Calcium sensor kinase activates potassium uptake systems in gland cells of Venus flytraps

The Darwin plant Dionaea muscipula is able to grow on mineral-poor soil, because it gains essential nutrients from captured animal prey. Given that no nutrients remain in the trap when it opens after the consumption of an animal meal, we here asked the question of how Dionaea sequesters prey-derived potassium. We show that prey capture triggers expression of a K+ uptake system in the Venus flytrap. In search of K+ transporters endowed with adequate properties for this role, we screened a Dionaea expressed sequence tag (EST) database and identified DmKT1 and DmHAK5 as candidates. On insect and touch hormone stimulation, the number of transcripts of these transporters increased in flytraps. After cRNA injection of K+-transporter genes into Xenopus oocytes, however, both putative K+ transporters remained silent. Assuming that calcium sensor kinases are regulating Arabidopsis K+ transporter 1 (AKT1), we coexpressed the putative K+ transporters with a large set of kinases and identified the CBL9-CIPK23 pair as the major activating complex for both transporters in Dionaea K+ uptake. DmKT1 was found to be a K+-selective channel of voltage-dependent high capacity and low affinity, whereas DmHAK5 was identified as the first, to our knowledge, proton-driven, high-affinity potassium transporter with weak selectivity. When the Venus flytrap is processing its prey, the gland cell membrane potential is maintained around -120 mV, and the apoplast is acidified to pH 3. These conditions in the green stomach formed by the closed flytrap allow DmKT1 and DmHAK5 to acquire prey-derived K+, reducing its concentration from millimolar levels down to trace levels

A Species-Wide Inventory of NLR Genes and Alleles in Arabidopsis thaliana

Infectious disease is both a major force of selection in nature and a prime cause of yield loss in agriculture. In plants, disease resistance is often conferred by nucleotide-binding leucine-rich repeat (NLR) proteins, intracellular immune receptors that recognize pathogen proteins and their effects on the host. Consistent with extensive balancing and positive selection, NLRs are encoded by one of the most variable gene families in plants, but the true extent of intraspecific NLR diversity has been unclear. Here, we define a nearly complete species-wide pan-NLRome in Arabidopsis thaliana based on sequence enrichment and long-read sequencing. The pan-NLRome largely saturates with approximately 40 well-chosen wild strains, with half of the pan-NLRome being present in most accessions. We chart NLR architectural diversity, identify new architectures, and quantify selective forces that act on specific NLRs and NLR domains. Our study provides a blueprint for defining pan-NLRomes. In plants, NLR proteins are important intracellular receptors with roles in innate immunity and disease resistance. This work provides a panoramic view of this diverse and complicated gene family in the model species A. thaliana and provides a foundation for the identification and functional study of disease-resistance genes in agronomically important species with complex genomes

Evolutionary distances in the twilight zone -- a rational kernel approach

Phylogenetic tree reconstruction is traditionally based on multiple sequence alignments (MSAs) and heavily depends on the validity of this information bottleneck. With increasing sequence divergence, the quality of MSAs decays quickly. Alignment-free methods, on the other hand, are based on abstract string comparisons and avoid potential alignment problems. However, in general they are not biologically motivated and ignore our knowledge about the evolution of sequences. Thus, it is still a major open question how to define an evolutionary distance metric between divergent sequences that makes use of indel information and known substitution models without the need for a multiple alignment. Here we propose a new evolutionary distance metric to close this gap. It uses finite-state transducers to create a biologically motivated similarity score which models substitutions and indels, and does not depend on a multiple sequence alignment. The sequence similarity score is defined in analogy to pairwise alignments and additionally has the positive semi-definite property. We describe its derivation and show in simulation studies and real-world examples that it is more accurate in reconstructing phylogenies than competing methods. The result is a new and accurate way of determining evolutionary distances in and beyond the twilight zone of sequence alignments that is suitable for large datasets.Comment: to appear in PLoS ON

The kinome of Phytophthora infestans reveals oomycete-specific innovations and links to other taxonomic groups

<p>Abstract</p> <p>Background</p> <p>Oomycetes are a large group of economically and ecologically important species. Its most notorious member is <it>Phytophthora infestans</it>, the cause of the devastating potato late blight disease. The life cycle of <it>P. infestans </it>involves hyphae which differentiate into spores used for dispersal and host infection. Protein phosphorylation likely plays crucial roles in these stages, and to help understand this we present here a genome-wide analysis of the protein kinases of <it>P. infestans </it>and several relatives. The study also provides new insight into kinase evolution since oomycetes are taxonomically distant from organisms with well-characterized kinomes.</p> <p>Results</p> <p>Bioinformatic searches of the genomes of <it>P. infestans</it>, <it>P. ramorum</it>, and <it>P. sojae </it>reveal they have similar kinomes, which for <it>P. infestans </it>contains 354 eukaryotic protein kinases (ePKs) and 18 atypical kinases (aPKs), equaling 2% of total genes. After refining gene models, most were classifiable into families seen in other eukaryotes. Some ePK families are nevertheless unusual, especially the tyrosine kinase-like (TKL) group which includes large oomycete-specific subfamilies. Also identified were two tyrosine kinases, which are rare in non-metazoans. Several ePKs bear accessory domains not identified previously on kinases, such as cyclin-dependent kinases with integral cyclin domains. Most ePKs lack accessory domains, implying that many are regulated transcriptionally. This was confirmed by mRNA expression-profiling studies that showed that two-thirds vary significantly between hyphae, sporangia, and zoospores. Comparisons to neighboring taxa (apicomplexans, ciliates, diatoms) revealed both clade-specific and conserved features, and multiple connections to plant kinases were observed. The kinome of <it>Hyaloperonospora arabidopsidis</it>, an oomycete with a simpler life cycle than <it>P. infestans</it>, was found to be one-third smaller. Some differences may be attributable to gene clustering, which facilitates subfamily expansion (or loss) through unequal crossing-over.</p> <p>Conclusion</p> <p>The large sizes of the <it>Phytophthora </it>kinomes imply that phosphorylation plays major roles in their life cycles. Their kinomes also include many novel ePKs, some specific to oomycetes or shared with neighboring groups. Little experimentation to date has addressed the biological functions of oomycete kinases, but this should be stimulated by the structural, evolutionary, and expression data presented here. This may lead to targets for disease control.</p

Comparative genomics of the tardigrades <i>Hypsibius dujardini</i> and <i>Ramazzottius varieornatus</i>

Tardigrada, a phylum of meiofaunal organisms, have been at the center of discussions of the evolution of Metazoa, the biology of survival in extreme environments, and the role of horizontal gene transfer in animal evolution. Tardigrada are placed as sisters to Arthropoda and Onychophora (velvet worms) in the superphylum Panarthropoda by morphological analyses, but many molecular phylogenies fail to recover this relationship. This tension between molecular and morphological understanding may be very revealing of the mode and patterns of evolution of major groups. Limnoterrestrial tardigrades display extreme cryptobiotic abilities, including anhydrobiosis and cryobiosis, as do bdelloid rotifers, nematodes, and other animals of the water film. These extremophile behaviors challenge understanding of normal, aqueous physiology: how does a multicellular organism avoid lethal cellular collapse in the absence of liquid water? Meiofaunal species have been reported to have elevated levels of horizontal gene transfer (HGT) events, but how important this is in evolution, and particularly in the evolution of extremophile physiology, is unclear. To address these questions, we resequenced and reassembled the genome of H. dujardini, a limnoterrestrial tardigrade that can undergo anhydrobiosis only after extensive pre-exposure to drying conditions, and compared it to the genome of R. varieornatus, a related species with tolerance to rapid desiccation. The 2 species had contrasting gene expression responses to anhydrobiosis, with major transcriptional change in H. dujardini but limited regulation in R. varieornatus. We identified few horizontally transferred genes, but some of these were shown to be involved in entry into anhydrobiosis. Whole-genome molecular phylogenies supported a Tardigrada+Nematoda relationship over Tardigrada+Arthropoda, but rare genomic changes tended to support Tardigrada+Arthropoda

Transcriptome Analysis in Hybrid Plants using Platinum Genomes

Heterosis refers to the deviation of the F1 progeny from the phenotypic mean of the parental plants, espe- cially in cases where both parents are inbred and homozygous throughout the genome. When comparing certain traits of a F1 hybrid to the corresponding parental plants one can compare the performance of the hybrid to (1) the midparent value (MPV), which refers to the mean of both parental plants for the given trait. Furthermore the F1 hybrid can be compared to (2) the best-parent value (BPV), referring to the value of the superior parental plant. Mainly three genetic models, explaining heterosis have been proposed. However, there is no consensus about the diversity of molecular principles of heterosis. Recent studies suggested structural genome variation, such as copy-number variation (CNV) as well as presence absence variation (PAV), among maize inbred lines, underlying complementary contributions of genes from both parents as an important factor (Springer et al. 2009). Gene expression with regard to heterosis has been analyzed in several plants includingA. thaliana (Alonso-Peral et al. 2017). How- ever, the majority of these studies relied either on microarray data, which are limited to transcripts present on the chip, or on RNA-seq data that have been processed using a single reference genome, thus not accounting for structural variation among different genotypes (accessions). We now have access to high quality full length genome sequences of different A. thaliana accessions. Two of these accessions have been used in a reciprocal crossing. The root and shoot transcriptomes of F1 hybrids as well as from the corresponding parental plants have been short read sequenced. RNA-seq reads have been processed using either one parental genome as a reference or a trans-reference genome, containing full length genome sequences of both parental plants and accounting for orthologous assignment. DESeq2 has been used to detect transcripts with non parental expression patterns (below or above MPV) in the F1 hybrid plants. When using a single reference genome we found various transposable elements among the differentially expressed genes (p < 0.01). However, whole genome alignment data among parental plants indicate that many of these are due to a reference bias. Here we present a way of how to process a double reference genome in order to improve the analysis of hybrid transcriptomes regarding heterosis

Building usable full genome variation graphs

The 1001 Genomes Project generated a polymorphism (SNP) and short structural variant (short SV) map for well over 1000 wild strains (accessions) of Arabidopsis thaliana. In addition transcriptome, methylation and phenotypic data for most of the accessions were collected. By utilising long read se- quencing technologies to generate de novo assemblies of different diverse A. thaliana accessions, we are launching the next phase of this project, in which we will detect and genotype large SVs. First we will shift from a single reference based approach to a multiple genome graph, representing a set of highly diverse A. thaliana accessions. Based on this we will detect SVs and subsequently genotype these in the 1001 Genomes Project short read data set. Most genome graphs are constructed from a multiple whole genome alignment (WGA). Building a WGA however is not trivial and its quality depends on the excess of shared regions to form informative nodes and (super-)bubbles (PNGH) in the graph. The quality of the WGA depends on several factors, with the similarity and the repetitiveness of the aligned sequences being the major ones. The diver- sity will result in less and smaller alignment blocks, whereas the repetitiveness will lead to multiple alignments. Such a WGA will convert into a highly connected, partially circularized graph that contain almost no usable information as nodes are too short and edges are too abundant to reliably and uniquely anchor superbubbles around interesting structural variants. Here we propose ways to cope with diverse sequences for graph construction. Our main target is to create a low complexity graph. We alter previous graph construction approaches by focusing on local alignment anchors. The approach reduces the alignment fragmentation by only considering re- gions near useful alignment anchors (MUMs (DKF+99)/ Minimizers (RHH+04)) and thus prohibits self alignments, which would result in the circularization of the graph. In a second approach we only focus on regions of interest and resolve them to the highest possible resolution and skip non informative parts around them. We further show that in a finished graph, variation can be removed by pruning thus taking information, such as allele frequencies within a population data set, into account. Although our approaches result in loss of information they enable us to generate genome graphs that help to understand variation of SNPs, short and long SVs as well as TEs at an unprecedented resolution when combined with previously generated short read data

Understanding global patterns of structural variation in Arabidopsis thaliana: Future of the 1001 Genome project

The recently released map of polymorphism of 1,135 re-sequenced A. thaliana natural inbred lines provides an invaluable resource to understand global genetic patterns in a large collection of wild individuals that are products of natural selection under diverse ecological conditions. Our work clarified prior hypotheses such as the strong impact of the last ice age on population structure and in addition revealed that modern A. thaliana is a mixture of stationary relicts and fast expanding survivors from different glacial refugia. In the future the resource will enable researchers to decipher more accurately how genetic variation translates into phenotypic variation. However, connecting hits from genome-wide association studies (GWAS) to causal sequences is still challenging since previous analysis approaches mostly rely on mapping to a single reference, which insufficiently captures structural variations (SVs) or the presence of sequences not found in the reference. Discovery and genotyping of such sequences remains computationally difficult with short read data, since they are often in repetitive regions and because they can the changes can be complex. The next objective for the 1001 Genomes project is to discover and genotype major classes of SVs in the global set of A. thaliana accessions. By reanalyzing the 1001 Genomes Project whole-genome sequencing (WGS) data together with long-read DNA technologies, we will survey SV mutation hotspots throughout the worldwide population and target so far undescribed patterns and classes of SV complexity. Here we present strategies, pilot studies and first results of our upcoming work that will provide struc- tural variations and polymorphisms as an integrated resource alongside with detailed information about epigenomes as well as molecular and nonmolecular phenotypes to understand how traits are connected to genomes and epigenomes

A kinome of 2600 in the ciliate Paramecium tetraurelia

Protein kinases play a crucial role in the regulation of cellular processes. Most eukaryotes reserve about 2.5% of their genes for protein kinases. We analysed the genome of the single-celled ciliate Paramecium tetraurelia and identified 2606 kinases, about 6.6% of its genes, representing the largest kinome to date. A gene tree combined with human kinases revealed a massive expansion of the calcium calmodulin regulated subfamily, underlining the importance of calcium in the physiology of P. tetraurelia. The kinases are embedded in only 40 domain architectures, contrasting 134 in human. This might indicate different mechanisms to achieve target specificity