Functional Analysis of Coilin in Virus Resistance and Stress Tolerance of Potato Solanum tuberosum using CRISPR-Cas9 Editing.

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.

Double-Stranded RNAs in Plant Protection Against Pathogenic Organisms and Viruses in Agriculture.

Recent studies have shown that plants are able to express the artificial genes responsible for the synthesis of double-stranded RNAs (dsRNAs) and hairpin double-stranded RNAs (hpRNAs), as well as uptake and process exogenous dsRNAs and hpRNAs to suppress the gene expression of plant pathogenic viruses, fungi, or insects. Both endogenous and exogenous dsRNAs are processed into small interfering RNAs (siRNAs) that can spread locally and systemically through the plant, enter pathogenic microorganisms, and induce RNA interference-mediated pathogen resistance in plants. There are numerous examples of the development of new biotechnological approaches to plant protection using transgenic plants and exogenous dsRNAs. This review summarizes new data on the use of transgenes and exogenous dsRNAs for the suppression of fungal and insect virulence genes, as well as viruses to increase the resistance of plants to these pathogens. We also analyzed the current ideas about the mechanisms of dsRNA processing and transport in plants.

Guide RNA Design for CRISPR/Cas9-Mediated Potato Genome Editing.

The activity of the pool of sgRNA molecules designed for different regions of potato coilin and phytoene desaturase genes was compared in vitro. Due to the presence of nucleotides unpaired with DNA, sgRNA is able not only to inhibit but also to stimulate the activity of the Cas9-sgRNA complex in vitro. Although the first six nucleotides located in the DNA substrate proximally to the PAM site at the 3' end are the binding sites for cas9, they had no significant effect on the activity of the Cas9-sgRNA complex.

Non-structural Functions of Hordeivirus Capsid Protein Identified in Plants Infected by a Chimeric Tobamovirus.

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).

Application of the CRISPR/Cas System for Generation of Pathogen-Resistant Plants.

The use of the CRISPR/Cas9 prokaryotic adaptive immune system has led to a breakthrough in targeted genome editing in eukaryotes. The CRISPR/Cas technology allows to generate organisms with desirable characteristics by introducing deletions/insertions into selected genome loci resulting in the knockout or modification of target genes. This review focuses on the current state of the CRISPR/Cas use for the generation of plants resistant to viruses, bacteria, and parasitic fungi. Resistance to DNA- and RNA-containing viruses is usually provided by expression in transgenic plants of the Cas endonuclease gene and short guide RNAs (sgRNAs) targeting certain sites in the viral or the host plant genomes to ensure either direct cleavage of the viral genome or modification of the plant host genome in order to decrease the efficiency of virus replication. Editing of plant genes involved in the defense response to pathogens increases plants resistance to bacteria and pathogenic fungi. The review explores strategies and prospects of the development of pathogen-resistant plants with a focus on the generation of non-transgenic (non-genetically modified) organisms, in particular, by using plasmid (DNA)-free systems for delivery of the Cas/sgRNA editing complex into plant cells.

Structure and Noncanonical Activities of Coat Proteins of Helical Plant Viruses.

The main function of virus coat protein is formation of the capsid that protects the virus genome against degradation. However, besides the structural function, coat proteins have many additional important activities in the infection cycle of the virus and in the defense response of host plants to viral infection. This review focuses on noncanonical functions of coat proteins of helical RNA-containing plant viruses with positive genome polarity. Analysis of data on the structural organization of coat proteins of helical viruses has demonstrated that the presence of intrinsically disordered regions within the protein structure plays an important role in implementation of nonstructural functions and largely determines the multifunctionality of coat proteins.

Structural Properties of Potexvirus Coat Proteins Detected by Optical Methods.

It has been shown by X-ray analysis that cores of coat proteins (CPs) from three potexviruses, flexible helical RNA-containing plant viruses, have similar α-helical structure. However, this similarity cannot explain structural lability of potexvirus virions, which is believed to determine their biological activity. Here, we used circular dichroism (CD) spectroscopy in the far UV region to compare optical properties of CPs from three potexviruses with the same morphology and similar structure. CPs from Alternanthera mosaic virus (AltMV), potato aucuba mosaic virus (PAMV), and potato virus X (PVX) have been studied in a free state and in virions. The CD spectrum of AltMV virions was similar to the previously obtained CD spectrum of papaya mosaic virus (PapMV) virions, but differed significantly from the CD spectrum of PAMV virions. The CD spectrum of PAMV virions resembled in its basic characteristics the CD spectrum of PVX virions characterized by molar ellipticity that is abnormally low for α-helical proteins. Homology modeling of the CP structures in AltMV, PAMV, and PVX virions was based on the known high-resolution structures of CPs from papaya mosaic virus and bamboo mosaic virus and confirmed that the structures of the CP cores in all three viruses were nearly identical. Comparison of amino acid sequences of different potexvirus CPs and prediction of unstructured regions in these proteins revealed a possible correlation between specific features in the virion CD spectra and the presence of disordered N-terminal segments in the CPs.

"Green" nanotechnologies: synthesis of metal nanoparticles using plants.

While metal nanoparticles are being increasingly used in many sectors of the economy, there is growing interest in the biological and environmental safety of their production. The main methods for nanoparticle production are chemical and physical approaches that are often costly and potentially harmful to the environment. The present review is devoted to the possibility of metal nanoparticle synthesis using plant extracts. This approach has been actively pursued in recent years as an alternative, efficient, inexpensive, and environmentally safe method for producing nanoparticles with specified properties. This review provides a detailed analysis of the various factors affecting the morphology, size, and yield of metal nanoparticles. The main focus is on the role of the natural plant biomolecules involved in the bioreduction of metal salts during the nanoparticle synthesis. Examples of effective use of exogenous biomatrices (peptides, proteins, and viral particles) to obtain nanoparticles in plant extracts are discussed.

In vitro phosphorylation of the N-terminal half of hordeivirus movement protein.

The N-terminal half of TGB1 movement protein of poa semilatent hordeivirus, which forms a ribonucleoprotein complex involved in movement of the viral genome in the plant, and its two domains, NTD and ID, are phosphorylated in vitro by a fraction enriched in cell walls from Nicotiana benthamiana. Using a set of protein kinase inhibitors with different specificities, it was found that enzymes possessing activities of casein kinase 1, protein kinase A, and protein kinase C are involved in phosphorylation. Commercial preparations of protein kinases A and C are able to phosphorylate in vitro recombinant proteins corresponding to the N-terminal half of the protein and its domains NTD and ID. Phosphorylation of the NTD has no effect on the efficiency and character of its binding to RNA. However, phosphorylation of the ID leads to a decrease in its RNA-binding activity and in the ability for homological protein-protein interactions.

Two RNA-binding sites in plant fibrillarin provide interactions with various RNA substrates.

Fibrillarin, one of the major proteins of the nucleolus, plays several essential roles in ribosome biogenesis including pre-rRNA processing and 2'-O-ribose methylation of rRNA and snRNAs. Recently, it has been shown that fibrillarin plays a role in virus infections and is associated with viral RNPs. Here, we demonstrate the ability of recombinant fibrillarin 2 from Arabidopsis thaliana (AtFib2) to interact with RNAs of different lengths and types including rRNA, snoRNA, snRNA, siRNA and viral RNAs in vitro. Our data also indicate that AtFib2 possesses two RNA-binding sites in the central (138-179 amino acids) and C-terminal (225-281 amino acids) parts of the protein, respectively. The conserved GCVYAVEF octamer does not bind RNA directly as suggested earlier, but may assist with the proper folding of the central RNA-binding site.

Involvement of the plant nucleolus in virus and viroid infections: parallels with animal pathosystems.

The nucleolus is a dynamic subnuclear body with roles in ribosome subunit biogenesis, mediation of cell-stress responses, and regulation of cell growth. An increasing number of reports reveal that similar to the proteins of animal viruses, many plant virus proteins localize in the nucleolus to divert host nucleolar proteins from their natural functions in order to exert novel role(s) in the virus infection cycle. This chapter will highlight studies showing how plant viruses recruit nucleolar functions to facilitate virus translation and replication, virus movement and assembly of virus-specific ribonucleoprotein (RNP) particles, and to counteract plant host defense responses. Plant viruses also provide a valuable tool to gain new insights into novel nucleolar functions and processes. Investigating the interactions between plant viruses and the nucleolus will facilitate the design of novel strategies to control plant virus infections.

The internal domain of hordeivirus movement protein TGB1 forms in vitro filamentous structures.

The 63 kDa hordeivirus movement protein TGB1 of poa semilatent virus (the PSLV TGB1 protein) forms viral ribonucleoprotein for virus transport within a plant. It was found using the dynamic laser light scattering technique that the internal domain of TGB1 protein forms in vitro high molecular weight complexes. According to results of atomic force microscopy, a part of these complexes is represented by globules of different sizes, while another part consists of extended filamentous structures. Similar properties are also characteristic of the N-terminal half of the protein and are obviously due to its internal domain moiety. The data support the hypothesis that upon viral ribonucleoprotein complex formation, the N-terminal half of the PSLV TGB1 protein plays a structural role and exhibits the ability to form multimeric filamentous structures (the ability for self-assembly).

Oligomerization of the potato virus X 25-kD movement protein.

A 25-kD movement protein (25K protein) encoded by the first gene of the potexvirus Potato virus X triple gene block of transport genes is essential for the viral movement in infected plants. The 25K protein belongs to superfamily 1 of NTPase/helicases and exhibits in vitro RNA helicase, Mg2+-dependent NTPase, and RNA-binding activities. In the present work, the ability of 25K protein for homologous interactions was studied using the yeast two-hybrid system, protein chemical cross-linking in the presence of glutaraldehyde, far-Western blotting, and ultracentrifugation in sucrose density gradients. The 25K protein was shown to form homodimers and homooligomers. Sites of homologous protein-protein interactions were found in both the N- and C-terminal portions of the protein.

Immunodetection and fluorescent microscopy of transgenically expressed hordeivirus TGBp3 movement protein reveals its association with endoplasmic reticulum elements in close proximity to plasmodesmata.

The subcellular localization of the hydrophobic TGBp3 protein of Poa semilatent virus (PSLV, genus Hordeivirus) was studied in transgenic plants using fluorescent microscopy to detect green fluorescent protein (GFP)-tagged protein and immunodetection with monoclonal antibodies (mAbs) raised against the GFP-based fusion expressed in E. coli. In Western blot analysis, mAbs efficiently recognized the wild-type and GFP-fused PSLV TGBp3 proteins expressed in transgenic Nicotiana benthamiana, but failed to detect TGBp3 in hordeivirus-infected plants. It was found that PSLV TGBp3 and GFP-TGBp3 had a tendency to form large protein complexes of an unknown nature. Fractionation studies revealed that TGBp3 represented an integral membrane protein and probably co-localized with an endoplasmic reticulum-derived domain. Microscopy of epidermal cells in transgenic plants demonstrated that GFP-TGBp3 localized to cell wall-associated punctate bodies, which often formed pairs of opposing discrete structures that co-localized with callose, indicating their association with the plasmodesmata-enriched cell wall fields. After mannitol-induced plasmolysis of the leaf epidermal cells in the transgenic plants, TGBp3 appeared within the cytoplasm and not at cell walls. Although TGBp3-induced bodies were normally static, most of them became motile after plasmolysis and displayed stochastic motion in the cytoplasm.

[Virions and membrane proteins of the potato X virus interact with microtubules and enables tubulin polymerization in vitro]. / Viriony i belok obolochki X-virusa kartofelia vzaimodeistvuiut s mikrotrubochkami i sposobstvuiut polimeriaztsii tubulina in vitro.

A study was made of the in vitro interactions of virions and the coat protein (CP) of the potato virus X (PVX) with microtubules (MT). Both virions and CP cosedimented with taxol-stabilized MT. In the presence of PVX CP, tubulin polymerized to produce structures resistant to chilling. Electron microscopy revealed the aberrant character of the resulting tubulin polymers (protofilaments and their sheets), which differed from MT assembled in the presence of cell MAP2. In contrast, PVX virions induced the assembly of morphologically normal MT sensitive to chilling. Virions were shown to compete with MAP2 for MT binding, suggesting an overlap for the MT sites interacting with MAP2 and with PVX virions. It was assumed that PVX virions interact with MT in vivo and that, consequently, cytoskeleton elements participate in intracellular compartmentalization of the PVX genome.

RNA helicase activity of the plant virus movement proteins encoded by the first gene of the triple gene block.

Cell-to-cell and long-distance transport of some plant viruses requires coordinated action of three movement proteins encoded by triple gene block (TGB). The largest of TGB proteins, TGBp1, is a member of the superfamily I of DNA/RNA helicases and possesses a set of conserved helicase sequence motifs necessary for virus movement. A recombinant His-tagged form of TGBp1 of two hordeiviruses and potato virus X, a potexvirus, produced in Escherichia coli had unwinding activity on a partially duplexed RNA, but not DNA substrate. The helicase activity of these proteins was dependent on Mg2+ and ATP. The isolated C-terminal half of the PSLV TGBp1 retaining all helicase motifs was also able to unwind RNA duplex.

RNA-binding properties of the 63 kDa protein encoded by the triple gene block of poa semilatent hordeivirus.

The 63 kDa '63K' movement protein encoded by the triple gene block of poa semilatent virus (PSLV) comprises the C-terminal NTPase/helicase domain and the N-terminal extension domain, which contains two positively charged sequence motifs, A and B. In this study, the in vitro RNA-binding properties of PSLV 63K and its mutants were analysed. Membrane-immobilized 63K and N-63K (isolated N-terminal extension domain) bound RNA at high NaCl concentrations. In contrast, C-63K (isolated NTPase/helicase domain) was able to bind RNA only at NaCl concentrations of up to 50 mM. In gel-shift assays, C-63K bound RNA to form complexes that were unable to enter an agarose gel, whereas complexes formed by N-63K could enter the gel. Full-length 63K formed both types of complexes. Visualization of the RNA-protein complexes formed by 63K, N-63K and C-63K by atomic force microscopy demonstrated that each complex had a different shape. Collectively, these data indicate that 63K has two distinct RNA-binding activities associated with the NTPase/helicase domain and the N-terminal extension domain. Mutations in either of the positively charged sequence motifs A and B had little effect on the RNA binding of the N-terminal extension domain, whereas mutations in both motifs together inhibited RNA binding. Hybrid viruses with mutations in motifs A and B were able to infect inoculated leaves of Nicotiana benthamiana plants, but were unable to move systemically to uninoculated leaves, suggesting that the RNA-binding activity of the N-terminal extension domain of PSLV 63K is associated with virus long-distance movement.

Evidence for two nonoverlapping functional domains in the potato virus X 25K movement protein.

To study subdomain organization of the potato virus X (PVX) movement protein (MP) encoded by the first gene in the triple gene block (TGB), we mutated the 25-kDa TGBp1 protein. The N-terminal deletion of the helicase motifs I, IA, and II resulted in loss of the ATPase activity and RNA binding. A frameshift mutation truncating the C-terminal motifs V and VI gave rise to increase of the TGBp1 ATPase activity and had little effect on RNA binding in vitro. Fusions of the green fluorescent protein with 25-kDa MP and its derivative lacking motifs V-VI exhibited similar fluorescence patterns in epidermal cells of Nicotiana benthamiana leaves. Cell-to-cell movement of the 25K-deficient PVX genome was not complemented by the TGBp1 of Plantago asiatica mosaic potexvirus (PlAMV) but was efficiently complemented by a chimeric TGBp1 consisting of the N-terminal part of PlAMV protein (motifs I-IV) and the PVX-specific C-terminal part (motifs V-VI). These results suggest that NTP hydrolysis, RNA binding, and targeting to the specific cellular compartment(s) are associated with the N-terminal domain of the TGBp1 including the helicase motifs I-IV and that the C-terminal domain is involved in specific interactions with other virus proteins.