Biology subjects

van der Hoorn, R. A. L.

Publications and source records attributed to van der Hoorn, R. A. L..

At least 19 recordsLinked to original sources

bioRxiv2026

NbRD21 protease controls receptor kinase homeostasis in Nicotiana benthamiana

RD21-like proteases are papain-like cysteine proteases with a C-terminal granulin domain that are abundant and ubiquitous in angiosperms and have often been implicated in immunity. We previously found that the activity of RD21 in Nicotiana benthamiana (NbRD21) is suppressed during infection with Pseudomonas syringae. Here, we studied the role of NbRD21 in immunity and proteome processing. NbRD21 was disrupted by genome editing and rd21 mutants were subjected to disease assays and shot-gun proteomics. Dipeptide substrate zLR-AMC was used in protease assays and agroinfiltration was used to transiently express NbRD21 and candidate substrates. Genome edited lines lacking NbRD21 develop normally but have drastically reduced zLRase activity and are significantly more susceptible to P. syringae. Shot gun proteomics revealed an increased accumulation of [~]20 diverse receptor-like kinases (RLKs) in untreated rd21 knockout lines, but their transcript levels are unaltered when compared to wild-type plants. 35S-driven GFP-tagged RLKs accumulate more upon transient expression in rd21 plants than in wild- type plants. These data indicate that NbRD21 post-translationally controls RLK homeostasis, either by directly degrading RLKs, or indirectly by regulating endocytic RLK recycling.

plant biology↗

bioRxiv2025

Polyphenol oxidase mutant Nicotiana benthamiana plants increase yield and purity of recombinant proteins and enable studies of proteins in their native state.

Agroinfiltration of Nicotiana benthamiana is widely used for recombinant protein production in plant science and molecular pharming, but enzymatic browning and native protein crosslinking during extraction may limit protein integrity and purification efficiency. We generated genome-edited N. benthamiana lines lacking two polyphenol oxidases (PPOs) and analysed protein integrity, enzymatic activity profiles, and recombinant protein purification under non-denaturing extraction conditions. PPO-deficient plants showed reduced browning and native protein crosslinking, preserved endogenous proteins at their predicted molecular weights, displayed increased detectable enzyme activities, and achieved a significantly higher recovery and improved purity of a transiently expressed recombinant protein. These findings identify PPO-mediated oxidation as a major bottleneck during protein extraction and demonstrate that PPO depletion enhances recombinant protein purification while preserving native protein integrity.

plant biology↗

bioRxiv2025

Expanding and applying reactivity-based protein profiling (RBPP) in Arabidopsis

Chemical probes that label surface-exposed amino acid residues in native proteomes serve as powerful tools for studying native reactivities, yet their use has primarily been limited to drug discovery. In this study, we expand the application of reactivity proteomics by monitoring ATP-induced changes over 21,000 reactive lysine, serine, threonine, and tyrosine residues across native Arabidopsis proteomes using biotinylated N-hydroxysuccinimide (BioNHS) ester probes. ATP caused significant differential labelling. Labelling was reduced in ATP-binding pockets, consistent with ligand occupancy, whereas many sites outside ATP-binding regions showed either reduced or increased labelling in response to ATP. Structural modelling revealed that these differentially labelled sites are associated with ATP-induced conformational changes, altering lysine exposure. Notable examples of ATP-driven structural shifts include adenylate kinase ADK4, acyl-activating enzyme AAE3, cell division control protein CDC48A and its interacting partner PUX1, and ATP-induced 26S proteasome assembly. Furthermore, we show that reactivity proteomics using phosphonic acid-NHS (PhoNHS) followed by IMAC enrichment offers a complementary and expanded list of labelling sites. Our findings establish reactivity proteomics as a versatile and powerful platform for exploring protein conformational dynamics, metabolite-protein interactions, and regulatory mechanisms directly in native proteomes.

plant biology↗

bioRxiv2025

Polyphenol oxidase silencing avoids protein crosslinking and enzymatic browning in Nicotiana benthamiana leaf extracts

Oxidation in extracts from agroinfiltrated leaves leads to browning and protein precipitation. Here we show that silencing of polyphenol oxidase (PPO) in Nicotiana benthamiana suppresses enzymatic browning and avoids crosslinking of endogenous proteins such as RuBisCo. PPO silencing does not impact plant growth or development and may also enhance transient gene expression. These results show that depletion of PPO activity could be highly beneficial for plant science applications and molecular pharming.

plant biology↗

bioRxiv2025

Bacterial pathogen deploys iminosugar galactosyrin to manipulate plant glycobiology

The extracellular space (apoplast) of plants is an important molecular battleground during infection by many pathogens. We previously found that a plant-secreted {beta}-galactosidase BGAL1 acts in immunity by facilitating the release of immunogenic peptides from bacterial flagellin and that Pseudomonas syringae suppresses this enzyme by producing a small molecule inhibitor called galactosyrin. Here, we elucidated the structure and biosynthesis of galactosyrin and uncovered its multifunctional roles during infection. Structural elucidation by cryo-EM and chemical synthesis revealed that galactosyrin is an iminosugar featuring a unique geminal diol attached to the pyrrolidine moiety that mimics galactose binding to the {beta}-galactosidase active site. Galactosyrin biosynthesis branches off from purine biosynthesis and involves three enzymes of which the first is a reductase that is unique in iminosugar biosynthesis. Besides inhibiting BGAL1 to avoid detection, galactosyrin also changes the glycoproteome and metabolome of the apoplast. The manipulation of host glycobiology may be common to plant-associated bacteria that carry putative iminosugar biosynthesis clusters.

plant biology↗

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↗

bioRxiv2024

Aphid effectors suppress plant immunity via recruiting defence proteins to processing bodies

Aphids are small insects that have developed specialized mouthparts and effector proteins to establish long-term relationships with plants. The peach-potato aphid, Myzus persicae, is a generalist, feeding on many plant species and capable of transmitting numerous pathogens. This study reveals how host-responsive cathepsins B (CathB) in the oral secretions of M. persicae facilitate aphid survival by modulating plant immune responses. Aphid CathB localize to processing bodies (p-bodies) and recruit key immune regulators EDS1, PAD4, and ADR1 to these bodies, suppressing plant defenses. A plant protein, Acd28.9 (Hsp20 family), counteracts this CathB activity and contributes to plant resistance to aphids. These findings highlight a novel role for p-bodies in plant immunity and uncover a plant resistance mechanism to aphid infestation.

plant biology↗

bioRxiv2024

Extracellular plant subtilases dampen cold shock peptide elicitor levels

Recognizing pathogen-associated molecular patterns (PAMPs) on the cell surface is crucial for plant immunity. The proteinaceous nature of many of these patterns suggests that secreted proteases play important roles in their formation and stability. Here, we demonstrate that the apoplastic subtilase SBT5.2a inactivates the immunogenicity of cold-shock proteins of the bacterial plant pathogen Pseudomonas syringae by cleaving within the immunogenic csp22 epitope. Consequently, mutant plants lacking SBT5.2a activity retain higher levels of csp22, leading to enhanced immune responses and reduced pathogen growth. SBT5.2 sensitivity is influenced by sequence variation surrounding the cleavage site and likely extends to CSPs from other bacterial species. These findings suggest that variations in csp22 stability among bacterial pathogens are a crucial factor in plant-bacteria interactions and that pathogens might exploit plant proteases to avoid pattern recognition.

plant biology↗

bioRxiv2024

Using AlphaFold Multimer to discover interkingdom protein-protein interactions

Structural prediction by artificial intelligence (AI) can be powerful new instruments to discover novel protein-protein interactions, but the community still grapples with the implementation, opportunities and limitations. Here, we discuss and re-analyse our in-silico screen for novel pathogen-secreted inhibitors of immune hydrolases to illustrate the power and limitations of structural predictions. We discuss strategies of curating sequences, including controls, and reusing sequence alignments and highlight important limitations originating from platforms, sequence depth and computing times. We hope these experiences will support similar interactomic screens by the research community.

plant biology↗

bioRxiv2024

Subtilase SBT5.2 counterbalances flagellin immunogenicity in the plant apoplast

Most angiosperm plants recognise the flg22 epitope in bacterial flagellin via homologs of cell surface receptor FLS2 and mount pattern-triggered immune responses. However, flg22 is buried within the flagellin protein indicating that proteases might be required for flg22 release. Here, we demonstrate the extracellular subtilase SBT5.2 not only releases flg22, but also inactivates the immunogenicity of flagellin and flg22 by cleaving within the flg22 epitope, consistent with previous reports that flg22 is unstable in the apoplast. The prolonged lifetime of flg22 in sbt5.2 mutant plants results in increased bacterial immunity in priming assays, indicating that SBT5.2 counterbalances flagellin immunogenicity to provide spatial-temporal control and restrict costly immune responses and that bacteria take advantage of the host proteolytic machinery to avoid detection by flagellin having a protease-sensitive flg22 epitope.

plant biology↗

bioRxiv2024

Discovery of active mouse, plant and fungal cytochrome P450s in endogenous proteomes and upon expression in planta.

Eukaryotes produce a large number of cytochrome P450s that mediate the synthesis and degradation of diverse endogenous and exogenous metabolites. Yet, most of these P450s are uncharacterized and global tools to study these challenging, membrane-resident enzymes remain to be exploited. Here, we applied activity profiling of plant, mouse and fungal P450s with chemical probes that become reactive when oxidized by P450 enzymes. Identification by mass spectrometry revealed labeling of a wide range of active P450s, including six plant P450s, 40 mouse P450s and 13 P450s of the fungal wheat pathogen Zymoseptoria tritici. We next used transient expression of GFP-tagged P450s by agroinfiltration to show ER-targeting and NADPH-dependent, activity-based labeling of plant, mouse and fungal P450s. Both global profiling and transient expression can be used to detect a broad range of active P450s to study e.g. their regulation and discover selective inhibitors.

plant biology↗

bioRxiv2024

Bioengineering secreted proteases converts divergent Rcr3 orthologs and paralogs into extracellular immune co-receptors

Secreted immune proteases Rcr3 and Pip1 of tomato are both inhibited by Avr2 from the fungal plant pathogen Cladosporium fulvum but only Rcr3 act as a decoy co-receptor that detects Avr2 in the presence of the Cf-2 immune receptor. Here, we identified crucial residues from tomato Rcr3 required for Cf-2-mediated signalling and bioengineered various proteases to trigger Avr2/Cf-2 dependent immunity. Despite substantial divergences in Rcr3 orthologs from eggplant and tobacco, only minimal alterations were sufficient to trigger Avr2/Cf-2-triggered immune signalling. Tomato Pip1, by contrast, was bioengineered with 16 Rcr3-specific residues to initiate Avr2/Cf-2-triggered immune signalling. These residues cluster on one side next to the substrate binding groove, indicating a potential Cf-2 interaction site. Our findings also revealed that Rcr3 and Pip1 have distinct substrate preferences determined by two variant residues and that both are suboptimal for binding Avr2. This study advances our understanding of Avr2 perception and opens avenues to bioengineer proteases to broaden pathogen recognition in other crops.

plant biology↗

bioRxiv2023

Protein migration and topography mapping reveals endogenous protein processing and homomers in Nicotiana benthamiana.

Processing by proteases irreversibly regulates the fate of plant proteins and hampers the production of recombinant protein in plants, yet only few processing events have been described in agroinfiltrated Nicotiana benthamiana, which has emerged as a favorite transient protein expression platform in plant science and molecular pharming. Here, we used in-gel digests and mass spectrometry to monitor the migration and topography of 5,040 plant proteins of agroinfiltrated N. benthamiana within a protein gel. By plotting the peptides over the gel slices, we generated peptographs that reveal where which part of each protein was detected within the protein gel. These data uncovered that 60% of the detected proteins have proteoforms that migrate at lower than predicted molecular weights, implicating extensive proteolytic processing. For instance, this analysis confirms the proteolytic removal and degradation of autoinhibitory prodomains of most but not all proteases, and revealed differential processing within pectinemethylesterase and lipase families. This analysis also uncovered intricate processing of glycosidases and uncovered that ectodomain shedding might be common for a diverse range of receptor-like kinases. Transient expression of double-tagged candidate proteins confirmed various processing events in vivo. This extensive proteomic dataset can be investigated further and demonstrates that most plant proteins are proteolytically processed and implicates an extensive proteolytic machinery shaping the proteome of agroinfiltrated N. benthamiana.

plant biology↗

bioRxiv2023

Enhanced late blight resistance by engineering an EpiC2B-insensitive immune protease

Crop protection strategies relying on the improvement of the natural plant immune system via genetic engineering are sustainable solutions against the pathogen thread on food security. Here we describe a novel way to improve the plant immune system by immune protease engineering. As proof of concept, we increased resistance against the late blight pathogen Phytopththora infestans by rendering the tomato secreted immune protease Pip1 insensitive to the P. infestans-secreted inhibitor Epic2B. This concept can be applied to secreted immune proteases in crops by precision breeding.

plant biology↗

bioRxiv2023

Alphafold-multimer predicts cross-kingdom interactions at the plant-pathogen interface

Adapted plant pathogens from various microbial kingdoms produce hundreds of unrelated small secreted proteins (SSPs) with elusive roles. Some of these SSPs might be inhibitors targeting the most harmful hydrolases secreted by the host. Here, we used Alphafold-Multimer (AFM) to screen 1,879 SSPs of seven tomato pathogens for interacting with six defence-related hydrolases of tomato that accumulate to high levels in the apoplast during infection. This screen of 11,274 protein pairs identified 15 SSPs that are predicted to obstruct the active site of chitinases and proteases with an intrinsic fold. Four SSPs were experimentally verified to be inhibitors of pathogenesis-related subtilase P69B, including extracellular protein-36 (Ecp36) and secreted-into-xylem-15 (Six15) of the fungal tomato pathogens Cladosporium fulvum and Fusarium oxysporum, respectively. Together with a novel P69B inhibitor from the bacterial pathogen Xanthomonas perforans and the previously reported Kazal-like inhibitors of the oomycete pathogen Phytophthora infestans, P69B emerges as an important effector hub targeted by different microbial kingdoms, consistent with the presence of a hyper-variant residue in P69B orthologs and gene duplication and diversification of P69B paralogs that could avoid inhibitor binding. This study demonstrates the power of artificial intelligence to accurately predict novel cross-kingdom interactions at the plant-pathogen interface.

plant biology↗

bioRxiv2023

Depletion of the NbCORE receptor drastically improves agroinfiltration productivity in older Nicotiana benthamiana plants.

Nicotiana benthamiana is increasingly used for transient gene expression to produce antibodies, vaccines, and other pharmaceutical proteins but transient gene expression is low in fully developed, 6-8wk old plants. This low gene expression is thought to be caused by the perception the cold shock protein (CSP) of Agrobacterium tumefaciens. The CSP receptor is contested because both NbCSPR and NbCORE have been claimed to perceive CSP. Here, we demonstrate that CSP perception is abolished in 6wk-old plants silenced for NbCORE but not NbCSPR. Importantly, older NbCORE-silenced plants support a drastically increased level of GFP fluorescence and protein upon agroinfiltration. The drastic increase in transient protein production in NbCORE depleted plants offers new opportunities for molecular farming, where older plants with larger biomass can now be used for efficient protein expression.

plant biology↗

bioRxiv2023

Glutathione transferase photoaffinity labeling demonstrates GST activation by safeners and NPR1-independent activation by BTH

Glutathione transferases (GSTs) represent a large and diverse enzyme family involved in detoxification of small molecules by glutathione conjugation in crops, weeds and model plants. Here, we introduce an easy and quick assay for photoaffinity labeling of GSTs to study global GST activation in various plant species. The small molecule probe contains glutathione, a photoreactive group, and a minitag for coupling to reporter tags via click chemistry. Under UV irradiation, this probe quickly and robustly labels GSTs in crude protein extracts. Enrichment and MS analysis of labeled proteins from Arabidopsis identified ten GSTs from the Phi(F) and Tau(U) classes. Photoaffinity labeling of GSTs demonstrated GST activation in wheat seedlings upon treatment with safeners, and in Arabidopsis leaves upon infection with avirulent bacteria. Photoaffinity labeling and proteomics identified six Phi- and Tau-class GSTs that are induced upon treatment with salicylic acid (SA) analog benzothiadiazole (BTH) and these were tested for enhancing immunity in disease assays. Our data confirm that BTH-induced GST activation is independent of NPR1, the master regulator of SA signaling.

plant biology↗

bioRxiv2022

Activity-based proteomics uncovers suppressed hydrolases and a neo-functionalised antibacterial enzyme at the plant-pathogen interface

The extracellular space of plant tissues contains hundreds of hydrolases that might harm colonizing microbes. Successful pathogens may suppress these hydrolases to enable disease. Here, we report the dynamics of extracellular hydrolases in leaves upon infection with Pseudomonas syringae. Using activity-based proteomics with a cocktail of biotinylated probes we simultaneously monitored 171 active hydrolases, including 109 serine hydrolases (SHs), 49 glycosidases (GHs) and 13 cysteine proteases (CPs). The activity of 82 of these hydrolases (mostly SHs) increases during infection, whilst the activity of 60 hydrolases (mostly GHs and CPs) is suppressed during infection. Active {beta}-galactosidase-1 (BGAL1) is amongst the suppressed hydrolases, consistent with production of the BGAL1 inhibitor by P. syringae. One of the other suppressed hydrolases, the pathogenesis-related NbPR3, decreases bacterial growth when transiently overexpressed. This is dependent on its active site, revealing a role for NbPR3 activity in antibacterial immunity. Despite being annotated as a chitinase, NbPR3 does not possess chitinase activity, and contains a E112Q active site substitution that is essential for antibacterial activity and is conserved only in Nicotiana species. This study introduces a powerful approach to reveal novel components of extracellular immunity, exemplified by the discovery of the suppression of neo-functionalised Nicotiana-specific antibacterial NbPR3.

plant biology↗