Biology subjects

Valerius, O.

Publications and source records attributed to Valerius, O..

16 recordsLinked to original sources

bioRxiv2026

Verticillium dahliae ubiquitin-specific proteases coordinate key developmental processes and pathogenicity

Ubiquitin is a posttranslational modifier that is conserved among eukaryotes. Ubiquitination alters stability and folding of cellular proteins. Deubiquitinases (DUBs) reverse ubiquitination and often function as part of protein complexes. There are 32 predicted DUB-encoding genes present in the soil-borne phytopathogenic fungus Verticillium dahliae. Nuclear ubiquitin-specific protease 3 (Usp3) is a member of the Spt-Ada-Gcn5 acetyltransferase (SAGA) complex, whereas Usp1 is predicted to associate with the COP9 signalosome (CSN), which controls specificities of cellular E3 ubiquitin ligase activities. A proteomics approach using biotin capture and identification (BioID) supports that Usp3 regulates gene expression beyond the transcription level. Western experiments showed a dysregulation in ubiquitinated cellular proteins in corresponding deletion strains. Usp3 and Usp1 are both required for fungal development. They regulate microsclerotia formation based on different environmental cues and provide redundant functions in controlling conidiation. Absence of both corresponding genes resulted in significant impairment of conidiospore formation, which is required for fungal propagation within the plant vascular system. This paralysed spreading ability reduced virulence on tomato plants (Solanum lycopersicum). In summary, V. dahliae responds to environmental cues by Usp3- and Usp1-mediated adjustment of gene expression and protein stability. This is important for key developmental processes of the V. dahliae disease cycle and its virulence towards the host plant. Author summaryUbiquitination and deubiquitination of proteins enable cells to rapidly react to environmental cues and adjust protein stabilities and subsequently transcriptomic profiles. Usp3 is a nuclear deubiquitinase subunit of the Spt-Ada-Gcn5 acetyltransferase (SAGA) transcriptional coactivator complex. Usp1 is predicted to be associated with the COP9 signalosome that regulates substrate specificities of the ubiquitin-proteasome system. BioID experiments suggest that other SAGA complex subunits, histone proteins, spliceosomal proteins, ribosomal proteins, a protein that tackles transcriptionally stalled RNAPII, and a protein that degrades mRNA with premature stop codons locate proximal to Usp3 within the cell. Deletion of USP3 led to the dysregulation of protein ubiquitination. A single deletion of USP1 did not significantly change ubiquitination profiles, however, a double deletion of USP1/3 significantly affected the ubiquitin-proteasome system. The altered ubiquitination profile correlated with a dysregulation of key developmental processes. Microsclerotia formation was decoupled from environmental cues in the {Delta}USP3 strain, whereas an additional deletion of USP1 reconnected it in a media-dependent manner. USP3 and USP1 contribute to a common governing process in conidiation, and the defect in spreading of the {Delta}USP1/3 strain is reflected by a significant reduction in plant pathogenicity.

microbiology↗

bioRxiv2026

Homologous ABA-independent kinase tracks coalesced into osmotic stress circuits during plant terrestrialization

Land plants possess a unique system for responding to environmental stressors. How this system evolved during plant terrestrialization remains one of the major questions in plant evolutionary biology. To retrace this process, it is essential to study both land plants and their closest algal relatives, the zygnematophytes. Using the single-celled zygnematophyte Mesotaenium, we integrated physiological stress experiments with phosphoproteomics, genome-wide transcription factor binding analyses, and protein-protein interaction studies to investigate the architecture of a key stress response pathway: the signaling cascade homologous to the plant abscisic acid (ABA)-mediated pathway. Our results highlight the roles of histidine kinases (HKs) and calcium-dependent protein kinases (CDPKs) in osmotic stress signaling. Focusing on SnRK2 and ABF, key components at the downstream end of the canonical ABA signaling pathway, we provide evidence that ABF plays a central role in osmotic stress responses even in the absence of ABA. Together, our data reveal the coordinated action of parallel functional modules that were likely integrated into the ABA response cascade during plant terrestrialization.

evolutionary biology↗

bioRxiv2026

A fungal effector targets the chloroplast to support biotrophy by balancing disease and plant health

Fungal pathogens are responsible for substantial crop losses worldwide. There is a pressing need to develop crops with improved disease resistance, especially given that climate change and human activities are exacerbating crop diseases. Our understanding of the molecular mechanisms by which fungi cause disease is incomplete. To address this limitation, we employed proteomics to identify candidate effector proteins from the pathogenic fungus Ustilago maydis that co-purified with the chloroplasts of maize host plants during infection. We specifically characterized the role of one putative chloroplast-associated effector, UmPce3, using heterologous expression in the non-host plant Arabidopsis thaliana. We discovered that UmPce3 interacts with the chloroplast DEAD-box RNA helicase, AtRH3. Phenotypes associated with the expression of UmPce3 in Arabidopsis mirrored those of plants with impaired AtRH3 function and included interference with chloroplast assembly, an impact on photosynthesis, and altered resistance to biotic and abiotic stresses. Support for RH3 as a bona fide effector target was obtained by identifying parallel phenotypic influences of UmPce3 in maize and by demonstrating an interaction between UmPce3 and maize ZmRH3b, an ortholog of AtRh3. Notably, UmPce3 contributes to biotrophy by promoting the virulence of U. maydis on maize seedlings and dampening virulence in plants challenged with salinity as an abiotic stress. Overall, this work highlights the chloroplast as a target of fungal pathogenesis and identifies RH3 as a potential hub for pathogen manipulation of organelle function to balance fungal proliferation and host health in support of biotrophy. Short summaryThe chloroplast plays a key role in plant immunity, in addition to its central contributions to photosynthesis, metabolism, and tolerance of abiotic stresses. The effector UmPce3 of the maize pathogen Ustilago maydis targets the DEAD-box RNA helicase RH3 in host plants to manipulate chloroplast function and enhance fungal pathogenesis. Unexpectedly, UmPce3 also influences host tolerance to salt stress thereby balancing the plant response to biotic and abiotic stressors in support of biotrophic development.

plant biology↗

bioRxiv2025

Capture of the SmSTRIPAK proxiome identifies the greenbeard proteins SmDOC1/2 as regulators of the MAK2 pathway to control sexual development in Sordaria macrospora

Hyphal fusion and sexual development in filamentous fungi rely on coordinated signaling of numerous conserved nodes such as the striatin interacting phosphatase and kinase (STRIPAK) complex or the pheromone response (PR) MAP kinase cascade (MIK2, MEK2, MAK2, HAM5). Here we used the homothallic ascomycete Sordaria macrospora (Sm) to screen for putative protein interactors of the SmSTRIPAK complex. Using the STRIPAK complex interactor 1 (SCI1) subunit of the complex as bait, we enriched and identified canonical SmSTRIPAK components and a determinant of communication (DOC) protein. The DOC proteins were previously described in the closely related and heterothallic species Neurospora crassa, functioning in allorecognition of germlings and hyphal fusions. We generated {Delta}Smdoc1, {Delta}Smdoc2 single deletion strains and the double deletion mutant {Delta}Smdoc1{Delta}Smdoc2 in S. macrospora. Deletion phenotypes were paradoxical: single knockouts ({Delta}Smdoc1 or {Delta}Smdoc2) were nearly sterile and sexual development was impaired, yet the double mutant ({Delta}Smdoc1{Delta}Smdoc2) exhibited wild-type fertility and development, demonstrating non-redundant and mutually antagonistic roles. Similarly, we demonstrated an impairment of the N. crassa {Delta}doc-2 mutant in sexual development. Using gene tagging at the native locus, we performed TurboID-based proximity mapping with SmDOC1 and SmDOC2 as bait proteins. This proximity mapping demonstrated close ties of SmDOC1/2 to components of the PR MAP kinase pathway and revealed mutual SmDOC1 - SmDOC2 proximity. Yeast Two-Hybrid experiments with SmDOC1 confirmed the direct interaction with the MAP kinases MEK2 and MAK2. Fluorescence microscopy revealed that SmDOC1-TagRFP-T localized to ring-like structures around septal pores. Our results demonstrate that the DOC system is not restricted to heterothallic N. crassa, but also plays an essential role in the development of fruiting bodies in the homothallic fungus S. macrospora. These findings suggest the DOC1/2 proteins as a novel system that integrates STRIPAK and PR pathways, providing a possible mechanistic explanation for their non-additive deletion strain phenotypes.

cell biology↗

bioRxiv2025

Myeloid cell-mediated killing of B-ALL by CD38 and CD20IgA antibody variants is enhanced by CD47/SIRPα interference

Enhancing myeloid effector cell recruitment may improve immunotherapy by monoclonal antibodies - including that against acute lymphoblastic leukemia (ALL). To assess expression of target antigens in B-ALL, we compared mRNA profiling of 559 patient leukemia samples across 18 molecular subtypes with that of representative cell lines. The latter served as target cells to compare human IgG1 or IgA2 variants against CD19, CD20 or CD38 in antibody-dependent cellular phagocytosis (ADCP) by macrophages and antibody-dependent cell-mediated cytotoxicity (ADCC) by polymorphonuclear leukocytes (PMN). Interestingly, antibodies against broadly expressed CD19 were negligibly effective in mediating ADCP or ADCC. Antibodies against CD20 or CD38, the former variably expressed across subtypes, triggered ADCP by macrophages both as IgG1 and IgA2. However, PMN mediated ADCC against CD20 or CD38 was only observed with IgA2 variants, but not with respective IgG1 antibodies. Blocking the myeloid checkpoint molecule CD47 with a CD47 antibody or a soluble SIRP-Fc fusion protein enhanced ADCP and ADCC by IgA2 antibodies. The binding site for SIRP on CD47 contains an N-terminal pyroglutamate (pGlu), whose formation is catalyzed by glutaminyl-peptide cyclotransferase like (QPCTL). The direct involvement of pGlu in CD47/SIRP interactions was shown by using engineered CD47 variants. Both CD47 and QPCTL were broadly expressed across BCP-ALL subtypes, indicating QPCTL inhibitors as additional therapeutic option. Importantly, the combination of anti-CD38 IgA2 and CD47 blockade was effective against xenografted B-ALL cells in human FcRI (CD89) transgenic (tg) NXG mice. Together, these studies support the combination of anti-CD38 IgA2 with CD47 interference to improve myeloid effector cell recruitment for immunotherapy of B-ALL. Data sharing statementRNA-Seq data from BCP-ALL patients and B cells from healthy donors are available in the European Genome-Phenome Archive (EGA) accession numbers EGAS00001006107 and EGAS00001007305, respectively. Mass spectrometry data of analyzed proteins will be made available after manuscript acceptance on PRIDE - PRoteomics IDEntifications Database. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=174 HEIGHT=200 SRC="FIGDIR/small/669665v1_ufig1.gif" ALT="Figure 1"> View larger version (36K): org.highwire.dtl.DTLVardef@15220daorg.highwire.dtl.DTLVardef@eace04org.highwire.dtl.DTLVardef@17b87daorg.highwire.dtl.DTLVardef@1142822_HPS_FORMAT_FIGEXP M_FIG C_FIG Key pointsO_LIIgG1 and IgA2 antibodies against CD38 or CD20 were effective in recruiting macrophages for ADCP, but only IgA2 triggered ADCC by PMN C_LIO_LIMyeloid effector cell activation was enhanced by interfering with the CD47/SIRP axis, especially when IgA2 antibodies were applied C_LI

cancer biology↗

bioRxiv2025

The VelB intrinsically disordered domain promotes selective heterodimer formation of velvet domain regulatory proteins for fungal development

Fungi possess several transcription factors with a characteristic velvet domain for DNA-binding and homo- or heterodimerization, which is structurally similar to the mammalian NF-B Rel homology domain. Velvet dimers control fungal development, virulence and mycotoxin formation. VelB is the only regulator, which carries an intrinsically disordered domain (IDD) within the velvet domain. The IDD as well as the positioning within VelB is conserved in the fungal kingdom. Intrinsically disordered regions contribute to transcription activation and DNA binding and frequently appear in eukaryotic transcription factors. The VelB IDD provides selective heterodimerization as well as protein stability control. The IDD is not required for the formation of the VelB-VeA heterodimer of Aspergillus nidulans or Verticillium dahliae, but promotes the formation of the VelB-VosA heterodimer. The IDD destabilizes VelB single molecules and also balances its distribution and ratio between both velvet heterodimers. These balances contribute to control appropriate mycotoxin production and sexual development. Herewith, the VelB IDD represents a novel control mechanism of velvet protein stability and heterodimer formation for precise priming of fungal development.

molecular biology↗

bioRxiv2025

Systems acclimation to osmotic stress in zygnematophyte cells

Zygnematophytes are the closest algal relatives of land plants. They hold key information to infer how the earliest land plants overcame the barrage of terrestrial stressors, prime of which is osmotic stress. Here, we applied two osmotic stressors on a unicellular and a multicellular representative of zygnematophytes and studied their response over a 25-hour time course generating 130, 60, and 30 of transcriptomic, proteomic, and metabolomic samples combined with photophysiology, sugar analysis, immunocytochemical glycoprotein analysis, and microscopy. Our data highlight a shared protein chassis that shows divergent responses with the same outcome: successful acclimation to osmotic challenges. We establish a model of how the algal sisters of land plants can overcome a prime stressor in the terrestrial habitat and highlight components of the plant terrestrialization toolkit.

evolutionary biology↗

bioRxiv2025

A single pathogen-secreted protein reprograms plants for drought resilience

Climate change-enforced drought stress conditions and diseases caused by pathogens often co-occur and represent one of the greatest challenges in plant science1-3. Wilt pathogens that colonize water-conducting plant tissues can aggravate the problem and affect a wide range of agricultural crops4,5. However, whilst fungal infections with the vascular pathogen Verticillium dahliae are typically associated with wilt symptoms due to occlusion of xylem tissues, the related V. longisporum induces de novo formation of tracheary elements6,7. This promotes not only its virulence but also enables elevated water storage capacity of the infected host plant and resilience against drought stress conditions6,7. Here, we identified a secreted Verticillium protein, TRANSDIFFERENTIATION EFFECTOR (TRADE), which triggers cell identity switches of bundle sheath cells into tracheary elements. We show that TRADE interacts with the intracellular plant protein VARICOSE (VCS), a conserved component of the mRNA turnover machinery and ortholog of the metazoan protein ENHANCER OF DECAPPING 4 (EDC4/HEDLS/Ge-1)8. The TRADE-VCS interaction induces SUCROSE NON-FERMENTING 1 (SNF1)-related protein kinase (SRK)-dependent phosphorylation and thus dysfunction of VCS. This affects the abundance of mRNAs encoding master regulators of xylem differentiation and demonstrates how a single pathogen effector protein triggers complex tissue-specific developmental reprogramming and thus promotes abiotic stress resilience.

plant biology↗

bioRxiv2024

Plasticity of the Arabidopsis leaf lipidome and proteome in response to pathogen infection and heat stress

Plants must cope with a variety of stressors during their life cycle, and the adaptive responses to these environmental cues involve all cellular organelles. Among them, comparatively little is known about the contribution of cytosolic lipid droplets (LDs) and their core set of neutral lipids and associated surface proteins to the rewiring of cellular processes in response to stress. Here, we analyzed the changes that occur in the lipidome and proteome of Arabidopsis leaves after pathogen infection with Botrytis cinerea or Pseudomonas syringae, or after heat stress. Analyses were carried out in wild-type plants and the oil-rich double mutant tgd1-1 sdp1-4 that allowed for an allied study of the LD proteome in stressed leaves. Using liquid chromatography-tandem mass spectrometry-based methods, we show that a hyperaccumulation of the primary LD core lipid triacylglycerol is a general response to stress and that acyl chain and sterol composition are remodeled during cellular adaptation. Likewise, comparative analysis of the LD protein composition in stress-treated leaves highlighted the plasticity of the LD proteome as part of the general stress response. We further identified at least two additional LD-associated proteins, whose localization to LDs in leaves was confirmed by confocal microscopy of fluorescent protein fusions. Taken together, these results highlight LDs as dynamic contributors to the cellular adaptation processes that underlie how plants respond to environmental stress. One sentence summaryBiotic and heat stress strongly alters the lipidome and proteome of Arabidopsis leaves including the proteome of lipid droplets.

plant biology↗

bioRxiv2022

Environmental gradients reveal stress hubs predating plant terrestrialization

Plant terrestrialization brought forth the land plants (embryophytes). Embryophytes account for most of the biomass on land and evolved from streptophyte algae in a singular event. Recent advances have unraveled the first full genomes of the closest algal relatives of land plants; among the first such species was Mesotaenium endlicherianum. Here, we used fine-combed RNAseq in tandem with photophysiological assessment on Mesotaenium exposed to a continuous range of temperature and light cues. Our data establish a grid of 42 different conditions, resulting in 128 transcriptomes and ~1.5 Tbp (~9.9 billion reads) of data to study combinatory effects of stress response using clustering along gradients. We describe major hubs in genetic networks underpinning stress response and acclimation in the molecular physiology of Mesotaenium. Our data suggest that lipid droplet formation, plastid and cell wall-derived signals denominate molecular programs since more than 600 million years of streptophyte evolution--before plants made their first steps on land.

evolutionary biology↗

bioRxiv2022

SLDP and LIPA mediate lipid droplet-plasma membrane tethering in Arabidopsis thaliana

Membrane contact sites (MCS) are inter-organellar connections that allow for the direct exchange of molecules, such as lipids or Ca2+ between organelles, but can also serve to tether organelles at specific locations within cells. Here we identified and characterised three proteins that form a lipid droplet (LD)-plasma membrane (PM) tethering complex in plant cells, namely LD-localised SEED LD PROTEIN (SLDP) 1 and 2 and PM-localised LD-PLASMA MEMBRANE ADAPTOR (LIPA). Using proteomics and different protein-protein interaction assays, we show that both SLDPs associate with LIPA. Disruption of either SLDP1 and 2 expression, or that of LIPA, leads to an aberrant clustering of LDs in Arabidopsis seedlings. Ectopic co-expression of one of the SLDPs with LIPA on the other hand is sufficient to reconstitute LD-PM tethering in Nicotiana tabacum pollen tubes, a cell type characterised by dynamically moving LDs in the cytosolic streaming. Further, confocal laser scanning microscopy revealed both SLDP2.1 and LIPA to be enriched at LD-PM contact sites in seedlings. These and other results suggest that SLDP and LIPA interact to form a tethering complex that anchors a subset of LDs to the PM during post-germinative seedling growth in Arabidopsis thaliana. One-sentence summarySEED LIPID DROPLET PROTEIN1 and 2 and LIPID DROPLET PLASMA MEMBRANE ADAPTOR tether lipid droplets to the plasma membrane in seedlings of Arabidopsis thaliana.

plant biology↗

bioRxiv2021

ultraID: a compact and efficient enzyme for proximity-dependent biotinylation in living cells

Proximity-dependent biotinylation (PDB) combined with mass spectrometry analysis has established itself as a key technology to study protein-protein interactions in living cells. A widespread approach, BioID, uses an abortive variant of the E. coli BirA biotin protein ligase, a quite bulky enzyme with slow labeling kinetics. To improve PDB versatility and speed, various enzymes have been developed by different approaches. Here we present a novel small-size engineered enzyme: ultraID. We show its practical use to probe the interactome of Argonaute-2 after a 10 min labeling pulse and expression at physiological levels. Moreover, using ultraID, we provide a membrane-associated interactome of coatomer, the coat protein complex of COPI vesicles. To date, ultraID is the smallest and most efficient biotin ligase available for PDB and offers the possibility of investigating interactomes at a high temporal resolution.

biochemistry↗

bioRxiv2021

Multi-omics analysis of xylem sap uncovers dynamic modulation of poplar defenses by ammonium and nitrate

Xylem sap is the major transport route for nutrients from roots to shoots. Here, we investigated how variations in nitrogen (N) nutrition affected the metabolome and proteome of xylem sap, growth of the xylem endophyte Brennaria salicis and report transcriptional re-wiring of leaf defenses in poplar (Populus x canescens). We supplied poplars with high, intermediate or low concentrations of ammonium or nitrate. We identified 288 unique proteins in xylem sap. About 85% of the xylem sap proteins were shared among ammonium- and nitrate-supplied plants. The number of proteins increased with increasing N supply but the major functional categories (catabolic processes, cell wall-related enzymes, defense) were unaffected. Ammonium nutrition caused higher abundances of amino acids and carbohydrates, while nitrate caused higher malate levels in xylem sap. Pipecolic acid and N-hydroxy-pipecolic acid increased whereas salicylic acid and jasmonoyl-isoleucine decreased with increasing N nutrition. Untargeted metabolome analyses revealed 2179 features in xylem sap, of which 863 were differentially affected by N treatments. We identified 122 metabolites, mainly from specialized metabolism of the groups of salicinoids, phenylpropanoids, phenolics, flavonoids, and benzoates. Their abundances increased with decreasing N. Endophyte growth was stimulated in xylem sap of high N- and suppressed in that of low N-fed plants. The drastic changes in xylem sap composition caused massive changes in the transcriptional landscape of leaves and recruited defense pathways against leaf feeding insects and biotrophic fungi, mainly under low nitrate. Our study uncovers unexpected complexity and variability of xylem composition with consequences for plant defenses. Significance statementThis study reports the largest, currently available plant xylem sap proteome and metabolome databases and highlights novel discoveries of specialized metabolites and phytohormones in the xylem sap. This is the first multi-omics study linking differences in nitrogen supply with changes xylem sap composition, endophyte growth and transcriptional defenses in leaves.

plant biology↗

bioRxiv2021

Hülle-cell-mediated protection of fungal reproductive and overwintering structures against fungivorous animals

Fungal Hulle cells with nuclear storage and developmental backup functions are reminiscent of multipotent stem cells. In the soil, Hulle cells nurse the overwintering fruiting bodies of Aspergillus nidulans. The genome of A. nidulans harbors genes for the biosynthesis of xanthones. We show that enzymes and metabolites of this biosynthetic pathway accumulate in Hulle cells under the control of the regulatory velvet complex, which coordinates development and secondary metabolism. Deletion strains blocked in the conversion of anthraquinones to xanthones are delayed in maturation and growth of fruiting bodies. Xanthones are not required for sexual development but exert antifeedant effects on fungivorous animals such as springtails and woodlice. These findings reveal a novel role of Hulle cells in establishing secure niches for A. nidulans by accumulating metabolites with antifeedant activity that protect reproductive structures from animal predators.

biochemistry↗

bioRxiv2020

Co-option of a seed-like proteome by oil-rich tubers

Co-option is an important aspect of evolution that can occur on several levels. Genes, whose function was molded by selection in the evolutionary past, are readily observed to serve a new function when acting in a different context in an extant system. Whole organs can be co-opted for new roles as well. For example, roots that evolved from shoot-like axes. Finally a framework of genes and its coded proteins can be co-opted to serve a similar molecular function but in a completely different organ, drastically changing its properties. Here, we describe such an example, where a set of proteins important for desiccation tolerance and oil accumulation in seeds of most angiosperms was co-opted in the tubers of yellow nutsedge (Cyperus esculentus). These tubers are not only desiccation tolerant but also store a large amount of lipids--especially TAG, similar to seeds. We generated nanoLC-MS/MS-based proteomes in five replicates of four stages of tuber development and compared them to the proteomes of roots and leaves, yielding 2257 distinct protein groups. Our data reveal a striking upregulation of hallmark proteins of seeds in the tubers. A deeper comparison to a previously published proteome of Arabidopsis seeds and seedlings indicate that indeed a seed-like proteome was co-opted. This was further supported by an analysis of the proteome of a lipid-droplet enriched fraction of yellow nutsedge, which also displayed seed-like characteristics.

evolutionary biology↗

bioRxiv2020

V. longisporum elicits media-dependent secretome responses with a further capacity to distinguish between plant-related environments

Verticillia cause a vascular wilt disease affecting a broad range of economically valuable crops. The fungus enters its host plants through the roots and colonizes the vascular system. It requires extracellular proteins for a successful plant colonization. The exoproteome of the allodiploid Verticillium longisporum was analyzed upon cultivation in different media. Secreted fungal proteins were identified by label free LC-MS/MS screening. V. longisporum induced two main secretion patterns. One response pattern was elicited in various non-plant related environments. The second pattern includes the exoprotein responses to the plant-related media, pectin-rich simulated xylem medium and pure xylem sap, which exhibited similar but additional distinct features. These exoproteomes include a shared core set of 223 secreted and similarly enriched fungal proteins. The pectin-rich medium significantly induced the secretion of 144 proteins including a number of pectin degrading enzymes, whereas xylem sap triggered a smaller but unique fungal exoproteome pattern with 32 enriched proteins. The latter pattern included proteins with domains of known effectors, metallopeptidases and carbohydrate-active enzymes. The most abundant and uniquely enriched proteins of these different groups are the necrosis and ethylene inducing-like proteins Nlp2 and Nlp3, the cerato-platanin proteins Cp1 and Cp2, the metallopeptidases Mep1 and Mep2 and the CAZys Gla1, Amy1 and Cbd1. Deletion of the majority of the corresponding genes caused no phenotypic changes during ex planta growth or invasion and colonization of tomato plants. However, we discovered that the NLP2 and NLP3 deletion strains were compromised in plant infections. Overall, our exoproteome approach revealed that the fungus induces specific secretion responses in different environments. The fungus has a general response to non-plant related media whereas it is able to fine-tune its exoproteome in the presence of plant material. Importantly, the xylem sap-specific exoproteome pinpointed Nlp2 and Nlp3 as single effectors required for successful V. dahliae colonization. Author SummaryVerticillium spp. infect hundreds of different plants world-wide leading to enormous economic losses. Verticillium wilt is a disease of the vasculature. The fungus colonizes the xylem of its host plant where it exploits the vascular system to colonize the whole plant. Therefore, the fungus spends part of its lifetime in this nutrient-low and imbalanced environment where it is inaccessible for disease control treatments. This lifestyle as well requires the fungus to react to plant defense responses by secreting specific effector molecules to establish a successful infection. We addressed the differences in media-dependent secretion responses of Verticillium longisporum. We identified a broad response pattern induced by several media, and a similar response (but with some distinct differences) for the plant-related environments: the pectin-rich medium SXM and xylem sap from the host rapeseed. Importantly, we show that the necrosis and ethylene inducing-like proteins Nlp2 and Nlp3 are xylem sap-specific proteins that are required for full V. dahliae pathogenicity on tomato. These factors play a role during the colonization phase and represent potential targets for new control strategies for Verticillium wilt.

genetics↗