Professor Renier van der Hoorn
Apoplast manipulation by plant pathogens
We investigate the molecular mechanisms underpinning host manipulation by plant pathogens, with a particular focus on apoplast manipulation of Solanaceous plants by bacterial microbes. One intruiging aspect of this work is the recruitment of an protease in the extracellular recognition of pathogen-derived inhibitors at the plant cell surface by the Cf-2 resistance protein.
Our research activities also aim at improved molecular pharming of secreted (glyco)proteins through the depletion of extracellular proteases. Unique is our approach to display protein activities by pioneering activity-based protein profiling (ABPP), a powerful functional proteomics technology that offers an exciting platform for collaborations. Please contact us if you are interested to join or collaborate!
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Agromonas: a rapid disease assay for Pseudomonas syringae growth in agroinfiltrated leaves.
October 2020|Journal article|Plant JThe lengthy process to generate transformed plants is a limitation in current research on the interactions of the model plant pathogen Pseudomonas syringae with plant hosts. Here we present an easy method called agromonas, where we quantify P. syringae growth in agroinfiltrated leaves of Nicotiana benthamiana using a cocktail of antibiotics to select P. syringae on plates. As a proof of concept, we demonstrate that transient expression of PAMP receptors reduces bacterial growth and that transient depletion of a host immune gene and transient expression of a type-III effector increase P. syringae growth in agromonas assays. We show that we can rapidly achieve structure-function analysis of immune components and test the function of immune hydrolases. The agromonas method is easy, fast and robust for routine disease assays with various Pseudomonas strains without transforming plants or bacteria. The agromonas assay offers a reliable approach for further comprehensive analysis of plant immunity. Supporting Information Table S1 Selection of bacterial strains on medium supplemented with CFC or gentamicin. Table S2 Binary vectors generated in this study. Table S3 Primers and nucleic acid sequences used in this study. Table S4 Bacterial strains used in this study.Agrobacterium, Nicotiana benthamiana, Pseudomonas syringae, disease assay, plant immunity -
Proteome-wide Profiling of RNA-Binding Protein Responses to flg22 Reveals Novel Components of Plant Immunity
September 2020|Journal article<jats:p>RNA-binding proteins (RBPs) play critical roles in post-transcriptional gene regulation and are known to contribute to plant immunity. To understand the responses of cellular RBPs to an immune elicitor, we applied RNA interactome capture to Arabidopsis leaves treated with flg22. Strikingly, flg22 induced a pervasive remodelling of the cellular RBPome affecting 186 proteins. Flg22-responsive RBPs included classical RBPs involved in RNA metabolism as well as non-canonical RBPs. RBP responders detected after 2h of treatment are enriched in putative sites for post-translational modifications, which may play a regulatory role. By contrast, changes in RBP abundance becomes increasingly important for the RBPome responses to flg22 after 12h. Plant resistance to <jats:italic>Pseudomonas syringae</jats:italic> is strongly altered in mutant lines lacking individual flg22-responsive RBPs, supporting the importance of RBP dynamics in plant immunity. This study provides a comprehensive and systematic census of flg22 responsive plant RBPs, discovering novel components of plant immunity.</jats:p> -
Evolution of a guarded decoy protease and its receptor in solanaceous plants.
September 2020|Journal article|Nat CommunRcr3 is a secreted protease of tomato that is targeted by fungal effector Avr2, a secreted protease inhibitor of the fungal pathogen Cladosporium fulvum. The Avr2-Rcr3 complex is recognized by receptor-like protein Cf-2, triggering hypersensitive cell death (HR) and disease resistance. Avr2 also targets Rcr3 paralog Pip1, which is not required for Avr2 recognition but contributes to basal resistance. Thus, Rcr3 acts as a guarded decoy in this interaction, trapping the fungus into a recognition event. Here we show that Rcr3 evolved > 50 million years ago (Mya), whereas Cf-2 evolved <6Mya by co-opting the pre-existing Rcr3 in the Solanum genus. Ancient Rcr3 homologs present in tomato, potato, eggplants, pepper, petunia and tobacco can be inhibited by Avr2 with the exception of tobacco Rcr3. Four variant residues in Rcr3 promote Avr2 inhibition, but the Rcr3 that co-evolved with Cf-2 lacks three of these residues, indicating that the Rcr3 co-receptor is suboptimal for Avr2 binding. Pepper Rcr3 triggers HR with Cf-2 and Avr2 when engineered for enhanced inhibition by Avr2. Nicotiana benthamiana (Nb) is a natural null mutant carrying Rcr3 and Pip1 alleles with deleterious frame-shift mutations. Resurrected NbRcr3 and NbPip1 alleles were active proteases and further NbRcr3 engineering facilitated Avr2 inhibition, uncoupled from HR signalling. The evolution of a receptor co-opting a conserved pathogen target contrasts with other indirect pathogen recognition mechanisms.Cladosporium, Disease Resistance, Evolution, Molecular, Fungal Proteins, Genes, Plant, Host-Parasite Interactions, Peptide Hydrolases, Phylogeny, Plant Diseases, Plant Immunity, Plant Proteins, Protease Inhibitors, Solanum, Tobacco -
Extracellular proteolytic cascade in tomato activates immune protease Rcr3.
July 2020|Journal article|Proceedings of the National Academy of Sciences of the United States of AmericaProteolytic cascades regulate immunity and development in animals, but these cascades in plants have not yet been reported. Here we report that the extracellular immune protease Rcr3 of tomato is activated by P69B and other subtilases (SBTs), revealing a proteolytic cascade regulating extracellular immunity in solanaceous plants. Rcr3 is a secreted papain-like Cys protease (PLCP) of tomato that acts both in basal resistance against late blight disease (Phytophthora infestans) and in gene-for-gene resistance against the fungal pathogen Cladosporium fulvum (syn. Passalora fulva) Despite the prevalent model that Rcr3-like proteases can activate themselves at low pH, we found that catalytically inactive proRcr3 mutant precursors are still processed into mature mRcr3 isoforms. ProRcr3 is processed by secreted P69B and other Asp-selective SBTs in solanaceous plants, providing robust immunity through SBT redundancy. The apoplastic effector EPI1 of P. infestans can block Rcr3 activation by inhibiting SBTs, suggesting that this effector promotes virulence indirectly by preventing the activation of Rcr3(-like) immune proteases. Rcr3 activation in Nicotiana benthamiana requires a SBT from a different subfamily, indicating that extracellular proteolytic cascades have evolved convergently in solanaceous plants or are very ancient in the plant kingdom. The frequent incidence of Asp residues in the cleavage region of Rcr3-like proteases in solanaceous plants indicates that activation of immune proteases by SBTs is a general mechanism, illuminating a proteolytic cascade that provides robust apoplastic immunity.Cladosporium, Lycopersicon esculentum, Peptide Hydrolases, Plant Proteins, Protein Isoforms, Virulence, Plant Diseases, Phytophthora infestans, Plant Immunity, Proteolysis -
BGAL1 depletion boosts the level of β-galactosylation of N- and O-glycans in N. benthamiana.
July 2020|Journal article|Plant biotechnology journalGlyco-design of proteins is a powerful tool in fundamental studies of structure-function relationship and in obtaining profiles optimized for efficacy of therapeutic glycoproteins. Plants, particularly Nicotiana benthamiana, are attractive hosts to produce recombinant glycoproteins, and recent advances in glyco-engineering facilitate customized N-glycosylation of plant-derived glycoproteins. However, with exception of monoclonal antibodies, homogenous human-like β1,4-galactosylation is very hard to achieve in recombinant glycoproteins. Despite significant efforts to optimize the expression of β1,4-galactosyltransferase, many plant-derived glycoproteins still exhibit incomplete processed N-glycans with heterogeneous terminal galactosylation. The most obvious suspects to be involved in trimming terminal galactose residues are β-galactosidases (BGALs) from the glycosyl hydrolase family GH35. To elucidate the so far uncharacterized mechanisms leading to the trimming of terminal galactose residues from glycans of secreted proteins, we studied a N. benthamiana BGAL known to be active in the apoplast (NbBGAL1). Here, we determined the NbBGAL1 subcellular localization, substrate specificity and in planta biological activity. We show that NbBGAL1 can remove β1,4- and β1,3-galactose residues on both N- and O-glycans. Transient BGAL1 down-regulation by RNA interference (RNAi) and BGAL1 depletion by genome editing drastically reduce β-galactosidase activity in N. benthamiana and increase the amounts of fully galactosylated complex N-glycans on several plant-produced glycoproteins. Altogether, our data demonstrate that NbBGAL1 acts on galactosylated complex N-glycans of plant-produced glycoproteins.Humans, Tobacco, Glycoproteins, Polysaccharides, Recombinant Proteins, Glycosylation -
Plant Biology: Distinct New Players in Processing Peptide Hormones during Abscission.
June 2020|Journal article|Current biology : CBFlower organ abscission in Arabidopsis is regulated by a peptide hormone that is released from its precursor by a network of redundant subtilases. An exciting new study describes how drought-induced flower abscission in tomato is regulated similarly, but distinctly via a single, different subtilase that releases a very different peptide hormone. -
Discovering the RNA-Binding Proteome of Plant Leaves with an Improved RNA Interactome Capture Method.
April 2020|Journal article|BiomoleculesRNA-binding proteins (RBPs) play a crucial role in regulating RNA function and fate. However, the full complement of RBPs has only recently begun to be uncovered through proteome-wide approaches such as RNA interactome capture (RIC). RIC has been applied to various cell lines and organisms, including plants, greatly expanding the repertoire of RBPs. However, several technical challenges have limited the efficacy of RIC when applied to plant tissues. Here, we report an improved version of RIC that overcomes the difficulties imposed by leaf tissue. Using this improved RIC method in Arabidopsis leaves, we identified 717 RBPs, generating a deep RNA-binding proteome for leaf tissues. While 75% of these RBPs can be linked to RNA biology, the remaining 25% were previously not known to interact with RNA. Interestingly, we observed that a large number of proteins related to photosynthesis associate with RNA in vivo, including proteins from the four major photosynthetic supercomplexes. As has previously been reported for mammals, a large proportion of leaf RBPs lack known RNA-binding domains, suggesting unconventional modes of RNA binding. We anticipate that this improved RIC method will provide critical insights into RNA metabolism in plants, including how cellular RBPs respond to environmental, physiological and pathological cues. -
Classification and Nomenclature of Metacaspases and Paracaspases: No More Confusion with Caspases
March 2020|Journal article|Molecular Cell
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