Biology subjects

Ostendorp, S.

Publications and source records attributed to Ostendorp, S..

6 recordsLinked to original sources

bioRxiv2026

Pathogen cell wall degrading enzymes facilitate extracellular vesicles to deliver RNA into plants

Extracellular vesicles (EVs) can deliver RNA and proteins into host cells to manipulate immunity; but how EVs transverse the cell wall is unknown. Using the fungal pathogen Botrytis cinerea that induces cross-kingdom RNA interference in plants, we uncovered that EV-mediated RNA delivery was dependent on cell wall degrading enzymes. Through fluorescence and transmission electron microscopy, and molecular genetic techniques, we demonstrate that EV-associated proteins compromise the plant cell wall, thereby facilitating RNA delivery. Notably, cell wall degrading enzymes are commonly associated with EVs across plant-colonizing bacterial, fungal and oomycete species, indicating a conserved role in EV transport across cell walls. These findings uncover a formerly unknown mechanism by which cell wall degrading enzymes facilitate EVs to transverse the cell wall for cargo delivery in cross-kingdom communication.

plant biology↗

bioRxiv2026

VIRP1 bromodomain shapes nuclear condensate formation and has a positive effect on PSTVd accumulation

O_LIViroids are small, non-coding RNAs that rely on host factors for replication, intracellular trafficking and systemic movement. VIRP1, a Bromodomain and Extra-terminal domain (BET) protein, has previously been implicated in Potato spindle tuber viroid (PSTVd) infection, yet its precise role and mode of action remain unresolved. C_LIO_LIIn this work, we highlight VIRP1 as the only Solanaceae BET protein containing a proline-rich domain, which overlaps with the PSTVd-binding site. VIRP1-deficient plants exhibit delayed flowering and increased ABA sensitivity, with differentially expressed genes enriched in stress-related pathways. C_LIO_LIVIRP1 forms condensates in planta and in vitro, consistent with phase-separation behaviour. Condensate morphology is altered by PSTVd RNA, deletion of the intrinsically disordered CTD and bromodomain mutations. C_LIO_LIVIRP1 is particularly important for the early establishment of PSTVd infection, while nuclear localization and bromodomain integrity are required for efficient viroid accumulation. By contrast, the disordered CTD region is dispensable for complementation of PSTVd accumulation. C_LIO_LIOur results support a model in which VIRP1 acts as a host nuclear factor that links stress-related functions, nuclear condensate formation and early viroid infection. C_LI

plant biology↗

bioRxiv2024

Targeted engineering of the phase separating PARCL protein

PARCL is a plant-specific RNA-binding protein (RBP) that exhibits chaperone activity, is abundant in the phloem, intrinsically disordered, and contains a prion-like domain (PLD). PARCL proteins have been observed to form large biomolecular condensates in vivo and in vitro. Biomolecular condensates are membraneless compartments, wherein biomolecules become partitioned from their surrounding liquid environment into liquid droplets with their own composition, dynamics, and function. Which molecular properties drive phase separation is of great interest for targeted engineering efforts. Here, we present results on residue interactions derived from simulations of PARCL using course-grained molecular dynamics with the HPS-Urry model. We adjust the parameters of the simulations to allow the inclusion of folded eYFP tags, since fluorescent tags are often used in phase separation experiments for visualising droplets, yet have not been included in simulations to date. While still simulating phase separation, these trajectories suggest minor changes to droplet and network structure when proteins contain eYFP. By analysing the residues of the PARCL molecules that come within contact distance in the simulations, we identify which individual residues drive phase separation. To experimentally validate these findings, we introduced mutations of the most contacted residues and could indeed confirm that these mutations prevent the formation of condensate droplets. To investigate the RNA-binding of PARCL, we added microRNA to the simulation and find a short region of PARCL consistently making contact with the miRNA, which is also in agreement with predictions and experiments. We discuss the implications of our findings in terms of model-guided engineering of biomolecular condensates.

biophysics↗

bioRxiv2024

Systemic mRNA transport depends on m5C methylation, nuclear mRNA export factors and developmental phase changes

In grafted plants mRNAs can be transported from shoot to root, however, how their mobility is regulated remains poorly understood. Recent work has shown that m5C methylation plays an essential role in systemic mRNA mobility in vegetative plants, but its role at later developmental stages remains unclear. To address this, we examined the mobility of three mobile mRNAs, TCTP1, HSC70.1 and GRP7 in mutants deficient in m5C mRNA methylation. By comparing vegetative and flowering stages, we found that in contrast to GRP7, the reduced mobility of TCTP1 and HSC70.1 observed in the vegetative growth phase is restored upon flowering, indicating that m5C-dependent regulation of RNA transport is developmentally controlled. We further identified two RNA-binding proteins, the nuclear mRNA export factors ALY2 and ALY4, as phloem mobile. ALY2 and ALY4 deficient plants show a similar developmental effect on mRNA mobility as m5C mRNA methylation mutants, suggesting that they contribute to the regulation of systemic mRNA transport. Notably, combined perturbation of m5C methylation and ALY2 or ALY4 results in reduced mRNA mobility even in flowering plants. Together, our findings reveal an unexpected developmental layer of control over mRNA transport and identify the nuclear mRNA export factors ALY2 and ALY4 as potential regulators of long-distance RNA movement. SIGNIFICANCE STATEMENTmRNA m5C methylation is essential for shoot-to-root mRNA transport during plant growth; however, whether and how mRNA mobility is regulated, and whether it changes during development remains unclear. We show that TCTP1 and HSC70.1 mRNA transport is reduced in vegetatively growing m5C-deficient mutants but remains unaffected upon flowering, and identify ALY2 and ALY4 proteins, two nuclear export factors, as additional regulators of long-distance mRNA transport, providing new layer of mRNA transport.

plant biology↗

bioRxiv2024

Rapid, direct, and sequence-specific identification of RNA viruses in various crop plants using CRISPR/Cas13a

Plant viruses are destructive pathogens causing significant damage to various crop species. Rapid, sensitive, and specific detection is crucial for the effective containment of emerging and resistance-breaking viruses. CRISPR/Cas has been established as a useful tool for plant virus identification. However, its application for on-site, direct detection of viruses from plant tissues is still limited. In this study, we present a rapid method for detecting viruses directly from RNA of different crop species using CRISPR/Cas13a. We successfully applied this method to identify tomato brown rugose fruit virus (ToBRFV) in infected tomato plants and differentiate it from closely related tobamoviruses. ToBRFV could be identified in a 100-fold dilution and early during infection, prior to the onset of viral symptoms. Moreover, CRISPR/Cas13a was used to directly identify cucumber green mottle mosaic virus (CGMMV) in cucumber plants and turnip mosaic virus (TuMV) in Brassica napus plants. Finally, we developed a user-friendly, extraction-free, 15-minute protocol for on-site ToBRFV identification using a portable fluorescent viewer and a mobile phone camera. This protocol was successfully applied for ToBRFV detection in a commercial greenhouse. These results demonstrate that CRISPR/Cas13a is a robust technology for direct, rapid, sensitive, and specific identification of multiple viruses in different crop plants that can be easily implemented for on-site detection.

plant biology↗

bioRxiv2022

Non-cell-autonomous HSC70.1 chaperone displays homeostatic feed-back regulation by binding its own mRNA

Heat shock proteins of the HSC70/HSP70 family are evolutionarily conserved chaperones that are involved in protein folding, protein transport and RNA binding. Arabidopsis HSC70 chaperones are thought to act as housekeeping chaperones and as such are involved in many growth-related pathways. Whether Arabidopsis HSC70 binds RNA and its function has remained an open question. Here, we show that the HSC70.1 chaperone binds its own mRNA via its C-terminal Short Variable Region (SVR) and inhibits its own translation. We propose that this negative protein-transcript feedback loop may establish an on-demand chaperone pool that allows for a rapid response to stress. Furthermore, we show that the SVR encoding RNA region is necessary for HSC70.1 transcript mobility to distant tissues and that HSC70.1 transcript and not protein mobility is required to rescue root growth and flowering time of hsc70 mutants. In summary, it seems that the Arabidopsis HSC70.1 chaperone can form a complex with its own transcript to regulate its translation and that both protein and transcript can act in a non-cell-autonomous manner maintaining chaperone homestasis between tissues.

plant biology↗