Fast-forward genetics identifies plant CPL phosphatases as regulators of miRNA processing factor HYL1.

MicroRNAs (miRNAs) are processed from primary transcripts that contain partially self-complementary foldbacks. As in animals, the core microprocessor in plants is a Dicer protein, DICER-LIKE1 (DCL1). Processing accuracy and strand selection is greatly enhanced through the RNA binding protein HYPONASTIC LEAVES 1 (HYL1) and the zinc finger protein SERRATE (SE). We have combined a luciferase-based genetic screen with whole-genome sequencing for rapid identification of new regulators of miRNA biogenesis and action. Among the first six mutants analyzed were three alleles of C-TERMINAL DOMAIN PHOSPHATASE-LIKE 1 (CPL1)/FIERY2 (FRY2). In the miRNA processing complex, SE functions as a scaffold to mediate CPL1 interaction with HYL1, which needs to be dephosphorylated for optimal activity. In the absence of CPL1, HYL1 dephosphorylation and hence accurate processing and strand selection from miRNA duplexes are compromised. Our findings thus define a new regulatory step in plant miRNA biogenesis.

Pangenome graph layout by Path-Guided Stochastic Gradient Descent.

MOTIVATION: The increasing availability of complete genomes demands for models to study genomic variability within entire populations. Pangenome graphs capture the full genomic similarity and diversity between multiple genomes. In order to understand them, we need to see them. For visualization, we need a human-readable graph layout: a graph embedding in low (e.g. two) dimensional depictions. Due to a pangenome graph's potential excessive size, this is a significant challenge. RESULTS: In response, we introduce a novel graph layout algorithm: the Path-Guided Stochastic Gradient Descent (PG-SGD). PG-SGD uses the genomes, represented in the pangenome graph as paths, as an embedded positional system to sample genomic distances between pairs of nodes. This avoids the quadratic cost seen in previous versions of graph drawing by SGD. We show that our implementation efficiently computes the low-dimensional layouts of gigabase-scale pangenome graphs, unveiling their biological features. AVAILABILITY AND IMPLEMENTATION: We integrated PG-SGD in ODGI which is released as free software under the MIT open source license. Source code is available at https://github.com/pangenome/odgi.

Impaired KIN10 function restores developmental defects in the Arabidopsis trehalose 6-phosphate synthase1 (tps1) mutant.

Sensing carbohydrate availability is essential for plants to coordinate their growth and development. In Arabidopsis thaliana, TREHALOSE 6-PHOSPHATE SYNTHASE 1 (TPS1) and its product, trehalose 6-phosphate (T6P), are important for the metabolic control of development. tps1 mutants are embryo-lethal and unable to flower when embryogenesis is rescued. T6P regulates development in part through inhibition of SUCROSE NON-FERMENTING1 RELATED KINASE1 (SnRK1). Here, we explored the role of SnRK1 in T6P-mediated plant growth and development using a combination of a mutant suppressor screen and genetic, cellular and transcriptomic approaches. We report nonsynonymous amino acid substitutions in the catalytic KIN10 and regulatory SNF4 subunits of SnRK1 that can restore both embryogenesis and flowering of tps1 mutant plants. The identified SNF4 point mutations disrupt the interaction with the catalytic subunit KIN10. Contrary to the common view that the two A. thaliana SnRK1 catalytic subunits act redundantly, we found that loss-of-function mutations in KIN11 are unable to restore embryogenesis and flowering, highlighting the important role of KIN10 in T6P signalling.

RPW8/HR repeats control NLR activation in Arabidopsis thaliana.

In many plant species, conflicts between divergent elements of the immune system, especially nucleotide-binding oligomerization domain-like receptors (NLR), can lead to hybrid necrosis. Here, we report deleterious allele-specific interactions between an NLR and a non-NLR gene cluster, resulting in not one, but multiple hybrid necrosis cases in Arabidopsis thaliana. The NLR cluster is RESISTANCE TO PERONOSPORA PARASITICA 7 (RPP7), which can confer strain-specific resistance to oomycetes. The non-NLR cluster is RESISTANCE TO POWDERY MILDEW 8 (RPW8) / HOMOLOG OF RPW8 (HR), which can confer broad-spectrum resistance to both fungi and oomycetes. RPW8/HR proteins contain at the N-terminus a potential transmembrane domain, followed by a specific coiled-coil (CC) domain that is similar to a domain found in pore-forming toxins MLKL and HET-S from mammals and fungi. C-terminal to the CC domain is a variable number of 21- or 14-amino acid repeats, reminiscent of regulatory 21-amino acid repeats in fungal HET-S. The number of repeats in different RPW8/HR proteins along with the sequence of a short C-terminal tail predicts their ability to activate immunity in combination with specific RPP7 partners. Whether a larger or smaller number of repeats is more dangerous depends on the specific RPW8/HR autoimmune risk variant.

The rate and potential relevance of new mutations in a colonizing plant lineage.

By following the evolution of populations that are initially genetically homogeneous, much can be learned about core biological principles. For example, it allows for detailed studies of the rate of emergence of de novo mutations and their change in frequency due to drift and selection. Unfortunately, in multicellular organisms with generation times of months or years, it is difficult to set up and carry out such experiments over many generations. An alternative is provided by "natural evolution experiments" that started from colonizations or invasions of new habitats by selfing lineages. With limited or missing gene flow from other lineages, new mutations and their effects can be easily detected. North America has been colonized in historic times by the plant Arabidopsis thaliana, and although multiple intercrossing lineages are found today, many of the individuals belong to a single lineage, HPG1. To determine in this lineage the rate of substitutions-the subset of mutations that survived natural selection and drift-, we have sequenced genomes from plants collected between 1863 and 2006. We identified 73 modern and 27 herbarium specimens that belonged to HPG1. Using the estimated substitution rate, we infer that the last common HPG1 ancestor lived in the early 17th century, when it was most likely introduced by chance from Europe. Mutations in coding regions are depleted in frequency compared to those in other portions of the genome, consistent with purifying selection. Nevertheless, a handful of mutations is found at high frequency in present-day populations. We link these to detectable phenotypic variance in traits of known ecological importance, life history and growth, which could reflect their adaptive value. Our work showcases how, by applying genomics methods to a combination of modern and historic samples from colonizing lineages, we can directly study new mutations and their potential evolutionary relevance.

A Role for the F-Box Protein HAWAIIAN SKIRT in Plant microRNA Function.

As regulators of gene expression in multicellular organisms, microRNAs (miRNAs) are crucial for growth and development. Although a plethora of factors involved in their biogenesis and action in Arabidopsis (Arabidopsis thaliana) has been described, these processes and their fine-tuning are not fully understood. Here, we used plants expressing an artificial miRNA target mimic (MIM) to screen for negative regulators of miR156. We identified a new mutant allele of the F-box gene HAWAIIAN SKIRT (HWS; At3G61590), hws-5, as a suppressor of the MIM156-induced developmental and molecular phenotypes. In hws plants, levels of some endogenous miRNAs are increased and their mRNA targets decreased. Plants constitutively expressing full-length HWS-but not a truncated version lacking the F-box domain-display morphological and molecular phenotypes resembling those of mutants defective in miRNA biogenesis and activity. In combination with such mutants, hws loses its delayed floral organ abscission ("skirt") phenotype, suggesting epistasis. Also, the hws transcriptome profile partially resembles those of well-known miRNA mutants hyl1-2, se-3, and ago1-27, pointing to a role in a common pathway. We thus propose HWS as a novel, F-box dependent factor involved in miRNA function.

KH domain protein RCF3 is a tissue-biased regulator of the plant miRNA biogenesis cofactor HYL1.

The biogenesis of microRNAs (miRNAs), which regulate mRNA abundance through posttranscriptional silencing, comprises multiple well-orchestrated processing steps. We have identified the Arabidopsis thaliana K homology (KH) domain protein REGULATOR OF CBF GENE EXPRESSION 3 (RCF3) as a cofactor affecting miRNA biogenesis in specific plant tissues. MiRNA and miRNA-target levels were reduced in apex-enriched samples of rcf3 mutants, but not in other tissues. Mechanistically, RCF3 affects miRNA biogenesis through nuclear interactions with the phosphatases C-TERMINAL DOMAIN PHOSPHATASE-LIKE1 and 2 (CPL1 and CPL2). These interactions are essential to regulate the phosphorylation status, and thus the activity, of the double-stranded RNA binding protein and DICER-LIKE1 (DCL1) cofactor HYPONASTIC LEAVES1 (HYL1).

Modulation of Ambient Temperature-Dependent Flowering in Arabidopsis thaliana by Natural Variation of FLOWERING LOCUS M.

Plants integrate seasonal cues such as temperature and day length to optimally adjust their flowering time to the environment. Compared to the control of flowering before and after winter by the vernalization and day length pathways, mechanisms that delay or promote flowering during a transient cool or warm period, especially during spring, are less well understood. Due to global warming, understanding this ambient temperature pathway has gained increasing importance. In Arabidopsis thaliana, FLOWERING LOCUS M (FLM) is a critical flowering regulator of the ambient temperature pathway. FLM is alternatively spliced in a temperature-dependent manner and the two predominant splice variants, FLM-ß and FLM-δ, can repress and activate flowering in the genetic background of the A. thaliana reference accession Columbia-0. The relevance of this regulatory mechanism for the environmental adaptation across the entire range of the species is, however, unknown. Here, we identify insertion polymorphisms in the first intron of FLM as causative for accelerated flowering in many natural A. thaliana accessions, especially in cool (15°C) temperatures. We present evidence for a potential adaptive role of this structural variation and link it specifically to changes in the abundance of FLM-ß. Our results may allow predicting flowering in response to ambient temperatures in the Brassicaceae.

Century-scale methylome stability in a recently diverged Arabidopsis thaliana lineage.

There has been much excitement about the possibility that exposure to specific environments can induce an ecological memory in the form of whole-sale, genome-wide epigenetic changes that are maintained over many generations. In the model plant Arabidopsis thaliana, numerous heritable DNA methylation differences have been identified in greenhouse-grown isogenic lines, but it remains unknown how natural, highly variable environments affect the rate and spectrum of such changes. Here we present detailed methylome analyses in a geographically dispersed A. thaliana population that constitutes a collection of near-isogenic lines, diverged for at least a century from a common ancestor. Methylome variation largely reflected genetic distance, and was in many aspects similar to that of lines raised in uniform conditions. Thus, even when plants are grown in varying and diverse natural sites, genome-wide epigenetic variation accumulates mostly in a clock-like manner, and epigenetic divergence thus parallels the pattern of genome-wide DNA sequence divergence.

Spontaneous epigenetic variation in the Arabidopsis thaliana methylome.

Heritable epigenetic polymorphisms, such as differential cytosine methylation, can underlie phenotypic variation. Moreover, wild strains of the plant Arabidopsis thaliana differ in many epialleles, and these can influence the expression of nearby genes. However, to understand their role in evolution, it is imperative to ascertain the emergence rate and stability of epialleles, including those that are not due to structural variation. We have compared genome-wide DNA methylation among 10 A. thaliana lines, derived 30 generations ago from a common ancestor. Epimutations at individual positions were easily detected, and close to 30,000 cytosines in each strain were differentially methylated. In contrast, larger regions of contiguous methylation were much more stable, and the frequency of changes was in the same low range as that of DNA mutations. Like individual positions, the same regions were often affected by differential methylation in independent lines, with evidence for recurrent cycles of forward and reverse mutations. Transposable elements and short interfering RNAs have been causally linked to DNA methylation. In agreement, differentially methylated sites were farther from transposable elements and showed less association with short interfering RNA expression than invariant positions. The biased distribution and frequent reversion of epimutations have important implications for the potential contribution of sequence-independent epialleles to plant evolution.

Evolution of DNA methylation patterns in the Brassicaceae is driven by differences in genome organization.

DNA methylation is an ancient molecular modification found in most eukaryotes. In plants, DNA methylation is not only critical for transcriptionally silencing transposons, but can also affect phenotype by altering expression of protein coding genes. The extent of its contribution to phenotypic diversity over evolutionary time is, however, unclear, because of limited stability of epialleles that are not linked to DNA mutations. To dissect the relative contribution of DNA methylation to transposon surveillance and host gene regulation, we leveraged information from three species in the Brassicaceae that vary in genome architecture, Capsella rubella, Arabidopsis lyrata, and Arabidopsis thaliana. We found that the lineage-specific expansion and contraction of transposon and repeat sequences is the main driver of interspecific differences in DNA methylation. The most heavily methylated portions of the genome are thus not conserved at the sequence level. Outside of repeat-associated methylation, there is a surprising degree of conservation in methylation at single nucleotides located in gene bodies. Finally, dynamic DNA methylation is affected more by tissue type than by environmental differences in all species, but these responses are not conserved. The majority of DNA methylation variation between species resides in hypervariable genomic regions, and thus, in the context of macroevolution, is of limited phenotypic consequence.

FYVE1 is essential for vacuole biogenesis and intracellular trafficking in Arabidopsis.

The plant vacuole is a central organelle that is involved in various biological processes throughout the plant life cycle. Elucidating the mechanism of vacuole biogenesis and maintenance is thus the basis for our understanding of these processes. Proper formation of the vacuole has been shown to depend on the intracellular membrane trafficking pathway. Although several mutants with altered vacuole morphology have been characterized in the past, the molecular basis for plant vacuole biogenesis has yet to be fully elucidated. With the aim to identify key factors that are essential for vacuole biogenesis, we performed a forward genetics screen in Arabidopsis (Arabidopsis thaliana) and isolated mutants with altered vacuole morphology. The vacuolar fusion defective1 (vfd1) mutant shows seedling lethality and defects in central vacuole formation. VFD1 encodes a Fab1, YOTB, Vac1, and EEA1 (FYVE) domain-containing protein, FYVE1, that has been implicated in intracellular trafficking. FYVE1 localizes on late endosomes and interacts with Src homology-3 domain-containing proteins. Mutants of FYVE1 are defective in ubiquitin-mediated protein degradation, vacuolar transport, and autophagy. Altogether, our results show that FYVE1 is essential for plant growth and development and place FYVE1 as a key regulator of intracellular trafficking and vacuole biogenesis.

Trowel: a fast and accurate error correction module for Illumina sequencing reads.

MOTIVATION: The ability to accurately read the order of nucleotides in DNA and RNA is fundamental for modern biology. Errors in next-generation sequencing can lead to many artifacts, from erroneous genome assemblies to mistaken inferences about RNA editing. Uneven coverage in datasets also contributes to false corrections. RESULT: We introduce Trowel, a massively parallelized and highly efficient error correction module for Illumina read data. Trowel both corrects erroneous base calls and boosts base qualities based on the k-mer spectrum. With high-quality k-mers and relevant base information, Trowel achieves high accuracy for different short read sequencing applications.The latency in the data path has been significantly reduced because of efficient data access and data structures. In performance evaluations, Trowel was highly competitive with other tools regardless of coverage, genome size read length and fragment size. AVAILABILITY AND IMPLEMENTATION: Trowel is written in C++ and is provided under the General Public License v3.0 (GPLv3). It is available at http://trowel-ec.sourceforge.net. CONTACT: euncheon.lim@tue.mpg.de or weigel@tue.mpg.de SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Accurate indel prediction using paired-end short reads.

BACKGROUND: One of the major open challenges in next generation sequencing (NGS) is the accurate identification of structural variants such as insertions and deletions (indels). Current methods for indel calling assign scores to different types of evidence or counter-evidence for the presence of an indel, such as the number of split read alignments spanning the boundaries of a deletion candidate or reads that map within a putative deletion. Candidates with a score above a manually defined threshold are then predicted to be true indels. As a consequence, structural variants detected in this manner contain many false positives. RESULTS: Here, we present a machine learning based method which is able to discover and distinguish true from false indel candidates in order to reduce the false positive rate. Our method identifies indel candidates using a discriminative classifier based on features of split read alignment profiles and trained on true and false indel candidates that were validated by Sanger sequencing. We demonstrate the usefulness of our method with paired-end Illumina reads from 80 genomes of the first phase of the 1001 Genomes Project ( http://www.1001genomes.org) in Arabidopsis thaliana. CONCLUSION: In this work we show that indel classification is a necessary step to reduce the number of false positive candidates. We demonstrate that missing classification may lead to spurious biological interpretations. The software is available at: http://agkb.is.tuebingen.mpg.de/Forschung/SV-M/.

Paralemmin-1 is expressed in lymphatic endothelial cells and modulates cell migration, cell maturation and tumor lymphangiogenesis.

The lymphatic system, the network of lymphatic vessels and lymphoid organs, maintains the body fluid balance and ensures the immunological surveillance of the body. In the adult organism, the de novo formation of lymphatic vessels is mainly observed in pathological conditions. In contrast to the molecular mechanisms governing the generation of the lymphatic vasculature during embryogenesis, the processes underlying pathological lymphangiogenesis are less well understood. A genome-wide screen comparing the transcriptome of tumor-derived lymphatic endothelial cells with that of blood vessel endothelial cells identified paralemmin-1 as a protein prominently expressed in lymphatic endothelial cells. Paralemmin-1 is a lipid-anchored membrane protein that in fibroblasts and neurons plays a role in the regulation of cell shape, plasma membrane dynamics and cell motility. Here, we show that paralemmin-1 is expressed in tumor-derived lymphatic endothelial cells as well as in lymphatic endothelial cells of normal, non-tumorigenic tissue. Paralemmin-1 represses cell migration and delays the formation of tube-like structures of lymphatic endothelial cells in vitro by modulating cell-substrate adhesion, filopodia formation and plasma membrane blebbing. While constitutive genetic ablation of paralemmin-1 expression in mice has no effect on the development and physiological function of the lymphatic system, the loss of paralemmin-1 impaired tumor-associated lymphangiogenesis. Together, these results newly identify paralemmin-1 as a protein highly expressed in lymphatic endothelial cells. Similar to its function in neurons, it may link the cytoskeleton to the plasma membrane and thereby modulate lymphatic endothelial cell adhesion, migration and lymphangiogenesis.

Pangenome graph layout by Path-Guided Stochastic Gradient Descent.

Motivation: The increasing availability of complete genomes demands for models to study genomic variability within entire populations. Pangenome graphs capture the full genomic similarity and diversity between multiple genomes. In order to understand them, we need to see them. For visualization, we need a human readable graph layout: A graph embedding in low (e.g. two) dimensional depictions. Due to a pangenome graph's potential excessive size, this is a significant challenge. Results: In response, we introduce a novel graph layout algorithm: the Path-Guided Stochastic Gradient Descent (PG-SGD). PG-SGD uses the genomes, represented in the pangenome graph as paths, as an embedded positional system to sample genomic distances between pairs of nodes. This avoids the quadratic cost seen in previous versions of graph drawing by Stochastic Gradient Descent (SGD). We show that our implementation efficiently computes the low dimensional layouts of gigabase-scale pangenome graphs, unveiling their biological features. Availability: We integrated PG-SGD in ODGI which is released as free software under the MIT open source license. Source code is available at https://github.com/pangenome/odgi.

Building pangenome graphs.

Pangenome graphs can represent all variation between multiple genomes, but existing methods for constructing them are biased due to reference-guided approaches. In response, we have developed PanGenome Graph Builder (PGGB), a reference-free pipeline for constructing unbi-ased pangenome graphs. PGGB uses all-to-all whole-genome alignments and learned graph embeddings to build and iteratively refine a model in which we can identify variation, measure conservation, detect recombination events, and infer phylogenetic relationships.

MethylScore, a pipeline for accurate and context-aware identification of differentially methylated regions from population-scale plant whole-genome bisulfite sequencing data.

Whole-genome bisulfite sequencing (WGBS) is the standard method for profiling DNA methylation at single-nucleotide resolution. Different tools have been developed to extract differentially methylated regions (DMRs), often built upon assumptions from mammalian data. Here, we present MethylScore, a pipeline to analyse WGBS data and to account for the substantially more complex and variable nature of plant DNA methylation. MethylScore uses an unsupervised machine learning approach to segment the genome by classification into states of high and low methylation. It processes data from genomic alignments to DMR output and is designed to be usable by novice and expert users alike. We show how MethylScore can identify DMRs from hundreds of samples and how its data-driven approach can stratify associated samples without prior information. We identify DMRs in the A. thaliana 1,001 Genomes dataset to unveil known and unknown genotype-epigenotype associations .

Methylation of CpG 5962 in L1 of the human papillomavirus 16 genome as a potential predictive marker for viral persistence: A prospective large cohort study using cervical swab samples.

Several studies have demonstrated that the viral genome can be methylated by the host cell during progression from persistent infection to cervical cancer. The aim of this study was to investigate whether methylation at a specific site could predict the development of viral persistence and whether viral load shows a correlation with specific methylation patterns. HPV16-positive samples from women aged 20-29 years (n = 99) with a follow-up time of 13 years, were included from a Danish cohort comprising 11 088 women. Viral load was measured by real-time PCR and methylation status was determined for 39 CpG sites in the upstream regulatory region (URR), E6/E7, and L1 region of HPV16 by next-generation sequencing. Participants were divided into two groups according to whether they were persistently (≥ 24 months) or transiently HPV16 infected. The general methylation status was significantly different between women with a persistent and women with a transient infection outcome (P = .025). One site located in L1 (nt. 5962) was statistically significantly (P = .00048) different in the methylation status after correction using the Holm-Sidak method (alpha = 0.05). Correlation analyses of samples from HPV16 persistently infected women suggest that methylation is higher although viral load is lower. This study indicates that methylation at position 5962 of the HPV16 genome within the L1 gene might be a predictive marker for the development of a persistent HPV16 infection.

Long-term balancing selection drives evolution of immunity genes in Capsella.

Genetic drift is expected to remove polymorphism from populations over long periods of time, with the rate of polymorphism loss being accelerated when species experience strong reductions in population size. Adaptive forces that maintain genetic variation in populations, or balancing selection, might counteract this process. To understand the extent to which natural selection can drive the retention of genetic diversity, we document genomic variability after two parallel species-wide bottlenecks in the genus Capsella. We find that ancestral variation preferentially persists at immunity related loci, and that the same collection of alleles has been maintained in different lineages that have been separated for several million years. By reconstructing the evolution of the disease-related locus MLO2b, we find that divergence between ancient haplotypes can be obscured by referenced based re-sequencing methods, and that trans-specific alleles can encode substantially diverged protein sequences. Our data point to long-term balancing selection as an important factor shaping the genetics of immune systems in plants and as the predominant driver of genomic variability after a population bottleneck.