AtHVA22a, a plant-specific homologue of Reep/DP1/Yop1 family proteins is involved in turnip mosaic virus propagation.

The movement of potyviruses, the largest genus of single-stranded, positive-sense RNA viruses responsible for serious diseases in crops, is very complex. As potyviruses developed strategies to hijack the host secretory pathway and plasmodesmata (PD) for their transport, the goal of this study was to identify membrane and/or PD-proteins that interact with the 6K2 protein, a potyviral protein involved in replication and cell-to-cell movement of turnip mosaic virus (TuMV). Using split-ubiquitin membrane yeast two-hybrid assays, we screened an Arabidopsis cDNA library for interactors of TuMV6K2. We isolated AtHVA22a (Hordeum vulgare abscisic acid responsive gene 22), which belongs to a multigenic family of transmembrane proteins, homologous to Receptor expression-enhancing protein (Reep)/Deleted in polyposis (DP1)/Yop1 family proteins in animal and yeast. HVA22/DP1/Yop1 family genes are widely distributed in eukaryotes, but the role of HVA22 proteins in plants is still not well known, although proteomics analysis of PD fractions purified from Arabidopsis suspension cells showed that AtHVA22a is highly enriched in a PD proteome. We confirmed the interaction between TuMV6K2 and AtHVA22a in yeast, as well as in planta by using bimolecular fluorescence complementation and showed that TuMV6K2/AtHVA22a interaction occurs at the level of the viral replication compartment during TuMV infection. Finally, we showed that the propagation of TuMV is increased when AtHVA22a is overexpressed in planta but slowed down upon mutagenesis of AtHVA22a by CRISPR-Cas9. Altogether, our results indicate that AtHVA22a plays an agonistic effect on TuMV propagation and that the C-terminal tail of the protein is important in this process.

Heavy Metal-Associated Isoprenylated Plant Proteins (HIPPs) at Plasmodesmata: Exploring the Link between Localization and Function.

Heavy metal-associated isoprenylated plant proteins (HIPPs) are a metallochaperone-like protein family comprising a combination of structural features unique to vascular plants. HIPPs possess both one or two heavy metal-binding domains and an isoprenylation site, facilitating a posttranslational protein lipid modification. Recent work has characterized individual HIPPs across numerous different species and provided evidence for varied functionalities. Interestingly, a significant number of HIPPs have been identified in proteomes of plasmodesmata (PD)-nanochannels mediating symplastic connectivity within plant tissues that play pivotal roles in intercellular communication during plant development as well as responses to biotic and abiotic stress. As characterized functions of many HIPPs are linked to stress responses, plasmodesmal HIPP proteins are potentially interesting candidate components of signaling events at or for the regulation of PD. Here, we review what is known about PD-localized HIPP proteins specifically, and how the structure and function of HIPPs more generally could link to known properties and regulation of PD.

Cell-to-Cell Connectivity Assays for the Analysis of Cytoskeletal and Other Regulators of Plasmodesmata.

The actin cytoskeleton has close but so far incompletely understood connections to plasmodesmata, the cell junctions of plants. Plasmodesmata are essential for plant development and responses to biotic and abiotic stresses and facilitate the intercellular exchange of metabolites and hormones but also macromolecules such as proteins and RNAs. The molecular size exclusion limited of plasmodesmata is dynamically regulated, including by actin-associated proteins. Therefore, experimental analysis of plasmodesmal regulation can be relevant to plant cytoskeleton research. This chapter presents two simple imaging-based protocols for analyzing macromolecular cell-to-cell connectivity in leaves.

Tomato SlGSTU38 interacts with the PepMV coat protein and promotes viral infection.

Pepino mosaic virus (PepMV) is pandemic in tomato crops, causing important economic losses world-wide. No PepMV-resistant varieties have been developed yet. Identification of host factors interacting with PepMV proteins is a promising source of genetic targets to develop PepMV-resistant varieties. The interaction between the PepMV coat protein (CP) and the tomato glutathione S-transferase (GST) SlGSTU38 was identified in a yeast two-hybrid (Y2H) screening and validated by directed Y2H and co-immunoprecipitation assays. SlGSTU38-knocked-out Micro-Tom plants (gstu38) generated by the CRISPR/Cas9 technology together with live-cell imaging were used to understand the role of SlGSTU38 during infection. The transcriptomes of healthy and PepMV-infected wild-type (WT) and gstu38 plants were profiled by RNA-seq analysis. SlGSTU38 functions as a PepMV-specific susceptibility factor in a cell-autonomous manner and relocalizes to the virus replication complexes during infection. Besides, knocking out SlGSTU38 triggers reactive oxygen species accumulation in leaves and the deregulation of stress-responsive genes. SlGSTU38 may play a dual role: On the one hand, SlGSTU38 may exert a proviral function depending on its specific interaction with the PepMV CP; and on the other hand, SlGSTU38 may delay PepMV-infection sensing by participating in the redox intracellular homeostasis in a nonspecific manner.

Reticulons 3 and 6 interact with viral movement proteins.

Plant reticulon (RTN) proteins are capable of constricting membranes and are vital for creating and maintaining tubules in the endoplasmic reticulum (ER), making them prime candidates for the formation of the desmotubule in plasmodesmata (PD). RTN3 and RTN6 have previously been detected in an Arabidopsis PD proteome and have been shown to be present in primary PD at cytokinesis. It has been suggested that RTN proteins form protein complexes with proteins in the PD plasma membrane and desmotubule to stabilize the desmotubule constriction and regulate PD aperture. Viral movement proteins (vMPs) enable the transport of viruses through PD and can be ER-integral membrane proteins or interact with the ER. Some vMPs can themselves constrict ER membranes or localize to RTN-containing tubules; RTN proteins and vMPs could be functionally linked or potentially interact. Here we show that different vMPs are capable of interacting with RTN3 and RTN6 in a membrane yeast two-hybrid assay, coimmunoprecipitation, and Förster resonance energy transfer measured by donor excited-state fluorescence lifetime imaging microscopy. Furthermore, coexpression of the vMP CMV-3a and RTN3 results in either the vMP or the RTN changing subcellular localization and reduces the ability of CMV-3a to open PD, further indicating interactions between the two proteins.

RNA Imaging with RNase-Inactivated Csy4 in Plants and Filamentous Fungi.

Subcellular localizations of RNAs can be imaged in vivo with genetically encoded reporters consisting of a sequence-specific RNA-binding protein (RBP) fused to a fluorescent protein. Several such reporter systems have been described based on RBPs that recognize RNA stem-loops. Here we describe RNA tagging for imaging with an inactive mutant of the bacterial endonuclease Csy4, which has a significantly higher affinity for its cognate stem-loop than alternative systems. This property allows for sensitive imaging with only few tandem copies of the target stem-loop inserted into the RNA of interest.

Creating Contacts Between Replication and Movement at Plasmodesmata - A Role for Membrane Contact Sites in Plant Virus Infections?

To infect their hosts and cause disease, plant viruses must replicate within cells and move throughout the plant both locally and systemically. RNA virus replication occurs on the surface of various cellular membranes, whose shape and composition become extensively modified in the process. Membrane contact sites (MCS) can mediate non-vesicular lipid-shuttling between different membranes and viruses co-opt components of these structures to make their membrane environment suitable for replication. Whereas animal viruses exit and enter cells when moving throughout their host, the rigid wall of plant cells obstructs this pathway and plant viruses therefore move between cells symplastically through plasmodesmata (PD). PD are membranous channels connecting nearly all plant cells and are now viewed to constitute a specialized type of endoplasmic reticulum (ER)-plasma membrane (PM) MCS themselves. Thus, both replication and movement of plant viruses rely on MCS. However, recent work also suggests that for some viruses, replication and movement are closely coupled at ER-PM MCS at the entrances of PD. Movement-coupled replication at PD may be distinct from the main bulk of replication and virus accumulation, which produces progeny virions for plant-to-plant transmission. Thus, MCS play a central role in plant virus infections, and may provide a link between two essential steps in the viral life cycle, replication and movement. Here, we provide an overview of plant virus-MCS interactions identified to date, and place these in the context of the connection between viral replication and cell-to-cell movement.

Multiple C2 domains and transmembrane region proteins (MCTPs) tether membranes at plasmodesmata.

In eukaryotes, membrane contact sites (MCS) allow direct communication between organelles. Plants have evolved a unique type of MCS, inside intercellular pores, the plasmodesmata, where endoplasmic reticulum (ER)-plasma membrane (PM) contacts coincide with regulation of cell-to-cell signalling. The molecular mechanism and function of membrane tethering within plasmodesmata remain unknown. Here, we show that the multiple C2 domains and transmembrane region protein (MCTP) family, key regulators of cell-to-cell signalling in plants, act as ER-PM tethers specifically at plasmodesmata. We report that MCTPs are plasmodesmata proteins that insert into the ER via their transmembrane region while their C2 domains dock to the PM through interaction with anionic phospholipids. A Atmctp3/Atmctp4 loss of function mutant induces plant developmental defects, impaired plasmodesmata function and composition, while MCTP4 expression in a yeast Δtether mutant partially restores ER-PM tethering. Our data suggest that MCTPs are unique membrane tethers controlling both ER-PM contacts and cell-to-cell signalling.

Hitchhikers, highway tolls and roadworks: the interactions of plant viruses with the phloem.

The phloem is of central importance to plant viruses, providing the route by which they spread throughout their host. Compared with virus movement in non-vascular tissue, phloem entry, exit, and long-distance translocation usually involve additional viral factors and complex virus-host interactions, probably, because the phloem has evolved additional protection against these molecular 'hitchhikers'. Recent progress in understanding phloem trafficking of endogenous mRNAs along with observations of membranous viral replication 'factories' in sieve elements challenge existing conceptions of virus long-distance transport. At the same time, the central role of the phloem in plant defences against viruses and the sophisticated viral manipulation of this host tissue are beginning to emerge.

Efficient Detection of Long dsRNA in Vitro and in Vivo Using the dsRNA Binding Domain from FHV B2 Protein.

Double-stranded RNA (dsRNA) plays essential functions in many biological processes, including the activation of innate immune responses and RNA interference. dsRNA also represents the genetic entity of some viruses and is a hallmark of infections by positive-sense single-stranded RNA viruses. Methods for detecting dsRNA rely essentially on immunological approaches and their use is often limited to in vitro applications, although recent developments have allowed the visualization of dsRNA in vivo. Here, we report the sensitive and rapid detection of long dsRNA both in vitro and in vivo using the dsRNA binding domain of the B2 protein from Flock house virus. In vitro, we adapted the system for the detection of dsRNA either enzymatically by northwestern blotting or by direct fluorescence labeling on fixed samples. In vivo, we produced stable transgenic Nicotiana benthamiana lines allowing the visualization of dsRNA by fluorescence microscopy. Using these techniques, we were able to discriminate healthy and positive-sense single-stranded RNA virus-infected material in plants and insect cells. In N. benthamiana, our system proved to be very potent for the spatio-temporal visualization of replicative RNA intermediates of a broad range of positive-sense RNA viruses, including high- vs. low-copy number viruses.

Architecture and permeability of post-cytokinesis plasmodesmata lacking cytoplasmic sleeves.

Plasmodesmata are remarkable cellular machines responsible for the controlled exchange of proteins, small RNAs and signalling molecules between cells. They are lined by the plasma membrane (PM), contain a strand of tubular endoplasmic reticulum (ER), and the space between these two membranes is thought to control plasmodesmata permeability. Here, we have reconstructed plasmodesmata three-dimensional (3D) ultrastructure with an unprecedented level of 3D information using electron tomography. We show that within plasmodesmata, ER-PM contact sites undergo substantial remodelling events during cell differentiation. Instead of being open pores, post-cytokinesis plasmodesmata present such intimate ER-PM contact along the entire length of the pores that no intermembrane gap is visible. Later on, during cell expansion, the plasmodesmata pore widens and the two membranes separate, leaving a cytosolic sleeve spanned by tethers whose presence correlates with the appearance of the intermembrane gap. Surprisingly, the post-cytokinesis plasmodesmata allow diffusion of macromolecules despite the apparent lack of an open cytoplasmic sleeve, forcing the reassessment of the mechanisms that control plant cell-cell communication.

Stitching Organelles: Organization and Function of Specialized Membrane Contact Sites in Plants.

The coordination of multiple metabolic activities in plants relies on an interorganelle communication network established through membrane contact sites (MCS). The MCS are maintained in transient or durable configurations by tethering structures which keep the two membranes in close proximity, and create chemical microdomains that allow localized and targeted exchange of small molecules and possibly proteins. The past few years have witnessed a dramatic increase in our understanding of the structural and molecular organization of plant interorganelle MCS, and their crucial roles in plant specialized functions including stress responses, cell to cell communication, and lipid transport. In this review we summarize recent advances in understanding the molecular components, structural organization, and functions of different plant-specific MCS architectures.

'2A-Like' Signal Sequences Mediating Translational Recoding: A Novel Form of Dual Protein Targeting.

We report the initial characterization of an N-terminal oligopeptide '2A-like' sequence that is able to function both as a signal sequence and as a translational recoding element. Owing to this translational recoding activity, two forms of nascent polypeptide are synthesized: (i) when 2A-mediated translational recoding has not occurred: the nascent polypeptide is fused to the 2A-like N-terminal signal sequence and the fusion translation product is targeted to the exocytic pathway, and, (ii) a translation product where 2A-mediated translational recoding has occurred: the 2A-like signal sequence is synthesized as a separate translation product and, therefore, the nascent (downstream) polypeptide lacks the 2A-like signal sequence and is localized to the cytoplasm. This type of dual-functional signal sequence results, therefore, in the partitioning of the translation products between the two sub-cellular sites and represents a newly described form of dual protein targeting.

Staying Tight: Plasmodesmal Membrane Contact Sites and the Control of Cell-to-Cell Connectivity in Plants.

Multicellularity differs in plants and animals in that the cytoplasm, plasma membrane, and endomembrane of plants are connected between cells through plasmodesmal pores. Plasmodesmata (PDs) are essential for plant life and serve as conduits for the transport of proteins, small RNAs, hormones, and metabolites during developmental and defense signaling. They are also the only pathways available for viruses to spread within plant hosts. The membrane organization of PDs is unique, characterized by the close apposition of the endoplasmic reticulum and the plasma membrane and spoke-like filamentous structures linking the two membranes, which define PDs as membrane contact sites (MCSs). This specialized membrane arrangement is likely critical for PD function. Here, we review how PDs govern developmental and defensive signaling in plants, compare them with other types of MCSs, and discuss in detail the potential functional significance of the MCS nature of PDs.

Putting the Squeeze on Plasmodesmata: A Role for Reticulons in Primary Plasmodesmata Formation.

Primary plasmodesmata (PD) arise at cytokinesis when the new cell plate forms. During this process, fine strands of endoplasmic reticulum (ER) are laid down between enlarging Golgi-derived vesicles to form nascent PD, each pore containing a desmotubule, a membranous rod derived from the cortical ER. Little is known about the forces that model the ER during cell plate formation. Here, we show that members of the reticulon (RTNLB) family of ER-tubulating proteins in Arabidopsis (Arabidopsis thaliana) may play a role in the formation of the desmotubule. RTNLB3 and RTNLB6, two RTNLBs present in the PD proteome, are recruited to the cell plate at late telophase, when primary PD are formed, and remain associated with primary PD in the mature cell wall. Both RTNLBs showed significant colocalization at PD with the viral movement protein of Tobacco mosaic virus, while superresolution imaging (three-dimensional structured illumination microscopy) of primary PD revealed the central desmotubule to be labeled by RTNLB6. Fluorescence recovery after photobleaching studies showed that these RTNLBs are mobile at the edge of the developing cell plate, where new wall materials are being delivered, but significantly less mobile at its center, where PD are forming. A truncated RTNLB3, unable to constrict the ER, was not recruited to the cell plate at cytokinesis. We discuss the potential roles of RTNLBs in desmotubule formation.

Importin-α-mediated nucleolar localization of potato mop-top virus TRIPLE GENE BLOCK1 (TGB1) protein facilitates virus systemic movement, whereas TGB1 self-interaction is required for cell-to-cell movement in Nicotiana benthamiana.

Recently, it has become evident that nucleolar passage of movement proteins occurs commonly in a number of plant RNA viruses that replicate in the cytoplasm. Systemic movement of Potato mop-top virus (PMTV) involves two viral transport forms represented by a complex of viral RNA and TRIPLE GENE BLOCK1 (TGB1) movement protein and by polar virions that contain the minor coat protein and TGB1 attached to one extremity. The integrity of polar virions ensures the efficient movement of RNA-CP, which encodes the virus coat protein. Here, we report the involvement of nuclear transport receptors belonging to the importin-α family in nucleolar accumulation of the PMTV TGB1 protein and, subsequently, in the systemic movement of the virus. Virus-induced gene silencing of two importin-α paralogs in Nicotiana benthamiana resulted in significant reduction of TGB1 accumulation in the nucleus, decreasing the accumulation of the virus progeny in upper leaves and the loss of systemic movement of RNA-CP. PMTV TGB1 interacted with importin-α in N. benthamiana, which was detected by bimolecular fluorescence complementation in the nucleoplasm and nucleolus. The interaction was mediated by two nucleolar localization signals identified by bioinformatics and mutagenesis in the TGB1 amino-terminal domain. Our results showed that while TGB1 self-interaction is needed for cell-to-cell movement, importin-α-mediated nucleolar targeting of TGB1 is an essential step in establishing the efficient systemic infection of the entire plant. These results enabled the identification of two separate domains in TGB1: an internal domain required for TGB1 self-interaction and cell-to-cell movement and the amino-terminal domain required for importin-α interaction in plants, nucleolar targeting, and long-distance movement.

Pumilio-based RNA in vivo imaging.

Subcellular, sequence-specific detection of RNA in vivo is a powerful tool to study the macromolecular transport that occurs through plasmodesmata. The RNA-binding domain of Pumilio proteins can be engineered to bind RNA sequences of choice and fused to fluorescent proteins for RNA imaging. This chapter describes the construction of a Pumilio-based imaging system to track the RNA of Tobacco mosaic virus in vivo, and practical aspects of RNA live-cell imaging.

Techniques for RNA in vivo imaging in plants.

Since the discovery of small RNAs and RNA silencing, RNA biology has taken a centre stage in cell and developmental biology. Small RNAs, but also mRNAs and other types of cellular and viral RNAs are processed at specific subcellular localizations. To fully understand cellular RNA metabolism and the various processes influenced by it, techniques are required that permit the sequence-specific tracking of RNAs in living cells. A variety of methods for RNA visualization have been developed since the 1990s, but plant cells pose particular challenges and not all approaches are applicable to them. On the other hand, plant RNA metabolism is particularly diverse and RNAs are even transported between cells, so RNA imaging can potentially provide many valuable insights into plant function at the cellular and tissue level. This Short Review briefly introduces the currently available techniques for plant RNA in vivo imaging and discusses their suitability for different biological questions.

Potato virus Y HCPro localization at distinct, dynamically related and environment-influenced structures in the cell cytoplasm.

Potyvirus HCPro is a multifunctional protein that, among other functions, interferes with antiviral defenses in plants and mediates viral transmission by aphid vectors. We have visualized in vivo the subcellular distribution and dynamics of HCPro from Potato virus Y and its homodimers, using green, yellow, and red fluorescent protein tags or their split parts, while assessing their biological activities. Confocal microscopy revealed a pattern of even distribution of fluorescence throughout the cytoplasm, common to all these modified HCPros, when transiently expressed in Nicotiana benthamiana epidermal cells in virus-free systems. However, in some cells, distinct additional patterns, specific to some constructs and influenced by environmental conditions, were observed: i) a small number of large, amorphous cytoplasm inclusions that contained α-tubulin; ii) a pattern of numerous small, similarly sized, dot-like inclusions distributing regularly throughout the cytoplasm and associated or anchored to the cortical endoplasmic reticulum and the microtubule (MT) cytoskeleton; and iii) a pattern that smoothly coated the MT. Furthermore, mixed and intermediate forms from the last two patterns were observed, suggesting dynamic transports between them. HCPro did not colocalize with actin filaments or the Golgi apparatus. Despite its association with MT, this network integrity was required neither for HCPro suppression of silencing in agropatch assays nor for its mediation of virus transmission by aphids.