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.

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.

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.

Plant-specific 4/1 polypeptide interacts with an endoplasmic reticulum protein related to human BAP31.

MAIN CONCLUSION: The plant-specific 4/1 protein interacts, both in yeast two-hybrid system and in vitro, and co-localizes in plant cells with plant BAP-like protein, the orthologue of human protein BAP31. In yeast two-hybrid system, we identified a number of Nicotiana benthamiana protein interactors of Nt-4/1, the protein known to affect systemic transport of potato spindle tuber viroid. For one of these interactors, an orthologue of human B-cell receptor-associated protein 31 (BAP31) termed plant BAP-like protein (PBL), the ability to interact with Nt-4/1 was studied in greater detail. Analyses of purified proteins expressed in bacterial cells carried out in vitro with the surface plasmon resonance (SPR) spectroscopy revealed that the N. tabacum PBL (NtPBL) was able to interact with Nt-4/1 with high-affinity, and that their complex can form at physiologically relevant concentrations of both proteins. Subcellular localization studies of 4/1-GFP and NtPBL-mRFP transiently co-expressed in plant cells revealed the co-localization of the two fusion proteins in endoplasmic reticulum-associated bodies, suggesting their interaction in vivo. The N-terminal region of the Nt-4/1 protein was found to be required for the specific subcellular targeting of the protein, presumably due to a predicted amphipathic helix mediating association of the Nt-4/1 protein with cell membranes. Additionally, this region was found to contain a trans-activator domain responsible for the Nt-4/1 ability to activate transcription of a reporter gene in yeast.

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.

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.

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.

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.

RNA phloem transport mediated by pre-miRNA and viral tRNA-like structures.

Phloem-mobile mRNAs are assumed to contain sequence elements directing RNA to the phloem translocation pathway. One of such elements is represented by tRNA sequences embedded in untranslated regions of many mRNAs, including those proved to be mobile. Genomic RNAs of a number of plant viruses possess a 3'-terminal tRNA-like structures (TLSs) only distantly related to genuine tRNAs, but nevertheless aminoacylated and capable of interaction with some tRNA-binding proteins. Here, we elaborated an experimental system for analysis of RNA phloem transport based on an engineered RNA of Potato virus X capable of replication, but not encapsidation and movement in plants. The TLSs of Brome mosaic virus, Tobacco mosaic virus and Turnip yellow mosaic virus were demonstrated to enable the phloem transport of foreign RNA. A miRNA precursor, pre-miR390b, was also found to render RNA competent for the phloem transport. In line with this, sequences of miRNA precursors were identified in a Cucurbita maxima phloem transcriptome, supporting the hypothesis that, at least in some cases, miRNA phloem signaling can involve miRNA precursors. Collectively, the data presented here suggest that RNA molecules can be directed into the phloem translocation pathway by structured RNA elements such as those of viral TLSs and miRNA precursors.

Mechanical stress-induced subcellular re-localization of N-terminally truncated tobacco Nt-4/1 protein.

The Nicotiana tabacum 4/1 protein (Nt-4/1) of unknown function expressed in plant vasculature has been shown to localize to cytoplasmic bodies associated with endoplasmic reticulum. Here, we analyzed molecular interactions of an Nt-4/1 mutant with a deletion of 90 N-terminal amino acid residues (Nt-4/1d90) having a diffuse GFP-like localization. Upon transient co-expression with VAP27, a membrane protein known to localize to the ER, ER-plasma membrane contact sites and plasmodesmata, Nt-4/1d90 was concentrated around the cortical ER tubules, forming a network matching the shape of the cortical ER. Additionally, in response to mechanical stress, Nt-4/1d90 was re-localized to small spherical bodies, whereas the subcellular localization of VAP27 remained essentially unaffected. The Nt-4/1d90-containing bodies associated with microtubules, which underwent noticeable bundling under the conditions of mechanical stress. The Nt-4/1d90 re-localization to spherical bodies could also be induced by incubation at an elevated temperature, although under heat shock conditions the re-localization was less efficient and incomplete. An Nt-4/1d90 mutant, which had phosphorylation-mimicking mutations in a predicted cluster of four potentially phosphorylated residues, was found to both inefficiently re-localize to spherical bodies and tend to revert back to the initial diffuse localization. The presented data show that Nt-4/1 has a potential for response to stresses that is manifested by its deletion mutant Nt-4/1d90, and this response can be mediated by protein dephosphorylation.

Data on the exon-intron organization of genes coding for B-cell receptor-like proteins.

B-cell receptor-associated protein (BAP) family plays important roles in the ER homeostasis and stress responses of eukaryotic cells [1]. We reported the analysis of plant BAP-like (PBL) genes and the encoded proteins of higher land plants [2]. The origin and functional divergence of these genes among all eukaryotes, however, are poorly studied, which impedes our understanding of the functional relationships and diversity among BAP-like proteins. One possible reason for the potential functional diversity may be the differences in the exon-intron structure of PBL genes. In this study, we first performed analysis of the exon-intron organization of these genes in the genome sequences of the Viridiplantae species in addition to previously published data on Angiosperms [2]. To further address the distribution of BAP-like genes in other eukaryotes, we extended our dataset to include the representative genes encoded by non-plant bikonts and unikonts [3].

Phylogenetic and functional analyses of a plant protein related to human B-cell receptor-associated proteins.

Human B-cell receptor-associated protein BAP31 (HsBAP31) is the endoplasmic reticulum-resident protein involved in protein sorting and transport as well as pro-apoptotic signaling. Plant orthologs of HsBAP31 termed 'plant BAP-like proteins' (PBL proteins) have thus far remained unstudied. Recently, the PBL protein from Nicotiana tabacum (NtPBL) was identified as an interactor of Nt-4/1, a plant protein known to interact with plant virus movement proteins and affect the long-distance transport of potato spindle tuber viroid (PSTVd) via the phloem. Here, we have compared the sequences of PBL proteins and studied the biochemical properties of NtPBL. Analysis of a number of fully sequenced plant genomes revealed that PBL-encoding genes represent a small multigene family with up to six members per genome. Two conserved motifs were identified in the C-terminal region of PBL proteins. The NtPBL C-terminal hydrophilic region (NtPBL-C) was expressed in bacterial cells, purified, and used for analysis of its RNA binding properties in vitro. In gel shift experiments, NtPBL-C was found to bind several tested RNAs, showing the most efficient binding to microRNA precursors (pre-miRNA) and less efficient interaction with PSTVd. Mutational analysis suggested that NtPBL-C has a composite RNA-binding site, with two conserved lysine residues in the most C-terminal protein region being involved in binding of pre-miRNA but not PSTVd RNA. Virus-mediated transient expression of NtPBL-C in plants resulted in stunting and leaf malformation, developmental abnormalities similar to those described previously for blockage of miRNA biogenesis/function. We hypothesize that the NtPBL protein represents a previously undiscovered component of the miRNA pathway.