Biology subjects

Doehlemann, G.

Publications and source records attributed to Doehlemann, G..

At least 19 recordsLinked to original sources

bioRxiv2026

Salicylic acid-triggered apoplastic proteolysis releases cryptic phytocytokines with distinct immunogenic functions

Plants rely on an innate immune system to defend against pathogens through various molecular responses. In addition to classical damage- and pathogen-associated molecular patterns (DAMPs and PAMPs), plants produce endogenous signaling peptides termed phytocytokines that amplify and regulate immune responses following stress. Although most characterized phytocytokines originate from dedicated precursor proteins, the contribution of multifunctional proteins to phytocytokine generation remains poorly understood. Here, we show that salicylic acid (SA) rapidly remodels the maize apoplastic peptidome through an early, transient proteolytic program driven by apoplastic serine hydrolases. Time course peptidomics identified fourteen candidate phytocytokines, including two cryptic peptides, PC13 and PC14, released from the stress-associated zinc-finger protein ZmSAP7 and the migration inhibitory factor-like protein ZmMDL1, respectively. Both peptides activated immune-associated gene expression but triggered distinct transcriptional responses and exerted opposing effects on Ustilago maydis infection, with PC13 enhancing resistance and PC14 promoting susceptibility. Biochemical analysis demonstrated that PMSF-sensitive apoplastic serine proteases directly process ZmMDL1 to release PC14. Together, our findings uncover a SA-responsive proteolytic pathway that generates functionally distinct phytocytokines from multifunctional proteins, expanding the repertoire of immune signaling peptides and revealing an additional layer of regulation in plant defense.

plant biology↗

bioRxiv2026

LUstiGE, Light responsive Ustilago maydis Gene Expression: Optogenetic control of morphogenesis and pathogenesis in the corn fungal pathogen Ustilago maydis

The basidiomycete Ustilago maydis is a well-characterized model organism for studying pathogen-host interactions and of great interest for a broad spectrum of biotechnological applications. We set here to develop light inducible molecular tools to enable dynamic studies on signaling networks and fungi-host communication, and for metabolic engineering approaches. In particular, light-controlled, optogenetic switches provide quantitative, spatio-temporal control capabilities, are minimal invasive and reversible. We engineered two blue light-inducible LOV-domain-based gene expression switches, to up- (Blue-ON) and down-regulate (Blue-OFF) gene expression, and performed a functional characterization in sporidia and hyphae of U. maydis. Profiting from the dynamic control ranges and rapid kinetics, we implemented the optoswitches to control cell morphology by initiating the transition from a haploid sporidial cellular morphotype to filaments upon regulation of the levels of the polarity factor Rac1 and its constitutive active mutant Q61L. In addition to showing how expression level of effectors can be precisely regulated as an approach to understand fungi-plants interaction, we show in two proof-of-principle applications targeted control over U. maydis filamentous fungal invasion of plant tissue and the mechanisms of tumor formation. For this we placed under Blue-ON and Blue-OFF control two U. maydis effectors, See1 (Seedling efficient effector 1) and TIN2 (Tumor inducing 2), and tumor formation was assayed on maize leaves. Taken together, this study established blue-light switches as effective tools to control morphogenesis and pathogenesis in U. maydis.

synthetic biology↗

bioRxiv2026

A membrane-anchored inhibitor of papain-like cysteine proteases promotes Pseudomonas root colonization

Pseudomonas species, spanning both beneficial and pathogenic lifestyles, possess conserved mechanisms to modulate plant immunity. Nevertheless, the mechanisms by which commensal bacteria establish and maintain host colonization remain poorly understood. Here, we report the characterization of a Pseudomonas chagasin-like protease inhibitor (Cpi1), conserved across pseudomonads representing a novel class of membrane-anchored PLCP inhibitor. Unlike previously described secreted protease inhibitors, P. putida Cpi1 is a lipoprotein localized to the bacterial surface and outer membrane vesicles (OMVs), positioning it to selectively inhibit immune-related papain-like cysteine proteases (PLCPs) during host interactions. Functional assays demonstrated inhibition of maize PLCP activity in the nanomolar range, while cpi1 deletion and chagasin motif mutants exhibited significantly impaired early root colonization, particularly in the meristematic and elongation zones. Besides, lack of cpi1 resulted in an altered structure of a maize root-associated synthetic community. We hypothesize that, Cpi1 may protect critical bacterial surface proteins from cleavage by inhibiting plant proteases and thereby modulate the release of MAMPs, dampening host immune responses. Moreover, the release of Cpi1 via OMVs could further extend its function within the root periphery and the apoplast. Together, our results uncover a conserved, membrane-anchored mechanism among pseudomonads for subverting plant immunity and establishing host-microbe interactions.

microbiology↗

bioRxiv2026

Infection of maize by Ustilago maydis remodels the phyllosphere microbiome and requires the activity of antimicrobial effectors

Plant-associated microbial communities play a critical role in plant health and disease resistance, but the mechanisms which reshape these communities during pathogen infection are poorly understood. In this study, we investigated how infection of maize by the smut fungus Ustilago maydis is functionally linked with the bacterial phyllosphere microbiome and explored the role of an antimicrobial effector GH25 in fungal infection. Using a combination of culture-dependent and culture-independent approaches, we compared the leaf microbiomes of infected and uninfected plants. We observed a significant increase in microbial abundance and pronounced shifts in community composition and identified distinct health-associated (HCom) and disease-associated (DCom) bacterial communities. To assess whether U. maydis directly manipulates the microbiome, we tested the antimicrobial activity of the antimicrobial effector GH25 against isolated strains. Notably, all HCom bacteria were sensitive to GH25 and co-inoculation of HCom bacteria with a U. maydis {Delta}gh25 knockout mutant significantly reduced fungal virulence. In contrast, DCom exhibited minimal sensitivity to U. maydis and did not affect the virulence of U. maydis {Delta}gh25. Genome-scale metabolic community modelling coupled with functional profiling revealed infection-associated shifts in predicted metabolic potential, consistent with U. maydis induced leaf tumors being strong sink tissues. Together, this work shows that U. maydis infection reshapes the maize phyllosphere microbiome through a combination of effector-mediated antimicrobial activity and host metabolic reprogramming.

microbiology↗

bioRxiv2026

Cis-regulatory elements orchestrate phase-specific effector gene expression in Ustilago maydis

Plant pathogenic fungi secrete small proteins, termed effectors, to reprogram host metabolism and suppress immune responses during infection. Although transcriptional waves of effector expression have been described in several pathosystems, the cis-regulatory elements encoding infection-stage specificity remain largely unknown. Here, we investigate the temporal regulation of effector genes in the biotrophic smut fungus Ustilago maydis, a model organism for fungal plant pathogenesis. By integrating transcriptome reanalysis with comparative promoter motif enrichment across biotrophic fungi, we identify distinct promoter motifs associated with defined infection phases. In U. maydis, three candidate cis-regulatory elements correlate with early, proliferative, and late infection stages, respectively. Positional enrichment relative to transcription start sites supports their regulatory relevance. Functional promoter mutagenesis demonstrates that the early-phase motif GTGGG significantly contributes to effector gene expression in planta and is sufficient to drive stage-restricted gene expression in synthetic minimal promoters. Collectively, our findings demonstrate that temporal deployment of the effector repertoire is at least partially encoded at the promoter level. The identified cis-regulatory elements provide a framework for dissecting transcriptional control during biotrophic infection and offer tools for infection-stage-specific gene expression in synthetic biology applications.

microbiology↗

bioRxiv2026

Targeting of the barley cell-surface receptor SRF3 by the Blumeria hordei effector AVRA13 overlaps with AVRA13 recognition by MLA and the induction of NLR-mediated cell death.

Pathogens secrete effector proteins to promote virulence. Despite their recognition by barley Mla resistance genes, the structurally-related Blumeria hordei (Bh) AVRA effectors are maintained in the Bh genome, suggesting virulence functions critical for fungal pathogenicity. Using proximity-dependent protein labelling in transgenic barley, we detected distinct host protein interactomes for five AVRAs despite their structural homology and convergence on MLAs. We report the specific interaction of the highly conserved AVRA13 effector with the barley cell-surface receptor SRF3. AVRA13 disrupts HvSRF3-HvBAK1 interaction and alters HvSRF3 plasma membrane levels. AVRa13-expression desensitizes iron-induced restriction of Bh growth, suggesting that AVRA13 facilitates fungal proliferation by manipulating SRF3-mediated iron homeostasis. Our results suggest that MLAs have diversified to specifically detect the residues that underly Bh effector neo-functionalization and intrinsic AVRA virulence functions. Together, these findings identify SRF3 as molecular link between pathogen virulence, immune recognition, and iron homeostasis.

plant biology↗

bioRxiv2025

Processing and release of the maize phytocytokine Zip1

Phytocytokines are endogenous peptides that modulate plant immunity outcomes, yet how their maturation and spatial deployment are controlled remains unclear. Here we show that the maize phytocytokine precursor PROZIP1 is controlled by a spatially separated, two-stage proteolytic pathway that mechanistically uncouples signal activation from extracellular attenuation. PROZIP1 associates with the endoplasmic reticulum and undergoes intracellular, arginine-dependent processing by type II metacaspases, generating a C-terminal PROZIP1 fragment (Ct-PROZIP1). This processing licenses PROZIP1 for export to the apoplast via an ER-Golgi-independent route. Proteomic mapping and mutational analyses identify arginine residues flanking the Zip1 peptide as critical for efficient processing and secretion. The calcium-dependent metacaspase ZmMC9 specifically processes PROZIP1, thereby efficiently generating the bioactive Ct-PROZIP1 fragment. In the apoplast, Ct-PROZIP1 is further processed by papain-like cysteine proteases and additional extracellular proteases, contributing to Zip1 turnover and signal clearance. While the free Zip1 peptide is detected at later stages, Ct-PROZIP appears to be the primary signaling entity in modulating pathogen-induced immune responses. Together, these findings demonstrate a previously unknown complexity in peptide signaling, suggesting a multilayered control of phytocytokine activity that provides spatial and temporal precision to disease modulation in maize.

plant biology↗

bioRxiv2025

GH25 lysozyme mediates tripartite interkingdom interactions and microbial competition on the plant leaf surface

Microbial communities inhabiting plants have emerged as crucial factors in regulating plant health and defense against disease-causing pathogens. The basidiomycete yeast Moesziomyces bullatus ex. Albugo on Arabidopsis (MbA) releases Glycoside Hydrolase 25 (GH25) protein which regulates the leaf microbiome by antagonizing an oomycete A. laibachii biotrophic pathogen MbA. Application of both MbA and GH25 protein rescued fresh shoot weight of A. thaliana upon A. laibachii infection, showing its potential in plant protection. Tripartite interaction assays did not reveal antagonistic activity of GH25 towards other plant pathogenic oomycetes or fungi besides A. laibachii. We identified a core set of bacteria are closely associated with A. laibachii and established that GH25 inhibits members of this core group. Among A. laibachii-associated bacteria that were inhibited by GH25, Curtobacterium sp. could override the inhibition of A. laibachii by MbA. We describe a tripartite antagonistic interaction in which bacterium and oomycete protect each other from growth inhibition by MbA. Curtobacterium sp., in turn, exhibits specific inhibition of A. laibachii-associated bacteria that are not targeted by MbA but themselves antagonize A. laibachii. Our study reveals an inter-kingdom interaction network in which a GH25 lysozyme shapes the antagonistic relationship between yeast, a pathogenic oomycete and an oomycete-associated bacterium.

microbiology↗

bioRxiv2025

A recombinant hybrid provides insights into gene regulation, pathogenesis and tumorigenesis of phytopathogenic smut fungi

The maize smut fungi Ustilago maydis and Sporisorium reilianum are closely related and have similar genomes in terms of size and synteny. While U. maydis induces tumors locally at sites of infection, S. reilianum systemically colonizes the host and causes symptoms in the inflorescences. To investigate the genetic basis of these differences, an interspecific recombinant hybrid (rUSH) with the mating type system of S. reilianum was generated. rUSH exhibited extensive in-planta proliferation, showing a S. reilianum-like phenotype at all developmental stages except teliospore formation. Transcriptome profiling revealed that expression of pathogenicity-related effector gene orthologs was induced in rUSH, but not in a wild-type hybrid control. Multiple transcriptome comparisons identified 253 differentially expressed one-to-one effector orthologs with distinct regulatory patterns, including cis-, trans-, and rUSH-specific regulation. Functional analysis via CRISPR/Cas9 mutagenesis uncovered three novel virulence factors among the rUSH-specific regulated effectors. Ultimately, rUSH facilitated to identify the transcription factor UmHdp2 as key regulator of U. maydis-induced tumorigenesis. Together, these findings highlight the utility of a recombinant, interspecific hybrid in unraveling the molecular mechanisms underlying pathogenic differences in closely related fungal pathogens.

microbiology↗

bioRxiv2024

Ustilago maydis disrupts carbohydrate signaling networks to induce hypertrophy in host cells

Ustilago maydis is a biotrophic fungus infecting maize, secreting effector proteins to manipulate host cellular processes and create nutrient-rich environments for its growth. Three effectors, Hap1-3 (hypertrophy-associated proteins), were identified as virulence factors promoting hypertrophic mesophyll tumor cells (HTT). Immunoprecipitation and mass spectrometry revealed interactions among Hap effectors, suggesting potential effector complex formation. CRISPR-Cas9 triple knockout of hap1-3 demonstrated Hap1s role in HTT formation. Infection assays identified Hap1 as a key virulence factor interacting with maize Snf1-related kinase 1 (SnRK1), a central energy regulator. RNA-seq analysis showed that Hap1 promotes cell cycle and starch biosynthesis genes, while CR-hap1 induced defense-related WRKY transcription factors. Phosphoproteomics revealed increased phosphorylation of SnRK1 and metabolic enzymes during SG200 infection. Our findings support a model where U. maydis induces hypertrophy through Hap1, targeting the SnRK1 subunit to prevent SnRK1 inhibition by high trehalose-6-phosphate (T6P), disrupting the antagonistic relationship between T6P and SnRK1. This reprograms host transcription to enhance starch metabolism and induce endoreduplication, leading to hypertrophy induction, while suppressing sugar-induced immune signaling.

plant biology↗

bioRxiv2024

Quantitative detection of plant signaling peptides utilizing ELISA

Plant signaling peptides, also known as phytocytokines, are involved in a number of signaling mechanisms, including cell-to-cell communication during plant development and immunity. The detection of small peptides in plant tissues is challenging and often relies on time-consuming and cost-intensive approaches. Here, we present an ELISA-based assay as a rapid and cost-effective method for the detection of naturally released peptides in plant tissues. Our ELISA-based method was developed to detect Zip1, a 17-amino-acid phytocytokine derived from Zea mays that elicits salicylic acid signaling in maize leaves. Using a custom peptide-antibody, we designed an experimental pipeline to achieve peptide specificity, selectivity and sensitivity allowing the detection of the Zip1 peptide in complex biological samples. As a proof of concept, we transfected maize protoplasts to overexpress the precursor molecule PROZIP1 and treated maize leaves with salicylic acid to induce native PROZIP1 expression and Zip1 release. Using ELISA, we were able to quantify native Zip1 signals with a detection limit in the nanogram range, which allowed us to detect different Zip1-containing peptides in plant material. This method can be adapted for the detection and quantification of a variety of plant signaling peptides.

plant biology↗

bioRxiv2023

CRISPR/Cas9 ribonucleoprotein-mediated mutagenesis in Sporisorium reilianum

Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein 9 (CRISPR/Cas9) has become the state of art for mutagenesis in filamentous fungi. Here, we describe a RNP-mediated CRISPR/Cas9 for mutagenesis in Sporisorium reilianum. The efficiency of the method was tested in vitro with a cleavage assay as well as in vivo with a GFP-expressing S. reilianum strain. We applied this method to generate frameshift-, knock-in- and knock-out mutants in S. reilianum without a resistance marker by using an auto-replicating plasmid for selection. The RNP-mediated CRISPR/Cas9 increased the mutagenesis efficiency and firstly enables a marker-free genome editing in S. reilianum.

genetics↗

bioRxiv2023

The fungal pathogen Ustilago maydis targets the maize corepressor TPL2 to modulate host transcription for tumorigenesis

Ustilago maydis is a biotrophic fungus that causes tumor formation on all aerial parts of maize. U. maydis secretes effector proteins during penetration and colonization to successfully overcome the plant immune response and reprogram host physiology to promote infection. In this study, we functionally characterized the U. maydis effector protein Topless (TPL) interacting protein 6 (Tip6). We found that Tip6 interacts with the N-terminus of ZmTPL2 through its two EAR (Ethylene-responsive element binding factor-associated amphiphilic repression) motifs. We show that the EAR motifs are essential for the virulence function of Tip6 and critical for altering the nuclear distribution pattern of ZmTPL2. We propose that Tip6 mimics the recruitment of ZmTPL2 by plant repressor proteins, thus disrupting host transcriptional regulation. We show that a large group of AP2/ERF B1 subfamily transcription factors are misregulated in the presence of Tip6. Our study suggests a regulatory mechanism where the U. maydis effector Tip6 utilizes repressive domains to recruit the corepressor ZmTPL2 to disrupt the transcriptional networks of the host plant.

plant biology↗

bioRxiv2023

A conserved extracellular ribonuclease with broad-spectrum cytotoxic activity enables smut fungi to compete with host-associated bacteria

Ribotoxins are secreted ribonucleases that specifically target and cleave the universally conserved sarcin-ricin loop sequence of rRNA, which leads to inhibition of protein biosynthesis and subsequently to cell death. We have identified and characterized a secreted Ribo1 protein of plant pathogenic smut fungi. Heterologous expression in different model systems showed that smut Ribo1 has cytotoxic activity against bacteria, yeast, host and non-host plants. Recombinant expression of Ribo1 in Nicotiana benthamiana induced plant cell death; however, an active site mutant induced cell death only when expressed as a secreted protein. In the maize smut Ustilago maydis, transcription of Ribo1 is specifically induced in early infection stages. While a knock-out mutant revealed that Ribo1 is dispensable for U. maydis virulence, the overexpression of Ribo1 in-planta had a strong dominant negative effect on virulence and induced host defense responses including cell death. This suggests a function of Ribo1 during the epiphytic development rather than for invasive colonization of the host. Accordingly, in presence of the biocontrol bacteria Pantoea sp., which were isolated from maize leaves, the ribo1 knock-out mutant was significantly impaired in virulence. Together, we conclude that Ribo1 enables smut fungi to compete with host-associated bacteria during epiphytic development.

plant biology↗

bioRxiv2023

A transcriptional activator effector of Ustilago maydis regulates hyperplasia in maize during pathogen-induced tumor formation

Ustilago maydis causes common smut in maize, which is characterized by tumor formation in aerial parts of the host. Tumors result from the de novo cell division of highly developed bundle sheath and subsequent cell enlargement. However, the molecular mechanisms underlying tumorigenesis are still largely unknown. Here, we characterize the U. maydis effector Sts2 (Small tumor on seedlings 2), which promotes the division of hyperplasia tumor cells. Upon infection, Sts2 is translocated into the maize cell nucleus, where it acts as a transcriptional activator, and the transactivation activity is crucial for its virulence function. Sts2 interacts with ZmNECAP1, a yet undescribed plant transcriptional activator, and it activates the expression of several leaf developmental regulators to potentiate tumor formation. Contrary, fusion of a suppressive SRDX-motif to Sts2 causes dominant negative inhibition of tumor formation, underpinning the central role of Sts2 for tumorigenesis. Our results not only disclose the virulence mechanism of a tumorigenic effector, but also reveal the essential role of leaf developmental regulators in pathogen-induced tumor formation.

plant biology↗

bioRxiv2022

Combination of transcriptomic, proteomic and degradomic profiling reveals common and distinct patterns of pathogen-induced cell death in maize

Regulated cell death (RCD) is crucial for plant development, as well as in decision-making in plant-microbe interactions. Previous studies revealed components of the molecular network controlling RCD, including different proteases. However, the identity, the proteolytic network as well as molecular components involved in the initiation and execution of distinct plant RCD processes, still remain largely elusive. In this study, we analyzed the transcriptome, proteome and N-terminome of Z. mays leaves treated with the Xanthomonas effector avrRxo1, the mycotoxin Fumonisin B1 (FB1), or the phytohormone salicylic acid (SA) to dissect plant cellular processes related to cell death and plant immunity. We found highly distinct and time-dependent biological processes being activated on transcriptional and proteome levels in response to avrRxo1, FB1 and SA. A correlation analysis of the transcriptome and proteome identified general, as well as trigger-specific markers for cell death in Z. mays. We found that proteases, particularly papain-like cysteine proteases, are specifically regulated during RCD. Collectivley, this study characterizes distinct RCD responses in Z. mays and provides a framework for the mechanistic exploration of components involved in the initiation and execution of cell death.

plant biology↗

bioRxiv2022

Maize phytocytokines and microbial-patterns trigger antagonistic features in co-incidence with wounding and fungal pathogens

Phytocytokines are signalling peptides that activate immune responses and alarm bystander cells of danger. The pathways of phytocytokine perception and activation are thought to be shared between exogenous danger signals such as microbe-associated molecular patterns (MAMPs) and endogenous, passively released, damage-associated molecular patterns (DAMPs). However, downstream responses triggered by danger molecules and their effect in plant survival is still largely unknown. Here, we have identified three biologically active maize orthologues of phytocytokines previously described in other plants. The maize phytocytokines show common features with MAMPs, including the induction of immune related genes and activation of papain-like cysteine proteases. In contrast to MAMPs, the phytocytokines do not promote cell death in the presence of wounding. In infection assays with fungal pathogens of two different life styles we found that phytocytokines affect the development of disease symptoms, likely due to the activation of phytohormonal pathways. Collectively, our results show that phytocytokines and MAMPs trigger unique and antagonistic features of immunity. We propose a model in which phytocytokines activate immune responses partially similar to MAMPs but in contrast to microbial signals, they act as danger and survival molecules to the surrounding cells. Future studies will focus on the components determining the divergence of signalling outputs upon phytocytokine activation.

plant biology↗

bioRxiv2022

Exploiting effector - host interactions using TurboID-based proximity labeling

Plant pathogens secrete effectors, which target host proteins to facilitate infection. The Ustilago maydis effector UmSee1 is required for tumor formation in the leaf during infection of maize. UmSee1 interacts with maize SGT1 and blocks its phosphorylation in-vivo. In the absence of UmSee1, U. maydis cannot trigger tumor formation in the bundle sheath. However, it remains unclear which host processes are manipulated by UmSee1 and the UmSee1-SGT1 interaction to cause the observed phenotype. Proximity-dependent protein labeling involving the turbo biotin ligase tag (TurboID) for proximal labeling of proteins is a powerful tool for identifying the protein interactome. We have generated transgenic U. maydis that secretes biotin ligase-fused See1 effector (UmSee1-TurboID-3HA) directly into maize cells. This approach, in combination with conventional co-immunoprecipitation allowed to identify additional UmSee1 interactors in maize cells. Collectively, our data identified three ubiquitin-proteasome pathway-related proteins (ZmSIP1, ZmSIP2, ZmSIP3) that either interact with or are close to UmSee1 during host infection of maize with U. maydis. ZmSIP3 represents a cell cycle regulator which degradation appears to be promoted in the presence of UmSee1. Our data provide a possible explanation for the requirement of UmSee1 in tumor formation during U. maydis - Zea mays interaction.

plant biology↗