A Non-Canonical Pathway Induced by Externally Applied Virus-Specific dsRNA in Potato Plants.

The external application of double-stranded RNA (dsRNA) has recently been developed as a non-transgenic approach for crop protection against pests and pathogens. This novel and emerging approach has come to prominence due to its safety and environmental benefits. It is generally assumed that the mechanism of dsRNA-mediated antivirus RNA silencing is similar to that of natural RNA interference (RNAi)-based defence against RNA-containing viruses. There is, however, no direct evidence to support this idea. Here, we provide data on the high-throughput sequencing (HTS) analysis of small non-coding RNAs (sRNA) as hallmarks of RNAi induced by infection with the RNA-containing potato virus Y (PVY) and also by exogenous application of dsRNA which corresponds to a fragment of the PVY genome. Intriguingly, in contrast to PVY-induced production of discrete 21 and 22 nt sRNA species, the externally administered PVY dsRNA fragment led to generation of a non-canonical pool of sRNAs, which were present as ladders of ~18-30 nt in length; suggestive of an unexpected sRNA biogenesis pathway. Interestingly, these non-canonical sRNAs are unable to move systemically and also do not induce transitive amplification. These findings may have significant implications for further developments in dsRNA-mediated crop protection.

The Temporal and Geographical Dynamics of Potato Virus Y Diversity in Russia.

Potato virus Y, an important viral pathogen of potato, has several genetic variants and geographic distributions which could be affected by environmental factors, aphid vectors, and reservoir plants. PVY is transmitted to virus-free potato plants by aphids and passed on to the next vegetative generations through tubers, but the effects of tuber transmission in PVY is largely unknown. By using high-throughput sequencing, we investigated PVY populations transmitted to potato plants by aphids in different climate zones of Russia, namely the Moscow and Astrakhan regions. We analyzed sprouts from the tubers produced by field-infected plants to investigate the impact of tuber transmission on PVY genetics. We found a significantly higher diversity of PVY isolates in the Astrakhan region, where winters are shorter and milder and summers are warmer compared to the Moscow region. While five PVY types, NTNa, NTNb, N:O, N-Wi, and SYR-I, were present in both regions, SYRI-II, SYRI-III, and 261-4 were only found in the Astrakhan region. All these recombinants were composed of the genome sections derived from PVY types O and N, but no full-length sequences of such types were present. The composition of the PVY variants in the tuber sprouts was not always the same as in their parental plants, suggesting that tuber transmission impacts PVY genetics.

Cajal bodies: Evolutionarily conserved nuclear biomolecular condensates with properties unique to plants.

Proper orchestration of the thousands of biochemical processes that are essential to the life of every cell requires highly organized cellular compartmentalization of dedicated microenvironments. There are 2 ways to create this intracellular segregation to optimize cellular function. One way is to create specific organelles, enclosed spaces bounded by lipid membranes that regulate macromolecular flux in and out of the compartment. A second way is via membraneless biomolecular condensates that form due to to liquid-liquid phase separation. Although research on these membraneless condensates has historically been performed using animal and fungal systems, recent studies have explored basic principles governing the assembly, properties, and functions of membraneless compartments in plants. In this review, we discuss how phase separation is involved in a variety of key processes occurring in Cajal bodies (CBs), a type of biomolecular condensate found in nuclei. These processes include RNA metabolism, formation of ribonucleoproteins involved in transcription, RNA splicing, ribosome biogenesis, and telomere maintenance. Besides these primary roles of CBs, we discuss unique plant-specific functions of CBs in RNA-based regulatory pathways such as nonsense-mediated mRNA decay, mRNA retention, and RNA silencing. Finally, we summarize recent progress and discuss the functions of CBs in responses to pathogen attacks and abiotic stresses, responses that may be regulated via mechanisms governed by polyADP-ribosylation. Thus, plant CBs are emerging as highly complex and multifunctional biomolecular condensates that are involved in a surprisingly diverse range of molecular mechanisms that we are just beginning to appreciate.

Psoroptes ovis-Early Immunoreactive Protein (Pso-EIP-1) a novel diagnostic antigen for sheep scab.

AIMS: Serodiagnosis of sheep scab is an established diagnostic method and has become popular in recent years. However, the current diagnostic antigen, Pso o 2, has shown promise as a component of a recombinant vaccine for scab, making it incompatible with discriminating between infected and vaccinated animals (DIVA). Here, we describe the discovery and characterization of a novel Psoroptes ovis immunodiagnostic antigen, P. ovis-Early Immunoreactive Protein-1 (Pso-EIP-1). METHODS AND RESULTS: Pso-EIP-1 is a highly abundant member of a six-gene family with no known homologs, indicating its potential uniqueness to P. ovis. Expression of recombinant Pso-EIP-1 (rPso-EIP-1) required a C-terminal fusion protein for stability and specific IgG immunoreactivity against rPso-EIP-1 was observed in sheep serum from 1 to 2 weeks post-infestation, indicating its highly immunogenic nature. Two of the three in silico-predicted B-cell epitopes of Pso-EIP-1 were confirmed by in vitro epitope mapping and, in a direct comparison by ELISA, Pso-EIP-1 performed to the same levels as Pso o 2 in terms of sensitivity, specificity and ability to diagnose P. ovis on sheep within 2 weeks of infestation. CONCLUSION: Pso-EIP-1 represents a novel diagnostic antigen for sheep scab with comparable levels of sensitivity and specificity to the existing Pso o 2 antigen.

CRISPR Applications in Plant Virology: Virus Resistance and Beyond.

Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated genes (Cas) is a prokaryotic adaptive immune system which has been reprogrammed into a precise, simple, and efficient gene targeting technology. This emerging technology is revolutionizing various areas of life sciences, medicine, and biotechnology and has raised significant interest among plant biologists, both in basic science and in plant protection and breeding. In this review, we describe the basic principles of CRISPR/Cas systems, and how they can be deployed to model plants and crops for the control, monitoring, and study of the mechanistic aspects of plant virus infections. We discuss how Cas endonucleases can be used to engineer plant virus resistance by directly targeting viral DNA or RNA, as well as how they can inactivate host susceptibility genes. Additionally, other applications of CRISPR/Cas in plant virology such as virus diagnostics and imaging are reviewed. The review also provides a systemic comparison between CRISPR/Cas technology and RNA interference approaches, the latter of which has also been used for development of virus-resistant plants. Finally, we outline challenges to be solved before CRISPR/Cas can produce virus-resistant crop plants which can be marketed.

Interaction of a plant virus protein with the signature Cajal body protein coilin facilitates salicylic acid-mediated plant defence responses.

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.

Virus-Like Particle Facilitated Deposition of Hydroxyapatite Bone Mineral on Nanocellulose after Exposure to Phosphate and Calcium Precursors.

We produced and isolated tobacco mosaic virus-like particles (TMV VLPs) from bacteria, which are devoid of infectious genomes, and found that they have a net negative charge and can bind calcium ions. Moreover, we showed that the TMV VLPs could associate strongly with nanocellulose slurry after a simple mixing step. We sequentially exposed nanocellulose alone or slurries mixed with the TMV VLPs to calcium and phosphate salts and utilized physicochemical approaches to demonstrate that bone mineral (hydroxyapatite) was deposited only in nanocellulose mixed with the TMV VLPs. The TMV VLPs confer mineralization properties to the nanocellulose for the generation of new composite materials.

Propagation and Isolation of Tobacco Mosaic Virus That Surface Displays Metal Binding and Reducing Peptides for Generation of Gold Nanoparticles.

In this chapter we describe an approach for propagating, isolating and characterizing tobacco mosaic virus (TMV) genetically modified to surface display a metal binding and reducing peptide, and its utilization for the production of free gold metal nanoparticles from metal salt precursors.

Data on a delivery of biomolecules into Nicothiana benthamiana leaves using different nanoparticles.

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.

Phytaspase-mediated precursor processing and maturation of the wound hormone systemin.

Peptide hormones are implicated in many important aspects of plant life and are usually synthesized as precursor proteins. In contrast to animals, data for plant peptide hormone maturation are scarce and the specificity of processing enzyme(s) is largely unknown. Here we tested a hypothesis that processing of prosystemin, a precursor of tomato (Solanum lycopersicum) wound hormone systemin, is performed by phytaspases, aspartate-specific proteases of the subtilase family. Following the purification of phytaspase from tomato leaves, two tomato phytaspase genes were identified, the cDNAs were cloned and the recombinant enzymes were obtained after transient expression in Nicotiana benthamiana. The newly identified tomato phytaspases hydrolyzed prosystemin at two aspartate residues flanking the systemin sequence. Site-directed mutagenesis of the phytaspase cleavage sites in prosystemin abrogated not only the phytaspase-mediated processing of the prohormone in vitro, but also the ability of prosystemin to trigger the systemic wound response in vivo. The data show that the prohormone prosystemin requires processing for signal biogenesis and biological activity. The identification of phytaspases as the proteases involved in prosystemin maturation provides insight into the mechanisms of wound signaling in tomato. Our data also suggest a novel role for cell death-related proteases in mediating defense signaling in plants.

Cajal bodies and their role in plant stress and disease responses.

Cajal bodies (CBs) are distinct sub-nuclear structures that are present in eukaryotic living cells and are often associated with the nucleolus. CBs play important roles in RNA metabolism and formation of RNPs involved in transcription, splicing, ribosome biogenesis, and telomere maintenance. Besides these primary roles, CBs appear to be involved in additional functions that may not be directly related to RNA metabolism and RNP biogenesis. In this review, we assess possible roles of plant CBs in RNA regulatory pathways such as nonsense-mediated mRNA decay and RNA silencing. We also summarize recent progress and discuss new non-canonical functions of plant CBs in responses to stress and disease. It is hypothesized that CBs can regulate these responses via their interaction with poly(ADP ribose)polymerase (PARP), which is known to play an important role in various physiological processes including responses to biotic and abiotic stresses. It is suggested that CBs and their components modify PARP activities and functions.

A Genetically Modified Tobacco Mosaic Virus that can Produce Gold Nanoparticles from a Metal Salt Precursor.

We genetically modified tobacco mosaic virus (TMV) to surface display a characterized peptide with potent metal ion binding and reducing capacity (MBP TMV), and demonstrate that unlike wild type TMV, this construct can lead to the formation of discrete 10-40 nm gold nanoparticles when mixed with 3 mM potassium tetrachloroaurate. Using a variety of analytical physicochemical approaches it was found that these nanoparticles were crystalline in nature and stable. Given that the MBP TMV can produce metal nanomaterials in the absence of chemical reductants, it may have utility in the green production of metal nanomaterials.

Biosynthesis of stable iron oxide nanoparticles in aqueous extracts of Hordeum vulgare and Rumex acetosa plants.

We report the synthesis and characterization of amorphous iron oxide nanoparticles from iron salts in aqueous extracts of monocotyledonous (Hordeum vulgare) and dicotyledonous (Rumex acetosa) plants. The nanoparticles were characterized by TEM, absorbance spectroscopy, SAED, EELS, XPS, and DLS methods and were shown to contain mainly iron oxide and iron oxohydroxide. H. vulgare extracts produced amorphous iron oxide nanoparticles with diameters of up to 30 nm. These iron nanoparticles are intrinsically unstable and prone to aggregation; however, we rendered them stable in the long term by addition of 40 mM citrate buffer pH 3.0. In contrast, amorphous iron oxide nanoparticles (diameters of 10-40 nm) produced using R. acetosa extracts are highly stable. The total protein content and antioxidant capacity are similar for both extracts, but pH values differ (H. vulgare pH 5.8 vs R. acetosa pH 3.7). We suggest that the presence of organic acids (such oxalic or citric acids) plays an important role in the stabilization of iron nanoparticles, and that plants containing such constituents may be more efficacious for the green synthesis of iron nanoparticles.

Coilin, the signature protein of Cajal bodies, differentially modulates the interactions of plants with viruses in widely different taxa.

Cajal bodies (CBs) are distinct nuclear bodies physically and functionally associated with the nucleolus. In addition to their traditional function in coordinating maturation of certain nuclear RNAs, CBs participate in cell cycle regulation, development, and regulation of stress responses. A key "signature" component of CBs is coilin, the scaffolding protein essential for CB formation and function. Using an RNA silencing (loss-of-function) approach, we describe here new phenomena whereby coilin also affects, directly or indirectly, a variety of interactions between host plants and viruses that have RNA or DNA genomes. Moreover, the effects of coilin on these interactions are manifested differently: coilin contributes to plant defense against tobacco rattle virus (tobravirus), tomato black ring virus (nepovirus), barley stripe mosaic virus (hordeivirus), and tomato golden mosaic virus (begomovirus). In contrast, with potato virus Y (potyvirus) and turnip vein clearing virus (tobamovirus), coilin serves to increase virus pathogenicity. These findings show that interactions with coilin (or CBs) may involve diverse mechanisms with different viruses and that these mechanisms act at different phases of virus infection. Thus, coilin (CBs) has novel, unexpected natural functions that may be recruited or subverted by plant viruses for their own needs or, in contrast, are involved in plant defense mechanisms that suppress host susceptibility to the viruses.

The use of tobacco mosaic virus and cowpea mosaic virus for the production of novel metal nanomaterials.

Due to the nanoscale size and the strictly controlled and consistent morphologies of viruses, there has been a recent interest in utilizing them in nanotechnology. The structure, surface chemistries and physical properties of many viruses have been well elucidated, which have allowed identification of regions of their capsids which can be modified either chemically or genetically for nanotechnological uses. In this review we focus on the use of such modifications for the functionalization and production of viruses and empty viral capsids that can be readily decorated with metals in a highly tuned manner. In particular, we discuss the use of two plant viruses (Cowpea mosaic virus and Tobacco mosaic virus) which have been extensively used for production of novel metal nanoparticles (<100nm), composites and building blocks for 2D and 3D materials, and illustrate their applications.

Dynamic localization of two tobamovirus ORF6 proteins involves distinct organellar compartments.

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.

The extreme N-terminal domain of a hordeivirus TGB1 movement protein mediates its localization to the nucleolus and interaction with fibrillarin.

The hordeiviral movement protein encoded by the first gene of the triple gene block (TGBp1) of Poa semilatent virus (PSLV), interacts with viral genomic RNAs to form RNP particles which are considered to be a form of viral genome capable of cell-to-cell and long-distance transport in infected plants. The PSLV TGBp1 contains a C-terminal NTPase/helicase domain (HELD) and an N-terminal extension region consisting of two structurally and functionally distinct domains: an extreme N-terminal domain (NTD) and an internal domain (ID). This study demonstrates that transient expression of TGBp1 fused to GFP in Nicotiana benthamiana leaves results in faint but obvious fluorescence in the nucleolus in addition to cytosolic distribution. Mutagenesis of the basic amino acids inside the NTD clusters A (116)KSKRKKKNKK(125) and B (175)KKATKKESKKQTK(187) reveals that these clusters are indispensable for nuclear and nucleolar targeting of PSLV TGBp1 and may contain nuclear and nucleolar localization signals or their elements. The PSLV TGBp1 is able to bind to fibrillarin, the major nucleolar protein (AtFib2 from Arabidopsis thaliana) in vitro. This protein-protein interaction occurs between the glycine-arginine-rich (GAR) domain of fibrillarin and the first 82 amino acid residues of TGBp1. The interaction of TGBp1 with fibrillarin is also visualized in vivo by bimolecular fluorescence complementation (BiFC) during co-expression of TGBp1 or its deletion mutants, and fibrillarin as fusions to different halves of YFP in N. benthamiana plants. The sites responsible for nuclear/nucleolar localization and fibrillarin binding, have been located within the intrinsically disordered TGBp1 NTD. These data could suggest that specific functions of hordeivirus TGBp1 may depend on its interaction with nucleolar components.

Domain organization of the N-terminal portion of hordeivirus movement protein TGBp1.

Three 'triple gene block' proteins known as TGBp1, TGBp2 and TGBp3 are required for cell-to-cell movement of plant viruses belonging to a number of genera including Hordeivirus. Hordeiviral TGBp1 interacts with viral genomic RNAs to form ribonucleoprotein (RNP) complexes competent for translocation between cells through plasmodesmata and over long distances via the phloem. Binding of hordeivirus TGBp1 to RNA involves two protein regions, the C-terminal NTPase/helicase domain and the N-terminal extension region. This study demonstrated that the extension region of hordeivirus TGBp1 consists of two structurally and functionally distinct domains called the N-terminal domain (NTD) and the internal domain (ID). In agreement with secondary structure predictions, analysis of circular dichroism spectra of the isolated NTD and ID demonstrated that the NTD represents a natively unfolded protein domain, whereas the ID has a pronounced secondary structure. Both the NTD and ID were able to bind ssRNA non-specifically. However, whilst the NTD interacted with ssRNA non-cooperatively, the ID bound ssRNA in a cooperative manner. Additionally, both domains bound dsRNA. The NTD and ID formed low-molecular-mass oligomers, whereas the ID also gave rise to high-molecular-mass complexes. The isolated ID was able to interact with both the NTD and the C-terminal NTPase/helicase domain in solution. These data demonstrate that the hordeivirus TGBp1 has three RNA-binding domains and that interaction between these structural units can provide a basis for remodelling of viral RNP complexes at different steps of cell-to-cell and long-distance transport of virus infection.

Cylindrical inclusion protein of potato virus A is associated with a subpopulation of particles isolated from infected plants.

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.

Caspase-resistant VirD2 protein provides enhanced gene delivery and expression in plants.

Agrobacterium tumefaciens VirD2 protein is one of the key elements of Agrobacterium-mediated plant transformation, a process of transfer of T-DNA sequence from the Agrobacterium tumour inducing plasmid into the nucleus of infected plant cells and its integration into the host genome. The VirD2 protein has been shown to be a substrate for a plant caspase-like protease activity (PCLP) in tobacco. We demonstrate here that mutagenesis of the VirD2 protein to prevent cleavage by PCLP increases the efficiency of reporter gene transfer and expression. These results indicate that PCLP cleavage of the Agrobacterium VirD2 protein acts to limit the effectiveness of T-DNA transfer and is a novel resistance mechanism that plants utilise to combat Agrobacterium infection.