Abstract 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.

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.

In plants, long-distance transport and phloem-mediated signal distribution play crucial roles in regulating stress adaptation and development. Phloem sap contains various types of RNAs, including small RNAs (sRNAs), messenger RNAs (mRNAs), and long noncoding RNAs (lncRNAs). Recently, endogenous circular RNAs have been identified in phloem sap of apple trees. Some phloem RNAs have been shown to have long-distance signaling functions, but for most, no functions have yet been determined. Due to their stability, circRNAs are interesting candidates with potential functions in long-distance signaling. Therefore, we aimed to characterize the circRNA content in the phloem sap of the crop plant Brassica napus. To achieve this, we performed Illumina sequencing of rRNA-depleted, circRNA-enriched, and sRNA libraries. The analysis revealed 1,734 distinct circRNAs in the phloem sap of B. napus. Of these, we validated ten circRNAs by PCR amplification and Sanger sequencing of their back-splicing junctions (BSJs). Using circ-Panel Nanopore sequencing, we investigated the full-length sequences of 14 circRNAs from phloem sap and leaf samples, identifying seven high-confidence candidates that exhibit potential intron retention and isoform variation across the two tissues. The investigation of potential interaction partners from phloem circRNA identified miRNA target sites on multiple circRNAs, particularly for known phloem-mobile miRNAs like miR156, miR169, and miR395. With Microscale Thermophoresis (MST), we were able to show the ability of the abundant RNA-binding protein BnGRP7 to bind the phloem circRNA circBnaANL2(7,8) with a dissociation constant of around 1 µM, raising questions about the involvement of RBPs in circRNA transport, stabilization, and function in phloem sap.

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.

BACKGROUND:Plant viral movement protein (MP) function is decisive for virus cell-to-cell movement. Often, MPs also induce membrane alterations, which are believed to play a role for the establishment of viral replication compartments. Despite these central roles in virus infection, knowledge of the underlying molecular mechanisms by which MPs cause changes in plasmodesmata (PD) size exclusion limit and contribute to the formation of viral replication compartments remain far from being complete. METHODS:To further identify host processes subverted by viral MPs, we here characterized the MP of Japanese soil-borne wheat mosaic virus (JSBWMV). We used confocal fluorescence microscopy to study the subcellular localization of MPJSBWMV and to address its functionality in promoting virus cell-to-cell movement. Using the biochemical and biophysical methods co-immunoprecipitation, fluorescence lifetime imaging, microscale thermophoresis and RNA immunoprecipitation we investigate the capacity of MPJSBWMV to multimerize and to bind viral and cellular RNAs. RESULTS:MPJSBWMV localized to PD, promoted cell-to-cell movement by complementing a movement-deficient unrelated virus, formed multimers in-vivo and bound to viral RNA with high affinity. Using RNA immunoprecipitation, we identified host RNAs associated with the viral MP. Within the MP-RNA complexes we found RNAs encoding proteins with key functions in membrane modification, signaling, protein folding, and degradation. We propose that binding of MP to these RNAs during infection and regulation of their spatio-temporal translation may represent a mechanism for MPs to achieve PD and host control during replication and movement. CONCLUSION:This study provides new insight into the complex interactions between viral MPs and host cellular processes.

CRISPR/Cas technology is an emerging tool for the identification of nucleic acids sequences. Here, we present a user-friendly, extraction-free, rapid protocol for specific on-site detection of plant viruses using CRISPR/Cas13a. Version 2: corrected the concentration of RNaseAlert in step 9 (from 200μM to 200nM).

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.

The Phloem-Associated RNA-Chaperone-Like (PARCL) protein is a plant-specific RNA-binding protein (RBP) that is highly abundant in the phloem. PARCL has been observed to form large biomolecular condensates that move within the phloem stream, potentially being involved in RNA transport. Here, we present results on unraveling drivers for PARCL’s phase separation. We used coarse-grained molecular dynamics simulations to compute a residue interaction map that identifies candidate residues involved in phase separation. Subsequent simulations with mutations of candidate residues resulted in disrupted condensation, supporting their involvement in phase separation. We performed in vitro and in vivo experiments to validate these predictions. To investigate the RNA-binding of PARCL, we added microRNA to the simulations and identified a short region of PARCL that consistently made contact with the miRNA in agreement with bioinformatics predictions and experiments. We discuss the implications of our findings in terms of model-guided engineering of biomolecular condensates.

Short-read RNA-seq studies of grafted plants have led to the proposal that thousands of messenger RNAs (mRNAs) move over long distances between plant tissues1–7, potentially acting as signals8–12. Transport of mRNAs between cells and tissues has been shown to play a role in several physiological and developmental processes in plants, such as tuberization13, leaf development14 and meristem maintenance15; yet for most mobile mRNAs, the biological relevance of transport remains to be determined16–19. Here we perform a meta-analysis of existing mobile mRNA datasets and examine the associated bioinformatic pipelines. Taking technological noise, biological variation, potential contamination and incomplete genome assemblies into account, we find that a high percentage of currently annotated graft-mobile transcripts are left without statistical support from available RNA-seq data. This meta-analysis challenges the findings of previous studies and current views on mRNA communication. This study reveals that a substantial number of transcripts that are currently annotated as graft-mobile lack statistical support from available RNA-seq data.

Short-read RNA-Seq analyses of grafted plants have led to the proposal that large numbers of mRNAs move over long distances between plant tissues, acting as potential signals. The detection of transported transcripts by RNA-Seq is both experimentally and computationally challenging, requiring successful grafting, delicate harvesting, rigorous contamination controls and data processing approaches that can identify rare events in inherently noisy data. Here, we perform a meta-analysis of existing datasets and examine the associated bioinformatic pipelines. Our analysis reveals that technological noise, biological variation and incomplete genome assemblies give rise to features in the data that can distort the interpretation. Taking these considerations into account, we find that a substantial number of transcripts that are currently annotated as mobile are left without support from the available RNA-Seq data. Whilst several annotated mobile mRNAs have been validated, we cannot exclude that others may be false positives. The identified issues may impact also other RNA-Seq studies, in particular those using single nucleotide polymorphisms (SNPs) to detect variants. ### Competing Interest Statement The authors have declared no competing interest.

In recent years, extracellular vesicles (EVs) have emerged as novel key players in plant-microbe interactions. While it is immensely useful to draw on the established "minimal information for studies of extracellular vesicles" (MISEV) guidelines and precedents in mammalian systems, working with plants and their associated microbes poses specific challenges. To navigate researchers through these obstacles, we offer detailed step-by-step suggestions for those embarking on EV research in the context of plant-microbe interactions. The advice is based on recent publications and our collective experience from the diverse plant and microbe systems studied in a dedicated research consortium. We provide considerations for experimental design, optimization, quality control, and recommendations on how to increase yield, purity, and reproducibility of EV isolation. With this perspective article, we aim not only to assist researchers in our field but also to promote discussions on plant and microbe EVs in the broader EV community.

In Arabidopis a high number of distinct mRNAs move from shoot to root. We previously reported on the correlation of m5C-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 m5C 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 m5C-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 m5C 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. ### Competing Interest Statement The authors have declared no competing interest.

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. ### Competing Interest Statement The authors have declared no competing interest.

Summary 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.

RNA interference (RNAi) is a crucial mechanism in immunity against infectious microbes through the action of DICER-LIKE (DCL) and ARGONAUTE (AGO) proteins. In the case of the taxonomically diverse fungal pathogen Botrytis cinerea and the oomycete Hyaloperonospora arabidopsidis, plant DCL and AGO proteins have proven roles as negative regulators of immunity, suggesting functional specialization of these proteins. To address this aspect in a broader taxonomic context, we characterized the colonization pattern of an informative set of DCL and AGO loss-of-function mutants in Arabidopsis thaliana upon infection with a panel of pathogenic microbes with different lifestyles, and a fungal mutualist. Our results revealed that, depending on the interacting pathogen, AGO1 acts as a positive or negative regulator of immunity, while AGO4 functions as a positive regulator. Additionally, AGO2 and AGO10 positively modulated the colonization by a fungal mutualist. Therefore, analyzing the role of RNAi across a broader range of plant-microbe interactions has identified previously unknown functions for AGO proteins. For some pathogen interactions, however, all tested mutants exhibited wild-type-like infection phenotypes, suggesting that the roles of AGO and DCL proteins in these interactions may be more complex to elucidate.

Microscale thermophoresis (MST) is a well-established method to quantify protein-RNA interactions. In this study, we employed MST to analyze the RNA binding properties of glycine-rich RNA binding protein 7 (GRP7), which is known to have multiple biological functions related to its ability to bind different types of RNA. However, the exact mechanism of GRP7's RNA binding is not fully understood. While the RNA-recognition motif of GRP7 is known to be involved in RNA binding, the glycine-rich region (known as arginine-glycine-glycine-domain or RGG-domain) also influences this interaction. To investigate to which extend the RGG-domain of GRP7 is involved in RNA binding, mutation studies on putative RNA interacting or modulating sites were performed. In addition to MST experiments, we examined liquid-liquid phase separation of GRP7 and its mutants, both with and without RNA. Furthermore, we systemically investigated factors that might affect RNA binding selectivity of GRP7 by testing RNAs of different sizes, structures, and modifications. Consequently, our study revealed that GRP7 exhibits a high affinity for a variety of RNAs, indicating a lack of pronounced selectivity. Moreover, we established that the RGG-domain plays a crucial role in binding longer RNAs and promoting phase separation.

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.

Ribosome biogenesis is a key process in all eukaryotic cells that requires hundreds of ribosome biogenesis factors (RBFs), which are essential to build the mature ribosomes consisting of proteins and rRNAs. The processing of the required rRNAs has been studied extensively in yeast and mammals, but in plants much is still unknown. In this study, we focused on a RBF from A. thaliana that we named NUCLEOLAR RNA CHAPERONE-LIKE 1 (NURC1). NURC1 was localized in the nucleolus of plant cell nuclei, and other plant RBF candidates shared the same localization. SEC-SAXS experiments revealed that NURC1 has an elongated and flexible structure. In addition, SEC-MALLS experiments confirmed that NURC1 was present in its monomeric form with a molecular weight of around 28 kDa. RNA binding was assessed by performing microscale thermophoresis with the Arabidopsis internal transcribed spacer 2 (ITS2) of the polycistronic pre-rRNA precursor, which contains the 5.8S, 18S, and 25S rRNA. NURC1 showed binding activity to the ITS2 with a dissociation constant of 228 nM and exhibited RNA chaperone-like activity. Our data suggested that NURC1 may have a function in pre-rRNA processing and thus ribosome biogenesis.

There is now a wealth of data, from different plants and labs and spanning more than two decades, which unequivocally demonstrates that RNAs can be transported over long distances, from the cell where they are transcribed to distal cells in other tissues. Different types of RNA molecules are transported, including micro- and messenger RNAs. Whether these RNAs are selected for transport and, if so, how they are selected and transported remain, in general, open questions. This aspect is likely not independent of the biological function and relevance of the transported RNAs, which are in most cases still unclear. In this review, we summarize the experimental data supporting selectivity or nonselectivity of RNA translocation and review the evidence for biological functions. After discussing potential issues regarding the comparability between experiments, we propose criteria that need to be critically evaluated to identify important signaling RNAs.

The long-distance transport of messenger RNAs (mRNAs) has been shown to be important for several developmental processes in plants. A popular method for identifying travelling mRNAs is to perform RNA-Seq on grafted plants. This approach depends on the ability to correctly assign sequenced mRNAs to the genetic background from which they originated. The assignment is often based on the identification of single-nucleotide polymorphisms (SNPs) between otherwise identical sequences. A major challenge is therefore to distinguish SNPs from sequencing errors. Here, we show how Bayes factors can be computed analytically using RNA-Seq data over all the SNPs in an mRNA. We used simulations to evaluate the performance of the proposed framework and demonstrate how Bayes factors accurately identify graft-mobile transcripts. The comparison with other detection methods using simulated data shows how not taking the variability in read depth, error rates and multiple SNPs per transcript into account can lead to incorrect classification. Our results suggest experimental design criteria for successful graft-mobile mRNA detection and show the pitfalls of filtering for sequencing errors or focusing on single SNPs within an mRNA.