Biology subjects

Menke, F. L. H.

Publications and source records attributed to Menke, F. L. H..

13 recordsLinked to original sources

bioRxiv2026

A stage-resolved map of dynamic septin interactions required for infection by the rice blast fungus

Septin GTPases are essential cytoskeletal regulators that organize membranes and scaffold protein complexes to control cytokinesis, polarity, and morphogenesis. How septins execute these functions remains poorly understood, and comprehensive, stage-resolved interaction maps are lacking. Here, we define a quantitative, time-resolved septin interactome in the rice blast fungus Magnaporthe oryzae using immunoprecipitation coupled to mass spectrometry. We map more than 350 interactors of septins Sep3, Sep4, Sep5 and Sep6, revealing a dynamic network required for appressorium-mediated plant infection. Beyond canonical roles in cytoskeletal organisation and polarity, septins associate with proteins linked to membrane remodelling, metabolism, and virulence, deployed during host invasion. Integration with ultra-high-throughput yeast two-hybrid analysis defines a high-confidence septin interactome and identifies previously uncharacterised factors, including Msi1, a BAR domain protein required for invasive growth. Together, these findings establish septins as dynamic organisers of infection-related processes and provide a framework for understanding how cytoskeletal scaffolds coordinate fungal pathogenesis.

cell biology↗

bioRxiv2026

Structural basis for the broad recognition specificity of an Arabidopsis immune receptor

Plant nucleotide-binding leucine-rich repeat (NLR) immune receptors typically confer resistance through recognition of specific pathogen effectors. The Arabidopsis NLR WRR4A defies this paradigm by recognizing multiple sequence-divergent effectors from Albugo candida, conferring resistance to multiple pathogen races. Despite minimal sequence similarity, these effectors share a conserved N-terminal ferredoxin-like fold. Through cryo-EM structure determination of two WRR4A resistosomes bound to sequence-distinct effectors, combined with AlphaFold modelling, we reveal a shape-based recognition mechanism: WRR4A engages structurally conserved backbone features of the effectors in a mostly side chain-independent manner, enabling recognition of diverse effectors with similar three-dimensional architectures. These insights guided successful engineering of WRR4A to acquire novel recognition specificity. In addition, analysis of the monomeric WRR4A resting state reveals a distinct domain architecture characteristic of C-JID-containing TIR-NLRs and informs their activation mechanism. This work provides insights into NLR-mediated broad-spectrum recognition and the potential for structure-informed engineering of improved crop resistance.

plant biology↗

bioRxiv2024

Putative neofunctionalization of a Poales-specific EXO70 clade

EXO70s are uniquely expanded in land plants compared to all other eukaryotic lineages. The functional implications of this expansion and diversification on the conserved role of EXO70 as a subunit of the octameric exocyst complex have remained unresolved. We previously demonstrated barley (Hordeum vulgare) EXO70FX12, a member of the monocot-specific EXO70FX clade, is required for resistance to wheat stripe rust in conjunction with the leucine-rich repeat receptor kinase (LRR-RK) HvPUR1. Through phylogenetic analysis, we identified unique features of the EXO70FX clade, leading us to hypothesize that this clade experienced neofunctionalization. Using structural predictions and protein-protein interaction assays, we demonstrate that HvEXO70FX12 lost the ability to serve as a subunit within the exocyst complex. We predict that the EXO70FX clade has largely lost exocyst association and represents a novel acquisition that emerged during Poales diversification for immunity.

plant biology↗

bioRxiv2024

The blast effector Pwl2 is a virulence factor that modifies the cellular localisation of host protein HIPP43 to suppress immunity.

The rice blast fungus Magnaporthe oryzae secretes a battery of effector proteins to facilitate host infection. Among these effectors, Pwl2 was first identified as a host specificity determinant for infection of weeping lovegrass (Eragrostis curvula) and is also recognised by the barley Mla3 resistance gene. However, its biological activity is not known. Here we show that PWL2 expression is regulated by the Pmk1 MAP kinase during cell-to-cell movement by M. oryzae at plasmodesmata (PD)-containing pit field sites. Consistent with its regulation, we provide evidence that Pwl2 binds to a barley heavy metal-binding isoprenylated protein HIPP43, which results in its displacement from plasmodesmata. Transgenic barley lines overexpressing either PWL2 or HIPP43 exhibit attenuated immune responses and increased disease susceptibility. By contrast, a Pwl2SNDEYWY mutant that does not interact with HIPP43, fails to alter the PD localisation of HIPP43. Targeted deletion of three copies of PWL2 in M. oryzae results in a{Delta} pwl2 mutant showing gain-of-virulence to weeping lovegrass and barley Mla3 lines, but also a reduction in severity of blast disease on susceptible host plants. Taken together, our results provide evidence that Pwl2 is a virulence factor that acts by suppressing host immunity through perturbing the plasmodesmatal deployment of HIPP43.

plant biology↗

bioRxiv2023

The leucine-rich repeat receptor kinase QSK1 is a novel regulator of PRR-RBOHD complex and is employed by the bacterial effector HopF2Pto to modulate plant immunity

Plants detect pathogens using cell-surface pattern recognition receptors (PRRs) like EFR and FLS2, which recognize bacterial EF-Tu and flagellin, respectively. These PRRs, belonging to the leucine-rich repeat receptor kinase (LRR-RK) family, activate the production of reactive oxygen species via the NADPH oxidase RBOHD. The PRR-RBOHD complex is tightly regulated to prevent unwarranted or exaggerated immune responses. However, certain pathogenic effectors can subvert these regulatory mechanisms, thereby suppressing plant immunity. To elucidate the intricate dynamics of the PRR-RBOHD complex, we conducted a comparative co-immunoprecipitation analysis using EFR, FLS2, and RBOHD. We identified QSK1, an LRR-RK, as a novel component of the PRR-RBOHD complex. QSK1 functions as a negative regulator of PRR-triggered immunity (PTI) by downregulating the abundance of FLS2 and EFR. QSK1 is targeted by the bacterial effector HopF2Pto, a mono-ADP ribosyltransferase, resulting in the reduction of FLS2 and EFR levels through both transcriptional and transcription-independent pathways, thereby inhibiting PTI. Furthermore, HopF2Pto reduces transcript levels of PROSCOOP genes encoding important stress-regulated phytocytokines and their receptor MIK2. Importantly, HopF2Pto requires QSK1 for its accumulation and virulence functions within plants. In summary, our results provide novel insights into the mechanism by which HopF2Pto employs QSK1 to desensitize plants to pathogen attack. One Sentence SummaryQSK1, a novel component in the plant immune receptor complex, downregulates these receptors and phytocytokines, and is exploited by bacterial effector HopF2Pto to desensitize plants to pathogen attack.

plant biology↗

bioRxiv2023

The phosphorylation landscape of infection-related development by the rice blast fungus

Many of the worlds most devastating crop diseases are caused by fungal pathogens which elaborate specialized infection structures to invade plant tissue. Here we present a quantitative mass spectrometry-based phosphoproteomic analysis of infection-related development by the rice blast fungus Magnaporthe oryzae, which threatens global food security. We mapped 8,005 phosphosites on 2,062 fungal proteins, revealing major re-wiring of phosphorylation-based signaling cascades during fungal infection. Comparing phosphosite conservation across 41 fungal species reveals phosphorylation signatures specifically associated with biotrophic and hemibiotrophic fungal infection. We then used parallel reaction monitoring to identify phosphoproteins directly regulated by the Pmk1 MAP kinase that controls plant infection by M. oryzae. We define 33 substrates of Pmk1 and show that Pmk1-dependent phosphorylation of a newly identified regulator, Vts1, is required for rice blast disease. Defining the phosphorylation landscape of infection therefore identifies potential therapeutic interventions for control of plant diseases.

plant biology↗

bioRxiv2023

A GH81-type β-glucan-binding protein facilitates colonization by mutualistic fungi in barley

Cell walls are important interfaces of plant-fungal interactions. Host cell walls act as robust physical and chemical barriers against fungal invaders, making them an essential line of defense. Upon fungal colonization, plants deposit phenolics and callose at the sites of fungal penetration to reinforce their walls and prevent further fungal progression. Alterations in the composition of plant cell walls significantly impact host susceptibility. Furthermore, plants and fungi secrete glycan hydrolases acting on each others cell walls. These enzymes release a wide range of sugar oligomers into the apoplast, some of which trigger the activation of host immunity via host surface receptors. Recent characterization of cell walls from plant-colonizing fungi have emphasized the abundance of {beta}-glucans in different cell wall layers, which makes them suitable targets for recognition. To characterize host components involved in immunity against fungi, we performed a protein pull-down with the biotinylated {beta}-glucan laminarin. Thereby, we identified a glycoside hydrolase family 81-type glucan-binding protein (GBP) as the major {beta}-glucan interactor. Mutation of GBP1 and its only paralogue GBP2 in barley led to decreased colonization by the beneficial root endophytes Serendipita indica and S. vermifera, as well as the arbuscular mycorrhizal fungus Rhizophagus irregularis. The reduction of symbiotic colonization was accompanied by enhanced responses at the host cell wall. Moreover, GBP mutation in barley also increased resistance to fungal infections in roots and leaves by the hemibiotrophic pathogen Bipolaris sorokiniana and the obligate biotrophic pathogen Blumeria graminis f. sp. hordei, respectively. These results indicate that GBP1 is involved in the establishment of symbiotic associations with beneficial fungi, a role that has potentially been appropriated by barley-adapted pathogens. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=134 SRC="FIGDIR/small/536646v1_figu1.gif" ALT="Figure 1"> View larger version (39K): org.highwire.dtl.DTLVardef@c47957org.highwire.dtl.DTLVardef@fa6727org.highwire.dtl.DTLVardef@18a54d2org.highwire.dtl.DTLVardef@c6b103_HPS_FORMAT_FIGEXP M_FIG C_FIG In BriefGBP1, a family 81 glycoside hydrolase, is an important {beta}-glucan interactor in barley. Mutation of GBP1 and its sole paralogue GBP2 leads to reduced colonization by beneficial root endophytes, AM fungi and pathogens, accompanied by enhanced responses at the plant cell wall. This indicates that GBP1 and {beta}-glucans are compatibility factors involved in the establishment of symbiotic associations with beneficial fungi, a role possibly hijacked by pathogens.

plant biology↗

bioRxiv2021

Biophysical and proteomic analyses suggest functions of Pseudomonas syringae pv tomato DC3000 extracellular vesicles in bacterial growth during plant infection

Vesiculation is a process employed by Gram-negative bacteria to release extracellular vesicles (EVs) into the environment. Bacterial EVs contain molecular cargo from the donor bacterium and play important roles in bacterial survival and growth. Here, we describe EV production in plant-pathogenic Pseudomonas syringae pv. tomato DC3000 (Pto DC3000), the causal agent of bacterial speck disease. Cultured Pto DC3000 exhibited EV structures both on the cell surface and in the vicinity of bacterial cells, observed as outer membrane vesicle (OMV) release. We used in-solution trypsin digestion coupled to mass spectrometry to identify 369 proteins enriched in EVs recovered from cultured Pto DC3000. The predicted localization profile of EV proteins supports the production of EVs also in the form of outer-inner-membrane vesicles (OIMVs). EV production varied slightly between bacterial lifestyles and also occurred in planta. The potential contribution of EVs to Pto DC3000 plant infection was assessed using plant treatments and bioinformatic analysis of the EV-enriched proteins. While these results identify immunogenic activities of the EVs, they also point at roles for EVs in bacterial defences and nutrient acquisition by Pto DC3000.

plant biology↗

bioRxiv2021

A hetero-oligomeric remorin-receptor complex regulates plant development

Plant growth and development are modulated by both biotic and abiotic stress. Increasing evidence suggests that cellular integration of the corresponding signals occurs within preformed hubs at the plasma membrane called nanodomains. These membrane sub-compartments are organized by multivalent molecular scaffold proteins, such as remorins. Here, we demonstrate that Group 1 remorins form a hetero-oligomeric complex at the plasma membrane. While these remorins are functionally redundant for some pathways their multivalency also allows the recruitment of specific interaction partners. One of them, the receptor-like kinase REMORIN-INTERACTING RECEPTOR 1 (RIR1), that acts redundantly with the closely related receptor NILR2, is specifically recruited by REM1.2 in a phosphorylation-dependent manner. Overlapping developmental phenotypes suggest that the REM/RIR complex regulates key developmental pathways.

plant biology↗

bioRxiv2020

The phagocytosis oxidase/Bem1p (PB1) domain-containing protein PB1CP negatively regulates the NADPH oxidase RBOHD in plant immunity

O_LIPerception of pathogen-associated molecular patterns (PAMPs) by surface-localized pattern-recognition receptors activates RESPIRATORY BURST OXIDASE HOMOLOG D (RBOHD) through direct phosphorylation by BOTRYTIS-INDUCED KINASE 1 (BIK1) and induces the production of reactive oxygen species (ROS). ROS have direct antimicrobial properties but also serve as signaling molecules to activate additional defense responses such as stomatal closure. RBOHD activity must be tightly controlled to avoid the detrimental effects of ROS, but little is known about RBOHD downregulation. C_LIO_LITo better understand the regulation of RBOHD, we used co-immunoprecipitation of RBOHD coupled with mass spectrometry analysis to identify RBOHD-associated proteins. C_LIO_LIAmong RBOHD-associated proteins, we identified PHAGOCYTOSIS OXIDASE/ BEM1P (PB1) DOMAIN-CONTAINING PROTEIN (PB1CP). We found that PB1CP negatively regulates RBOHD and the resistance against the fungal pathogen Colletotrichum higginsianum. PB1CP directly interacts with RBOHD in vitro, and PAMP treatment increases the interaction in vivo. PB1CP is localized at the cell periphery and in cytoplasm, but PAMP treatment induces PB1CP relocalization to small endomembrane compartments. PB1CP overexpression reduces plasma membrane localization of RBOHD, suggesting that PB1CP down-regulates RBOHD function by relocalizing it away from the plasma membrane. C_LIO_LIWe reveal a novel negative regulation mechanism of ROS production through the relocalization of RBOHD by PB1CP. C_LI

plant biology↗

bioRxiv2020

Pathogen effector recognition-dependent association of NRG1 with EDS1 and SAG101 in TNL receptor immunity

Plants utilise intracellular nucleotide-binding, leucine-rich repeat (NLR) immune receptors to detect pathogen effectors and activate local and systemic defence. NRG1 and ADR1 "helper" NLRs (RNLs), cooperate with enhanced disease susceptibility 1 (EDS1), senescence-associated gene 101 (SAG101) and phytoalexin-deficient 4 (PAD4) lipase-like proteins to mediate signalling from TIR domain NLR receptors (TNLs). However, the mechanism of RNL/EDS1-family protein cooperation is poorly understood. Here, we provide genetic and molecular evidence for exclusive EDS1/SAG101/NRG1 and EDS1/PAD4/ADR1 co-functions in TNL immunity. Using immunoprecipitation and mass spectrometry, we show effector recognition-dependent association of NRG1 with EDS1 and SAG101, but not PAD4. An EDS1-SAG101 complex associates with NRG1, and EDS1-PAD4 associates with ADR1, only in an immune-activated state. NRG1 requires an intact nucleotide-binding P-loop motif, and EDS1 a functional EP domain and its partner SAG101, for induced association and immunity. Thus, two distinct modules (NRG1/EDS1/SAG101 and ADR1/EDS1/PAD4) are required to execute TNL receptor defence signalling.

plant biology↗

bioRxiv2020

Large-scale identification of ubiquitination sites on membrane-associated proteins in Arabidopsis thaliana seedlings

Protein phosphorylation and ubiquitination are two of the most abundant forms of post-translational modifications in eukaryotes, regulated by thousands of protein kinases, phosphatases, E3 ubiquitin ligases, and ubiquitin proteases. Although previous studies have catalogued several ubiquitinated proteins in plants (Walton et al., 2016), few membrane-localized proteins have been identified. Receptor kinases (RKs) initiate phosphorylation signal relays that regulate plant growth, development, and stress responses. While the regulatory role of phosphorylation on protein kinase function is well-documented (Couto and Zipfel, 2016), considerably less is known about the role of ubiquitination on protein kinase function, even though protein turnover is critical to their signaling competence and cellular homeostasis. Here we describe the large-scale identification of ubiquitination sites on Arabidopsis proteins associated with or integral to the plasma membrane, including over 100 protein kinases.

plant biology↗

bioRxiv2017

An Oomycete Effector Protein Induces Shade Avoidance In Arabidopsis And Attenuates Salicylate Signaling By Binding To Host Proteins Of The RADICAL-INDUCED CELL DEATH1 Family

The oomycete pathogen Hyaloperonospora arabidopsidis (Hpa) causes downy mildew disease on Arabidopsis. During infection, Hpa like other biotrophic pathogens, suppresses activation of plant innate immunity by translocating effector proteins into host cells. Some of these effectors localize to the host cell nucleus where they may manipulate transcriptional reprogramming of plant defense genes. Here we report that the nuclear-localized Hpa effector HaRxL106, when expressed in Arabidopsis, induces shade avoidance and attenuates the transcriptional response to the defense signaling molecule salicylic acid. HaRxL106 interacts with RADICAL-INDUCED CELL DEATH1 (RCD1) and loss of RCD1 function renders Arabidopsis resilient against HaRxL106-mediated suppression of immunity. To further characterize the molecular functions of RCD1 we solved a crystal structure of RCD1s Poly-(ADP-ribose)-Polymerase (PARP) domain and, based on non-conservation of amino acids constituting the active site of canonical PARPs, conclude that RCD1 has no PARP activity. We report that RCD1-type proteins are phosphorylated and identified histone-modifying Mut9-like kinases (MLKs) as RCD1-interacting proteins. A mlk1,3,4 triple mutant exhibits stronger SA-induced defense marker gene expression compared to wild-type plants. Our data suggest that HaRxL106 suppresses Arabidopsis innate immunity by manipulating the function(s) of RCD1 in the host cell nucleus and point towards a role of RCD1 as a transcriptional co-regulator that integrates signals from light and pathogen sensors.

plant biology↗