Biology subjects

Stam, R.

Publications and source records attributed to Stam, R..

29 records · Page 2Linked to original sources

bioRxiv2021

Intraspecific diversity observed in the wild tomato species Solanum chilense in initial immune responses towards a glucan elicitor.

Natural plant populations are polymorphic and show intraspecific variation in resistance properties against pathogens. The activation of the underlying defence responses can depend on variation in perception of pathogen-associated molecular patterns or elicitors. To dissect such variation, we evaluated the responses induced by laminarin, (a glucan, representing an elicitor from oomycetes) in the wild tomato species Solanum chilense and correlated this to observed infection frequencies of Phytophthora infestans. We measured reactive oxygen species burst and levels of diverse phytohormones upon elicitation in 83 plants originating from nine populations. We found high diversity in basal and elicitor-induced levels of each component. Further we generated linear models to explain the observed infection frequency of P. infestans. The effect of individual components differed dependent on the geographical origin of the plants. We found that the resistance in the southern coastal region, but not in the other regions is directly correlated to ethylene responses and confirmed this positive correlation using ethylene inhibition assays. Our findings reveal high diversity in the strength of defence responses within a species and the involvement of different components with a quantitatively different contribution of individual components to resistance in geographically separated populations of a wild plant species. HighlightLarge-scale screenings reveal geographically distinct intraspecific differences in the dominant physiological pathogen defence responses upon glucan elicitor treatment in a wild tomato species.

plant biology↗

bioRxiv2021

AB12PHYLO: an integrated pipeline for Maximum Likelihood phylogenetic inference from ABI trace data

Multi-gene phylogenies constructed from multiplexed and Sanger sequencing data are regularly used in mycology and other disciplines as a cost-effective way of species identification and as a first means to investigate genetic diversity samples. Today, a number of tools exist for each of the steps in this analysis, including quality control and trimming, the generation of a multiple sequence alignment (MSA), extraction of informative sites, and the construction of the final phylogenetic tree. A BLAST search in a reference database is often performed to identify sequences of type specimens to compare the samples with in the phylogeny. Made over the past decades, these tools are all independent from and often not perfectly adapted to one another. We present AB12PHYLO, an integrated pipeline that can perform all necessary steps from reading in raw Sanger sequencing data through visualizing and editing phylogenies. In addition, AB12PHYLO can calculate basic summary statistics for each gene in the phylogeny. AB12PHYLO is designed as a wrapper of several open access and commonly used tools for each of the intermediate stages, and intended to simplify the phylogenetic pipeline while still allowing a high degree of access. It comes as a command-line version for the highest reproducibility and an intuitive graphical user interface (GUI) for easy adoption by IT-agnostic end-users. The use of AB12PHYLO significantly reduces the hands-on working time for these analyses.

evolutionary biology↗

bioRxiv2020

Allelic variants of the NLR protein Rpi-chc1 differentially recognise members of the Phytophthora infestans PexRD12/31 effector superfamily through the leucine-rich repeat domain

O_LIPhytophthora infestans is a pathogenic oomycete that causes the infamous potato late blight disease. Resistance (R) genes from diverse Solanum species encode intracellular receptors that recognize P. infestans RXLR effector proteins and provide effective defence responses. To deploy these R genes in a durable fashion in agriculture, we need to understand the mechanism of effector recognition and the way the pathogen evades recognition. C_LIO_LIWe cloned sixteen allelic variants of the Rpi-chc1 gene from Solanum chacoense and other Solanum species, and identified the cognate P. infestans RXLR effectors. These tools were used to study receptor-ligand interactions and co-evolution. C_LIO_LIFunctional and non-functional alleles of Rpi-chc1 encode Coiled-Coil-Nucleotide Binding-Leucine-Rich-Repeat (CNL) proteins. Rpi-chc1.1 recognised multiple PexRD12 (AVRchc1.1) proteins while Rpi-chc1.2 recognised multiple PexRD31 (AVRchc1.2) proteins, both from the PexRD12/31 superfamily. Domain swaps between Rpi-chc1.1 and Rpi-chc1.2 revealed that overlapping subdomains in the LRR were responsible for the difference in effector recognition. C_LIO_LIThis study showed that Rpi-chc1.1 and Rpi-chc1.2, evolved to recognize distinct members of the same PexRD12/31 effector family via the LRR domain. The biased distribution of polymorphisms suggests that exchange of LRRs during host-pathogen co-evolution can lead to novel recognition specificities. These insights will help future strategies to breed for durable resistant varieties. C_LI

plant biology↗

bioRxiv2020

Multi-omics approach highlights differences between functional RLP classes in Arabidopsis thaliana

The receptor-like protein (RLP) family is a complex gene family with 57 members in Arabidopsis thaliana. Some members of the RLP family are known to be involved in basal developmental processes, whereas others have found to be involved in defence responses. However, functional data is to date, only available for a small subset of RLPs, leaving the remaining ones classified as RLPs of unknown function. Using publicly available datasets, we annotated those RLPs of unknown functions as either likely defence-related or likely fulfilling a more basal function in plants. Using these categories, we can identify important characteristics that differ between the RLP sub classes. We find the two classes differ in abundance on both transcriptome and proteome level, physical clustering in the genome and putative interaction partners. However, the classes do not differ in the genetic diversity of their individual members in accessible pan-genome data. Our work has several implications for work related to functional studies on RLPs as well as for the understanding of RLP gene family evolution. Using our annotations, we can make suggestions of which RLPs can be identified as potential immune receptors using genetics tools, which can be useful for disease studies. The lack of differences in nucleotide diversity between the two RLP subclasses further suggests that non-synonymous diversity of gene sequences alone cannot distinguish defence from developmental genes. By contrast, differences in transcript and protein abundance or clustering at genomic loci might also allow for functional annotations and characterisation in other plant species.

genomics↗

bioRxiv2020

Population studies in the wild tomato species Solanum chilense reveal maintenance and loss of resistance at geographically distinct locations

Natural plant populations encounter strong pathogen pressure and defense-associated genes are known to be under different selection pressure dependent on the pressure by the pathogens. Here we use wild tomato Solanum chilense populations to investigate natural resistance against Cladosporium fulvum, a well-known pathogenic fungus of domesticated tomatoes. We show that populations of S. chilense differ in resistance against the pathogen. Next, we explored the underlying molecular processes in a species wide-context. Then, focusing on recognition of the two prominent avirulence factors secreted by C. fulvum (Avr4 and Avr9) in central and northern populations of S. chilense we observed high complexity in the cognate homologues of Cladosporium resistance (Hcr9) locus underlying the recognition of these effectors. Presence of canonical genomic regions coding for Cf-4 and Cf-9, two major dominant resistance genes in the Hcr9 locus recognizing Avr4 and Avr9, respectively, does not meet prediction from Avr response phenotypes. We find both genes in varying fractions of the plant populations and we show possible co-existence of two functionally active resistance genes, previously thought to be allelic. Additionally, we observed the complete local absence of recognition of additional Avr proteins of C. fulvum. In the southern populations we attribute this to changes in the coregulatory network. As a result of loss of pathogen pressure or adaptation to extreme climatic conditions. This may ultimately explain the observed pathogen susceptibility in the southern populations. This work puts major gene mediated disease resistance in an ecological context.

ecology↗

bioRxiv2020

A complex resistance locus in Solanum americanum recognizes a conserved Phytophthora effector

Late blight caused by Phytophthora infestans greatly constrains potato production. Many Resistance (R) genes were cloned from wild Solanum species and/or introduced into potato cultivars by breeding. However, individual R genes have been overcome by P. infestans evolution; durable resistance remains elusive. We positionally cloned a new R gene, Rpi-amr1, from Solanum americanum, that encodes an NRC helper-dependent CC-NLR protein. Rpi-amr1 confers resistance in potato to all 19 P. infestans isolates tested. Using association genomics and long-read RenSeq, we defined eight additional Rpi-amr1 alleles from different S. americanum and related species. Despite only [~]90% identity between Rpi-amr1 proteins, all confer late blight resistance but differentially recognize Avramr1 orthologs and paralogs. We propose that Rpi-amr1 gene family diversity facilitates detection of diverse paralogs and alleles of the recognized effector, enabling broad-spectrum and durable resistance against P. infestans.

plant biology↗

bioRxiv2019

Partner-independent fusion gene detection by multiplexed CRISPR/Cas9 enrichment and long-read Nanopore sequencing

Fusion genes are hallmarks of various cancer types and important determinants for diagnosis, prognosis and treatment possibilities. The promiscuity of fusion genes with respect to partner choice and exact breakpoint-positions restricts their detection in the diagnostic setting, even for known and recurrent fusion gene configurations. To accurately identify these gene fusions in an unbiased manner, we developed FUDGE: a FUsion gene Detection assay from Gene Enrichment. FUDGE couples target-selected and strand-specific CRISPR/Cas9 activity for enrichment and detection of fusion gene drivers (e.g. BRAF, EWSR1, KMT2A/MLL) - without prior knowledge of fusion partner or breakpoint-location - to long-read Nanopore sequencing. FUDGE encompasses a dedicated bioinformatics approach (NanoFG) to detect fusion genes from Nanopore sequencing data. Our strategy is flexible with respect to target choice and enables multiplexed enrichment for simultaneous analysis of several genes in multiple samples in a single sequencing run. We observe on average a 508 fold on-target enrichment and identify fusion breakpoints at nucleotide resolution - all within two days. We demonstrate that FUDGE effectively identifies fusion genes in cancer cell lines, tumor samples and on whole genome amplified DNA irrespective of partner gene or breakpoint-position in 100% of cases. Furthermore, we show that FUDGE is superior to routine diagnostic methods for fusion gene detection. In summary, we have developed a rapid and versatile fusion gene detection assay, providing an unparalleled opportunity for pan-cancer detection of fusion genes in routine diagnostics.

genomics↗

bioRxiv2019

The de novo reference genome and transcriptome assemblies of the wild tomato species Solanum chilense

BackgroundWild tomato species, like Solanum chilense, are important germplasm resources for enhanced biotic and abiotic stress resistance in tomato breeding. In addition, S. chilense serves as a model system to study adaptation of plants to drought and to investigate the evolution of seed banks. However to date, the absence of a well annotated reference genome in this compulsory outcrossing, very diverse species limits in-depth studies on the genes involved.\n\nFindingsWe generated [~]134 Gb of DNA and 157 Gb of RNA sequence data of S chilense, which yielded a draft genome with an estimated length of 914 Mb in total encoding 25,885 high-confidence (hc) predicted gene models, which show homology to known protein-coding genes of other tomato species. Approximately 71% (18,290) of the hc gene models are additionally supported by RNA-seq data derived from leaf tissue samples. A benchmarking with Universal Single-Copy Orthologs (BUSCO) analysis of predicted gene models retrieved 93.3% BUSCO genes, which is in the current range of high-quality genomes for non-inbred plants. To further verify the genome annotation completeness and accuracy, we manually inspected the NLR resistance gene family and assessed its assembly quality. We revealed the existence of unique gene families of NLRs to S. chilense. Comparative genomics analyses of S. chilense, cultivated tomato S. lycopersicum and its wild relative S. pennellii revealed similar levels of highly syntenic gene clusters between the three species.\n\nConclusionsWe generated the first genome and transcriptome sequence assembly for the wild tomato species Solanum chilense and demonstrated its value in comparative genomics analyses. We make these genomes available for the scientific community as an important resource for studies on adaptation to biotic and abiotic stress in Solanaceae, on evolution of self-incompatibility, and for tomato breeding.

plant biology↗

bioRxiv2018

The multi-speed genome of Fusarium oxysporum reveals association of histone modifications with sequence divergence and footprints of past horizontal chromosome transfer events

Fusarium oxysporum is an economically important pathogen causing wilting or rotting disease symptoms in a large number of crops. It is proposed to have a structured, \"two-speed\" genome: i.e. regions containing genes involved in pathogenicity cluster with transposons on separate accessory chromosomes. This is hypothesized to enhance evolvability. Given the continuum of adaptation of all the genes encoded in a genome, however, one would expect a more complex genome structure. By comparing the genome of reference strain Fol4287 to those of 58 other Fusarium oxysporum strains, we found that some Fol4287 accessory chromosomes are lineage-specific, while others occur in multiple lineages with very high sequence similarity - but only in strains that infect the same host as Fol4287. This indicates that horizontal chromosome transfer has been instrumental in past host-switches. Unexpectedly, we found that the sequence of the three smallest core chromosomes (Chr. 11, 12 and 13) is more divergent than that of the other core chromosomes. Moreover, these chromosomes are enriched in genes involved in metabolism and transport and genes that are differentially regulated during infection. Interestingly, these chromosomes are -like the accessory chromosomes- marked by histone H3 lysine 27 trimethylation (H3K27me3) and depleted in histone H3 lysine 4 dimethylation (H3K4me2). Detailed genomic analyses revealed a complex, \"multi-speed genome\" structure in Fusarium oxysporum. We found a strong association of H3K27me3 with elevated levels of sequence divergence that is independent of the presence of repetitive elements. This provides new leads into how clustering of genes evolving at similar rates could increase evolvability.\n\nAuthor summaryFungi that cause disease on plants are an increasingly important threat to food security. New fungal diseases emerge regularly. The agricultural industry makes large investments to breed crops that are resistant to fungal infections, yet rapid adaptation enables fungal pathogens to overcome this resistance within a few years or decades. It has been proposed that genome compartmentalization of plant pathogenic fungi, in which infection-related genes are clustered with transposable elements (or jumping genes) into separate, fast-evolving regions, enhances their adaptivity. Here, we aimed to shed light on the possible interplay between genome organization and adaptation. We measured differences in sequence divergence and dispensability between and within individual chromosomes of the important plant pathogen Fusarium oxysporum. Based on these differences we defined four distinct chromosomal categories. We then mapped histone modifications and gene expression levels under different conditions for these four categories. We found a division of labor between chromosomes, where some are pathogenicity chromosomes - specialized towards infection of a specific host, while others are enriched in genes involved in more generic infection-related processes. Moreover, we confirmed that horizontal transfer of pathogenicity chromosomes likely plays an important role in gain of pathogenicity. Finally, we found that a specific histone modification is associated with increased sequence divergence.

genomics↗

bioRxiv2017

The evolutionary history of the current global Ramularia collo-cygni epidemic

Ramularia Leaf Spot (RLS) has emerged as a threat for barley production in many regions of the world. Late appearance of unspecific symptoms caused that Ramularia collo-cygni could only by molecular diagnostics be detected as the causal agent of RLS. Although recent research has shed more light on the biology and genomics of the pathogen, the cause of the recent global spread remains unclear.\n\nTo address urgent questions, especially on the emergence to a major disease, life-cycle, transmission, and quick adaptation to control measures, we de-novo sequenced the genome of R. collo-cygni (urug2 isolate). Additionally, we sequenced fungal RNA from 6 different conditions, which allowed for an improved genome annotation. This resulted in a high quality draft assembly of about 32 Mb, with only 78 scaffolds with an N50 of 2.1 Mb. The overall annotation enabled the prediction of 12.346 high confidence genes. Genomic comparison revealed that R. collo-cygni has significantly diverged from related Dothidiomycetes, including gain and loss of putative effectors, however without obtaining species-specific genome features.\n\nTo evaluate the species-wide genetic diversity, we sequenced the genomes of 19 R. collo-cygni isolates from multiple geographic locations and diverse hosts and mapped sequences to our reference genome. Admixture analyses show that R. collo-cygni is world-wide genetically uniform and that samples do not show a strong clustering on either geographical location or host species. To date, the teleomorph of R. collo-cygni has not been observed. Analysis of linkage disequilibrium shows that in the world-wide sample set there are clear signals of recombination and thus sexual reproduction, however these signals largely disappear when excluding three outliers samples, suggesting that the main global expansion of R. collo-cygni comes from mixed or clonally propagating populations. We further analysed the historic population size (Ne) of R. collo-cygni using Bayesian simulations.\n\nWe discuss how our genomic data and population genetics analysis can help understand the current R. collo-cygni epidemic and provide different hypothesis that are supported by our data. We specifically highlight how recombination, clonal spreading and lack of host-specificity could further support global epidemics of this increasingly recognized plant disease and suggest specific approaches to combat this pathogen.

plant biology↗

bioRxiv2017

A small subset of NLR genes drives local adaptation to pathogens in wild tomato

O_LINucleotide binding site, Leucine-rich repeat Receptors (NLRs), are canonical resistance (R) genes in plants, fungi and animals, functioning as central (helper) and peripheral (sensor) genes in a signalling network. We investigate NLR evolution during the colonisation of novel habitats in a model tomato species, Solanum chilense.\nC_LIO_LIWe used R-gene enrichment sequencing (RENSeq) to obtain polymorphism data at NLRs of 140 plants sampled across 14 populations covering the whole species range. We inferred the past demographic history of habitat colonisation by resequencing whole genomes from three S. chilense plants from three key populations, and performing Approximate Bayesian Computation using data from the 14 populations.\nC_LIO_LIUsing these parameters we simulated the genetic differentiation statistics distribution expected under neutral NLR evolution, and identified small subsets of outlier NLRs exhibiting signatures of selection across populations.\nC_LIO_LINLRs under selection between habitats are more often helper genes, while those showing signatures of adaptation in single populations are more often sensor-NLRs. Thus, centrality in the NLR network does not constrain NLR evolvability, and new mutations in central genes in the network are key for R gene adaptation during colonisation of different habitats.\nC_LI

evolutionary biology↗