Biology subjects

Joosten, M. H. A. J.

Publications and source records attributed to Joosten, M. H. A. J..

12 recordsLinked to original sources

bioRxiv2026

TurboID-based proximity-dependent labeling using SOBIR1 as a bait in potato leads to the identification of novel defense-related signaling partners

The plant immune system comprises a complex signaling network that is activated upon perceiving molecules derived from invading pathogens. The first line of defense at the plant cell surface is mediated by receptor-like proteins (RLPs) and receptor-like kinases (RLKs). RLPs, which lack a cytoplasmic signalling domain themselves, constitutively interact with the RLK SUPRESSOR OF BIR1-1 (SOBIR1), which is a key component initiating immune signal transduction upon pathogen perception. Therefore, elucidating the composition of the SOBIR1 protein complex will contribute to understanding the basic molecular mechanisms of plant disease resistance. Most of the studies focused on the identification of SOBIR1-interacting proteins are limited to model plants, due to technical challenges and lack of reliable genome and proteome databases in crop plants. Here, we evaluate the application of the biotin ligase TurboID (TbID)-based proximity-dependent labeling (PL) approach by transiently expressing SOBIR1 from Nicotiana benthamiana (NbSOBIR1), fused to TbID in leaves of the wild potato Solanum microdontum. We show that NbSOBIR1-YFP-TbID properly accumulates in potato and that proximal proteins are biotinylated. Quantitative proteomic analysis yielded over 130 candidate proteins to be in the proximity of the cytoplasmic kinase domain of NbSOBIR1, of which some could be linked to disease resistance by KEGG pathway and gene ontology (GO) molecular function analysis. We also studied the dynamics of the proteome in proximity of NbSOBIR1 upon perception of the INF1 elicitin of Phytophthora infestans that was co-expressed in potato with the elicitin receptor ELR, which is an RLP that constitutively interacts with SOBIR1. We found more than 80 proteins, including the NB-LRR REQUIRED FOR HR-ASSOCIATED CELL DEATH 1 (NRC1), putatively interacting with NbSOBIR1. In conclusion, we were able to successfully apply PL in potato and a future roadmap for further research on deciphering the composition of protein complexes involved in immune signaling has been established.

plant biology↗

bioRxiv2026

Cf-4- and Cf-5-triggered plant immunity: Similarities and differences

Plant immunity is, amongst others, mediated by receptor like proteins (RLPs), which are localized on the plasma membrane. RLPs recognize extracellular immunogenic patterns (ExIPs) originating from pathogens or derived from the host itself, which leads to extracellularly triggered immunity (ExTI). Cf proteins, which are well-known RLPs of tomato (Solanum lycopersicum) confer resistance against the fungal pathogen Fulvia fulva. Cf-9, Cf-4, Cf-2 and Cf-5 are well-known examples of Cf proteins, mediating recognition of the matching ExIPs Avr9, Avr4, Avr2 and Avr5, respectively, which are secreted effectors of F. fulva and trigger hypersensitive response (HR)-related cell death in tomato plants carrying these Cf proteins. Although all these Cf proteins confer proper resistance to the fungus, Cf-9 and Cf-4 trigger a stronger and faster cell death than Cf-5 and Cf-2. It is unknown whether these phenotypical differences arise from variations in the molecular mechanism of the cellular immune response that is initiated by the Cf proteins, and whether this phenotypic difference correlates with varying degrees in the intensity and timing of the triggered immune responses and robustness of the resistance. To try to answer these questions, in this study the immune responses triggered by Cf-4 and Cf-5 were compared. Cf-4 and Cf-5 share the same core upstream signaling components to trigger HR-related cell death in Nicotiana benthamiana. In tomato, both receptors induce rapid MAPK activation, which is more sustained for the Cf-5/Avr5 combination. Both Avr4 and Avr5 induce an apoplastic burst of reactive oxygen species (ROS), independently of the presence of their matching receptors, while remaining dependent on RBOHB for this ROS burst. Full transcriptome analysis at 3 and 7 hours after immune activation revealed a large shared set of differentially expressed genes, alongside qualitative and quantitative differences, with the Cf-5/Avr5 combination inducing a broader transcriptional reprogramming. Despite these differences, Cf-4 and Cf-5 confer a comparable level of resistance to F. fulva. These results demonstrate that Cf-4 and Cf-5 share conserved immune initiation mechanisms, but diverge in downstream signaling dynamics, and that the intensity and timing of the HR-related cell death do not affect the robustness of the resistance.

plant biology↗

bioRxiv2026

Effector-centred proximity-dependent labelling enables the discovery of cell-surface immune receptors in plants

Identifying plant disease resistance proteins remains challenging, particularly for cell-surface receptors that perceive apoplastic pathogen effectors through transient or indirect interactions. Here, we establish effector-centred proximity-dependent labelling using TurboID as a protein-level approach to identify immune receptors and guarded host targets in planta. We fused three apoplastic effectors, Avr2 and Avr4 from Fulvia fulva and XEG1 from Phytophthora sojae, to TurboID and transiently expressed these fusion proteins in leaves of Nicotiana benthamiana and tomato (Solanum lycopersicum). The fusions significantly biotinylate their matching receptors, including Cf-4 by Avr4-TurboID and Cf-2 by Avr2-TurboID, as well as the guarded virulence target of Avr2, the Rcr3 protease, demonstrating that both direct and indirect recognition systems can be captured. Application of this approach to XEG1 revealed SlEix1 as the functional ortholog of NbRXEG1. Functional assays and structural modelling support SlEix1 as a signalling-competent receptor contributing to XEG1-triggered immunity. Together with previous studies, these findings position the tomato EIX locus as a multi-effector immune hub encoding closely related receptor-like proteins with distinct ligand specificities. Collectively, this study establishes effector-TurboID-mediated proximity-dependent labelling as a versatile approach for identifying cell surface plant immune receptors and apoplastic virulence targets, providing a scalable route to accelerate resistance gene discovery in crop species.

plant biology↗

bioRxiv2025

Mutation or deletion of the Avr6 avirulence effector gene of Fulvia fulva leads to breakdown of the Cf-6 leaf mold resistance locus in tomato

Genetic resistance mediated by Cf cell-surface receptors is a cornerstone of tomato breeding against leaf mold disease caused by the fungal pathogen Fulvia fulva. After widespread deployment of the Cf-9 resistance locus, breeders increasingly relied on Cf-6, yet Cf-6-mediated resistance has already been overcome in multiple regions. The molecular basis of this breakdown, and the identity of the matching avirulence (Avr) effector, have remained unresolved. Here, we use comparative genomics and functional genetics to identify the F. fulva effector Avr6 as the previously described apoplastic protein Ecp5. Sequencing of Cf-6-breaking strains revealed either deletion of the Ecp5 locus or non-synonymous mutations in its coding sequence. Using transient expression, targeted gene knockout, and complementation assays, we show that Ecp5 is both necessary and sufficient for Cf-6-mediated recognition and defence activation. We then use Avr6-triggered cell death as a phenotypic marker in a bulked segregant analysis combined with Comparative Subsequence Sets analysis (CoSSa) to map Cf-6 to a [~]2 Mb interval on the short arm of chromosome 12 that overlaps the previously described Cf-Ecp5.12 locus. Sequencing of Avr6 in a broad strain collection reveals a predominantly conserved allele under apparent purifying selection, with recent independent loss-of-function variants in Europe and South America that confer virulence on Cf-6 plants. Together, these findings establish the Avr6-Cf-6 gene-for-gene pair, explain the erosion of Cf-6-mediated resistance, and illustrate how effector loss or mutation provides a rapid route for F. fulva adaptation, with important implications for designing more durable resistance strategies against tomato leaf mold.

microbiology↗

bioRxiv2025

Immunity gene silencing increases transient protein expression in Nicotiana benthamiana

Agroinfiltration of Nicotiana benthamiana is routinely used for transient gene expression in plant sciences and molecular pharming. Here, we depleted transcripts of 21 different immunity-related genes through virus-induced gene silencing (VIGS) to identify genes that hamper transient expression in juvenile plants. These experiments uncovered that silencing of ethylene insensitive- 2 (EIN2), receptor-like kinase CERK1, transcription factor regulator nonexpressor of pathogenesis-related genes 1 (NPR1) and isochorismate synthase (ICS), increases transient GFP expression by 2-, 4-, 4- and 11-fold, respectively. Accordingly, the npr1a/npr1b double mutant of N. benthamiana does indeed facilitate increased protein accumulation when transiently expressed. These results indicate that glycan perception through CERK1, and ET and SA signaling pathways via EIN2 and NPR1/ICS, respectively, contribute to suppressed transient gene expression in frequently used juvenile N. benthamiana plants.

plant biology↗

bioRxiv2025

The dominant lineage of Phakopsora pachyrhizi in the United States of America does not have a Brazilian origin

O_LIAsian soybean rust (ASR), caused by the obligate biotrophic fungus, Phakopsora pachyrhizi, was first reported in the continental United States of America (USA) in 2004 and over the years has been of concern to soybean production in the USA. The prevailing hypothesis is that P. pachyrhizi spores were introduced into the USA via hurricanes originating from South America, particularly Hurricane Ivan. C_LIO_LITo investigate the genetic diversity and global population structure of P. pachyrhizi, we employed exome-capture based sequencing on 84 field isolates collected from different geographic regions worldwide. We compared the gene-encoding regions from all these field isolates and found that four major haplotypes are prevalent worldwide. Here, we provide genetic evidence supporting multiple incursions that have led to the currently established P. pachyrhizi population of the USA. Phylogenetic analysis of mitochondrial genes further supports this hypothesis. C_LIO_LINotably, we observed limited genetic diversity in P. pachyrhizi populations in Brazil, suggesting a clonal population structure in that country that contrasts to populations from the USA and Africa. C_LIO_LIThis study provides the first comprehensive characterization of P. pachyrhizi population structures defined by genetic evidence from populations across major soybean growing regions. C_LI

evolutionary biology↗

bioRxiv2023

Sequential breakdown of the complex Cf-9 leaf mould resistance locus in tomato by Fulvia fulva

O_LILeaf mould, caused by Fulvia fulva, is a devastating disease of tomato plants. In many commercial tomato cultivars, resistance to this disease is governed by the Cf-9 locus, which comprises five paralogous genes (Cf-9A-9E) that encode receptor-like proteins. Two of these proteins contribute to resistance: Cf-9C recognizes the previously identified F. fulva effector Avr9 and provides resistance during all plant growth stages, while Cf-9B recognises the yet-unidentified F. fulva effector Avr9B and provides mature plant resistance only. In recent years, F. fulva strains have emerged that have overcome the Cf-9 locus, with Cf-9C circumvented through Avr9 deletion. To understand how Cf-9B is circumvented, we set out to identify Avr9B. C_LIO_LIComparative genomics, in planta transient expression assays and gene complementation experiments were used to identify Avr9B, while gene sequencing was used to assess Avr9B allelic variation across a worldwide strain collection. C_LIO_LIA strict correlation between Avr9 deletion and resistance-breaking mutations in Avr9B was observed in strains recently collected from Cf-9 cultivars, whereas Avr9 deletion but no mutations in Avr9B were observed in older strains. C_LIO_LIThis research showcases how F. fulva has evolved to sequentially break down the two functional resistance genes of the complex Cf-9 locus and highlights that this locus now has limited value for controlling leaf mould disease in worldwide commercial tomato production. C_LI

microbiology↗

bioRxiv2020

Arabidopsis cell surface LRR immune receptor signaling through the EDS1-PAD4-ADR1 node

Plants use both cell surface and intracellular immune receptors with leucine rich-repeat (LRRs) to detect pathogens. LRR receptor kinases (LRR-RKs) and LRR receptor-like proteins (LRR-RPs) recognize extracellular microbe-derived molecules to confer pattern-triggered immunity (PTI), while nucleotide-binding LRR (NLR) proteins detect microbial effectors inside the cell to confer effector-triggered immunity (ETI). Despite PTI and ETI signaling being initiated in different compartments, both rely on the transcriptional activation of similar sets of genes, suggesting convergence in signaling upstream of nuclear events. Here we report that two sets of molecules, helper NLRs from the ADR1 (ACTIVATED DISEASE RESISTANCE 1) family as well as lipase-like proteins EDS1 (ENHANCED DISEASE SUSCEPTIBILITY 1) and PAD4 (PHYTOALEXIN DEFICIENT 4), are required not only for ETI, but also for PTI. A further similarity is seen in the evolutionary patterns of some PTI and ETI receptor genes, with both often being highly polymorphic, and with nevertheless distinct roles of LRR-RK and LRR-RP receptors in immunity. We find that the LRR-RK SOBIR1 directly links LRR-RPs with the ADR1 helper NLR as well as EDS1 and PAD4, suggesting the formation of constitutive supramolecular signalosome complexes at the inner side of the plasma membrane. We propose that the EDS1-PAD4-ADR1 node is an essential component and convergence point for immune signaling cascades activated by both surface-resident LRR-RP receptors and intracellular NLR receptors.

plant biology↗

bioRxiv2020

ELR is a true pattern recognition receptor that associates with elicitins from diverse Phytophthora species

The first layer of plant immunity against pathogens is mediated by cell surface pattern recognition receptors (PRRs) that recognize pathogen molecules in the apoplast. Several pairs of PRRs and their matching extracellular ligands have been described but, in many cases, actual evidence for ligand binding by the PRR is lacking. The receptor-like protein ELR from Solanum microdontum, which triggers cell death upon co-expression with elicitins of various Phytophthora species and enhances resistance to late blight caused by Phytophthora infestans, was previously identified as the elicitin receptor by forward genetic screenings employing the INF1 elicitin of P. infestans. In this study, we investigated whether ELR associates with INF1 and other elicitins that are secreted by diverse Phytophthora spp. We performed in planta and in vitro co-immunoprecipitation of ELR with several affinity-tagged elicitins, as well as in planta transient co-expression assays. We found that ELR physically interacts with the class I elicitins INF1 and ParA1, from P. infestans and Phytophthora parasitica, respectively, which is in line with their ability to cause cell death when co-expressed with ELR in potato. Together, we demonstrate that ELR is a genuine PRR that binds elicitins of Phytophthora species.

plant biology↗

bioRxiv2020

Knocking out SOBIR1 in Nicotiana benthamiana abolishes functionality of transgenic receptor-like protein Cf-4

The first layer of plant immunity is formed by pattern recognition receptors (PRRs) that are present at the cell surface and perceive extracellular immunogenic patterns. Receptor-like proteins (RLPs), such as the tomato (Solanum lycopersicum) PRR Cf-4 that provides resistance to the fungus Cladosporium fulvum secreting the matching avirulence factor Avr4, have an extracellular receptor domain consisting of leucine-rich repeats, but lack a cytoplasmic kinase domain for downstream signaling. RLPs constitutively interact with the receptor-like kinase SUPPRESSOR OF BIR1-1 (SOBIR1), thereby providing the receptor with a kinase domain, and recruit the co-receptor BRI-ASSOCIATED KINASE 1 (BAK1) upon their activation by a matching ligand. Trans-phosphorylation events, which can take place between the kinase domains of SOBIR1 and BAK1 after their association with the RLP, are thought to initiate downstream defense signaling. Currently, our knowledge on RLP/SOBIR1/BAK1-mediated defence initiation is limited and to understand the role of SOBIR1 in RLP function, we knocked out SOBIR1 and its close homolog SOBIR1-like in the model plant Nicotiana benthamiana, as well as in transgenic N. benthamiana stably expressing Cf-4. We observed that Cf-4 function is completely abolished in the knock-out mutants, and we show that these plants can be used to perform transient complementation studies with SOBIR1 mutants. Thereby, these mutants are an important tool to study the fundamentals of plant immunity mediated by RLPs.

plant biology↗

bioRxiv2020

Red-light imaging for programmed cell death visualization and quantification in plant-pathogen interactions

Studies on plant-pathogen interactions often involve monitoring disease symptoms or responses of the host plant to pathogen-derived immunogenic patterns, either visually or by staining the plant tissue. Both these methods have limitations with respect to resolution, reproducibility and the ability to quantify the results. In this study we show that red light detection in a multi-purpose fluorescence imaging system that is probably available in many labs can be used to visualize plant tissue undergoing cell death. Red light emission is the result of chlorophyll fluorescence upon thylakoid membrane disassembly during the development of a programmed cell death process. The activation of programmed cell death can occur either during a hypersensitive response to a biotrophic pathogen or an apoptotic cell death triggered by a necrotrophic pathogen. Quantifying the intensity of the red light signal enables to evaluate the magnitude of programmed cell death and provides a non-invasive readout of the plant immune response in a faster and safer manner as compared to chemical staining methodologies previously developed. This application can be implemented to screen for differences in symptom severity in plant-pathogen interactions, and to visualize and quantify in a sensitive and objective manner the intensity of a plant response upon perception of a given immunological pattern. We illustrate the utility and versatility of the method using diverse immunogenic patterns and pathogens.

plant biology↗

bioRxiv2020

Population studies in the wild tomato species Solanum chilense reveal maintenance and loss of resistance at geographically distinct locations

Natural plant populations encounter strong pathogen pressure and defense-associated genes are known to be under different selection pressure dependent on the pressure by the pathogens. Here we use wild tomato Solanum chilense populations to investigate natural resistance against Cladosporium fulvum, a well-known pathogenic fungus of domesticated tomatoes. We show that populations of S. chilense differ in resistance against the pathogen. Next, we explored the underlying molecular processes in a species wide-context. Then, focusing on recognition of the two prominent avirulence factors secreted by C. fulvum (Avr4 and Avr9) in central and northern populations of S. chilense we observed high complexity in the cognate homologues of Cladosporium resistance (Hcr9) locus underlying the recognition of these effectors. Presence of canonical genomic regions coding for Cf-4 and Cf-9, two major dominant resistance genes in the Hcr9 locus recognizing Avr4 and Avr9, respectively, does not meet prediction from Avr response phenotypes. We find both genes in varying fractions of the plant populations and we show possible co-existence of two functionally active resistance genes, previously thought to be allelic. Additionally, we observed the complete local absence of recognition of additional Avr proteins of C. fulvum. In the southern populations we attribute this to changes in the coregulatory network. As a result of loss of pathogen pressure or adaptation to extreme climatic conditions. This may ultimately explain the observed pathogen susceptibility in the southern populations. This work puts major gene mediated disease resistance in an ecological context.

ecology↗