dsRNA-induced immunity targets plasmodesmata and is suppressed by viral movement proteins.

Emerging evidence indicates that in addition to its well-recognized functions in antiviral RNA silencing, dsRNA elicits pattern-triggered immunity (PTI), likely contributing to plant resistance against virus infections. However, compared to bacterial and fungal elicitor-mediated PTI, the mode-of-action and signaling pathway of dsRNA-induced defense remain poorly characterized. Here, using multicolor in vivo imaging, analysis of GFP mobility, callose staining, and plasmodesmal marker lines in Arabidopsis thaliana and Nicotiana benthamiana, we show that dsRNA-induced PTI restricts the progression of virus infection by triggering callose deposition at plasmodesmata, thereby likely limiting the macromolecular transport through these cell-to-cell communication channels. The plasma membrane-resident SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE 1, the BOTRYTIS INDUCED KINASE1/AVRPPHB SUSCEPTIBLE1-LIKE KINASE1 kinase module, PLASMODESMATA-LOCATED PROTEINs 1/2/3, as well as CALMODULIN-LIKE 41 and Ca2+ signals are involved in the dsRNA-induced signaling leading to callose deposition at plasmodesmata and antiviral defense. Unlike the classical bacterial elicitor flagellin, dsRNA does not trigger a detectable reactive oxygen species (ROS) burst, substantiating the idea that different microbial patterns trigger partially shared immune signaling frameworks with distinct features. Likely as a counter strategy, viral movement proteins from different viruses suppress the dsRNA-induced host response leading to callose deposition to achieve infection. Thus, our data support a model in which plant immune signaling constrains virus movement by inducing callose deposition at plasmodesmata and reveals how viruses counteract this layer of immunity.

A chaperonin containing T-complex polypeptide-1 facilitates the formation of the PbWoxT1-PbPTB3 ribonucleoprotein complex for long-distance RNA trafficking in Pyrus betulaefolia.

Numerous plant endogenous mRNAs move via phloem and thus affect the growth and development of long-distant organs. mRNAs are transported with RNA-binding proteins forming a ribonucleoprotein complex. However, it remains elusive how such RNP complex assembles and facilitates mRNA trafficking. Protease digestion and RNA immunoprecipitation were used to investigate the RNP assembly function of the complete Chaperonin Containing T-complex Polypeptide-1. In situ hybridization, hairy root transformation, microprojectile bombardment, and grafting experiments demonstrate the role of CCT complex in the transport of a PbWoxT1-PbPTB3 RNP complex in Pyrus betulaefolia. PbCCT5 silenced caused defective movement of GFP-PbPTB3 and GFP-PbWoxT1 from hairy roots to new leaves via the phloem. PbCCT5 is shown to interact with PbPTB3. PbCCT complex enhanced PbPTB3 stabilization and permitted assembly of PbWoxT1 and PbPTB3 into an RNP complex. Furthermore, silencing of individual CCT subunits inhibited the intercellular movement of GFP-PbPTB3 and long-distance trafficking of PbWoxT1 and PbPTB3 in grafted plants. Taken together, the CCT complex assembles PbPTB3 and PbWoxT1 into an RNP complex in the phloem in order to facilitate the long-distance trafficking of PbWoxT1 in P. betulaefolia. This study therefore provides important insights into the mechanism of RNP complex formation and transport.

Interaction between Movement Proteins of Hibiscus green spot virus.

Movement proteins (MPs) of plant viruses enable the translocation of viral genomes from infected to healthy cells through plasmodesmata (PD). The MPs functions involve the increase of the PD permeability and routing of viral genome both to the PD entrance and through the modified PD. Hibiscus green spot virus encodes two MPs, termed BMB1 and BMB2, which act in concert to accomplish virus cell-to-cell transport. BMB1, representing an NTPase/helicase domain-containing RNA-binding protein, localizes to the cytoplasm and the nucleoplasm. BMB2 is a small hydrophobic protein that interacts with the endoplasmic reticulum (ER) membranes and induces local constrictions of the ER tubules. In plant cells, BMB2 localizes to PD-associated membrane bodies (PAMBs) consisting of modified ER tubules and directs BMB1 to PAMBs. Here, we demonstrate that BMB1 and BMB2 interact in vitro and in vivo, and that their specific interaction is essential for BMB2-directed targeting of BMB1 to PAMBs. Using mutagenesis, we show that the interaction involves the C-terminal BMB1 region and the N-terminal region of BMB2.

Function of Plasmodesmata in the Interaction of Plants with Microbes and Viruses.

Plasmodesmata (PD) are gated plant cell wall channels that allow the trafficking of molecules between cells and play important roles during plant development and in the orchestration of cellular and systemic signaling responses during interactions of plants with the biotic and abiotic environment. To allow gating, PD are equipped with signaling platforms and enzymes that regulate the size exclusion limit (SEL) of the pore. Plant-interacting microbes and viruses target PD with specific effectors to enhance their virulence and are useful probes to study PD functions.

In Vivo Aniline Blue Staining and Semiautomated Quantification of Callose Deposition at Plasmodesmata.

The deposition and turnover of callose (beta-1,3 glucan polymer) in the cell wall surrounding the neck regions of plasmodesmata (PD) controls the cell-to-cell diffusion rate of molecules and, therefore, plays an important role in the regulation of intercellular communication in plants.Here we describe a simple and fast in vivo staining procedure for the imaging and quantification of callose at PD. We also introduce calloseQuant, a plug-in for semiautomated image analysis and non-biased quantification of callose levels at PD using ImageJ.

Virus Genome-Based Reporter for Analyzing Viral Movement Proteins and Plasmodesmata Permeability.

Plant virus movement proteins (MPs) mediate cell-to-cell movement of the virus genome through plasmodesmata (PD). MPs target PD to increase their size exclusion limit (SEL), and this MP function is essential for virus intercellular trafficking. In this chapter, we describe the use of a Potato virus X genome-derived reporter for agroinfiltration-based identification of virus genome-encoded MPs and analysis of the ability of individual viral MPs or plant proteins to increase the PD SEL.

In Vivo Visualization of Mobile mRNA Particles in Plants Using BglG.

Cells have developed mechanisms for cytoplasmic RNA transport and localization that participate in the regulation and subcellular localization of protein synthesis. In addition, plants can exchange RNA molecules between cells through plasmodesmata and to distant tissues in the phloem. These mechanisms are hijacked by RNA viruses to establish their replication complexes and to disseminate their genomes throughout the plant organism with the help of virus-encoded movement proteins (MP). Live imaging of RNA molecules is a fundamental approach to understand the regulation and molecular basis of these processes. The most widely used experimental systems for the in vivo visualization of genetically encoded RNA molecules are based on fluorescently tagged RNA binding proteins that bind to specific motifs inserted into the RNA, thus allowing the tracking of the specific RNA molecule by fluorescent microscopy. Recently, we developed the use of the E. coli RNA binding protein BglG for the imaging of RNAs tagged with BglG-binding sites in planta. We describe here the detailed method by which we use this in vivo RNA tagging system for the real-time imaging of Tobacco mosaic virus (TMV) MP mRNA.

Potential Impact of Global Warming on Virus Propagation in Infected Plants and Agricultural Productivity.

The increasing pace of global warming and climate instability will challenge the management of pests and diseases of cultivated plants. Several reports have shown that increases in environmental temperature can enhance the cell-to-cell and systemic propagation of viruses within their infected hosts. These observations suggest that earlier and longer periods of warmer weather may cause important changes in the interaction between viruses and their host's plants, thus posing risks of new viral diseases and outbreaks in agriculture and the wild. As viruses target plasmodesmata (PD) for cell-to-cell spread, these cell wall pores may play yet unknown roles in the temperature-sensitive regulation of intercellular communication and virus infection. Understanding the temperature-sensitive mechanisms in plant-virus interactions will provide important knowledge for protecting crops against diseases in a warmer climate.

Reticulon-like properties of a plant virus-encoded movement protein.

Plant viruses encode movement proteins (MPs) that ensure the transport of viral genomes through plasmodesmata (PD) and use cell endomembranes, mostly the endoplasmic reticulum (ER), for delivery of viral genomes to PD and formation of PD-anchored virus replication compartments. Here, we demonstrate that the Hibiscus green spot virus BMB2 MP, an integral ER protein, induces constrictions of ER tubules, decreases the mobility of ER luminal content, and exhibits an affinity to highly curved membranes. These properties are similar to those described for reticulons, cellular proteins that induce membrane curvature to shape the ER tubules. Similar to reticulons, BMB2 adopts a W-like topology within the ER membrane. BMB2 targets PD and increases their size exclusion limit, and these BMB2 activities correlate with the ability to induce constrictions of ER tubules. We propose that the induction of ER constrictions contributes to the BMB2-dependent increase in PD permeability and formation of the PD-associated replication compartments, therefore facilitating the virus intercellular spread. Furthermore, we show that the ER tubule constrictions also occur in cells expressing TGB2, one of the three MPs of Potato virus X (PVX), and in PVX-infected cells, suggesting that reticulon-like MPs are employed by diverse RNA viruses.

In vivo imaging of tagged mRNA in plant tissues using the bacterial transcriptional antiterminator BglG.

RNA transport and localization represent important post-transcriptional mechanisms to determine the subcellular localization of protein synthesis. Plants have the capacity to transport messenger (m)RNA molecules beyond the cell boundaries through plasmodesmata and over long distances in the phloem. RNA viruses exploit these transport pathways to disseminate their infections and represent important model systems to investigate RNA transport in plants. Here, we present an in vivo plant RNA-labeling system based on the Escherichia coli RNA-binding protein BglG. Using the detection of RNA in mobile RNA particles formed by viral movement protein (MP) as a model, we demonstrate the efficiency and specificity of mRNA detection by the BglG system as compared with MS2 and λN systems. Our observations show that MP mRNA is specifically associated with MP in mobile MP particles but hardly with MP localized at plasmodesmata. MP mRNA is clearly absent from MP accumulating along microtubules. We show that the in vivo BglG labeling of the MP particles depends on the presence of the BglG-binding stem-loop aptamers within the MP mRNA and that the aptamers enhance the coprecipitation of BglG by MP, thus demonstrating the presence of an MP:MP mRNA complex. The BglG system also allowed us to monitor the cell-to-cell transport of the MP mRNA, thus linking the observation of mobile MP mRNA granules with intercellular MP mRNA transport. Given its specificity demonstrated here, the BglG system may be widely applicable for studying mRNA transport and localization in plants.

Turnip mosaic virus in oilseed rape activates networks of sRNA-mediated interactions between viral and host genomes.

Virus-induced plant diseases in cultivated plants cause important damages in yield. Although the mechanisms of virus infection are intensely studied at the cell biology level, only little is known about the molecular dialog between the invading virus and the host genome. Here we describe a combinatorial genome-wide approach to identify networks of sRNAs-guided post-transcriptional regulation within local Turnip mosaic virus (TuMV) infection sites in Brassica napus leaves. We show that the induction of host-encoded, virus-activated small interfering RNAs (vasiRNAs) observed in virus-infected tissues is accompanied by site-specific cleavage events on both viral and host RNAs that recalls the activity of small RNA-induced silencing complexes (RISC). Cleavage events also involve virus-derived siRNA (vsiRNA)-directed cleavage of target host transcripts as well as cleavage of viral RNA by both host vasiRNAs and vsiRNAs. Furthermore, certain coding genes act as virus-activated regulatory hubs to produce vasiRNAs for the targeting of other host genes. The observations draw an advanced model of plant-virus interactions and provide insights into the complex regulatory networking at the plant-virus interface within cells undergoing early stages of infection.

Visualization of Transiently Expressed mRNA in Plants Using MS2.

RNA transport and localization are evolutionarily conserved processes that allow protein translation to occur at specific subcellular sites and thereby having fundamental roles in the determination of cell fates, embryonic patterning, asymmetric cell division, and cell polarity. In addition to localizing RNA molecules to specific subcellular sites, plants have the ability to exchange RNA molecules between cells through plasmodesmata (PD). Plant RNA viruses hijack the mechanisms of intracellular and intercellular RNA transport to establish localized replication centers within infected cells and then to disseminate their infectious genomes between cells and throughout the plant organism with the help of their movement proteins (MP). In this chapter, we describe the transient expression of the tobacco mosaic virus movement protein (TMV-MP) and the application of the MS2 system for the in vivo labeling of the MP-encoding mRNA. The MS2 method is based on the binding of the bacteriophage coat protein (CP) to its origin of assembly (OAS) in the phage RNA. Thus, to label a specific mRNA in vivo, a tandem repetition of a 19-nucleotide-long stem-loop (SL) sequence derived from the MS2 OAS sequence (MSL) is transcriptionally fused to the RNA under investigation. The RNA is detected by the co-expression of fluorescent protein-tagged MS2 CP (MCP), which binds to each of the MSL elements. In providing a detailed protocol for the in vivo visualization of TMV-MP mRNA tagged with the MS2 system in Nicotiana benthamiana epidermal cells, we describe (1) the specific DNA constructs, (2) Agrobacterium tumefaciens-mediated transfection for their transient expression in plants, and (3) imaging conditions required to obtain high-quality mRNA imaging data.

Functionalized Tobacco Mosaic Virus Coat Protein Monomers and Oligomers as Nanocarriers for Anti-Cancer Peptides.

Components with self-assembly properties derived from plant viruses provide the opportunity to design biological nanoscaffolds for the ordered display of agents of diverse nature and with complementing functions. With the aim of designing a functionalized nanoscaffold to target cancer, the coat protein (CP) of Tobacco mosaic virus (TMV) was tested as nanocarrier for an insoluble, highly hydrophobic peptide that targets the transmembrane domain of the Neuropilin-1 (NRP1) receptor in cancer cells. The resulting construct CPL-K (CP-linker-"Kill") binds to NRP1 in cancer cells and disrupts NRP1 complex formation with PlexA1 as well as downstream Akt survival signaling. The application of CPL-K also inhibits angiogenesis and cell migration. CP was also fused to a peptide that targets the extracellular domain of NRP1 and this fusion protein (CPL-F, CP-Linker-"Find") is shown to bind to cultured cancer cells and to inhibit NRP1-dependent angiogenesis as well. CPL-K and CPL-F maintain their anti-angiogenic properties upon co-assembly to oligomers/nanoparticles together with CPL. The observations show that the CP of TMV can be employed to generate a functionalized nanoparticle with biological activity. Remarkably, fusion to CPL allowed us to solubilize the highly insoluble transmembrane NRP1 peptide and to retain its anti-angiogenic effect.

m5C Methylation Guides Systemic Transport of Messenger RNA over Graft Junctions in Plants.

In plants, transcripts move to distant body parts to potentially act as systemic signals regulating development and growth. Thousands of messenger RNAs (mRNAs) are transported across graft junctions via the phloem to distinct plant parts. Little is known regarding features, structural motifs, and potential base modifications of transported transcripts and how these may affect their mobility. We identified Arabidopsis thaliana mRNAs harboring the modified base 5-methylcytosine (m5C) and found that these are significantly enriched in mRNAs previously described as mobile, moving over graft junctions to distinct plant parts. We confirm this finding with graft-mobile methylated mRNAs TRANSLATIONALLY CONTROLLED TUMOR PROTEIN 1 (TCTP1) and HEAT SHOCK COGNATE PROTEIN 70.1 (HSC70.1), whose mRNA transport is diminished in mutants deficient in m5C mRNA methylation. Together, our results point toward an essential role of cytosine methylation in systemic mRNA mobility in plants and that TCTP1 mRNA mobility is required for its signaling function.

Perception of double-stranded RNA in plant antiviral immunity.

RNA silencing and antiviral pattern-triggered immunity (PTI) both rely on recognition of double-stranded (ds)RNAs as defence-inducing signals. While dsRNA recognition by dicer-like proteins during antiviral RNA silencing is thoroughly investigated, the molecular mechanisms involved in dsRNA perception leading to antiviral PTI are just about to be untangled. Parallels to antimicrobial PTI thereby only partially facilitate our view on antiviral PTI. PTI against microbial pathogens involves plasma membrane bound receptors; however, dsRNAs produced during virus infection occur intracellularly. Hence, how dsRNA may be perceived during this immune response is still an open question. In this short review, we describe recent discoveries in PTI signalling upon sensing of microbial patterns and endogenous 'danger' molecules with emphasis on immune signalling-associated subcellular trafficking processes in plants. Based on these studies, we develop different scenarios how dsRNAs could be sensed during antiviral PTI.

Citrus Psorosis Virus Movement Protein Contains an Aspartic Protease Required for Autocleavage and the Formation of Tubule-Like Structures at Plasmodesmata.

Plant virus cell-to-cell movement is an essential step in viral infections. This process is facilitated by specific virus-encoded movement proteins (MPs), which manipulate the cell wall channels between neighboring cells known as plasmodesmata (PD). Citrus psorosis virus (CPsV) infection in sweet orange involves the formation of tubule-like structures within PD, suggesting that CPsV belongs to "tubule-forming" viruses that encode MPs able to assemble a hollow tubule extending between cells to allow virus movement. Consistent with this hypothesis, we show that the MP of CPsV (MPCPsV) indeed forms tubule-like structures at PD upon transient expression in Nicotiana benthamiana leaves. Tubule formation by MPCPsV depends on its cleavage capacity, mediated by a specific aspartic protease motif present in its primary sequence. A single amino acid mutation in this motif abolishes MPCPsV cleavage, alters the subcellular localization of the protein, and negatively affects its activity in facilitating virus movement. The amino-terminal 34-kDa cleavage product (34KCPsV), but not the 20-kDa fragment (20KCPsV), supports virus movement. Moreover, similar to tubule-forming MPs of other viruses, MPCPsV (and also the 34KCPsV cleavage product) can homooligomerize, interact with PD-located protein 1 (PDLP1), and assemble tubule-like structures at PD by a mechanism dependent on the secretory pathway. 20KCPsV retains the protease activity and is able to cleave a cleavage-deficient MPCPsV in trans Altogether, these results demonstrate that CPsV movement depends on the autolytic cleavage of MPCPsV by an aspartic protease activity, which removes the 20KCPsV protease and thereby releases the 34KCPsV protein for PDLP1-dependent tubule formation at PD.IMPORTANCE Infection by citrus psorosis virus (CPsV) involves a self-cleaving aspartic protease activity within the viral movement protein (MP), which results in the production of two peptides, termed 34KCPsV and 20KCPsV, that carry the MP and viral protease activities, respectively. The underlying protease motif within the MP is also found in the MPs of other members of the Aspiviridae family, suggesting that protease-mediated protein processing represents a conserved mechanism of protein expression in this virus family. The results also demonstrate that CPsV and potentially other ophioviruses move by a tubule-guided mechanism. Although several viruses from different genera were shown to use this mechanism for cell-to-cell movement, our results also demonstrate that this mechanism is controlled by posttranslational protein cleavage. Moreover, given that tubule formation and virus movement could be inhibited by a mutation in the protease motif, targeting the protease activity for inactivation could represent an important approach for ophiovirus control.

Crosstalk between PTGS and TGS pathways in natural antiviral immunity and disease recovery.

Virus-induced diseases cause severe damage to cultivated plants, resulting in crop losses. Certain plant-virus interactions allow disease recovery at later stages of infection and have the potential to reveal important molecular targets for achieving disease control. Although recovery is known to involve antiviral RNA silencing1,2, the specific components of the many plant RNA silencing pathways 3 required for recovery are not known. We found that Arabidopsis thaliana plants infected with oilseed rape mosaic virus (ORMV) undergo symptom recovery. The recovered leaves contain infectious, replicating virus, but exhibit a loss of viral suppressor of RNA silencing (VSR) protein activity. We demonstrate that recovery depends on the 21-22 nt siRNA-mediated post-transcriptional gene silencing (PTGS) pathway and on components of a transcriptional gene silencing (TGS) pathway that is known to facilitate non-cell-autonomous silencing signalling. Collectively, our observations indicate that recovery reflects the establishment of a tolerant state in infected tissues and occurs following robust delivery of antiviral secondary siRNAs from source to sink tissues, and establishment of a dosage able to block the VSR activity involved in the formation of disease symptoms.