Biology subjects

Sotiropoulos, A. G.

Publications and source records attributed to Sotiropoulos, A. G..

13 recordsLinked to original sources

bioRxiv2026

Proteogenomics of Blumeria hordei supports RNA and protein coding innovative potential derived from transposable elements

Some filamentous plant-pathogenic fungi have comparably large genome sizes within the fungal kingdom due to the proliferation of transposable elements (TEs). Blumeria hordei (Bh), the causal agent of the powdery mildew disease on barley, is a filamentous obligate biotrophic fungus. Compared to other ascomycetes, it contains a low number of genes but a high genomic TE content of approximately 75%. Yet, a comprehensive understanding of the contribution of TEs to the RNA and protein landscape of Bh is lacking. Here, we use Bh as a model to study transcripts and proteins derived from genes and individual TEs. Therefore, we created two high-quality genome assemblies of the German Bh isolate TUM1 and the Australian Bh isolate AUS1. We applied deep proteomics with mass spectrometry, long-read and short-read sequencing on both DNA and RNA. Based on these multi-omic resources, we completed nearly gapless genome assemblies, new gene and TE annotations, and effector predictions. Using long-read RNA sequencing, we detected extensive co-transcription of TEs and genes as TE-gene chimeric transcripts. We identified previously unpredicted splice variants or genes, partially supported by proteomics. The intergenic and TE genomic space of Bh TUM1 gives rise to thousands of transcripts and several novel TE-derived proteins that lack from previous TE protein predictions. Together, this supports an existing potential for expression of novel transcripts and proteins from highly abundant TEs in the Bh genome.

plant biology↗

bioRxiv2025

Dual recognition of structurally unrelated mildew effectors underlies the broad-spectrum resistance of Pm3e in wheat

Broad-spectrum resistance genes are highly valuable for sustainable crop protection, yet the molecular basis of their activity is often unknown. The Pm3 allelic series in wheat encodes NLR receptors that recognize avirulence (AVR) effectors of wheat powdery mildew. Here, we show that near-identical Pm3 alleles vary greatly in resistance efficacy and broadness against a global mildew isolate collection and subsequently use this model system to study the mechanisms underlying broad-spectrum resistance. We demonstrate that two alleles, Pm3d and Pm3e, provide resistance against the majority of isolates worldwide, by each recognizing two AVR effectors from powdery mildew, thereby lowering the risk of resistance breakdown. While Pm3d recognizes two closely related RNase-like AVR effectors, Pm3e detects two structurally diverse AVRs, including an effector belonging to a large, uncharacterized protein family with a novel structural fold. Using chimeric Pm3 NLRs, we identify specificity-defining polymorphisms of Pm3d and Pm3e against their diverse effector targets. Lastly, we demonstrate that Pm3d and Pm3e activities can be combined in engineered Pm3 NLRs, thereby further extending their recognition spectrum. Our findings highlight the potential of Pm3 immune receptors for long-lasting wheat protection by demonstrating their versatility in recognizing structurally diverse effectors and their amenability to NLR engineering.

plant biology↗

bioRxiv2025

The CATION CALCIUM EXCHANGER 4 (CCX4) regulates LRX1-related root hair development through Ca2+ homeostasis

Calcium, as a cellular second messenger, is essential for plant growth. A tip-focused Ca2+ gradient in polarized cells is considered to drive cell expansion. The cell wall polysaccharide pectin is a major Ca2+ binding structure and Ca2+ homeostasis is influenced by the cell wall architecture. LRR-extensin (LRX) proteins are extracellular regulators of cell wall development that are anchored in the cell wall by their extensin domain. The extensin-less LRX1{Delta}E14 variant of the root hair-expressed LRX1 of Arabidopsis induces a dominant-negative effect resulting in aberrant root hairs. In an effort to identify the underlying mechanism of the root hair defect caused by LRX1{Delta}E14, we isolated a suppressor of dominant-negative effect mutant, sune42. It codes for the CATION CALCIUM EXCHANGER 4 (CCX4) that localizes to the Golgi apparatus and was shown to have Ca2+ transport activity. A detailed investigation of the Ca2+ dynamics revealed that LRX1{Delta}E14 coincides with a defect in tip-focused cytoplasmic Ca2+ oscillation, and this effect is alleviated by the sune42 mutation. Additionally, reducing Ca2+ availability influences the LRX1{Delta}E14-induced root hair defect. We conclude that sune42 suppresses the root hair defect in LRX1{Delta}E14 through modulating cytoplasmic Ca2+ dynamics, pointing at the importance of the Golgi apparatus for cellular Ca2+ homeostasis.

plant biology↗

bioRxiv2025

Combining pangenomics and population genetics finds chromosomal re-arrangements, accessory-like chromosome segments, copy number variations and transposon polymorphisms in wheat and rye powdery mildew

Grass powdery mildews (Blumeria spp.) include economically important fungal crop pathogens with complex and highly repetitive genomes. To investigate the diversity and genome evolution in Blumeria graminis, we combined population genetic and pangenomic analyses using a worldwide sample of 399 wheat powdery mildew isolates. Additionally, we produced high-quality genome assemblies for seven isolates from wheat and one from rye powdery mildew. Using these, we compiled the first grass powdery mildew pangenome comprising 11 Blumeria graminis isolates. We found multiple chromosomal rearrangements between the isolates that grow on wheat, rye and/or triticale hosts. Interestingly, chr-11 showed some characteristics of accessory chromosomes such as presence/absence of large chromosomal segments and higher sequence diversity. Additionally, we identified nearly 67,000 cases of copy number variations (CNVs), which were highly enriched within effector gene families. Furthermore, we found evidence for recent and high transposable element (TE) activity, such as high numbers of TE insertion polymorphisms. Analyses of TE families showed enrichment 1 kb to 2 kb up- and downstream of effector genes, and we also found high levels of TE insertion polymorphisms between populations. Our results demonstrate that chromosomal variations, gene family expansions and contractions, and TE activity are important sources of genome diversification and diversity in grass powdery mildews. Our findings indicate that a combination of pangenomic and population genetics analyses is needed to understand drivers of evolution in plant pathogenic fungi in a comprehensive way. Author SummaryFungi are a diverse group of organisms, with some of them (e.g. powdery mildews) having at times a devastating impact on important crops like wheat. Studying the genomes of these grass powdery mildew fungi can improve our understanding the pathogens patterns of genome evolution and virulence in order to find more efficient ways to protect crops. Using high quality genomes of a worldwide dataset of wheat and rye powdery mildews, we found multiple re-arrangements in chromosomes, as well as presence and absence of large chromosomal segments in different mildew strains. Along with some genes, important for making these pathogens infectious on various crop lines, some genomic regions may be deleted or duplicated, potentially affecting how the fungus survives and spreads. Additionally, we found that transposable elements are highly active in powdery mildews and that they are enriched near so-called effector genes, which are associated with fungal virulence. Our study shows how powdery mildew genomes have diversified during recent evolution, which has implications for future breeding of crops toward better resistance to fungal diseases.

genomics↗

bioRxiv2025

Multi-locus phylogenetic network analysis of Ampelomyces mycoparasites isolated from diverse powdery mildews in Australia and the generation of two de novo genome assemblies

The interactions between powdery mildews (Ascomycota, Erysiphaceae), obligate biotrophic pathogens of many plants, and pycnidial fungi belonging to the genus Ampelomyces, are classic examples of specific mycoparasitic relationships. These interactions are common and finely tuned tritrophic relationships amongst host plants, powdery mildews, and Ampelomyces mycoparasites wherever these organisms co-occur in the field. Selected Ampelomyces strains have already been developed as biocontrol agents of powdery mildew infections of some crops. In Australia, their study has received little attention so far. Only a single Ampelomyces strain, included in a whole-genome sequencing (WGS) project, was known from this continent. Here, we report the isolation of 20 more Ampelomyces strains from eight powdery mildew species in Australia. Multi-locus phylogenetic network analyses of all the 21 Australian Ampelomyces strains carried out in combination with 32 reference strains from overseas revealed that the Australian strains belonged to four molecular taxonomic units (MOTUs). All those MOTUs were delimited earlier based on Ampelomyces strains isolated in Europe, North America, and elsewhere. Analyses indicated possible recombination events amongst phylogenetically different Ampelomyces strains. Based on the phylogenetic analyses, two Australian strains belonging to different MOTUs were selected for WGS. Long-read (minION) and short-read (Illumina) technologies were used to provide genome assemblies with high completeness. Both assemblies reported here had bipartite structure, i.e., consisted of AT-rich, gene-sparse regions interspersed with GC-balanced, gene-rich regions. These new high-quality assemblies and evidence-based annotations are important resources for future analyses of mycoparasitic interactions to disentangle molecular mechanisms underlying mycoparasitism, possible new biocontrol applications, and natural tritrophic relationships.

microbiology↗

bioRxiv2024

A reference metagenome sequence of the lichen Cladonia rangiformis

[bullet] Lichens are an ancient symbiosis comprising the thalli of lichen-forming fungi, their photoautotrophic partners and their microbiome. So far, they were poorly studied at the genome sequence level. Here, we present a reference metagenome for the holobiont of Cladonia rangiformis. [bullet]Using long read sequences from an entire symbiotic complex, plus short read libraries from 28 additional diverse European lichen samples, we were able to separate genome sequences of 20 individual species. [bullet]We constructed chromosome-scale assemblies of the C. rangiformis fungus and its trebouxioid green algal photobiont Asterochloris mediterranea. The genome of the fungus comprises [~]22% transposable elements and is highly compartmentalized into genic regions and large TE-derived segments which show extensive signatures of repeat-induced point mutations (RIP). We found that A. mediterranea centromeres are predominantly derived from two interacting retrotransposon families. We also identified strong candidates for genes that were horizontally transferred from bacteria to both alga and fungus. Furthermore, we isolated 18 near-complete bacterial genomes, of which 13 are enriched in the lichen compared to surrounding soil. [bullet]Our study revealed that the thalli of C. rangiformis have a highly complex microbiome, comprising a mix of species that may include opportunists, ecologically obligate symbionts and possibly even lichen-beneficial bacteria.

evolutionary biology↗

bioRxiv2024

k-mer-based GWAS in a wheat collection reveals novel and diverse sources of powdery mildew resistance

BackgroundWheat landraces and cultivars stored in gene banks worldwide represent a valuable source of genetic diversity for discovering genes critical for agriculture, which is increasingly constrained by climate change and inputs reduction. We assembled and genotyped, using DArTseq technology, a panel of 461 accessions representative of the genetic diversity of Swiss wheat material. The collection was evaluated for powdery mildew resistance under field conditions for two consecutive years and at the seedling stage with 10 different wheat powdery mildew isolates. ResultsTo identify the genetic basis of mildew resistance in wheat, we developed a k-mer-based GWAS approach using multiple fully-assembled genomes including Triticum aestivum as well as four progenitor genomes. Compared to approaches based on single reference genomes, we unambiguously mapped an additional 25% resistance-associated k-mers. Our approach outperformed SNP-based GWAS in terms of number of loci identified and precision of mapping. In total, we detected 34 (Pm) powdery mildew resistance loci, including seven previously-described and more importantly 27 novel loci active at the seedling stage. Furthermore, we identified a region associated with adult plant resistance, which was not detected with SNP-based approaches. ConclusionsThe described non-reference-based approach highlights the potential of integrating multiple wheat reference genomes with k-mer GWAS to harness the untapped genetic diversity present in germplasm collections.

genomics↗

bioRxiv2024

Avirulence depletion assay: combining R gene-mediated selection with bulk sequencing for rapid avirulence gene identification in wheat powdery mildew

Wheat production is threatened by multiple fungal pathogens, such as the wheat powdery mildew fungus (Blumeria graminis f. sp. tritici, Bgt). Wheat resistance breeding frequently relies on the use of resistance (R) genes that encode diverse immune receptors which detect specific avirulence (AVR) effectors and subsequently induce an immune response. While R gene cloning has accelerated recently, AVR identification in many pathogens including Bgt lags behind, preventing pathogen-informed deployment of resistance sources. Here we describe a new "avirulence depletion (AD) assay" for rapid identification of AVR genes in Bgt. This assay relies on the selection of a segregating, haploid F1 progeny population on a resistant host, followed by bulk sequencing, thereby allowing rapid avirulence candidate gene identification with high mapping resolution. In a proof-of- concept experiment we mapped the AVR component of the wheat immune receptor Pm3a to a 25kb genomic interval in Bgt harboring a single effector, the previously described AvrPm3a2/f2. Subsequently, we applied the AD assay to map the unknown AVR effector recognized by the Pm60 immune receptor. We show that AvrPm60 is encoded by three tandemly arrayed, nearly identical effector genes that trigger an immune response upon co- expression with Pm60 and its alleles Pm60a and Pm60b. We furthermore provide evidence that Pm60 outperforms Pm60a and Pm60b through more efficient recognition of AvrPm60 effectors, suggesting it should be prioritized for wheat breeding. Finally, we show that virulence towards Pm60 is caused by simultaneous deletion of all AvrPm60 gene paralogs and that isolates lacking AvrPm60 are especially prevalent in the US thereby limiting the potential of Pm60 in this region. The AD assay is a powerful new tool for rapid and inexpensive AVR identification in Bgt with the potential to contribute to pathogen-informed breeding decisions for the use of novel R genes and regionally tailored gene deployment.

plant biology↗

bioRxiv2024

Histidine limitation causes alteration in the TOR network and plant development

Plant growth depends on growth regulators, nutrient availability, and amino acids levels. The TOR (Target of Rapamycin) network senses these parameters and influences cell wall formation and expansion accordingly. Cell wall integrity and structures are surveyed and modified by a complex array of cell wall integrity sensors, including LRR-extensins (LRXs), that function as hormone receptors and help to compact cell walls. Expressing the Arabidopsis root-hair specific LRX1 without the extensin domain, which anchors the protein to the cell wall, has a negative effect on root hair development. The mechanism of this negative effect was investigated by a suppressor screen, which led to the identification of a sune (suppressor of dominant-negative LRX1) mutant collection. The sune82 mutant was identified as an allele of HISN2 which encodes an enzyme essential for histidine biosynthesis. The sune82 mutation leads to reduced accumulation of histidine, and this influences the TOR network. The sune82 mutant reflects the impact of the TOR network on cell wall formation processes involving LRX proteins. It also represents an excellent tool to study the effects of reduced histidine levels on plant development, as it is a rare example of a viable partial loss-of-function allele in an essential biosynthetic pathway. HighlightPartial loss of function of HISN2 in sune82 results in a significant reduction in histidine content, which subsequently alters the TOR network.

cell biology↗

bioRxiv2022

Doomed by popularity: The broad use of the Pm8 resistance gene in wheat resulted in hypermutation of the AvrPm8 gene in the powdery mildew pathogen

O_LIThe Pm8 resistance gene against the powdery mildew disease has been introgressed from rye into wheat as part of a large 1BL.1RS chromosomal translocation. Due to its high agronomic value, this translocation has seen continuous global use since the 1960s on large growth areas, even after Pm8 resistance was overcome. This allows studying the effect of long and widespread resistance gene use on a pathogen population. C_LIO_LIUsing genome wide association studies in a global population of wheat mildew isolates, we identified the avirulence effector AvrPm8 specifically recognized by Pm8. C_LIO_LIHaplovariant mining in the global population revealed 17 virulent haplotypes of the AvrPm8 gene that grouped into two categories. The first one from geographically diverse regions comprised two single amino acid polymorphisms at the same position in the AvrPm8 protein, which we confirmed to be crucial for recognition by Pm8. The second category consisted of numerous destructive mutations to the AvrPm8 open reading frame. C_LIO_LIMost individual gain-of-virulence mutations were found in geographically restricted regions, indicating they occurred recently as a consequence of the frequent Pm8 use. We conclude that both standing genetic variation as well as locally occurring new mutations contributed to the global breakdown of the Pm8 resistance gene introgression. C_LI

plant biology↗

bioRxiv2022

A survey of lineage-specific genes in Triticeae reveals de novo gene evolution from genomic raw material

Plant genomes typically contain [~]35,000 genes, almost all belonging to highly-conserved gene families. Only a small fraction are lineage-specific, which are found in only one or few closely related species. Little is known about how genes arise de novo in plant genomes and how often this occurs, however they are believed to be important for plants diversification and adaptation. We developed a pipeline to identify lineage-specific genes in Triticeae, using newly available genome assemblies of wheat, barley and rye. Applying a set of stringent criteria, we identified 5,942 candidate Triticeae-specific genes (TSGs), of which 2,337 were validated as protein-coding genes in wheat. Differential gene expression analyses revealed that stress-induced wheat TSGs are strongly enriched in secreted proteins. Some were previously described to be involved in Triticeae non-host resistance and cold adaptation. Additionally, we show that 1,079 TSGs have sequence homology to transposable elements (TEs), [~]68% of them deriving from regulatory non-coding regions of Gypsy retrotransposons. Most importantly, we demonstrate that these TSGs are enriched in transmembrane domains and are among the most highly expressed wheat genes overall. To summarize, we conclude that de novo gene formation is relatively rare and that Triticeae probably possess [~]779 lineage-specific genes per haploid genome. TSGs which respond to pathogen and environmental stresses, may be interesting candidates for future targeted resistance breeding in Triticeae. Finally, we propose that non-coding regions of TEs might provide important genetic raw material for the functional innovation of TM domains and the evolution of novel secreted proteins.

evolutionary biology↗

bioRxiv2021

Standing genetic variation of the AvrPm17 avirulence gene in powdery mildew limits the effectiveness of an introgressed rye resistance gene in wheat

Introgressions of chromosomal segments from related species into wheat are important sources of resistance against fungal diseases. The durability and effectiveness of introgressed resistance genes upon agricultural deployment is highly variable - a phenomenon that remains poorly understood as the corresponding fungal avirulence genes are largely unknown. Until its breakdown, the Pm17 resistance gene introgressed from rye to wheat provided broad resistance against powdery mildew (Blumeria graminis). Here, we used QTL mapping to identify the corresponding wheat mildew avirulence effector AvrPm17. It is encoded by two paralogous genes that exhibit signatures of re-occurring gene conversion events and are members of a mildew sub-lineage specific effector cluster. Extensive haplovariant mining in wheat mildew and related sub-lineages identified several ancient virulent AvrPm17 variants that were present as standing genetic variation in wheat powdery mildew prior to the Pm17 introgression, thereby paving the way for the rapid breakdown of the Pm17 resistance. QTL mapping in mildew identified a second genetic component likely corresponding to an additional resistance gene present on the 1AL.1RS translocation carrying Pm17. This gene remained previously undetected due to suppressed recombination within the introgressed rye chromosomal segment. We conclude that the initial effectiveness of 1AL.1RS was based on simultaneous introgression of two genetically linked resistance genes. Our results demonstrate the relevance of pathogen-based genetic approaches to disentangle complex resistance loci in wheat. We propose that identification and monitoring of avirulence gene diversity in pathogen populations becomes an integral part of introgression breeding to ensure effective and durable resistance in wheat. Significance StatementDomesticated and wild wheat relatives provide an important source of new immune receptors for wheat resistance breeding against fungal pathogens. The durability of these resistance genes is variable and difficult to predict, yet it is crucial for effective resistance breeding. We identified a fungal effector protein recognised by an immune receptor introgressed from rye to wheat. We found that variants of the effector allowing the fungus to overcome the resistance are ancient. They were already present in the wheat powdery mildew gene pool before the introgression of the immune receptor and are therefore responsible for the rapid resistance breakdown. Our study demonstrates that the effort to identify new resistance genes should be accompanied by studies of avirulence genes on the pathogen side.

plant biology↗

bioRxiv2020

Bacteriophage deficiency characterizes respiratory virome dysbiosis in childhood asthma

Asthma development and persistence is tightly linked to respiratory viruses. Viral presence is usually interrogated with targeted approaches during periods of disease activity and/or infections, thus neglecting viral occurrence during steady states. We investigate the virome in the upper respiratory system of healthy and asthmatic preschool children during asymptomatic/non-infection periods using metagenomics. Children with asthma have a characteristically dysbiotic virome that correlates to disease severity and control. The major component of dysbiosis is bacteriophage deficiency, while eukaryotic viral presence is increased. At the metacommunity level, differential virus species co-occurrence patterns suggest a decrease of the microbiota community resilience in asthma. Viral dysbiosis is therefore a key characteristic of asthma pathophysiology.

microbiology↗