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ATP‐binding cassette sub‐family E member 1 (ABCE1) is recognized as a strongly conserved ribosome recycling factor, indispensable for translation in archaea and eukaryotes, however, its role in plants remains largely unidentified. Arabidopsis thaliana encodes two paralogous ABCE proteins (AtABCE1 and AtABCE2), sharing 81% identity. We previously reported that AtABCE2 functions as a suppressor of RNA silencing and that its gene is ubiquitously expressed. Here we describe the structural requirements of AtABCE2 for its suppressor function.Using agroinfiltration assays, we transiently overexpressed mutated versions of AtABCE2 together with GFP, to induce silencing in GFP transgenic Nicotiana benthamiana leaves. The influence of mutations was analysed at both local and systemic levels by in vivo imaging of GFP, Northern blot analysis of GFP siRNAs and observation of plants under UV light.Mutants of AtABCE2 with impaired ATP binding in either active site I or II failed to suppress GFP RNA silencing. Mutations disrupting ATP hydrolysis influenced the suppression of silencing differently at active site I or II. We also found that the N‐terminal iron–sulphur cluster domain of AtABCE2 is crucial for its suppressor function.Meaningfully, the observed structural requirements of AtABCE2 for RNA silencing suppression were found to be similar to those of archaeal ABCE1 needed for ribosome recycling. AtABCE2 might therefore suppress RNA silencing via supporting the competing RNA degradation mechanisms associated with ribosome recycling. [ABSTRACT FROM AUTHOR]

Abstract Background The positioning and dynamics of vesicles and organelles, and thus the growth of plant cells, is mediated by the acto-myosin system. In Arabidopsis there are 13 class XI myosins which mediate vesicle and organelle transport in different cell types. So far the involvement of five class XI myosins in cell expansion during the shoot and root development has been shown, three of which, XI-1, XI-2, and XI-K, are essential for organelle transport. Results Simultaneous depletion of Arabidopsis class XI myosins XI-K, XI-1, and XI-2 in double and triple mutant plants affected the growth of several types of epidermal cells. The size and shape of trichomes, leaf pavement cells and the elongation of the stigmatic papillae of double and triple mutant plants were affected to different extent. Reduced cell size led to significant size reduction of shoot organs in the case of triple mutant, affecting bolt formation, flowering time and fertility. Phenotype analysis revealed that the reduced fertility of triple mutant plants was caused by delayed or insufficient development of pistils. Conclusions We conclude that the class XI myosins XI-K, XI-1 and XI-2 have partially redundant roles in the growth of shoot epidermis. Myosin XI-K plays more important role whereas myosins XI-1 and XI-2 have minor roles in the determination of size and shape of epidermal cells, because the absence of these two myosins is compensated by XI-K. Co-operation between myosins XI-K and XI-2 appears to play an important role in these processes.

Abstract Background Actin is an ancient molecule that shows more than 90% amino acid homology between mammalian and plant actins. The regions of the actin molecule that are involved in F-actin assembly are largely conserved, and it is likely that mammalian actin is able to incorporate into microfilaments in plant cells but there is no experimental evidence until now. Results Visualization of microfilaments in onion bulb scale epidermis cells by different techniques revealed that rhodamine-phalloidin stained F-actin besides cytoplasm also in the nuclei whereas GFP-mouse talin hybrid protein did not enter the nuclei. Microinjection of fluorescently labeled actin was applied to study the presence of nuclear microfilaments in plant cells. Ratio imaging of injected fluorescent rabbit skeletal muscle actin and phalloidin staining of the microinjected cells showed that mammalian actin was able to incorporate into plant F-actin. The incorporation occurred preferentially in the nucleus and in the perinuclear region of plant cells whereas part of plant microfilaments, mostly in the periphery of cytoplasm, did not incorporate mammalian actin. Conclusions Microinjected mammalian actin is able to enter plant cell's nucleus, whereas incorporation of mammalian actin into plant F-actin occurs preferentially in the nucleus and perinuclear area.

High-throughput sequencing technologies were used to identify plant viruses in cereal samples surveyed from 2012 to 2017. Fifteen genome sequences of a tenuivirus infecting wheat, oats, and spelt in Estonia, Norway, and Sweden were identified and characterized by their distances to other tenuivirus sequences. Like most tenuiviruses, the genome of this tenuivirus contains four genomic segments. The isolates found from different countries shared at least 92% nucleotide sequence identity at the genome level. The planthopper Javesella pellucida was identified as a vector of the virus. Laboratory transmission tests using this vector indicated that wheat, oats, barley, rye, and triticale, but none of the tested pasture grass species ( Alopecurus pratensis , Dactylis glomerata , Festuca rubra , Lolium multiflorum , Phleum pratense , and Poa pratensis ), are susceptible. Taking into account the vector and host range data, the tenuivirus we have found most probably represents European wheat striate mosaic virus first identified about 60 years ago. Interestingly, whereas we were not able to infect any of the tested cereal species mechanically, Nicotiana benthamiana was infected via mechanical inoculation in laboratory conditions, displaying symptoms of yellow spots and vein clearing evolving into necrosis, eventually leading to plant death. Surprisingly, one of the virus genome segments (RNA2) encoding both a putative host systemic movement enhancer protein and a putative vector transmission factor was not detected in N. benthamiana after several passages even though systemic infection was observed, raising fundamental questions about the role of this segment in the systemic spread in several hosts.

The integrity and dynamics of actin cytoskeleton is necessary not only for plant cell architecture but also for membrane trafficking-mediated processes such as polar auxin transport, senescence, and cell death. In Arabidopsis , the inactivation of actin-based molecular motors, class XI myosins, affects the membrane trafficking and integrity of actin cytoskeleton, and thus causes defective plant growth and morphology, altered lifespan and reduced fertility. To evaluate the potential contribution of class XI myosins to the auxin response, senescence and cell death, we followed the flower and leaf development in the triple gene knockout mutant xi1 xi2 xik (3KO) and in rescued line stably expressing myosin XI-K:YFP (3KOR). Assessing the development of primary inflorescence shoots we found that the 3KO plants produced more axillary branches. Exploiting the auxin-dependent reporters DR5::GUS and IAA2::GUS, a significant reduction in auxin responsiveness was found throughout the development of the 3KO plants. Examination of the flower development of the plants stably expressing the auxin transporter PIN1::PIN1-GFP revealed partial loss of PIN1 polarization in developing 3KO pistils. Surprisingly, the stable expression of PIN1::PIN1-GFP significantly enhanced the semi-sterile phenotype of the 3KO plants. Further we investigated the localization of myosin XI-K:YFP in the 3KOR floral organs and revealed its expression pattern in floral primordia, developing pistils, and anther filaments. Interestingly, the XI-K:YFP and PIN1::PIN1-GFP shared partially overlapping but distinct expression patterns throughout floral development. Assessing the foliar development of the 3KO plants revealed increased rosette leaf production with signs of premature yellowing. Symptoms of the premature senescence correlated with massive loss of chlorophyll, increased cell death, early plasmolysis of epidermal cells, and strong up-regulation of the stress-inducible senescence-associated gene SAG13 in 3KO plants. Simultaneously, the reduced auxin responsiveness and premature leaf senescence were accompanied by significant anthocyanin accumulation in 3KO tissues. Collectively, our results provide genetic evidences that Arabidopsis class XI myosins arrange the flower morphogenesis and leaf longevity via contributing to auxin responses, leaf senescence, and cell death.

Solanum nodiflorum mottle virus (SNMoV) was isolated from a small-flowered nightshade (Solanum nodiflorum) in Queensland, Australia. It has been included in the genus Sobemovirus based on virion morphology and serological relationships. Here, we report the sequence of the complete genome of SNMoV. Sequence analysis confirmed that SNMoV has the characteristic genome organization of sobemoviruses. Phylogenetic analysis showed that it clusters most closely with velvet tobacco mottle virus (VTMoV), another sobemovirus native to Australia. Their genomes show 56.8 % sequence identity.

Sobemovirus P1 protein, characterized previously as a suppressor of posttranscriptional gene silencing, is required for systemic virus spread and infection in plants. Mutations in the ORF1 initiation codon do not affect viral replication indicating P1 is not necessary for this process. Wild type, recombinant and P1 deletion mutants of Cocksfoot mottle virus and Rice yellow mottle virus were used to infect oat, rice, wheat, barley, Arabidopsis thaliana and Nicotiana benthamiana plants. Wild type RYMV, RYMV without P1 and RYMV with CfMV P1 were detected in inoculated leaves of all tested plant species. We found that RYMV does not need P1 for replication and for local movement neither in host nor non-host species tested in this study. However, it is crucial for successful systemic spread of the virus in its host plant rice. Moreover, adding CfMV P1 into RYMV genome did not help it to overcome restriction to the inoculated leaf., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Myosins and actin filaments in the actomyosin system act in concert in regulating cell structure and dynamics and are also assumed to contribute to plant gravitropic response. To investigate the role of the actomyosin system in the inflorescence stem gravitropism, we used single and multiple mutants affecting each of the 17 Arabidopsis myosins of class VIII and XI. We show that class XI but not class VIII myosins are required for stem gravitropism. Simultaneous loss of function of myosins XI1, XI2, and XIK leads to impaired gravitropic bending that is correlated with altered growth, stiffness, and insufficient sedimentation of gravity sensing amyloplasts in stem endodermal cells. The gravitropic defect of the corresponding triple mutant xi1 xi2 xik could be rescued by stable expression of the functional XIK:YFP in the mutant background, indicating a role of class XI myosins in this process. Altogether, our results emphasize the critical contributions of myosins XI in stem gravitropism of Arabidopsis .

Members of the Caliciviridae family of positive sense RNA viruses cause a wide range of diseases in both humans and animals. The detailed characterization of the calicivirus life cycle had been hampered due to the lack of robust cell culture systems and experimental tools for many of the members of the family. However, a number of caliciviruses replicate efficiently in cell culture and have robust reverse genetics systems available, most notably feline calicivirus (FCV) and murine norovirus (MNV). These are therefore widely used as representative members with which to examine the mechanistic details of calicivirus genome translation and replication. The replication of the calicivirus RNA genome occurs via a double-stranded RNA intermediate that is then used as a template for the production of new positive sense viral RNA, which is covalently linked to the virus-encoded protein VPg. The covalent linkage to VPg occurs during genome replication via the nucleotidylylation activity of the viral RNA-dependent RNA polymerase. Using FCV and MNV, we used mass spectrometry-based approach to identify the specific amino acid linked to the 5' end of the viral nucleic acid. We observed that both VPg proteins are covalently linked to guanosine diphosphate (GDP) moieties via tyrosine positions 24 and 26 for FCV and MNV respectively. These data fit with previous observations indicating that mutations introduced into these specific amino acids are deleterious for viral replication and fail to produce infectious virus. In addition, we also detected serine phosphorylation sites within the FCV VPg protein with positions 80 and 107 found consistently phosphorylated on VPg-linked viral RNA isolated from infected cells. This work provides the first direct experimental characterization of the linkage of infectious calicivirus viral RNA to the VPg protein and highlights that post-translational modifications of VPg may also occur during the viral life cycle.

ABCE1 is a highly conserved protein universally present in eukaryotes and archaea, which is crucial for the viability of different organisms. First identified as RNase L inhibitor, ABCE1 is currently recognized as an essential translation factor involved in several stages of eukaryotic translation and ribosome biogenesis. The nature of vital functions of ABCE1, however, remains unexplained. Here, we study the role of ABCE1 in human cell proliferation and its possible connection to translation. We show that ABCE1 depletion by siRNA results in a decreased rate of cell growth due to accumulation of cells in S phase, which is accompanied by inefficient DNA synthesis and reduced histone mRNA and protein levels. We infer that in addition to the role in general translation, ABCE1 is involved in histone biosynthesis and DNA replication and therefore is essential for normal S phase progression. In addition, we analyze whether ABCE1 is implicated in transcript-specific translation via its association with the eIF3 complex subunits known to control the synthesis of cell proliferation-related proteins. The expression levels of a few such targets regulated by eIF3A, however, were not consistently affected by ABCE1 depletion.

The genus Sobemovirus, unassigned to any family, consists of viruses with single-stranded plus-oriented single-component RNA genomes and small icosahedral particles. Currently, 14 species within the genus have been recognized by the International Committee on Taxonomy of Viruses (ICTV) but several new species are to be recognized in the near future. Sobemovirus genomes are compact with a conserved structure of open reading frames and with short untranslated regions. Several sobemoviruses are important pathogens. Moreover, over the last decade sobemoviruses have become important model systems to study plant virus evolution. In the current review we give an overview of the structure and expression of sobemovirus genomes, processing and functions of individual proteins, particle structure, pathology and phylogenesis of sobemoviruses as well as of satellite RNAs present together with these viruses. Based on a phylogenetic analysis we propose that a new family Sobemoviridae should be recognized including the genera Sobemovirus and Polemovirus. Finally, we outline the future perspectives and needs for the research focusing on sobemoviruses.

Once considered a tentative member of the genus Sobemovirus, rottboellia yellow mottle virus (RoMoV) was excluded from the latest species list of the ICTV after the discovery of imperata yellow mottle virus (IYMV), which resembles RoMoV in host range and geographic origin. Here, sequence analysis of the complete genome of RoMoV suggested that it should be considered a distinct species within the genus Sobemovirus. It has the highest sequence identity (55 %) to ryegrass mottle virus (RGMoV), whereas its sequence identity to IYMV is lower (44 %). In a phylogenetic tree, RoMoV clusters together with RGMoV and artemisia virus A (ArtVA), a dicot-infecting sobemovirus.

ATP-binding cassette sub-family E member 1 (ABCE1) is a highly conserved protein among eukaryotes and archaea. Recent studies have identified ABCE1 as a ribosome-recycling factor important for translation termination in mammalian cells, yeast and also archaea. Here we report another conserved function of ABCE1. We have previously described AtRLI2, the homolog of ABCE1 in the plant Arabidopsis thaliana, as an endogenous suppressor of RNA silencing. In this study we show that this function is conserved: human ABCE1 is able to suppress RNA silencing in Nicotiana benthamiana plants, in mammalian HEK293 cells and in the worm Caenorhabditis elegans. Using co-immunoprecipitation and mass spectrometry, we found a number of potential ABCE1-interacting proteins that might support its function as an endogenous suppressor of RNA interference. The interactor candidates are associated with epigenetic regulation, transcription, RNA processing and mRNA surveillance. In addition, one of the identified proteins is translin, which together with its binding partner TRAX supports RNA interference.

Background: The Sobemovirus genome consists of polycistronic single-stranded positive-sense RNA. The first ORF encodes P1, a suppressor of RNA silencing required for virus movement. The coat protein (CP) is expressed from the 3' proximal ORF3 via subgenomic RNA. In addition to its structural role, the CP of some sobemoviruses has been reported to be required for systemic movement and to interact with P1. The aim of this study was to analyse the role of Cocksfoot mottle virus (CfMV) CP in the suppression of RNA silencing and virus movement., Methods: Agrobacterium-mediated transient expression method was used for testing CfMV CP capacity to suppress RNA silencing. CP substitution and deletion mutants were generated to examine the role of this protein in CfMV infection, using three host plants (oat, barley and wheat). The viral movement was characterised with CfMV expressing EGFP fused to the C-terminus of CP., Results: In the current study we show that CfMV CP is an additional RNA silencing suppressor. Interestingly, we observed that all CP mutant viruses were able to infect the three tested host plants systemically, although usually with reduced accumulation. CfMV expressing EGFP was detected in epidermal and mesophyll cells of inoculated leaves. Although EGFP fluorescence was not detected in upper leaves, some plants displayed CfMV symptoms. Analysis of the upper leaves revealed that the viruses had lost the EGFP sequence and sometimes also most of the CP gene., Conclusions: The present study demonstrates that CfMV CP suppresses RNA silencing but, surprisingly, is dispensable for systemic movement. Thus, CfMV does not move as virion in the tested host plants. The composition of the movement RNP complex remains to be elucidated.

Unlike other sobemoviruses, lucerne transient streak virus (LTSV) and turnip rosette virus (TRoV) have been reported to contain two successive ORF1s (denoted as ORF1a and ORF1b) instead of a single ORF1. Also, their next ORF (ORF2a/2a2b) has been mapped to a region ca. 200 nucleotides downstream from that of other sobemoviruses, leading to the lack of transmembrane segments at the N-termini of P2a/2a2b. In the current study, we resequenced this region for TRoV and LTSV. The hypothetical beginning of ORF1b was mapped as the beginning of ORF2a/2a2b for both TRoV and LTSV. Computional analysis revealed transmembrane segments at the N-termini of the TRoV and LTSV polyproteins.

Cocksfoot mottle virus (CfMV) localization in oat plants was analyzed during three weeks post infection by immunohistochemical staining to follow its spread through different tissues. In early stages of infection, the virus was first detectable in phloem parenchyma and bundle sheath cells of inoculated leaves. Bundle sheath and phloem parenchyma were also the cell types where the virus was first detected in stems and systemic leaves of infected plants. In later stages of infection, CfMV spread also into the mesophyll surrounding vascular bundles and was seldom detected in xylem parenchyma of inoculated leaves. In systemic leaves, CfMV was not detected from xylem. Moreover, sometimes it was found from phloem only. In straw and roots, CfMV was detected both from phloem and xylem. According to our observations, CfMV predominantly moves through phloem, which makes the systemic movement of CfMV different from that of another monocot-infecting sobemovirus, Rice yellow mottle virus (RYMV)., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Positive sense ssRNA virus genomes from several genera have a viral protein genome-linked (VPg) attached over a phosphodiester bond to the 5' end of the genome. The VPgs of Southern bean mosaic virus (SBMV) and Ryegrass mottle virus (RGMoV) were purified from virions and analyzed by mass spectrometry. SBMV VPg was determined to be linked to RNA through a threonine residue at position one, whereas RGMoV VPg was linked to RNA through a serine also at the first position. In addition, we identified the termini of the corresponding VPgs and discovered three and seven phosphorylation sites in SBMV and RGMoV VPgs, respectively. This is the first report on the use of threonine for linking RNA to VPg., (Copyright © 2011 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

Sobemoviruses possess a viral genome-linked protein (VPg) attached to the 5' end of viral RNA. VPg is processed from the viral polyprotein. In the current study, Cocksfoot mottle virus (CfMV) and Rice yellow mottle virus (RYMV) VPgs were purified from virions and analysed by mass spectrometry. The cleavage sites in the polyprotein and thereof the termini of VPg were experimentally proven. The lengths of the mature VPgs were determined to be 78 and 79 aa residues, respectively. The amino acid residues covalently linked to RNA in the two VPgs were, surprisingly, not conserved; it is a tyrosine at position 5 of CfMV VPg and serine at position 1 of RYMV VPg. Phosphorylations were identified in CfMV and RYMV VPgs with two positionally similar locations T20/S14 and S71/S72, respectively. RYMV VPg contains an additional phosphorylation site at S41.

Cocksfoot mottle virus (CfMV) coat protein (CP) localization was studied in plant and mammalian cells. Fusion of the full-length CP with enhanced green fluorescent protein (EGFP) localized to the cell nucleus whereas similar constructs lacking the first 33 N-terminal amino acids of CP localized to the cytoplasm. CP and EGFP fusions containing mutations in the arginine-rich motif of CP localized to the cytoplasm and to the nucleus in plant cells indicating the involvement of the motif in nuclear localization. In mammalian cells, mutations in the arginine-rich region were sufficient to completely abolish nuclear transport. The analysis of deletions of amino acid residues 1-11, 1-22, and 22-33 of CP demonstrated that there were two separate nuclear localization signals (NLS) within the N-terminus--a strong NLS1 in the arginine-rich region (residues 22-33) and a weaker NLS2 within residues 1-22. Analysis of point mutants revealed that the basic amino acid residues in the region of the two NLSs were individually not sufficient to direct CP to the nucleus. Additional microinjection studies with fluorescently labeled RNA and CP purified from CfMV particles demonstrated that the wild-type CP was capable of transporting the RNA to the nucleus. This feature was not sequence-specific in transient assays since both CfMV and GFP mRNA were transported to the cell nucleus by CfMV CP. Together the results suggest that the nucleus may be involved in CfMV infection.

The -1 programmed ribosomal frameshifting (-1 PRF) mechanism utilized by many viruses is dependent on a heptanucleotide slippery sequence and a downstream secondary structure element. In the current study, the RNA structure downstream from the slippery site of cocksfoot mottle sobemovirus (CfMV) was proven to be a 12bp stem-loop with a single bulge and a tetranucleotide loop. Several deletion and insertion mutants with altered stem-loop structures were tested in wheat germ extract (WGE) for frameshifting efficiency. The impact of the same mutations on virus infectivity was tested in oat plants. Mutations shortening or destabilizing the stem region reduced significantly but did not abolish -1 PRF in WGE. The same mutations proved to be deleterious for virus infection. However, extending the loop region to seven nucleotides had no significant effect on frameshifting efficiency in WGE and did not hamper virus replication in infected leaves. This is the first report about the experimentally proven RNA secondary structure directing -1 PRF of sobemoviruses.

This study investigated the effects of silencing suppressors derived from six different viruses (P1, P19, P25, HcPro, AC2 and 2b), expressed in transgenic Nicotiana tabacum and Nicotiana benthamiana plants, on the infection pattern of tobacco ringspot virus (TRSV) potato calico strain. In N. benthamiana, this virus produced an initial infection with severe systemic symptoms, but the infection was strongly reduced within a few weeks as the plant recovered from the infection. P25 and HcPro silencing suppressors effectively prevented recovery in this host, allowing continuous accumulation of the viral RNA as well as of the virus-specific small interfering RNAs, in the systemically infected leaves. In the P1-, P19-, AC2- or 2b-expressing transgenic N. benthamiana, the recovery was not complete. Susceptibility of N. tabacum to this virus was temperature sensitive. At lower temperatures, up to 25 degrees C, the plants became systemically infected, but at higher temperatures, the infections were limited to the inoculated leaves. In these preventative conditions, all silencing suppressor transgenes (except P25, which was expressed at very low levels) allowed the establishment of systemic infections. Very strong and consistent systemic infections were observed in HcPro- and AC2-expressing plants.

RNA silencing suppressor genes derived from six virus genera were transformed into Nicotiana benthamiana and N. tabacum plants. These suppressors were P1 of Rice yellow mottle virus (RYMV), P1 of Cocksfoot mottle virus, P19 of Tomato bushy stunt virus, P25 of Potato virus X, HcPro of Potato virus Y (strain N), 2b of Cucumber mosaic virus (strain Kin), and AC2 of African cassava mosaic virus (ACMV). HcPro caused the most severe phenotypes in both Nicotiana spp. AC2 also produced severe effects in N. tabacum but a much milder phenotype in N. benthamiana, although both HcPro and AC2 affected the leaf tissues of the two Nicotiana spp. in similar ways, causing hyperplasia and hypoplasia, respectively. P1-RYMV caused high lethality in the N. benthamiana plants but only mild effects in the N. tabacum plants. Phenotypic alterations produced by the other transgenes were minor in both species. Interestingly, the suppressors had very different effects on crucifer-infecting Tobamovirus (crTMV) infections. AC2 enhanced both spread and brightness of the crTMV-green fluorescent protein (GFP) lesions, whereas 2b and both P1 suppressors enhanced spread but not brightness of these lesions. P19 promoted spread of the infection into new foci within the infiltrated leaf, whereas HcPro and P25 suppressed the spread of crTMV-GFP lesions.

The crystal structure of the sobemovirus Ryegrass mottle virus (RGMoV) has been determined at 2.9 A resolution. The coat protein has a canonical jellyroll beta-sandwich fold. In comparison to other sobemoviruses the RGMoV coat protein is missing several residues in two of the loop regions. The first loop contributes to contacts between subunits around the quasi-threefold symmetry axis. The altered contact interface results in tilting of the subunits towards the quasi-threefold axis. The assembly of the T=3 capsid of sobemoviruses is controlled by the N-termini of C subunits forming a so-called beta-annulus. The other loop that is smaller in the RGMoV structure contains a helix that participates in stabilization of the beta-annulus in other sobemoviruses. The loss of interaction between the RGMoV loop and the beta-annulus has been compensated for by additional interactions between the N-terminal arms. As a consequence of these differences, the diameter of the RGMoV particle is 8 A smaller than that of the other sobemoviruses. The interactions of coat proteins in sobemovirus capsids involve calcium ions. Depletion of calcium ions results in particle swelling, which is considered a first step in disassembly. We could not identify any density for metal ions in the proximity of the conserved residues normally involved in calcium binding, but the RGMoV structure does not show any signs of swelling. A likely reason is the low pH (3.0) of the crystallization buffer in which the groups interacting with the calcium ions are not charged.

Movement protein binding 2C (MPB2C) is a plant endogenous microtubule-associated protein previously identified as an interaction partner of tobacco (Nicotiana tabacum) mosaic virus movement protein (TMV-MP). In this work, the role of MPB2C in cell-to-cell transport of TMV-MP, viral spread of TMV, and subcellular localization of TMV-MP was examined. To this end, plants with reduced MPB2C levels were generated by a gene-silencing strategy. Local and systemic spread of TMV and cell-to-cell movement of TMV-MP were unimpaired in MPB2C-silenced plants as compared to nonsilenced plants, indicating that MPB2C is not required for intercellular transport of TMV-MP itself or spread of TMV. However, a clear change in subcellular distribution of TMV-MP characterized by a nearly complete loss of microtubular localization was observed in MPB2C-silenced plants. This result shows that the MPB2C is a central player in determining the complex subcellular localization of TMV-MP, in particular its microtubular accumulation, a phenomenon that has been frequently observed and whose role is still under discussion. Clearly, MPB2C mediated accumulation of TMV-MP at microtubules is not required for intercellular spread but may be a means to withdraw the TMV-MP from the cell-to-cell transport pathway.

The Sobemovirus genome consists of positive sense, single-stranded polycistronic RNA. The 5'-terminal ORF, encoding the protein P1, is its most variable region. Sobemoviral P1 has been described as dispensable for replication but indispensable for systemic infection. The P1 of Rice yellow mottle virus-Nigerian isolate (RYMV-N) is the only RNA silencing suppressor reported for sobemoviruses until now. Using an agrobacterium-mediated transient assay, we demonstrate here that P1 of Cocksfoot mottle virus-Norwegian isolate (CfMV-NO) suppresses RNA silencing in Nicotiana benthamiana and Nicotiana tabacum, two non-host plants. CfMV-NO P1 was able to suppress the initiation and maintenance of silencing. The suppression of systemic silencing was weaker with CfMV-NO P1 than in the case of RYMV-N P1. In the case of suppression at the local level, the reduction in the amount of 25-nucleotide small interfering RNAs (siRNAs) was less pronounced for CfMV-NO P1 than it was when RYMV-N P1 was used. At the same time, we show that CfMV-NO P1 did not bind siRNAs.