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The main aim of this work was to develop a time-saving and cost-effective purification method of infectious plant viral nanoparticles. Virions of Tomato bushy stunt virus (TBSV), which is a member of Tombusvirus genus, were purified by one-step Biogel HT Hydroxyapatite (HA) column chromatography. Extracts from Nicotiana benthamiana plants infected with TBSV were directly loaded onto the HA column and eluted by 10 mM sodium phosphate buffer (pH 6.8). A specificity of virions has been confirmed by immunoblotting and electron microscopy. Homogeneity of virions was tested by SDS-PAGE, where only 41 kDa polypeptide bands referring to the capsid protein of TBSV were detected by Coomassie staining. The biological infectious activity of a purified material was demonstrated by observing TBSV-specific symptoms observed in N. benthamiana plants at 7-10 days of post-inoculation (dpi). Moreover, purified virions were used for immunization of the BALb/c mouse to raise primary antibodies against the TBSV virus. Our results show that in low concentrations of sodium phosphate buffer total proteins extracted from infected plants adsorb to HA sorbent, while viral particles do not adsorb to the HA matrix and flow throw column due to Ca2+ ions implicated in TBSV virions' structure. This highly effective and simple virus purification protocol can also be used for the isolation of other plant virions. [ABSTRACT FROM AUTHOR]

The advent of unique technologies of the developing Solar Energy (SE), actual energy, faces economic and environmental problems. The main obstacle to the widespread use of SE is the low value of the average annual efficiency of known solar installations. In a sharply continental climate, they are exploited only in the warm season, about 6-7 months. Known combined systems, where additional conventional water heaters duplicate the operation of solar units, require additional costs for energy carriers. These disadvantages are not offered by the integrated system of SE use. In the article, the system was studied using the example of a cattle-breeding farm. The new system performs these functions; it recycles heat, organizes their movement and accumulation, and smooths out the uneven SE. The main components of the system are: Solar Power Plant (SPP), milk cooler, climate unit, Heat Pump (HP), the battery heat, automatic control system, and device heating and hot water. The main goal, i.e. lower cost of the energy produced and the elimination of the uneven SE, compared to the known SPP, is achieved through the flow of energy from the sources mentioned above. [ABSTRACT FROM AUTHOR]

RNA interference (RNAi) plays multiple biological roles in eukaryotic organisms to regulate gene expression. RNAi also operates as a conserved adaptive molecular immune mechanism against invading viruses. The antiviral RNAi pathway is initiated with the generation of virus-derived short-interfering RNAs (siRNAs) that are used for subsequent sequence-specific recognition and degradation of the cognate viral RNA molecules. As an efficient counter-defensive strategy, most plant viruses evolved the ability to encode specific proteins capable of interfering with RNAi, and this process is commonly known as RNA silencing suppression. Virus-encoded suppressors of RNAi (VSRs) operate at different steps in the RNAi pathway and display distinct biochemical properties that enable these proteins to efficiently interfere with the host-defense system. Tombusvirus-encoded P19 is an important pathogenicity factor, required for symptom development and elicitation of a hypersensitive response in a host-dependent manner. Protein plays a crucial role of TBSV P19 in protecting viral RNA during systemic infection on Nicotiana benthamiana. The X-ray crystallographic studies conducted by two independent groups revealed the existence of a P19-siRNA complex; a conformation whereby caliper tryptophan residues on two subunits of P19 dimers measure and bind 21-nt siRNA duplexes. These structural studies provided the first details on the possible molecular mechanism of any viral suppressor to block RNAi. The association between P19 and siRNAs was also shown to occur in infected plants These and related studies revealed that in general the ability of P19 to efficiently sequester siRNAs influences symptom severity, however this is not a strict correlation in all hosts.The current working model is that during TBSV infection of plants, P19 appropriates abundantly circulating Tombusvirus-derived siRNAs thereby rendering these unavailable to program RISC, to prevent degradation of viral RNA and thus permit maintenance of viral RNA for systemic invasion. Evidence in support of this notion is that infection of N. benthamiana with P19-deficient tombusviral mutants was associated with the assembly of a discrete, high molecular weight RISC-like complex, which contains virus-derived siRNAs and exhibits specific ribonuclease activity.

The Tomato bushy stunt virus (TBSV)-encoded p19 protein (P19) is widely used as a robust tool to suppress RNA interference (RNAi) in various model organisms. P19 dimers appropriate 21-nucleotide (nt) duplex short interfering RNAs (siRNAs) generated by Dicer presumably to prevent programming of the RNA-induced silencing complex (RISC). In the context of virus infection, this model predicts that P19 mutants compromised for siRNA binding cannot prevent RISC-mediated degradation of TBSV RNA and thus reduce viral pathogenicity. To test this, we used P19/43 (R-->W), which is less pathogenic than wild-type P19 (wtP19), and P19/75-78 (RR-->GG), with pathogenicity properties (i.e., viral spread and symptom induction) comparable to those of a P19-null mutant. We demonstrate that P19/43 still suppresses RNAi-mediated viral RNA degradation in infected Nicotiana benthamiana, while P19/75-78 is unable to prevent this clearance of viral RNA, leading to an irreversible recovery phenotype. Gel filtration and immunoprecipitation assays show that at the onset of the infection, wtP19, P19/43, and P19/75-78 readily accumulate, and they form dimers. The wtP19 is stably associated with duplex approximately 21-nt TBSV siRNAs, while P19/75-78 does not bind these molecules, and the electrostatic interaction of P19/43 with siRNAs is perturbed for approximately 21-nt duplexes but not for longer siRNAs. This is the first clear demonstration of a direct correlation between a novel structurally orchestrated siRNA binding of an RNAi suppressor and its roles in viral pathogenesis. The findings should be particularly valuable for the RNAi field in general because the P19 mutants enable precise determination of siRNA appropriation effects.

SUMMARY This study identified a role for the Tomato bushy stunt virus (TBSV) p19 protein (P19) in local lesion expansion on cowpea (Vigna unguiculata), and cell-to-cell movement in pepper (Capsicum annuum). The contribution to short distance spread in both hosts was strongly influenced by a cluster of charged amino acids between positions 72 and 78 on the 172 amino acid P19. Charged amino acids near this region between positions 43 and 85 were required for long distance spread in pepper. These results indicate that the central domain of P19 plays a key role for its activities in TBSV movement and that additional regions on this protein contribute to virus spread in a host-specific manner.