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In addition to well-known roles in RNA metabolism, the nucleolus and Cajal bodies (CBs), both located within the nucleus, are involved in plant responses to biotic and abiotic stress. Previously we showed that plants in which expression of the CB protein coilin is downregulated are more susceptible to certain viruses including tobacco rattle virus (TRV), suggesting a role of coilin in antiviral defence. Experiments with coilin-deficient plants and the deletion mutant of the TRV 16K protein showed that both 16K and coilin are required for restriction of systemic TRV infection. The potential mechanisms of coilin-mediated antiviral defence were elucidated via experiments involving co-immunoprecipitation, use of NahG transgenic plants deficient in salicylic acid (SA) accumulation, measurement of endogenous SA concentrations and assessment of SA-responsive gene expression. Here we show that TRV 16K interacts with and relocalizes coilin to the nucleolus. In wild-type plants these events are accompanied by activation of SA-responsive gene expression and restriction of TRV systemic infection. By contrast, viral systemic spread was enhanced in NahG plants, implicating SA in these processes. Our findings suggest that coilin is involved in plant defence, responding to TRV infection by recognition of the TRV-encoded 16K protein and activating SA-dependent defence pathways., (© 2019 The Authors. New Phytologist © 2019 New Phytologist Trust.)

ORF6 is a small gene that overlaps the movement and coat protein genes of subgroup 1a tobamoviruses. The ORF6 protein of tomato mosaic virus (ToMV) strain L (L-ORF6), interacts in vitro with eukaryotic elongation factor 1α, and mutation of the ORF6 gene of tobacco mosaic virus (TMV) strain U1 (U1-ORF6) reduces the pathogenicity in vivo of TMV, whereas expression of this gene from two other viruses, tobacco rattle virus (TRV) and potato virus X (PVX), increases their pathogenicity. In this work, the in vivo properties of the L-ORF6 and U1-ORF6 proteins were compared to identify sequences that direct the proteins to different subcellular locations and also influence virus pathogenicity. Site-specific mutations in the ORF6 protein were made, hybrid ORF6 proteins were created in which the N-terminal and C-terminal parts were derived from the two proteins, and different subregions of the protein were examined, using expression either from a recombinant TRV vector or as a yellow fluorescent protein fusion from a binary plasmid in Agrobacterium tumefaciens. L-ORF6 caused mild necrotic symptoms in Nicotiana benthamiana when expressed from TRV, whereas U1-ORF6 caused severe symptoms including death of the plant apex. The difference in symptoms was associated with the C-terminal region of L-ORF6, which directed the protein to the endoplasmic reticulum (ER), whereas U1-ORF6 was directed initially to the nucleolus and later to the mitochondria. Positively charged residues at the N terminus allowed nucleolar entry of both U1-ORF6 and L-ORF6, but hydrophobic residues at the C terminus of L-ORF6 directed this protein to the ER.

Potato virus A (PVA) particles were purified by centrifugation through a 30 % sucrose cushion and the pellet (P1) was resuspended and sedimented through a 5-40 % sucrose gradient. The gradient separation resulted in two different virus particle populations: a virus fraction (F) that formed a band in the gradient and one that formed a pellet (P2) at the bottom of the gradient. All three preparations contained infectious particles that retained their integrity when visualized by electron microscopy (EM). Western blotting of the P1 particles revealed that the viral RNA helicase, cylindrical inclusion protein (CI), co-purified with virus particles. This result was confirmed with co-immunoprecipitation experiments. CI was detected in P2 particle preparations, whereas F particles were devoid of detectable amounts of CI. ATPase activity was detected in all three preparations with the greatest amount in P2. Results from immunogold-labelling EM experiments suggested that a fraction of the CI present in the preparations was localized to one end of the virion. Atomic force microscopy (AFM) studies showed that P1 and P2 contained intact particles, some of which had a protruding tip structure at one end, whilst F virions were less stable and mostly appeared as beaded structures under the conditions of AFM. The RNA of the particles in F was translated five to ten times more efficiently than RNA from P2 particles when these preparations were subjected to translation in wheat-germ extracts. The results are discussed in the context of a model for CI-mediated functions.

The particles of potato virus Y (PVY) and potato virus A (PVA) potyviruses are helically constructed filaments that are thought to contain a single type of coat protein subunit. Examination of negatively stained virions by electron microscopy reveals flexuous rod-shaped particles with no obvious terminal structures. It is known that some helically constructed rod-shaped virus particles incorporate additional minor protein components, which form stable complexes that mediate particle disassembly, movement or transmission by vectors. Some of this information has been obtained using imaging techniques such as atomic force microscopy. The particles of PVY and PVA were examined by atomic force microscopy and immunogold labelling electron microscopy. Our results show that some of the potyvirus particles contain a protruding tip at one end of the virus particles, which is presumably associated with the 5'-end of viral RNA. The tip contains the virus-encoded proteins genome-linked protein and helper-component proteinase. The composition and possible roles of the terminal tip structures in virus biology are discussed.

RNA-protein interactions are fundamental for different aspects of molecular biology such as gene expression, assembly of biomolecular complexes or macromolecular transport. The 3a movement protein (MP) of a plant virus, Cucumber mosaic virus (CMV), forms ribonucleoprotein (RNP) complexes with viral RNA, capable of trafficking from cell-to-cell throughout the infected plant only in the presence of the CMV capsid protein (CP). However, deletion of the C-terminal 33 amino acid residues of the CMV MP (in the mutant designated 3aDeltaC33 MP) resulted in CP-independent cell-to-cell movement. The biological differences in the behaviour of CMV wild type (wt) 3a MP and 3aDeltaC33 MP could have been a consequence of differences in the RNA-binding properties of the two MPs detected previously using biochemical assays on ensembles of molecules. To investigate the physical mechanisms of MP-RNA interactions at a single molecule level, we applied atomic force microscopy to measure for the first time unbinding forces between these individual binding partners. Minimal unbinding forces determined for individual interaction of the CMV RNA molecule with the CMV wt or truncated MPs were estimated to be approximately 45 pN and approximately 90 pN, respectively, suggesting that the distinct differences in the strength of MP-RNA interactions for the wt MP and truncated MP are attributable to the molecular binding mechanism. We also demonstrated that molecules of both CMV 3a MP and 3aDeltaC33 MP were capable of self-interaction with minimal unbinding forces of approximately 50 pN and approximately 70 pN, respectively, providing a physical basis for the cooperative mechanism of the RNA binding. The significance of intermolecular force measurements for understanding the structural and functional aspects of viral RNP formation and trafficking is discussed.

The capsid protein (CP) of Cucumber mosaic virus (CMV) is required for cell-to-cell movement, mediated by the 3a movement protein (MP). Deletion of the C-terminal 33 amino acids of the CMV 3a MP (in the mutant designated 3aDeltaC33 MP) resulted in CP-independent cell-to-cell movement, but not long-distance movement. RNA-binding studies done in vitro using isolated bacterially expressed MP showed that the 3aDeltaC33 MP bound RNA more strongly, with fewer regions sensitive to RNase and formed cooperatively bound complexes at lower ratios of protein : RNA than the wild-type (wt) 3a MP. Analysis of the architecture of the complexes by atomic force microscopy showed that the wt 3a MP formed a single type of complex with RNA, resembling beads on a string. By contrast, the 3aDeltaC33 MP formed several types of complexes, including complexes with virtually no MP bound or thicker layers of MP bound to the RNA. Assays showed that protein-RNA complexes containing high levels of either MP inhibited the infectivity and in vitro translatability of viral RNAs. The 3aDeltaC33 MP inhibited these processes at lower ratios of protein : RNA than the wt 3a MP, consistent with its stronger binding properties. The apparent contradiction between these inhibition data and the CP-independent cell-to-cell movement of CMV expressing the 3aDeltaC33 MP is discussed.

The genomes of umbraviruses differ from those of most other viruses in that they do not encode a coat protein, and thus no virus particles are formed in infected plants. Protection of umbraviral RNA outside the host plant, during vector transmission, utilizes the coat protein of an assistor luteovirus, but this review focuses on the mechanisms that compensate for the lack of a coat protein in processes within the host plant. As well as an RNA-dependent RNA polymerase, umbravirus genomes encode two other proteins from almost completely overlapping open reading frames. One of these is a cell-to-cell movement protein that can mediate the transport of homologous and heterologous viral RNAs through plasmodesmata without the participation of a coat protein. The other, the ORF3 protein, binds to viral RNA to form filamentous ribonucleoprotein particles that have elements of helical structure. It serves to stabilize the RNA and facilitates its transport through the vascular system of the plant. It may also be involved in protection of the viral RNA from the plant's defensive RNA-silencing response, although it is not a suppressor of silencing. The ORF3 protein also enters the cell nucleus, specifically targeting the nucleolus. Although the function of this localization is unknown, the ORF3 protein may provide a valuable tool for investigating plant nucleolar function.