Biology subjects

Sabelleck, B.

Publications and source records attributed to Sabelleck, B..

4 recordsLinked to original sources

bioRxiv2026

Powdery mildew fungi block plant vacuolar traffic to suppress immunity

Plant immunity can be activated by membrane-localized pattern recognition receptors or by cytosolic nucleotide-binding leucine-rich repeat (NLR) receptors, while it can be counteracted by pathogen secreted effectors. Manifestation of immunity often involves endomembrane traffic. However, limited evidence is available for such an involvement in NLR-mediated immunity, which typically includes a hypersensitive reaction (HR)-programmed cell death response. Here we show that the barley powdery mildew fungus uses several effectors to target and inhibit the vacuolar trafficking pathway, which causes endomembrane markers to stall in the endoplasmic reticulum (ER). We used this ER-stalling phenotype as a proxy to track fungal manipulation of the vacuolar pathway during different stages of the actual infection. Our data indicate that powdery mildew fungi interfere with the vacuolar pathway, but only temporarily, as the stalling is lifted once the fungal haustorial feeding structures are well-developed for nutrient uptake in epidermal cells. Notably, we show evidence that blocking the vacuolar pathway causes a general inhibition of NLR-mediated HR, and that this mechanism is taken advantage of by the pathogen. Finally, we provide an example that the fungus can secrete a different set of effectors at the haustorial stage that result in the re-opening of the vacuolar pathway by inhibiting the initial vacuolar traffic-suppressing effectors.

plant biology↗

bioRxiv2024

A powdery mildew core effector protein targets host endosome tethering complexes HOPS and CORVET

Powdery mildew fungi are serious pathogens of many plant species. Their genomes encode extensive repertoires of secreted effector proteins that suppress host immunity. Here, we revised and analyzed the candidate secreted effector protein (CSEP) effectome of the barley powdery mildew fungus, Blumeria hordei (Bh) and identified seven CSEPs that are broadly conserved in powdery mildew fungal species, rendering them core effectors of these phytopathogens. We show that one of these, CSEP0214, interacts with the barley VPS18 protein, a core component of the CORVET and HOPS endosomal tethering complexes, mediating fusions of early endosomes and multivesicular bodies with the central vacuole, respectively. Overexpression of CSEP0214 and knockdown of either VPS18, HOPS-specific VPS41 or CORVET-specific VPS8 caused a block of the vacuolar pathway and the accumulation of the fluorescent vacuolar marker protein (SP)-RFP-AFVY in the endoplasmic reticulum. Co-immunoprecipitation and yeast two-hybrid experiments suggest that CSEP0214 blocks the interaction of VPS18 and VPS16, which are both core components of CORVET as well as HOPS. Additionally, expression of CSEP0214 blocked the hypersensitive cell death response associated with resistance gene-mediated immunity in barley, indicating that endomembrane traffic is required for this process. It also prevented callose deposition in cell wall appositions at attack sites and encasements of fungal infection structures. Our results indicate that this powdery mildew core effector is an essential immunity suppressor. One sentence summaryThe Blumeria hordei effector protein CSEP0214 interacts with barley VPS18, a core component of the CORVET and HOPS endosomal tethering complexes, thereby interfering with host endomembrane trafficking.

plant biology↗

bioRxiv2023

Structures of Arabidopsis thaliana MDL Proteins and Synergistic Effects with the Cytokine MIF on Human Receptors

Vertebrates have developed effective immune mechanisms to fight microbial attacks, relying on a sophisticated network of innate and adaptive responses, a circulatory system, and numerous orchestrating soluble mediators such as cytokines. Mammalian macrophage migration inhibitory factor (MIF) and its paralog D-dopachrome tautomerase (D-DT/MIF-2) are multifunctional inflammatory cytokines with chemokine-like properties that modulate immunity. Plants possess orthologous MIF/D-DT-like (MDL) proteins, whose function is largely unexplored. Driven by the previous discovery of cross-kingdom mimicry of plant (Arabidopsis thaliana) MDL proteins and human MIF receptor signaling, we here characterized the structures of the three A. thaliana MDLs by X-ray crystallography and explored the mechanism underlying the interplay between plant MDLs, human MIF, and its receptors. We obtained high-resolution structures at 1.56 [A], 1.40 [A], and 2.00 [A] resolution for MDL1, MDL2, and MDL3, respectively, revealing a typical trimeric assembly and a high three-dimensional similarity to human MIF. Although residues at the catalytic site of the three MDLs show high identity to human MIF, the proteins showed low tautomerase activity for the substrate 4-hydroxyphenylpyruvate (HPP). Structural differences likely explain the enzymatic inactivity of plant MDLs for HPP. Strikingly, employing in vitro, in vivo, and in planta test systems, we found that MIF and MDL proteins interact with each other and have the capacity to form hetero-oligomeric complexes. The functional consequences of this interaction were demonstrated applying a yeast-based reporter system specific for the MIF chemokine receptors CXCR2 and CXCR4. MDLs not only triggered receptor signaling on their own, but exhibited pronounced synergism regarding the activation of the CXCR2- and CXCR4-dependent signaling pathways, when co-applied with MIF. These findings were substantiated by the co-administration of pharmacological inhibitors that either disrupt MIF receptor binding or block the catalytic cavity. Moreover, biochemical and biophysical experiments using an allosteric oligomer-specific MIF inhibitor established hexa-oligomer formation between MIF and MDLs as the putative basis for the synergistic effect. Our results are the starting point for a mechanistic understanding of the immunomodulatory activity of a family of highly conserved plant proteins. One Sentence SummaryA. thaliana MDLs and human macrophage inhibitory factor interact with each other and induce synergistic effects in activating CXCR2 and CXCR4.

immunology↗

bioRxiv2023

Comprehensive comparative assessment of the Arabidopsis thaliana MLO2-calmodulin interaction by various in vitro and in vivo protein-protein interaction assays

Mildew resistance locus o (MLO) proteins are heptahelical integral membrane proteins of which some isoforms act as susceptibility factors for the fungal powdery mildew pathogen. In many angiosperm plant species, loss-of-function mlo mutants confer durable broad-spectrum resistance against the powdery mildew disease. Barley Mlo is known to interact via a cytosolic carboxyl-terminal domain with the intracellular calcium sensor calmodulin (CAM) in a calcium-dependent manner. Site-directed mutagenesis has revealed key amino acid residues in the barley Mlo calcium-binding domain (CAMBD) that, when mutated, affect the MLO-CAM association. We here tested the respective interaction between Arabidopsis thaliana MLO2 and CAM2 using seven different types of in vitro and in vivo protein-protein interaction assays. In each assay, we deployed a wild-type version of either the MLO2 carboxyl terminus (MLO2CT), harboring the CAMBD, or the MLO2 full-length protein and corresponding mutant variants in which two key residues within the CAMBD were substituted by non-functional amino acids. We focused in particular on the substitution of two hydrophobic amino acids (LW/RR mutant) and found in most protein-protein interaction experiments reduced binding of CAM2 to the corresponding MLO2/MLO2CT LW/RR mutant variants in comparison to the respective wild-type versions. However, the Ura3-based yeast split-ubiquitin system and in planta bimolecular fluorescence complementation (BiFC) assays failed to indicate reduced CAM2 binding to the mutated CAMBD. Our data shed further light on the interaction of MLO and CAM proteins and provide a comprehensive comparative assessment of different types of protein-protein interaction assays with wild-type and mutant versions of an integral membrane protein.

plant biology↗