Fine Structure of Plasmodesmata-Associated Membrane Bodies Formed by Viral Movement Protein.

Cell-to-cell transport of plant viruses through plasmodesmata (PD) requires viral movement proteins (MPs) often associated with cell membranes. The genome of the Hibiscus green spot virus encodes two MPs, BMB1 and BMB2, which enable virus cell-to-cell transport. BMB2 is known to localize to PD-associated membrane bodies (PAMBs), which are derived from the endoplasmic reticulum (ER) structures, and to direct BMB1 to PAMBs. This paper reports the fine structure of PAMBs. Immunogold labeling confirms the previously observed localization of BMB1 and BMB2 to PAMBs. EM tomography data show that the ER-derived structures in PAMBs are mostly cisterns interconnected by numerous intermembrane contacts that likely stabilize PAMBs. These contacts predominantly involve the rims of the cisterns rather than their flat surfaces. Using FRET-FLIM (Förster resonance energy transfer between fluorophores detected by fluorescence-lifetime imaging microscopy) and chemical cross-linking, BMB2 is shown to self-interact and form high-molecular-weight complexes. As BMB2 has been shown to have an affinity for highly curved membranes at cisternal rims, the interaction of BMB2 molecules located at rims of adjacent cisterns is suggested to be involved in the formation of intermembrane contacts in PAMBs.

Properties of Plant Virus Protein Encoded by the 5'-Proximal Gene of Tetra-Cistron Movement Block.

To move from cell to cell through plasmodesmata, many plant viruses require the concerted action of two or more movement proteins (MPs) encoded by transport gene modules of virus genomes. A tetra-cistron movement block (TCMB) is a newly discovered transport module comprising four genes. TCMB encodes three proteins, which are similar to MPs of the transport module known as the "triple gene block", and a protein unrelated to known viral MPs and containing a double-stranded RNA (dsRNA)-binding domain similar to that found in a family of cell proteins, including AtDRB4 and AtHYL1. Here, the latter TCMB protein, named vDRB for virus dsRNA-binding protein, is shown to bind both dsRNA and single-stranded RNA in vitro. In a turnip crinkle virus-based assay, vDRB exhibits the properties of a viral suppressor of RNA silencing (VSR). In the context of potato virus X infection, vDRB significantly decreases the number and size of "dark green islands", regions of local antiviral silencing, supporting the VSR function of vDRB. Nevertheless, vDRB does not exhibit the VSR properties in non-viral transient expression assays. Taken together, the data presented here indicate that vDRB is an RNA-binding protein exhibiting VSR functions in the context of viral infection.

Predicted Membrane-Associated Domains in Proteins Encoded by Novel Monopartite Plant RNA Viruses Related to Members of the Family Benyviridae.

As a continuation of our previous work, in this paper, we examine in greater detail the genome organization and some protein properties of the members of a potential group named Reclovirids and belonging to Benyviridae-related viruses. It can be proposed that the single-component Reclovirid genomes encode previously undiscovered transport genes. Indeed, analysis of the coding potential of these novel viral genomes reveals one or more cistrons ranging in size from 40 to 80 to about 600 codons, located in the 3'-terminal region of the genomic RNA, encoding proteins with predicted hydrophobic segments that are structurally diverse among Reclovirids and have no analogues in other plant RNA viruses. Additionally, in many cases, the possible methyltransferase domain of Reclovirid replicases is preceded by membrane-embedded protein segments that are not present in annotated members of the Benyviridae family. These observations suggest a general association of most Reclovirid proteins with cell membranes.

Role of Plant Virus Movement Proteins in Suppression of Host RNAi Defense.

One of the systems of plant defense against viral infection is RNA silencing, or RNA interference (RNAi), in which small RNAs derived from viral genomic RNAs and/or mRNAs serve as guides to target an Argonaute nuclease (AGO) to virus-specific RNAs. Complementary base pairing between the small interfering RNA incorporated into the AGO-based protein complex and viral RNA results in the target cleavage or translational repression. As a counter-defensive strategy, viruses have evolved to acquire viral silencing suppressors (VSRs) to inhibit the host plant RNAi pathway. Plant virus VSR proteins use multiple mechanisms to inhibit silencing. VSRs are often multifunctional proteins that perform additional functions in the virus infection cycle, particularly, cell-to-cell movement, genome encapsidation, or replication. This paper summarizes the available data on the proteins with dual VSR/movement protein activity used by plant viruses of nine orders to override the protective silencing response and reviews the different molecular mechanisms employed by these proteins to suppress RNAi.

Regulatory miPEP Open Reading Frames Contained in the Primary Transcripts of microRNAs.

This review aims to consider retrospectively the available data on the coding properties of pri-microRNAs and the regulatory functions of their open reading frames (ORFs) and the encoded peptides (miPEPs). Studies identifying miPEPs and analyzing the fine molecular mechanisms of their functional activities are reviewed together with a brief description of the methods to identify pri-miRNA ORFs and the encoded protein products. Generally, miPEPs have been identified in many plant species of several families and in a few animal species. Importantly, molecular mechanisms of the miPEP action are often quite different between flowering plants and metazoan species. Requirement for the additional studies in these directions is highlighted by alternative findings concerning negative or positive regulation of pri-miRNA/miRNA expression by miPEPs in plants and animals. Additionally, the question of how miPEPs are distributed in non-flowering plant taxa is very important for understanding the evolutionary origin of such micropeptides. Evidently, further extensive studies are needed to explore the functions of miPEPs and the corresponding ORFs and to understand the full set of their roles in eukaryotic organisms. Thus, we address the most recent integrative views of different genomic, physiological, and molecular aspects concerning the expression of miPEPs and their possible fine functions.

Uncovering Plant Virus Species Forming Novel Provisional Taxonomic Units Related to the Family Benyviridae.

Based on analyses of recent open-source data, this paper describes novel horizons in the diversity and taxonomy of beny-like viruses infecting hosts of the plant kingdom (Plantae or Archaeplastida). First, our data expand the known host range of the family Benyviridae to include red algae. Second, our phylogenetic analysis suggests that the evolution of this virus family may have involved cross-kingdom host change events and gene recombination/exchanges between distant taxa. Third, the identification of gene blocks encoding known movement proteins in beny-like RNA viruses infecting non-vascular plants confirms other evidence that plant virus genomic RNAs may have acquired movement proteins simultaneously or even prior to the evolutionary emergence of the plant vascular system. Fourth, novel data on plant virus diversity highlight that molecular evolution gave rise to numerous provisional species of land-plant-infecting viruses, which encode no known potential movement genetic systems.

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.

In-Plant Persistence and Systemic Transport of Nicotiana benthamiana Retrozyme RNA.

Retrozymes are nonautonomous retrotransposons with hammerhead ribozymes in their long terminal repeats (LTRs). Retrozyme transcripts can be self-cleaved by the LTR ribozyme, circularized, and can undergo RNA-to-RNA replication. Here, we demonstrate that the Nicotiana benthamiana genome contains hundreds of retrozyme loci, of which nine represent full-length retrozymes. The LTR contains a promoter directing retrozyme transcription. Although retrozyme RNA is easily detected in plants, the LTR region is heavily methylated, pointing to its transcriptional silencing, which can be mediated by 24 nucleotide-long retrozyme-specific RNAs identified in N. benthamiana. A transcriptome analysis revealed that half of the retrozyme-specific RNAs in plant leaves have no exact matches to genomic retrozyme loci, containing up to 13% mismatches with the closest genomic sequences, and could arise as a result of many rounds of RNA-to-RNA replication leading to error accumulation. Using a cloned retrozyme copy, we show that retrozyme RNA is capable of replication and systemic transport in plants. The presented data suggest that retrozyme loci in the N. benthamiana genome are transcriptionally inactive, and that circular retrozyme RNA can persist in cells due to its RNA-to-RNA replication and be transported systemically, emphasizing functional and, possibly, evolutionary links of retrozymes to viroids-noncoding circular RNAs that infect plants.

Distinct Mechanisms of Endomembrane Reorganization Determine Dissimilar Transport Pathways in Plant RNA Viruses.

Plant viruses exploit the endomembrane system of infected cells for their replication and cell-to-cell transport. The replication of viral RNA genomes occurs in the cytoplasm in association with reorganized endomembrane compartments induced by virus-encoded proteins and is coupled with the virus intercellular transport via plasmodesmata that connect neighboring cells in plant tissues. The transport of virus genomes to and through plasmodesmata requires virus-encoded movement proteins (MPs). Distantly related plant viruses encode different MP sets, or virus transport systems, which vary in the number of MPs and their properties, suggesting their functional differences. Here, we discuss two distinct virus transport pathways based on either the modification of the endoplasmic reticulum tubules or the formation of motile vesicles detached from the endoplasmic reticulum and targeted to endosomes. The viruses with the movement proteins encoded by the triple gene block exemplify the first, and the potyviral system is the example of the second type. These transport systems use unrelated mechanisms of endomembrane reorganization. We emphasize that the mode of virus interaction with cell endomembranes determines the mechanism of plant virus cell-to-cell transport.

Bioinformatic Analysis Predicts a Novel Genetic Module Related to Triple Gene and Binary Movement Blocks of Plant Viruses: Tetra-Cistron Movement Block.

Previous studies have shown that the RNA genomes of some plant viruses encode two related genetic modules required for virus movement over the host body, containing two or three genes and named the binary movement block (BMB) and triple gene block (TGB), respectively. In this paper, we predict a novel putative-related movement gene module, called the tetra-cistron movement block (TCMB), in the virus-like transcriptome assemblies of the moss Dicranum scoparium and the Antarctic flowering plant Colobanthus quitensis. These TCMBs are encoded by smaller RNA components of putative two-component viruses related to plant benyviruses. Similar to the RNA2 of benyviruses, TCMB-containing RNAs have the 5'-terminal coat protein gene and include the RNA helicase gene which is followed by two small overlapping cistrons encoding hydrophobic proteins with a distant sequence similarity to the TGB2 and TGB3 proteins. Unlike TGB, TCMB also includes a fourth 5'-terminal gene preceding the helicase gene and coding for a protein showing a similarity to the double-stranded RNA-binding proteins of the DSRM AtDRB-like superfamily. Additionally, based on phylogenetic analysis, we suggest the involvement of replicative beny-like helicases in the evolution of the BMB and TCMB movement genetic modules.

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.

RNA Binding by Plant Serpins in vitro.

Serpins constitute a large family of protease inhibitors with regulatory functions found in all living organisms. Most plant serpins have not been functionally characterized, with the exception of Arabidopsis thaliana AtSerpin1, an inhibitor of pro-apoptotic proteases, which is involved in the regulation of the programmed cell death induction, and Cucurbita maxima CmPS1, a phloem protein, which presumably inhibits insect digestive proteases and binds RNA. CmPS1 interacts most efficiently with highly structured RNA; in particular, it forms a specific complex with tRNA. Here, we demonstrated that AtSerpin1 also forms a complex with tRNA. Analysis of tRNA species bound by AtSerpin1 and CmPS1 in the presence of tRNA excess revealed that both proteins have no strict selectivity for individual tRNAs, suggesting specific interaction of AtSerpin1 and CmPS1 proteins with elements of the secondary/tertiary structure universal for all tRNAs. Analysis of CmPS1 binding of the microRNA precursor pre-miR390 and its mutants demonstrated that the pre-miR390 mutant with a perfect duplex in the hairpin stem lost the ability to form a discrete complex with CmPS1, whereas another variant of pre-miR390 with the native unpaired nucleotide residues in the stem retained this ability. These data indicate that specific interactions of plant serpins with structured RNA are based on the recognition of structurally unique spatial motifs formed with the participation of unpaired nucleotide residues in the RNA duplexes.

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.

Small hydrophobic viral proteins involved in intercellular movement of diverse plant virus genomes.

Most plant viruses code for movement proteins (MPs) targeting plasmodesmata to enable cell-to-cell and systemic spread in infected plants. Small membrane-embedded MPs have been first identified in two viral transport gene modules, triple gene block (TGB) coding for an RNA-binding helicase TGB1 and two small hydrophobic proteins TGB2 and TGB3 and double gene block (DGB) encoding two small polypeptides representing an RNA-binding protein and a membrane protein. These findings indicated that movement gene modules composed of two or more cistrons may encode the nucleic acid-binding protein and at least one membrane-bound movement protein. The same rule was revealed for small DNA-containing plant viruses, namely, viruses belonging to genus Mastrevirus (family Geminiviridae) and the family Nanoviridae. In multi-component transport modules the nucleic acid-binding MP can be viral capsid protein(s), as in RNA-containing viruses of the families Closteroviridae and Potyviridae. However, membrane proteins are always found among MPs of these multicomponent viral transport systems. Moreover, it was found that small membrane MPs encoded by many viruses can be involved in coupling viral replication and cell-to-cell movement. Currently, the studies of evolutionary origin and functioning of small membrane MPs is regarded as an important pre-requisite for understanding of the evolution of the existing plant virus transport systems. This paper represents the first comprehensive review which describes the whole diversity of small membrane MPs and presents the current views on their role in plant virus movement.

Phloem transport of structured RNAs: A widening repertoire of trafficking signals and protein factors.

The conducting sieve tubes of the phloem consist of sieve elements (SEs), which are enucleate cells incapable of transcription and translation. Nevertheless, SEs contain a large variety of RNAs, and long-distance RNA trafficking via the phloem has been documented. The phloem transport of certain RNAs, as well as the further unloading of these RNAs at target tissues, is essential for plant individual development and responses to environmental cues. The translocation of such RNAs via the phloem is believed to be directed by RNA structural elements serving as phloem transport signals (PTSs), which are recognized by proteins that direct the PTS-containing RNAs into the phloem translocation pathway. The ability of phloem transport has been reported for several classes of structured RNAs including viroids, genuine tRNAs, mRNAs with tRNA sequences embedded into mRNA untranslated regions, tRNA-like structures in the genomic RNAs of plant viruses, and micro-RNA (miRNA) precursors (pri-miRNA). Here, three distinct types of such RNAs are discussed, along with the proteins that may specifically interact with these structures in the phloem. Three-dimensional (3D) motifs, which are characteristic of imperfect RNA duplexes, are discussed as elements of phloem-mobile structured RNAs specifically recognized by proteins involved in phloem transport, thus serving as PTSs.

Detection and in vitro studies of Cucurbita maxima phloem serpin-1 RNA-binding properties.

Apart from being a conduit for photoassimilate transport in plants, the phloem serves as a pathway for transport of proteins and RNAs from sites of their synthesis to distant plant parts. As demonstrated for mRNAs and small RNAs such as miRNA and siRNA, their phloem transport is largely involved in responses to environmental cues including stresses and pathogen attacks. RNA molecules are believed to be transported in the phloem in the form of complexes with RNA-binding proteins; however, proteins forming such complexes are generally poorly studied. Here, we demonstrate that the Cucurbita maxima phloem serpin-1 (CmPS1), which has been previously described as a functional protease inhibitor capable of long-distance transport via the phloem, is able to bind RNA in vitro. Among different RNAs tested, CmPS1 exhibits a preference for imperfect RNA duplexes and the highest affinity to tRNA. A characteristic complex formed by CmPS1 with tRNA is not observed upon CmPS1 binding to tRNA-like structures of plant viruses. Mutational analysis demonstrates that the CmPS1 N-terminal region is not involved in RNA binding. Since antithrombin-III, the human protease inhibitor of serpin family most closely sequence-related to CmPS1, is found to be unable to bind RNA, one can suggest that, in its evolution, CmPS1 has gained the RNA binding capability as an additional function likely relevant to its specific activities in the plant phloem.

TAS3 miR390-dependent loci in non-vascular land plants: towards a comprehensive reconstruction of the gene evolutionary history.

Trans-acting small interfering RNAs (ta-siRNAs) are transcribed from protein non-coding genomic TAS loci and belong to a plant-specific class of endogenous small RNAs. These siRNAs have been found to regulate gene expression in most taxa including seed plants, gymnosperms, ferns and mosses. In this study, bioinformatic and experimental PCR-based approaches were used as tools to analyze TAS3 and TAS6 loci in transcriptomes and genomic DNAs from representatives of evolutionary distant non-vascular plant taxa such as Bryophyta, Marchantiophyta and Anthocerotophyta. We revealed previously undiscovered TAS3 loci in plant classes Sphagnopsida and Anthocerotopsida, as well as TAS6 loci in Bryophyta classes Tetraphidiopsida, Polytrichopsida, Andreaeopsida and Takakiopsida. These data further unveil the evolutionary pathway of the miR390-dependent TAS3 loci in land plants. We also identified charophyte alga sequences coding for SUPPRESSOR OF GENE SILENCING 3 (SGS3), which is required for generation of ta-siRNAs in plants, and hypothesized that the appearance of TAS3-related sequences could take place at a very early step in evolutionary transition from charophyte algae to an earliest common ancestor of land plants.