Virus Elimination from Naturally Infected Field Cultivars of Potato (Solanum tuberosum) by Transgenic RNA Interference.

Tissue culture methods enable virus elimination from vegetatively propagated crop plants but cannot prevent new infections. Here we used a tissue culture transgenic approach for curing field cultivars of Solanum tuberosum through the stimulation of RNA interference (RNAi)-based antiviral defenses. Expression cassettes carrying inverted repeats of potato virus S (PVS, genus Carlavirus) movement or coat protein sequences were used for the transformation of potato cultivars naturally infected with PVS and/or a related carlavirus potato virus M (PVM), without or with potato virus Y (PVY, genus Potyvirus). A high proportion of transformants PCR-positive for transgenes were cured from both carlaviruses and PVY. After 3-year field trials, 22 transgenic lines representing seven cultivars remained free of any virus or became infected only with PVY. Vegetative progenies of the transgenic lines of cultivar Zeren (initially coinfected with PVS, PVM, and PVY), sampled after in vitro propagation or field trials, and other field cultivars accumulated transgene-derived 21, 22, and 24 nt small interfering (si)RNAs almost exclusively from the PVS inverted repeats. Additionally, some field progenies accumulated 21-22 nt siRNAs from the entire PVY genome, confirming PVY infection. Taken together, transgenic RNAi is effective for virus elimination from naturally infected potato cultivars and their sequence-specific immunization against new infections.

Phosphorylation of the alpha-subunit of plant eukaryotic initiation factor 2 prevents its association with polysomes but does not considerably suppress protein synthesis.

Phosphorylation of the α-subunit of eukaryotic initiation factor 2 (eIF2α) and subsequent inhibition of protein synthesis is a major survival response to different stresses in animal and yeast cells. However, the role of this regulatory mechanism in plants is not unambiguously established to date. Here we describe a slight reduction of polysome abundance in Nicotiana benthamiana after the transient expression of a cDNA, AteIF2α(S56D), encoding a phosphomimetic form of Arabidopsis thaliana eIF2α. In contrast, the expression of a cDNA, AteIF2α(S56A), that encodes a non-phosphorylatable form of AteIF2α caused slightly elevated polysome formation compared to the control. Recombinant AteIF2α(S56A) was detected in association with 40S ribosomal subunit-containing complexes and also in the polysomal fraction, while recombinant AteIF2α(S56D) was detected mainly in complex with 40S subunits. Intentional phosphorylation of TaeIF2α induced by L-histidinol in a wheat germ (Triticum aestivum) cell-free extract did not reduce the abundance of polysomes. Interestingly, the phosphorylated TaeIF2(αP) was not detected in the polysomal fraction, similar to AteIF2α(S56D) in the in vivo experiment. Using mRNAs with a 'Strepto-tag' in the 3' untranslated region, the 48S pre-initiation complexes isolated from histidinol-treated wheat germ extracts were shown to contain phosphorylated TaeIF2(αP). Thus, the phosphorylation of plant eIF2 does not greatly affect its ability to participate in the initiation of mRNA translation, in contrast to animals and yeast, in which eIF2α phosphorylation results in profound suppression of protein synthesis.

Production of the sheep pox virus structural protein SPPV117 in tobacco chloroplasts.

OBJECTIVE: A chloroplast transgenic approach was assessed in order to produce a structural protein SPPV117 of sheep pox virus in Nicotiana tabacum for the future development of a plant-based subunit vaccine against sheep pox. RESULTS: Two DNA constructs containing SPPV117 coding sequence under the control of chloroplast promoter and terminator of psbA gene or rrn promoter and rbcL terminator were designed and inserted into the chloroplast genome by a biolistic method. The transgenic plants were selected via PCR analysis. Northern and Western blot analysis showed expression of the transgene at transcriptional and translational levels, respectively. The recombinant protein accumulated to about 0.3% and 0.9% of total soluble protein in leaves when expressed from psbA and rrn promoter, respectively. Plant-produced SPPV117 protein was purified using metal affinity chromatography and the protein yield was 19.67  ±  1.25 µg g-1 (FW). The serum of a sheep infected with the virus recognised the chloroplast-produced protein indicating that the protein retains its antigenic properties. CONCLUSIONS: These results demonstrate that chloroplasts are a suitable system for the production of a candidate subunit vaccine against sheep pox.

Evidence That Phosphorylation of the α-Subunit of eIF2 Does Not Essentially Inhibit mRNA Translation in Wheat Germ Cell-Free System.

A mechanism based on reversible phosphorylation of the α-subunit of eukaryotic initiation factor 2 (eIF2α) has been confirmed as an important regulatory pathway for the inhibition of protein synthesis in mammalian and yeast cells, while plants constitute the significant exception. We studied the induction of TaeIF2α phosphorylation in germinated wheat (Triticum aestivum) embryos subjected to different adverse conditions. Data confirmed that formation of TaeIF2(αP) was not a general response, as no phosphorylation was observed under salt, oxidative, or heat stress. Nevertheless, treatment by salicylic acid, UV-light, cold shock and histidinol did induce phosphorylation of TaeIF2α of wheat as has been established previously for AteIF2α in Arabidopsis (Arabidopsis thaliana). The influence of TaeIF2α phosphorylation on translation of reporter mRNA with different 5'-untranslated regions (5'UTRs) was studied in wheat germ cell-free system (WG-CFS), in which TaeIF2α was first phosphorylated either by heterologous recombinant human protein kinase, HsPKR (activated by double-stranded (ds)RNA), or by endogenous protein kinase TaGCN2 (activated by histidinol). Pretreatment of WG-CFS with HsPKR in the presence of dsRNA or with histidinol resulted in intense phosphorylation of TaeIF2α; however, the translation levels of all tested mRNAs decreased by only 10-15% and remained relatively high. In addition, factor OceIF2 from rabbit (Oryctolagus cuniculus) bound GDP much more strongly than the homologous factor TaeIF2 from wheat germ. Furthermore, factor OceIF2B was able to stimulate guanine nucleotide exchange (GDP→GTP) on OceIF2 but had no effect on a similar exchange on TaeIF2. These results suggest that the mechanism of stress response via eIF2α phosphorylation is not identical in all eukaryotes, and further research is required to find and study in detail new plant-specific mechanisms that may inhibit overall protein synthesis in plants under stress.

The Effect of Translation Promoting Site (TPS) on Protein Expression in E. coli Cells.

The study of translation initiation in prokaryotes assumes that there should be a mechanism different from the canonical model, which postulates the formation of the pre-initiation complex through the interaction of the Shine-Dalgarno sequence (SD) at the 5'-end of mRNA and the anti-Shine-Dalgarno site at the 3'-end of 16S rRNA. In this paper we've studied the effect of TPS (Translation-initiation Promoting Site) on ß-glucuronidase expression in E. coli cells at different cultivation temperatures. The examined leader sequences were cloned into the pET23c plasmid upstream the ß-glucuronidase gene; protein expression was performed in E. coli BL21 (DE3) cells. ß-glucuronidase activity was measured in bacterial cell extracts via paranitrophenyl b-D-glucuronide assay. The quantity of expressed protein was measured by Western blotting with following densitometry. It was shown that TPS increases the level of protein expression at stressful conditions (10 °C and 44 °C) 5-8 times compared to control. The combination of TPS and SD sites in the 5'-leader sequence of the mRNA created an enhancer that increased the expression level 2-3.6 times compared to a single SD-sequence. Based on the obtained data and the computer modeling of interaction between 16S rRNA and TPS, we proposed an alternative variation of prokaryotic translation initiation.

Recombinant Sheep Pox Virus Proteins Elicit Neutralizing Antibodies.

The aim of this work was to evaluate the immunogenicity and neutralizing activity of sheep pox virus (SPPV; genus Capripoxvirus, family Poxviridae) structural proteins as candidate subunit vaccines to control sheep pox disease. SPPV structural proteins were identified by sequence homology with proteins of vaccinia virus (VACV) strain Copenhagen. Four SPPV proteins (SPPV-ORF 060, SPPV-ORF 095, SPPV-ORF 117, and SPPV-ORF 122), orthologs of immunodominant L1, A4, A27, and A33 VACV proteins, respectively, were produced in Escherichia coli. Western blot analysis revealed the antigenic and immunogenic properties of SPPV-060, SPPV-095, SPPV-117 and SPPV-122 proteins when injected with adjuvant into experimental rabbits. Virus-neutralizing activity against SPPV in lamb kidney cell culture was detected for polyclonal antisera raised to SPPV-060, SPPV-117, and SPPV-122 proteins. To our knowledge, this is the first report demonstrating the virus-neutralizing activities of antisera raised to SPPV-060, SPPV-117, and SPPV-122 proteins.

2'-OH of mRNA are critical for the binding of its codons at the 40S ribosomal P site but not at the mRNA entry site.

The roles of 2'-OH groups in the binding of mRNA to human ribosomes were studied using site-directed cross-linking. We found that both mRNA and mDNA analogues bearing a cross-linker can modify ribosomal proteins (rps) S3e and S2e at the mRNA entry site independently on tRNA presence, but only mRNA analogues were capable of a tRNA(Phe)-dependent binding to human ribosomes and cross-linking to rpS26e in the mRNA binding centre. Thus, 2'-OH groups of mRNA are unimportant for binding at the entry site but they are crucial for codon-anticodon interactions at the P site, implying the existence of mRNA-ribosome contacts that do not occur in bacteria.