Biology subjects

Hueckelhoven, R.

Publications and source records attributed to Hueckelhoven, R..

16 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↗

bioRxiv2026

The crop pathogen Blumeria hordei exhibits genome-wide pervasive selective and neutral sweepstakes reproduction signatures.

The fungal crop pathogen Blumeria hordei, causal agent of powdery mildew on barley, presents life-history and epidemiological characteristics, as well as and selective pressures due to modern agriculture leading to expected sweepstakes reproduction, that is highly skewed offspring distributions. Using genome-wide polymorphism data and population genomics inferences, we aim to 1) infer the past demographic history and the strength of sweepstakes reproduction in B. hordei, and 2) quantify the contributions of these selective and neutral processes in the genome. An new inference method based on Neural Posterior Estimation and diversity and linkage disequilibrium statistics was developed and tested on simulated and B. hordei genomic data. We confirm that B. hordei exhibits a moderate sweepstakes reproduction (-parameter of 1.6). We highlight that the Site Frequency Spectrum (SFS) appears sensitive to the joint occurrence of sweepstakes and recent demographic changes, which may caution on the reliability of the SFS to infer sweepstakes reproduction. We then scan the genome for selective sweeps, adjusting the significance thresholds of the methods for demographic history and sweepstakes reproduction, thereby yielding a counterintuitive result. When conditioning the significance threshold for sweep detection on simulations under sweepstakes and demography, a very large number of putatively selected regions is found (11.6% of the genome). We suggest that sweepstakes reproduction in B. hordei is due to 1) neutrality (clonal/sexual phases and Boom-and-Bust cycles) generating a genome-wide level of background noise in the coalescent genealogies, and 2) selective sweepstakes due to pervasive positive selection. Our findings have important implications for both population genomic methodology and our understanding of pathogen evolution.

evolutionary biology↗

bioRxiv2026

The Limits of Cross-Species WGCNA: Library Imbalance and Signal Dilution Constrain Effector Gene Recovery in Dual-Organism RNA-seq

Dual-organism RNA sequencing (RNA-seq) experiments, in which the transcriptomes of a host and a microbe are sequenced simultaneously, are increasingly used to study plant-microbe interactions. A central analytical goal is identifying effector proteins and their host targets through gene co-expression. Weighted Gene Co-expression Network Analysis (WGCNA) is the dominant tool for gene co-expression analyses, yet its ability to recover interaction-interface genes from a merged dual-organism matrix has not been systematically characterised. Here we present a simulation framework using real gene models from Hordeum vulgare (barley) and Blumeria graminis f. sp. Hordei M.Liu & Hambl (powdery mildew) to evaluate single-network WGCNA across a gradient of plant-to-fungal library size ratios (1:1-20:1), three levels of co-expression signal strength, and three WGCNA network construction types (signed, unsigned, signed hybrid). We embed 20 model effector genes (bridge genes) driven by a mixed host-pathogen eigengene and evaluate recovery using four metrics aligned with the biological objective: cross-species hub rank, top-decile hub enrichment, bridge gene detection rate, and bridge co-separation (the fraction of effector-target pairs co-assigned to the same detected module). Across 225 simulation runs (15 conditions x 5 replicates x 3 network types), bridge genes are robustly identifiable as cross-species connectivity hubs (mean rank 0.92 versus 0.50 for module genes) but co-assignment of effector-target pairs to the same module fails in 41% of runs due to scale-free topology collapse. Signal strength (2 = 0.12) and library ratio (2 = 0.22) are the primary determinants of co-separation, while network type choice accounts for less than 2%. A read-depth bias systematically inflates pathogen gene hub ranks relative to host genes at high ratios. These results establish that the method can identify effector candidates as cross-species hubs under a broad range of conditions, but reliable co-assignment requires adequate pathogen read depth and strong co-expression signal--properties that experimental design, not analytical parameterisation, must provide.

systems biology↗

bioRxiv2025

Network Inference Reveals Distinct Transcriptional Regulation in Barley against Drought and Fusarium Head Blight

We analyzed transcriptional networks in barley under single and combined Fusarium head blight (FHB) and drought stress. We applied complementary Weighted Gene Correlation Network Analysis (WGCNA) to identify stress-associated gene co-expression modules and GENIE3 to infer gene regulatory networks (GRNs). Integration of these frameworks revealed strong overlaps between co-expression modules and GRN clusters, highlighting robust regulatory patterns. Key transcription factors (TFs) were identified based on their weighted node degrees, reflecting their connectivity within the network. Independent analysis of paired transcription factor binding sites in promoter regions further supported predicted regulatory interactions. Notably, WRKY TFs emerged as central regulators of FHB response, consistent with their known roles in defense and secondary metabolite biosynthesis, but did not appear in drought-associated contexts. For bHLH or NAC TFs, individual family members steered FHB or drought responses but not both. Our findings demonstrate the power of combining network inference and motif enrichment to identify candidate TFs controlling stress responses, providing a solid foundation for targeted functional validation.

plant biology↗

bioRxiv2025

Transcriptome and hormone regulations shape drought stress-dependent Fusarium Head Blight susceptibility in different barley genotypes

Little is known about regulatory mechanisms that crop plants use to respond to combinations of abiotic and biotic stress. We analysed four barley genotypes under simultaneous Fusarium culmorum infection and drought stress by phenotyping of Fusarium Head Blight (FHB) disease, hormone profiling, and transcriptome analysis. FHB severity was host genotype-dependent, with moderately resistant cultivars Avalon and Barke showing increased FHB under drought, while drought did not further increase FHB severity in highly susceptible Morex and Palmella Blue. Transcriptome analysis revealed largely additive effects of single stresses, with drought-dominated regulation with increasing drought severity at later stages. Co-expression analysis connected abscisic acid and auxin to gene expression modules functionally enriched with stress-specific physiological responses. Stress-response genes, uniformly expressed across genotypes, were linked to pathogen defence, detoxification, and drought adaptation, whereas a cluster of hundreds of moderately Fusarium-responsive genes was limited in up-regulation under combined stress, possibly explaining enhanced FHB severity under drought. A multiple linear regression model accurately predicted combined stress expression from single stress responses, demonstrating modularity of barleys stress regulation under combined drought and Fusarium stress. Graphical AbstractSimplified working model illustrating drought effects on abscisic acid (ABA) levels and susceptibility to Fusarium Head Blight (FHB) of barley, and key processes such as storage and desiccation responses, photosynthesis, cell wall formation, metabolism, and transferase activity. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=117 SRC="FIGDIR/small/689882v1_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@15504adorg.highwire.dtl.DTLVardef@a62498org.highwire.dtl.DTLVardef@1acb1f5org.highwire.dtl.DTLVardef@d78a3b_HPS_FORMAT_FIGEXP M_FIG C_FIG

plant biology↗

bioRxiv2025

MIK2 clade receptor function in perception of a Fusarium-derived elicitor is conserved among different plant families

Plants continuously monitor their environment to adapt to threats affecting health and fitness. Cell-surface receptors detect conserved microbe-associated molecular patterns (MAMPs) or endogenous immunogenic signals to activate signaling pathways that induce broad-spectrum disease resistance, known as pattern-triggered immunity (PTI). In Arabidopsis thaliana, the leucine-rich repeat receptor kinase MIK2 emerged as a versatile receptor involved in perceiving endogenous SCOOP peptides as well as potential MAMPs derived from Fusarium and related fungi. While SCOOP perception appears restricted to Brassicales, the Fusarium-derived elicitor triggers PTI-responses across different plant lineages. Here, we demonstrate that Fusarium elicitor-responsiveness and MIK2-clade proteins are conserved among seed plants. We identified a tomato MIK2-clade receptor that shares AtMIK2 properties in perceiving the Fusarium elicitor, but not SCOOPs. Tomato mutants lacking the receptor show compromised PTI-responses to the fungal elicitor and increased susceptibility to Fusarium oxysporum. By integrating AI-assisted structural prediction with experimental data, we identify conserved and variable residues in Arabidopsis and tomato MIK2-clade receptors that may underlie shared and lineage-specific recognition specificities. Together, these findings establish MIK2-clade receptors as immune surveillance modules that have undergone lineage-specific diversification, supporting a model in which endogenous danger perception might have evolved from an ancestral role in microbial pattern recognition.

plant biology↗

bioRxiv2025

Endogenous RALF peptide function is required for powdery mildew host colonization

The receptor kinase FERONIA (FER) is a susceptibility factor for biotrophic powdery mildew fungal pathogens in Arabidopsis thaliana, but the underlying molecular mechanisms remain largely unknown. FER is required for the perception of endogenous RAPID ALKALINIZATION FACTOR (RALF) peptides to control various aspects of plant growth, development and immunity. RALFs are perceived by FER-LORELEI-LIKE GPI-ANCHORED PROTEIN (LLG) heterocomplexes to induce cellular responses and bind to LEUCINE-RICH REPEAT EXTENSIN (LRX) proteins as structural components of the cell wall. Combining genetics, cell biology and biochemistry, we found that FERs endogenous RALF ligands are necessary for full colonization success of the powdery mildew species Erysiphe cruciferarum. We reveal that LLGs and LRXs are also powdery mildew susceptibility factors. We show that cell wall remodeling and apoplastic pH homeostasis, hallmark features of RALF function, support powdery mildew reproductive success. Moreover, we provide data that RALF-dependent powdery mildew pathogenesis is partially independent of FER. Powdery mildew fungi likely do not produce RALF peptide mimics, suggesting their reliance on endogenous RALFs for successful host colonization. We propose that powdery mildew fungi require RALF-mediated modulation of apoplastic pH and pectin re-modelling for successful host colonization, highlighting a new susceptibility mechanism by obligate biotrophic fungi.

plant biology↗

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

Cross-family transfer of the Arabidopsis cell-surface immune receptor LORE to tomato confers sensing of 3-hydroxylated fatty acids and enhanced disease resistance

Plant pathogens pose a high risk of yield losses and threaten food security. Technological and scientific advances have improved our understanding of the molecular processes underlying host-pathogen interactions, which paves the way for new strategies in crop disease management beyond the limits of conventional breeding. Cross-family transfer of immune receptor genes is one such strategy that takes advantage of common plant immune signaling pathways to improve disease resistance in crops. Sensing of microbe- or host damage-associated molecular patterns (MAMPs/DAMPs) by plasma membrane-resident pattern recognition receptors (PRR) activates pattern-triggered immunity (PTI) and restricts the spread of a broad spectrum of pathogens in the host plant. In the model plant Arabidopsis thaliana, the S-domain receptor-like kinase LIPOOLIGOSACCHARIDE-SPECIFIC REDUCED ELICITATION (AtLORE, SD1-29) functions as PRR, which senses medium chain-length 3-hydroxylated fatty acids (mc-3-OH-FAs), such as 3-OH-C10:0, and 3-hydroxyalkanoates (HAAs) of microbial origin to activate PTI. In this study, we show that ectopic expression of the Brassicaceae-specific PRR AtLORE in the solanaceous crop species Solanum lycopersicum cv. M82 leads to the gain of 3-OH-C10:0 immune sensing without altering plant development. AtLORE-transgenic tomato shows enhanced resistance against Pseudomonas syringae pv. tomato DC3000 and Alternaria solani NL03003. Applying 3-OH-C10:0 to the soil before infection induces resistance against the oomycete pathogen Phytophthora infestans Pi100 and further enhances resistance to A. solani NL03003. Our study proposes a potential application of AtLORE-transgenic crop plants and mc-3-OH-FAs as resistance-inducing bio-stimulants in disease management.

plant biology↗

bioRxiv2023

Barley shows reduced Fusarium Head Blight under drought and modular expression of differential expressed genes under combined stress

Plants often face simultaneous abiotic and biotic stress conditions. However, physiological and transcriptional responses of plants under combined stress situations are little understood. Spring barley is susceptible to Fusarium Head Blight (FHB), which is strongly affected by weather conditions. We therefore studied the potential influence of drought on FHB severity and responses in three differently susceptible spring barley varieties and found strongly reduced FHB severity in susceptible varieties under drought. Quantity of differentially expressed genes (DEGs) and strength of transcriptomic regulation reflected the concentration of physiological stress markers such as abscisic acid or fungal DNA contents. Infection-related gene expression associated rather with susceptibility than resistance. Weighted gene correlation network analysis uncovered 18 modules of co-expressed genes, which reflect the pathogen or drought response in the used varieties. A generally infection-related module contained co-expressed genes for defence, programmed cell death and mycotoxin-detoxification indicating that diverse genotypes use a similar defence strategy towards FHB albeit with different success. Further DEGs showed co-expression in drought or genotype-associated modules correlating with measured phytohormones or the osmolyte proline. The combination of drought stress with infection lead to highest numbers of DEGs and provoked a modular composition of single stress responses rather than a specific transcriptional readout. HighlightCo-expression network analysis reveals association of physiological stress markers and gene expression modules under biotic and abiotic stress

plant biology↗

bioRxiv2022

Phytocytokine signaling integrates cell surface immunity and nitrogen limitation

Plant endogenous signaling peptides shape growth, development and adaptations to biotic and abiotic stress. Here, we identified C-TERMINALLY ENCODED PEPTIDEs (CEPs) as novel immune-modulatory peptides (phytocytokines) in Arabidopsis thaliana. Our data reveals that CEPs induce immune outputs and are required to mount resistance against the leaf-infecting bacterial pathogen Pseudomonas syringae pv. tomato. We show that effective immunity requires CEP perception by tissue-specific CEP RECEPTOR 1 (CEPR1) and CEPR2. Moreover, we identified the related RECEPTOR-LIKE KINASE 7 (RLK7) as a novel CEP4-specific CEP receptor contributing to CEP-mediated immunity, suggesting a complex interplay of multiple CEP ligands and receptors in different tissues during biotic stress. CEPs have a known role in the regulation of root growth and systemic nitrogen (N)-demand signaling. We now provide evidence that CEPs and their receptors promote immunity in an N status-dependent manner, suggesting a previously unknown molecular crosstalk between plant nutrition and cell surface immunity. We propose that CEPs and their receptors are central regulators for the adaptation of biotic stress responses to plant-available resources.

plant biology↗

bioRxiv2022

Convergent evolution of plant pattern recognition receptors sensing cysteine-rich patterns from three microbial kingdoms

The Arabidopsis thaliana receptor-like protein RLP30 contributes to immunity against the fungal pathogen Sclerotinia sclerotiorum. Here we identified the RLP30-ligand as a small cysteine-rich protein (SCP) that occurs in many fungi and oomycetes and is also recognized by the Nicotiana benthamiana RLP RE02. However, RLP30 and RE02 share little sequence similarity and respond to different parts of the native/folded protein. Interestingly, some Brassicaceae other than Arabidopsis also respond to a linear SCP peptide, suggesting that SCP is an eminent immune target that led to the convergent evolution of distinct immune receptors in plants. Surprisingly, RLP30 shows a second ligand specificity for a SCP-nonhomologous protein secreted by bacterial Pseudomonads. RLP30 expression in N. tabacum resulted in lower susceptibility to bacterial, fungal and oomycete pathogens, thus demonstrating that detection of immunogenic patterns by Arabidopsis RLP30 is involved in defense against pathogens from three microbial kingdoms.

plant biology↗

bioRxiv2021

Barley guanine nucleotide exchange factor HvGEF14 is an activator of the susceptibility factor HvRACB and supports host cell entry by Blumeria graminis f.sp. hordei

In barley (Hordeum vulgare), the function of ROPs appears central to polar cell development and the interaction outcome with parasitic fungi but little is known about ROP activation. Guanine nucleotide exchange factors (GEFs) facilitate the exchange of GDP with GTP and thereby turn ROPs into a signalling-activated ROP-GTP state. Plants possess a unique class of GEFs harbouring a plant specific ROP nucleotide exchanger domain (PRONE). We performed phylogenetic analyses and annotated barley PRONE GEFs. HvGEF14 is expressed in leaf epidermal tissue and downregulated after inoculation with Blumeria graminis f.sp. hordei. Protein-protein interaction assays indicate that HvGEF14 and the type I barley ROP protein HvRACB can interact in yeast and in planta. Overexpression of HvGEF14 further recruited the ROP-GTP downstream interactor HvRIC171 to the cell periphery and let to interaction of HvRACB with an extended CRIB (Cdc42/Rac Interactive Binding motif) peptide of HvRIC171 in a similar manner as constitutively activated HvRACB. Finally, the over expression of HvGEF14 led to enhanced susceptibility to fungal entry while HvGEF14 RNAi provoked a trend to more penetration resistance. HvGEF14 might therefore play a role in the activation of HvRACB in barley epidermal cells, which is crucial for fungal penetration success. HighlightThe activated GTPase HvRACB is a susceptibility factor of barley in response to Blumeria graminis f.sp. hordei. The newly discovered guanine nucleotide exchange factor HvGEF14 is a HvRACB activator.

plant biology↗

bioRxiv2021

RGI-GOLVEN signalling promotes FLS2 abundance to regulate plant immunity

Endogenous plant signalling peptides regulate developmental and growth-related processes. Recent research indicates that some of these peptides are classified as phytocytokines as they have regulatory functions during plant immune responses. However, the mechanistic basis for phytocytokine-mediated immune modulation remains largely elusive. Here, we identify GOLVEN2 (GLV2) peptides as novel phytocytokines in Arabidopsis thaliana. By peptide application, precursor overexpression and loss-of-function studies we show that GLV2 enhances sensitivity of plants to elicitation with the bacterial flagellin epitope flg22. GLV2 is perceived by ROOT MERISTEM GROWTH FACTOR 1 INSENSITIVE (RGI) receptors and RGI3 forms an flg22-induced complex with the flg22-receptor FLAGELLIN SENSITIVE 2, suggesting that RGIs are part of activated pattern recognition receptor signalling platforms. GLV2 perception increases posttranscriptional FLS2 abundance and RGIs promote FLS2 protein accumulation. Thus, GLV-RGI signalling controls above ground plant immunity via a novel mechanism of phytocytokine activity.

plant biology↗

bioRxiv2020

The secreted hypersensitive response inducing protein 1 from Botrytis cinerea displays non-canonical PAMP-activity

According to their lifestyle, plant pathogens are divided into biotrophic and necrotrophic organisms. While biotrophic pathogens establish a relationship with living host cells, necrotrophic pathogens rapidly kill host cells and feed on the cell debris. To this end, the necrotrophic ascomycete fungus Botrytis cinerea secretes large amounts of phytotoxic proteins and cell wall degrading enzymes. However, the precise role of these proteins during the infection process is unknown. Here we report on the identification and characterization of the previously unknown toxic protein hypersensitive response inducing protein 1 (Hip1), which induces plant cell death. We found the adoption of a folded protein structure to be a prerequisite for Hip1 to exert its necrosis-inducing activity in Nicotiana benthamiana. Localization and the induction of specific plant responses by Hip1 indicate recognition as pathogen-associated molecular pattern at the plant plasma membrane. Our results demonstrate that recognition of Hip1, even in the absence of obvious enzymatic or poreforming activity, induces strong plant defense reactions eventually leading to plant cell death.

plant biology↗

bioRxiv2017

The partial duplication of an E3-ligase gene in Triticeaespecies mediates resistance to powdery mildew fungi

In plant-pathogen interactions, components of the plant ubiquitination machinery are preferred targets of pathogen-encoded effectors suppressing defense responses or co-opting host cellular functions for accommodation. Here, we employed transient and stable gene silencing-and over-expression systems in Hordeum vulgare (barley) to study the function of HvARM1 (for H. vulgare Armadillo 1), a partial gene duplicate of the U-box/armadillo-repeat E3 ligase HvPUB15 (for H. vulgare Plant U-Box 15). The partial ARM1 gene was derived from an ancient gene-duplication event in a common ancestor of the Triticeae tribe of grasses comprising the major crop species H. vulgare, Triticum aestivum and Secale cereale. The barley gene HvARM1 contributed to quantitative host as well as nonhost resistance to the biotrophic powdery mildew fungus Blumeria graminis, and allelic variants were found to be associated with powdery mildew-disease severity. Both HvPUB15 and HvARM1 proteins interacted in yeast and plant cells with the susceptibility-related, plastid-localized barley homologs of THF1 (for Thylakoid formation 1) and of ClpS1 (for Clp-protease adaptor S1) of Arabidopsis thaliana. The results suggest a neo-functionalization HvARM1 to increase resistance against powdery mildew and provide a link to plastid function in susceptibility to biotrophic pathogen attack.

plant biology↗