Steffen Ostendorp

SUMMARY: Viroids 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. In 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. VIRP1 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. VIRP1 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. Our 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.

As obligate intracellular pathogens, plant viruses interact with host factors to achieve efficient replication and spread. Host factors also play important roles in virus detection and antiviral defence. Furoviruses cause substantial losses in cereals and efficient control measures are lacking. We therefore used Japanese soil-borne wheat mosaic furovirus movement protein (MP ^JSBWMV ) in immunoprecipitation experiments to identify host interaction partners in the model host plant Nicotiana benthamiana . We identified a P-type pentatricopeptide repeat-containing protein (NbPPR) as the interaction partner of the MP ^JSBWMV . Fluorescent protein-tagged NbPPR localized to chloroplasts and to the cytosol and plasmodesmata (PD) depending on the location of the fluorescent protein tag, suggesting that the protein may be dually localized to these cellular compartments. MP ^JSBWMV :RFP and GFP:NbPPR colocalized and interacted at PD as shown by fluorescence resonance energy transfer fluorescence lifetime imaging and co-immunoprecipitation. To functionally characterize the MP ^JSBWMV -interacting NbPPR, we immunoprecipitated GFP:NbPPR-interacting RNAs and identified a motif overrepresented in the GFP:NbPPR-bound RNAs. The motif is consistent with the PPR target sequence predicted using PPR code. While most of the RNA targets encode subunits of the organellar NADH-dehydrogenase complex, we also identified RNAs encoding cell wall proteins. Downregulation of NbPPR, using virus-induced gene silencing and subsequent infection with fluorescent protein-tagged viruses, resulted in increased numbers of infection sites for JSBWMV in plants silenced for NbPPR compared to controls. We propose a model to explain how virus infection may affect NbPPR function and how NbPPR may regulate the infection cycle. As PPRs are increasingly explored for the modulation of gene expression, virus-targeting PPRs may be developed into tools for virus control in future.

Plant viruses are destructive pathogens of 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 new tool for plant virus identification. However, its application for direct detection of viruses in the field is still limited. In this study, we present a CRISPR/Cas13a-based method for rapid detection of different viruses directly from RNA of several crop species, including tomato, cucumber, and rapeseed. This method was used to identify the emerging tomato brown rugose fruit virus (ToBRFV), a prominent pathogen in tomato cultivation, and distinguish it from closely related viruses in infected tomato plants. ToBRFV could be identified in a 100-fold dilution and early during infection, prior to the onset of viral symptoms. Finally, we developed a user-friendly, extraction-free, 15 min protocol for on-site virus detection using a portable fluorescence viewer and a mobile phone camera. This protocol was successfully applied to identify ToBRFV in several commercial greenhouses. These results demonstrate that CRISPR/Cas13a is a robust technology for on-site detection of multiple viruses in different crop plants. This method could be swiftly adapted to identify newly emerging pests, which threaten global food security.

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.

In Arabidopis a high number of distinct mRNAs move from shoot to root. We previously reported on the correlation of m ⁵ C-methylation and lack of mRNA transport in juvenile plants depending on the RNA methyltransferases DNMT2 NSUN2B . However, to our surprise we uncovered that lack of DNMT2 NSUN2B (writer) activity did not abolished transport of TCTP1 and HSC70.1 transcripts in flowering plants. We uncovered that transport of both transcripts is reinstated in dnmt2 nsun2b mutants after commitment to flowering. This finding suggests that additional factors are seemingly involved in regulating / mediating mRNA transport. In search of such candidates, we identified the two ALY2 and ALY4 nuclear mRNA export factors belonging to the ALYREF family as bona fide m ⁵ C readers mediating mRNA transport. We show that both proteins are allocated along the phloem and that they bind preferentially to mobile mRNAs. MST measurements indicate that ALY2 and ALY4 bind to mobile mRNAs with relative high affinity with ALY4 showing higher affinity towards m ⁵ C-methylated mobile mRNAs. An analysis of the graft-mobile transcriptome of juvenile heterografted-grafted wild type, dnmt2 nsun2b , aly2 and aly4 mutants revealed that the nuclear export factors are key regulators of mRNA transport. We suggest that depending on the developmental stage m ⁵ C methylation has a negative and positive regulatory function in mRNA transport and acts together with ALY2 and ALY4 to facilitate mRNA transport in both juvenile and flowering plants.

Summary: The HSC70/HSP70 family of heat shock proteins are evolutionarily conserved chaperones 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 whether this interaction is functional has remained an open question. We provide evidence that the HSC70.1 chaperone binds its own mRNA via its C‐terminal short variable region (SVR) and inhibits its own translation. The SVR encoding mRNA 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. We propose that this negative protein‐transcript feedback loop may establish an on‐demand chaperone pool that allows for a rapid response to stress. In summary, our data suggest 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 noncell‐autonomous manner, potentially maintaining chaperone homeostasis between tissues.

Summary: 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.