then signals through ADR1 to trigger immunity. Our work elucidates the long-sought-after function of SNC1 in defense, and also how plants can use dual strategies, sequence diversification, and a multi-layered guard-guardee system, to counteract pathogen's attack on core immunity functions.

Spatial metatranscriptomics resolves host-bacteria-fungi interactomes.

The interactions of microorganisms among themselves and with their multicellular host take place at the microscale, forming complex networks and spatial patterns. Existing technology does not allow the simultaneous investigation of spatial interactions between a host and the multitude of its colonizing microorganisms, which limits our understanding of host-microorganism interactions within a plant or animal tissue. Here we present spatial metatranscriptomics (SmT), a sequencing-based approach that leverages 16S/18S/ITS/poly-d(T) multimodal arrays for simultaneous host transcriptome- and microbiome-wide characterization of tissues at 55-µm resolution. We showcase SmT in outdoor-grown Arabidopsis thaliana leaves as a model system, and find tissue-scale bacterial and fungal hotspots. By network analysis, we study inter- and intrakingdom spatial interactions among microorganisms, as well as the host response to microbial hotspots. SmT provides an approach for answering fundamental questions on host-microbiome interplay.

Small proteins modulate ion-channel-like ACD6 to regulate immunity in Arabidopsis thaliana.

The multi-pass transmembrane protein ACCELERATED CELL DEATH 6 (ACD6) is an immune regulator in Arabidopsis thaliana with an unclear biochemical mode of action. We have identified two loci, MODULATOR OF HYPERACTIVE ACD6 1 (MHA1) and its paralog MHA1-LIKE (MHA1L), that code for ∼7 kDa proteins, which differentially interact with specific ACD6 variants. MHA1L enhances the accumulation of an ACD6 complex, thereby increasing the activity of the ACD6 standard allele for regulating plant growth and defenses. The intracellular ankyrin repeats of ACD6 are structurally similar to those found in mammalian ion channels. Several lines of evidence link increased ACD6 activity to enhanced calcium influx, with MHA1L as a direct regulator of ACD6, indicating that peptide-regulated ion channels are not restricted to animals.

The genetic and physiological basis of Arabidopsis thaliana tolerance to Pseudomonas viridiflava.

The opportunistic pathogen Pseudomonas viridiflava colonizes > 50 agricultural crop species and is the most common Pseudomonas in the phyllosphere of European Arabidopsis thaliana populations. Belonging to the P. syringae complex, it is genetically and phenotypically distinct from well-characterized P. syringae sensu stricto. Despite its prevalence, we lack knowledge of how A. thaliana responds to its native isolates at the molecular level. Here, we characterize the host response in an A. thaliana - P. viridiflava pathosystem. We measured host and pathogen growth in axenic infections and used immune mutants, transcriptomics, and metabolomics to determine defense pathways influencing susceptibility to P. viridiflava infection. Infection with P. viridiflava increased jasmonic acid (JA) levels and the expression of ethylene defense pathway marker genes. The immune response in a susceptible host accession was delayed compared with a tolerant one. Mechanical injury rescued susceptibility, consistent with an involvement of JA. The JA/ethylene pathway is important for suppression of P. viridiflava, yet suppression capacity varies between accessions. Our results shed light on how A. thaliana can suppress the ever-present P. viridiflava, but further studies are needed to understand how P. viridiflava evades this suppression to spread broadly across A. thaliana populations.

Pervasive under-dominance in gene expression underlying emergent growth trajectories in Arabidopsis thaliana hybrids.

BACKGROUND: Complex traits, such as growth and fitness, are typically controlled by a very large number of variants, which can interact in both additive and non-additive fashion. In an attempt to gauge the relative importance of both types of genetic interactions, we turn to hybrids, which provide a facile means for creating many novel allele combinations. RESULTS: We focus on the interaction between alleles of the same locus, i.e., dominance, and perform a transcriptomic study involving 141 random crosses between different accessions of the plant model species Arabidopsis thaliana. Additivity is rare, consistently observed for only about 300 genes enriched for roles in stress response and cell death. Regulatory rare-allele burden affects the expression level of these genes but does not correlate with F1 rosette size. Non-additive, dominant gene expression in F1 hybrids is much more common, with the vast majority of genes (over 90%) being expressed below the parental average. Unlike in the additive genes, regulatory rare-allele burden in the dominant gene set is strongly correlated with F1 rosette size, even though it only mildly covaries with the expression level of these genes. CONCLUSIONS: Our study underscores under-dominance as the predominant gene action associated with emergence of rosette growth trajectories in the A. thaliana hybrid model. Our work lays the foundation for understanding molecular mechanisms and evolutionary forces that lead to dominance complementation of rare regulatory alleles.