Biology subjects

van den Burg, H. A.

Publications and source records attributed to van den Burg, H. A..

11 recordsLinked to original sources

bioRxiv2025

Protocol for capturing the RNA-binding proteome from plants using orthogonal organic phase separation

RNA-binding proteins (RBPs) regulate many processes related to RNA biogenesis, localization, half-life and function. Here, we present a protocol for the en masse isolation of RBPs cross-linked to RNA and provide a strategy for proteomics analysis. We describe the steps for in vivo UV-crosslinking of RNA-protein complexes, tissue lysis, fractionation and purification of crosslinked RNA-protein adducts using organic solvents. Although the protocol was developed for Nicotiana benthamiana leaves, it can be optimized for use in different plants and tissues.

plant biology↗

bioRxiv2025

Asynchronous viral spread of two unrelated viruses determines Lettuce Big Vein Disease symptom development

Lettuce big-vein disease (LBVD) is a major disease affecting lettuce cultivation worldwide. LBVD is caused by two unrelated negative-stranded RNA viruses, that is, Mirafiori lettuce big-vein virus (MiLBVV) and Lettuce big-vein associated virus (LBVaV) both vectored by the soilborne fungus Olpidium virulentus. Despite extensive research, a synergistic effect between the two viruses has not been observed, while both viruses individually have been suggested to be the causal agent for the disease. By performing lettuce reinfections using a large soil sample collection carrying LBVD infested O. virulentus spores, the presence of LBVaV was consistently established in diseased lettuce heads, while MiLBVV infections were apparently less prevalent. Yet, aboveground infections with MiLBVV corresponded with strong disease symptoms. Strikingly, the spread of LBVaV from the root to shoot always preceded that of MiLBVV. The LBVaV systemic spread was highly synchronized between plants, while MiLBVV spread was always delayed and asynchronous. A pan-genome analysis revealed independent segment reassortments for both viruses indicative of mixed field infections over the sampled period. Yet, RNA segment abundance was highly conserved for both viruses between all re-infections, suggesting that segment abundance has a regulatory role for the two individual viruses, but that segment abundance is not impacted by the presence of the other two viruses. The pan-genome analysis also revealed different evolutionary rates of the viral ORFs suggesting that mutagenesis of certain ORFs compromises viral fitness and thus revealing a potential weak spot for both viruses. ImportanceLettuce big-vein disease (LBVD) is an important viral disease complex affecting lettuce cultivation worldwide. Here we reveal a synergistic interaction between the two principal associated segmented RNA viruses, Mirafiori lettuce big-vein virus (MiLBVV) and Lettuce big-vein associated virus (LBVaV). We show unequivocally that MiLBVV is the main virus responsible for severe disease symptoms in lettuce heads. Yet, MiLBVV root-to-shoot movement was in our conditions always preceded by LBVaV movement into the lettuce heads. Arguably, LBVaV thus facilitates the root-to-shoot movement of MiLBVV. Moreover, both viruses undergo segment reassortment increasing their genome plasticity and the reassortment events appeared to be independent events with mixed infections. Finally, we provide data that both viruses regulate gene expression via the copy number of their RNA segments, but that the genome formula does not change in dual infections. We thus provide evidence for a synergistic interaction needed for strong LBVD symptoms.

pathology↗

bioRxiv2024

Four cell wall-degrading enzymes of Xanthomonas campestris pv. campestris determine bacterial escape from hydathodes to the leaf vasculature

To colonize plants, pathogenic bacteria modulate the biology of the host employing different bacterial secretion systems. For example, the type II secretion system (T2SS) releases toxins, proteases, lipases and carbohydrate-degrading enzymes into the extracellular environment to promote tissue softening and soft rot. In this way, the T2SS promotes virulence of phytopathogenic Gram-negative bacteria. However, the role of the T2SS and its substrates for vascular disease remains enigmatic. Here, we show that the Xps-T2SS allows Xanthomonas campestris pv. campestris (Xcc) to breach the tissue barrier between hydathodes- the initial bacterial entry point - and xylem thereby gaining access to the leaf vasculature. Yet, Xps-T2SS was dispensable for bacterial multiplication in the leaf apoplast or inside the hydathode cavity suggesting a role beyond plant defense suppression or nutrient acquisition. Using comparative genomics, four plant cell wall-degrading enzymes (CWDEs) were found to be associated with vascular pathogenesis. Testing gene knockout combinations of those enzymes revealed that virulence of only the quadruple CWDE mutant was down to the level of the xps-T2SS mutant. Our results thus demonstrate that the Xps-T2SS and a set of CWDEs that is likely secreted by this system allow Xcc to break this tissue barrier enabling long-distance mobility of Xcc inside the host plant. We thus expand our understanding on how certain bacterial pathogens have specialized towards vascular pathogens.

plant biology↗

bioRxiv2024

Non-invasive, bioluminescence-based visualization and quantification of bacterial infections in Arabidopsis over time

Plant pathogenic bacteria use various entry strategies to colonize their host, like entering through natural openings and wounds in leaves and roots. The vascular pathogen Xanthomonas campestris pv. campestris (Xcc) enters through hydathodes, organs at the leaf margin involved in guttation. Subsequently, Xcc breaks out from infected hydathodes, progressing into the xylem vessels and causing systemic disease. To elucidate the mechanisms that underpin the different stages of Xcc pathogenesis, a need exists to image Xcc progression in planta in a non-invasive manner. Here, we describe a phenotyping setup and Python image analysis pipeline capturing the Xcc infection in 16 Arabidopsis thaliana plants in parallel over time. The setup used both an RGB to capture disease symptoms and an ultra-sensitive CCD camera to monitor bacterial progression inside the leaves using bioluminescence. We demonstrate that the image analysis pipeline reliably quantifies bacterial growth in planta for two bacterial species, that is vascular Xcc and the mesophyll pathogen Pseudomonas syringae pv. tomato. The resolution of the camera allowed early detection of Xcc in the hydathodes, yielding valuable information on this early stage of the Xcc infection process. The data obtained through the automated image analysis pipeline was robust and validated findings from other bioluminescence imaging methods, while requiring fewer samples. We can thus quantify the resistance level of a large number of Arabidopsis thaliana accessions and mutant lines to different bacterial strains in a non-invasive manner for phenotypic screenings.

plant biology↗

bioRxiv2024

Hydathode immunity against the vascular pathogen Xanthomonas campestris pv. campestris by the Arabidopsis CNL-type receptor SUT1

Bacterial plant pathogens exploit natural openings, such as pores or wounds, to enter the plant interior and cause disease. Plants guard these openings through defense mechanisms. However, bacteria from the genus Xanthomonas have specialized in that they enter their host via a special entry point, the hydathode--an organ at the leaf margin involved in xylem sap guttation. Hydathodes can mount an immune response against bacteria, including non-adapted and adapted pathogens like X. campestris pv. campestris (Xcc) that cause vascular disease. Previously, it was shown that the RKS1/ZAR1 immune complex confers vascular resistance against Xcc by recognizing XopAC activity, a type III effector (T3E). However, in absence of XopAC recognition, Arabidopsis Col-0 hydathodes still display resistance against Xcc. Here we mapped the causal gene using an inoculation method that promotes Xcc hydathode entry. Using a population of Recombinant Inbred Lines (RILs) of a cross between a susceptible (Oy-0) and resistant accession (Col-0), a major QTL for Xcc resistance was found on the right arm of Chromosome 5 in Col-0. Combining this result with a genome-wide association analysis yielded a single candidate gene encoding a coiled-coil nucleotide-binding leucine-rich repeat (CNL-type) immune receptor protein called SUPPRESSOR OF TOPP4 1 (SUT1). Expression of SUT1 was confirmed in hydathodes. We reveal that RKS1/ZAR1 and SUT1 confer different levels of Xcc resistance in different tissue types. Both RKS1/ZAR1 and SUT1 are alone sufficient for Xcc resistance in Col-0 hydathodes. However, RKS1/ZAR1 resistance is also effective in tissue types that represent late infection stages, i.e. xylem and mesophyll. In contrast, SUT1 resistance is not effective in the xylem, while weakly additive to RKS1/ZAR1 in the mesophyll. We thus identify a novel R gene, SUT1, that confers Xcc resistance primarily early in the infection during hydathode colonization. Author summaryBlack rot disease, caused by the bacterial pathogen Xanthomonas campestris pv. campestris (Xcc), is an economically important disease of cabbage crops. Xcc is unique in that it enters the plant interior through specialized organs at the leaf margin. These structures called hydathodes contain water pores and are involved in root pressure regulation. Although we know that hydathodes can mount an immune response against these bacteria, specific immune receptors still need to be discovered. Here we use the model plant Arabidopsis thaliana to map an hydathode-effective resistance gene. By screening two different Arabidopsis populations, we could map a single gene, SUT1, that is involved in this resistance. SUT1 restricts the early hydathode colonization by Xcc thus suppressing disease progression. Interestingly, SUT1 resistance was not effective in the plant vascular system, which the bacteria subsequently colonize. Therefore, this study provides a new insight into the role of hydathodes in anti-bacterial resistance in plants and opens the door for research on tissue- and organ-specific resistance mechanisms.

plant biology↗

bioRxiv2024

A SUMO interacting motif in the Replication initiator protein of Tomato yellow leaf curl virus is required for viral replication

CRESS-DNA viruses form a diverse group of viruses that use rolling-circle replication to replicate their genomes. They infect organisms in almost all branches of the eukaryotic tree of life. All CRESS-DNA viruses have one protein in common, the Replication initiator protein (Rep), which orchestrates viral replication using the host DNA replication machinery. In the case of the plant-infecting Geminiviridae, this multifunctional protein both recruits the host DNA replication machinery and manipulates posttranslational modification including Small ubiquitin-like modifier (SUMO) conjugation. In fact, Rep from two different geminiviruses, Tomato yellow leaf curl virus (TYLCV) and Tomato golden mosaic virus (TGMV), was shown to interact with the SUMO conjugating enzyme SCE1. Here, we demonstrate that also TYLCV Rep interacts with Arabidopsis SUMO1 and report on a SUMO interacting motif (SIM) in the SF3 helicase domain of Rep. Remarkably, an intact SIM proved to be important for the interaction of Rep with both SUMO1 and SCE1. The same motif was also essential for viral replication and Rep ATPase activity. Our findings thus connect the interaction between Rep and the SUMO machinery with viral replication of TYLCV. ImportanceThe identification of a non-canonical SUMO-interacting motif (SIM) within the Rep protein of Tomato yellow leaf curl virus (TYLCV) reveals a connection between viral replication and a protein modification, SUMOylation. Importantly, the motif was found to be conserved between Rep proteins from different geminiviruses. Functionally, the motif was critical for the interaction of Rep with proteins of the SUMO machinery, viral DNA replication, and Rep ATPase acitvity. In particular, the third position of the motif was important for each of these activities. We thus uncover a novel mechanism on how geminiviruses recruit the SUMO machinery likely to their own need.

plant biology↗

bioRxiv2023

Whitefly effector G4 interacts with tomato proteins of which MIPDB141 affects whitefly performance

The phloem-feeding insect Bemisia tabaci is an important pest, responsible for the transmission of several crop-threatening virus species. While feeding, the insect secretes a cocktail of effectors to modulate defense responses. Here, we present a set of proteins that was identified in artificial diet on which B. tabaci was salivating. We studied whether these candidate effectors can play a role in plant immune suppression. Effector G4 was the most robust suppressor of the flg22-induced ROS response when transiently expressed in Nicotiana benthamiana. In addition, G4 was able to supress ROS in Solanum lycopersicum (tomato) and Capsicum annuum (pepper). Fused to a fluorescence tag, G4 localized in the cytoplasm in N. benthamiana. A yeast two-hybrid screen combined with a luciferase bimolecular complementation and co-localization assays resulted in the identification of two target proteins in tomato: REF-like stress related protein 1 (RSP1) and meloidogyne-induced giant cell protein DB141 (MIPDB141). Silencing of MIPDB141 in tomato, using virus-induced gene silencing, reduced whitefly fecundity up to 40% demonstrating that the protein is involved in susceptibility to B. tabaci. Together our data demonstrate that effector G4 impairs tomato immunity to whiteflies by interfering with the ROS production and via a direct interaction with tomato susceptibility protein MIPDB141.

plant biology↗

bioRxiv2021

The protein modifier SUMO is critical for Arabidopsis shoot meristem maintenance at warmer ambient temperatures

Short heat waves (>37{degrees}C) are extremely damaging to non-acclimated plants and their capacity to recover from heat stress is key for their survival. To acclimate, the HEAT SHOCK TRANSCRIPTION FACTOR A1 (HSFA1) subfamily activates a transcriptional response that resolves incurred damages. In contrast, little is known how plants acclimate to sustained non-detrimental warm periods at 27-28{degrees}C. Plants respond to this condition with a thermomorphogenesis response. In addition, HSFA1 is critical for plant survival during these warm periods. We find that SUMO, a protein modification whose conjugate levels rise sharply during acute heat stress in eukaryotes, is critical too for plant longevity during warm periods, in particular for normal shoot meristem development. The known SUMO ligases were not essential to endure these warm periods, alone or in combination. Thermo-lethality was also not seen when plants lacked certain SUMO proteases or when SUMO chain formation was blocked. The SUMO-dependent thermo-resilience was as well independent of the autoimmune phenotype of the SUMO mutants. As acquired thermotolerance was normal in the sumo1/2 knockdown mutant, our data thus reveal a role for SUMO in heat acclimation that differs from HSFA1 and SIZ1. We conclude that SUMO is critical for shoot meristem integrity during warm periods. HighlightThe protein modifier SUMO governs shoot meristem maintenance in Arabidopsis allowing sustained rosette development when plants endure a sustained warm non-detrimental period of 28 degrees Celsius.

plant biology↗

bioRxiv2020

Contrasting functions of Arabidopsis SUMO1/2 isoforms with SUMO3 intersect to modulate innate immunity and global SUMOylome responses

Reversible covalent attachment of SMALL UBIQUITIN-LIKE MODIFIERS (SUMOs) on target proteins regulate diverse cellular process across all eukaryotes. In Arabidopsis thaliana, most mutants with perturbed global SUMOylome display severe impairments in growth and adaptations to physiological stresses. Since SUMOs self-regulate activities of SUMOylation-associated proteins, existence of multiple isoforms introduces possibilities of their functional intersections which remain unexplored especially in plant systems. Using well-established defense responses elicited against virulent and avirulent Pseudomonas syringae pv. tomato strains, we investigated crosstalks in individual and combinatorial Arabidopsis sum mutants. Here we report that while SUM1 and SUM2 additively, but not equivalently suppress basal and TNL-specific immunity via down-regulation of salicylic acid (SA)-dependent responses, SUM3 promotes these defenses genetically downstream of SA. Remarkably, the expression of SUM3 is transcriptionally suppressed by SUMO1 or SUMO2. The loss of SUM3 not only lowers basal or post-bacterial challenge responsive enhancements of SUMO1/2-congugates but also reduces upregulation dynamics of defensive proteins and SUMOylation-associated transcripts. Combining a sum3 mutation partially attenuates heightened immunity of sum1 or sum2 mutants suggesting intricate functional impingements among these isoforms in optimizing immune amplitudes. Similar SUM1-SUM3 intersections also affect global SUMOylome responses to heat-shock affecting most notably the induction of selective heat-shock transcription factors. Overall, our investigations reveal novel insights into auto-regulatory mechanisms among SUMO isoforms in host SUMOylome maintenance and adjustments to environmental challenges. Author SummaryIn plants, similar to animals, protein functions are regulated at multiple levels. One prevalent mode is to allow covalent linkage of small proteins to specific amino acids on targets thereby affecting its fate and function. One such kind of modification named as SUMOylation involves attachment of SUMO proteins. A plant maintains strict control over its pool of SUMOylated proteins (termed SUMOylome) which upon biotic or abiotic stresses are altered to facilitate appropriate responses, returning back to steady-state when the threat subsides. In mutants of the model plant Arabidopsis thaliana having disturbed steady-state SUMOylome, growth and developmental defects ensue. These mutants are auto-immune showing more resistance to infection by the bacterial pathogen Pseudomonas syringae. However, Arabidopsis SUMO-family are comprised of multiple members raising the question about their specificity or functional crosstalks. We discovered that two SUMO members function in coordination to suppress immunity including the repression of a third member which supports defenses. The expression of this third member during pathogen attack or heat-shock influences the responsive changes in the host SUMOylome likely suggesting SUMOs themselves play vital role in these adaptations. Overall, our work highlights novel intersections of SUMO members in mounting stress-specific responses.

plant biology↗

bioRxiv2018

The SUMO conjugation complex self-assembles into nuclear bodies independent of SIZ1 and COP1

One sentence SummarySUMO conjugation activity causes formation of SUMO nuclear bodies, which strongly overlap with COP1 bodies thanks to a substrate-binding (VP) motif in the E3 ligase SIZ1 that acts as bridge protein.\n\nAbstractAttachment of the small ubiquitin-like modifier SUMO to substrate proteins modulates their turnover, activity or interaction partners. An unresolved question is how this SUMO conjugation activity concentrates the enzymes involved and the substrates into uncharacterized nuclear bodies (NBs). We here define the requirements for the formation of SUMO NBs and for their subsequent co-localisation with the master regulator of growth, the E3 ubiquitin ligase COP1. COP1 activity results in degradation of transcription factors, which primes the transcriptional response that underlies elongation growth induced by night-time and high ambient temperatures (skoto- and thermomorphogenesis, respectively). SUMO conjugation activity itself is sufficient to target the SUMO machinery into NBs. Co-localization of these bodies with COP1 requires besides SUMO conjugation activity, a SUMO acceptor site in COP1 and the SUMO E3 ligase SIZ1. We find that SIZ1 docks in the substrate-binding pocket of COP1 via two VP motifs - a known peptide motif of COP1 substrates. The data reveal that SIZ1 physically connects COP1 and SUMO conjugation activity in the same NBs that can also contain the blue-light receptors CRY1 and CRY2. Our findings thus suggest that sumoylation apparently coordinates COP1 activity inside these NBs; a mechanism that potentially explains how SIZ1 and SUMO both control the timing and amplitude of the high-temperature growth response. The strong co-localization of COP1 and SUMO in these NBs might also explain why many COP1 substrates are sumoylated.\n\nFunding informationThe Netherlands Scientific Organisation (ALW-VIDI grant 864.10.004 to HvdB) and the Topsector T&U program Better Plants for Demands (grant 1409-036 to HvdB), including the partnering breeding companies, supported this work; FM is financially supported by Keygene N.V. (The Netherlands).

plant biology↗

bioRxiv2017

Phylogenomic Synteny Network Analysis Reveals an Ancient MADS-Box Transcription Factor Tandem Duplication and Lineage-Specific Transpositions

Conserved genomic context (or synteny) provides critical information for comparative evolutionary analysis, such as the inference of ancient polyploidy events, recurrent genomic rearrangements across species and gene ancestry. With the increase of sequenced and assembled plant genomes, we now have the opportunity to use synteny to analyze the dynamics of gene family expansion and contraction across broad phylogenetic groups. Here we present an integrated approach to organize plant kingdom-wide gene synteny networks using k-clique percolation. As an example, we analyzed the gene synteny network of the MADS-box transcription factor family based on fifty-one completed plant genomes. We conclude from two massive gene clusters that one of the two Type II MADS-box gene clades evolved from an ancient tandem gene duplication likely predating the radiation of seed plants, which then expanded by polyploidy events and sub-functionalization. This gene clade now contains key regulators of major phenotypes of angiosperms including flower development. Moreover, we find lineage-specific gene clusters derived from transposition events. For example, lineage-specific clusters in the Brassicales containing genes that are well-known for their function in controlling flower morphology (AP3 and PI). Our phylogenomic synteny network approach can be applied to any group of species to gain new insights into the evolution and dynamics of any set of genes.

genomics↗