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Genome editing using the CRISPR/Cas9 system is an innovation platform exploiting site-specific nuclease to create modifications (deletions or insertions) into a target region of the genome of eukaryotic organisms. Plasmid-free methods of editing via delivery of the preformed RNP complex consisting of Cas 9 endonuclease and short guide RNA are of particular interest in modern plant biology. The use of these methods rules out an integration of foreign DNA into the plant genome. We have recently developed the method of genome editing for the potato crop using delivery of the RNP editing complex immobilized on gold or chitosan microparticles into meristem cells. Here, using the gene of coilin, the structural protein of Cajal bodies which also controls plant stress resistance, we show that this method leads to the formation of large deletions of about 500 bp in the gene, which are not common for the CRISPR/Cas9 system possibly due to molecular-genetic peculiarities of meristem cells. Data presented are discussed in the context of practical applications of this method.

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.

The role of the nuclear protein coilin in the mechanisms of resistance of potato Solanum tuberosum cultivar Chicago to biotic and abiotic stresses was studied using the CRISPR/Cas9 technology. For the coilin gene editing, a complex consisting of the Cas9 endonuclease and a short guide RNA was immobilized on gold or chitosan microparticles and delivered into apical meristem cells by bioballistics or vacuum infiltration methods, respectively. Editing at least one allele of the coilin gene considerably increased the resistance of the edited lines to infection with the potato virus Y and their tolerance to salt and osmotic stress.

Plant–virus interactions are greatly influenced by environmental factors such as temperatures. In virus‐infected plants, enhanced temperature is frequently associated with more severe symptoms and higher virus content. However, the mechanisms involved in such regulatory effects remain largely uncharacterized. To provide more insight into the mechanisms whereby temperature regulates plant–virus interactions, we analysed changes in the proteome of potato cv. Chicago plants infected with potato virus Y (PVY) at normal (22 °C) and elevated temperature (28 °C), which is known to significantly increase plant susceptibility to the virus. One of the most intriguing findings is that the main enzymes of the methionine cycle (MTC) were down‐regulated at the higher but not at normal temperatures. With good agreement, we found that higher temperature conditions triggered consistent and concerted changes in the level of MTC metabolites, suggesting that the enhanced susceptibility of potato plants to PVY at 28 °C may at least be partially orchestrated by the down‐regulation of MTC enzymes and concomitant cycle perturbation. In line with this, foliar treatment of these plants with methionine restored accumulation of MTC metabolites and subverted the susceptibility to PVY at elevated temperature. These data are discussed in the context of the major function of the MTC in transmethylation processes., The work describes mechanisms whereby proteomic, transcriptional, and metabolic changes associated with the methionine cycle may modulate temperature‐sensitive plant–virus interactions.

Nanoparticles (NPs) have a number of unique properties associated with their ultrasmall size and exhibit many advantages compared with existing plant biotechnology platforms for delivery of proteins, RNA and DNA of various sizes into the plant cells (Arruda et al., 2015; Silva et al., 2010; Martin-Ortigosa et al., 2014; Mitter et al., 2017) [1], [2], [3], [4]. The data presented in this article demonstrate a delivery of biomolecules into Nicotiana benthamiana plant leaves using various types of NPs including gold, iron oxide and chitosan NPs and methods of biolistic bombardment and infiltration. The data demonstrate physical characteristics of NPs coated with fluorescently labeled protein and small RNA (size and zeta-potential) and visualization of nanocomplexes delivery into cells of N. benthamiana leaves by fluorescence microscopy.

Capsid proteins (CPs) of (+)RNA-containing plant viruses are multifunctional proteins involved in many stages of viral infection cycle, in addition to their main function of virus capsid formation. For example, the tobamoviral CP ensures virus systemic transport in plants and defines the virus-host interactions, thereby influencing the virus host range, virus infectivity, pathogenicity, and manifestation of infection symptoms. Hordeiviruses and tobamoviruses belong to the Virgaviridae family and have rod-shaped virions with a helical symmetry; their CPs are similar in structure. However, no non-structural functions of hordeiviral CPs have been described so far. In this study, we assayed possible non-structural functions of CP from the barley stripe mosaic virus (BSMV) (hordeivirus). To do this, the genome of turnip vein clearing virus (TVCV) (tobamovirus) was modified by substituting the TVCV CP gene with the BSMV CP gene or its mutants. We found that BSMV CP efficiently replaced TVCV CP at all stages of viral infection. In particular, BSMV CP performed the role of tobamoviral CP in the long-distance transport of the chimeric virus, acted as a hypersensitive response elicitor, and served as a pathogenicity determinant that influenced the symptoms of the viral infection. The chimeric tobamovirus coding for the C-terminally truncated BSMV CP displayed an increased infectivity and was transported in plants in a form of atypical virions (ribonucleoprotein complexes).

Hordeivirus movement protein encoded by the first gene of the triple gene block (TGB1 protein, TGBp1) interacts in vivo with viral genomic and subgenomic RNAs to form ribonucleoprotein (RNP) particles that are considered to be a form of viral genome (non-virion transport form) capable of cell-to-cell and long-distance transport in infected plants. The structures of these RNPs have not been elucidated. The poa semilatent virus (PSLV) TGBp1 contains a structured C-terminal NTPase/helicase domain and an N-terminal extension region consisting of two domains - a completely intrinsically disordered extreme N-terminal domain and an internal domain (ID) with structure resembling a partially disordered molten globule. Here, we characterized the structures assembled in vitro by the full-length PSLV TGBp1 alone or in the presence of viral RNA. The PSLV TGBp1 was capable of multimerization and self-assembly into extended high-molecular-mass complexes. These complexes disassembled to apparent monomers upon incubation with ATP. Upon incubation with viral RNA, the PSLV TGBp1 in vitro formed RNP structures that appeared as filamentous particles resembling virions of helical filamentous plant viruses in morphology and dimensions. By comparing the biophysical characteristics of PSLV TGBp1 and its domains in the presence and absence of RNA, we show that the ID plays the main structural role in the self-interactions and RNA interactions of TGBp1 leading to the assembly of virus-like RNP particles.

The nucleolus is the most conspicuous domain in the eukaryotic cell nucleus, whose main function is ribosomal RNA (rRNA) synthesis and ribosome biogenesis. However, there is growing evidence that the nucleolus is also implicated in many other aspects of cell biology, such as regulation of cell cycle, growth and development, senescence, telomerase activity, gene silencing, responses to biotic and abiotic stresses. In the first part of the review, we briefly assess the traditional roles of the plant nucleolus in rRNA synthesis and ribosome biogenesis as well as possible functions in other RNA regulatory pathways such as splicing, nonsense-mediated mRNA decay and RNA silencing. In the second part of the review we summarize recent progress and discuss already known and new hypothetical roles of the nucleolus in plant growth and development. In addition, this part will highlight studies showing new nucleolar functions involved in responses to pathogen attack and abiotic stress. Cross-talk between the nucleolus and Cajal bodies is also discussed in the context of their association with poly(ADP ribose)polymerase (PARP), which is known to play a crucial role in various physiological processes including growth, development and responses to biotic and abiotic stresses.

In infected plant cells, closterovirus replicative polyproteins 1a and 1ab drive membrane remodeling and formation of multivesicular replication platforms. Polyprotein 1a contains a variable Central Region (CR) between the methyltransferase and helicase domains. In a previous study, we have found that transient expression of the Beet yellows virus CR-2 segment (aa 1305-1494) in Nicotiana benthamiana induces the formation of ~1 µm mobile globules originating from the ER membranes. In the present study, sequence analysis has shown that a part of the CR named the “Zemlya region” (overlapping the CR-2), is conserved in all members of the Closterovirus genus and contains a predicted amphipathic helix (aa 1368-1385). By deletion analysis, the CR-2 region responsible for the induction of 1-μm globules has been mapped to aa 1368-1432. We suggest that the conserved membrane-modifying region of the BYV 1a may be involved in the biogenesis of closterovirus replication platforms.