Comparison of antigenic properties of the X-potato virus and its coat proteins. Effect of proteolytic cleavage on antigenic characteristics

Antigenic properties of intact potato virus X (PVX) particles and of PVX coat protein (CP) preparations were compared using different modifications of ELISA test. In the competitive ELISA test (reaction in solution) antibodies to intact virus react much stronger with PVX than with CP while antibodies to CP react much stronger with CP than with PVX. In the direct ELISA test (reaction on the solid support) the difference in reactions of antiCP antibodies with PVX and CP is eliminated while the one in reactions of antiPVX antibodies with these antigens remains. No difference was registered in reactivity of PVX absorbed directly on polystyrene or on immunoglobulin-coated wells (sandwich ELISA) to antiCP antibodies.     

Synthesis of the tobacco mosaic virus in blocked spreading of infection

The possibility of infection of tobacco upper leaves with tobacco mosaic virus (TMV) was examined in experiments where the inoculum was imbibed through the cut stem. The inoculum used were: a) a preparation of a virus-specific informosome-like ribonucleoproteins (vRNP) isolated from TMV-infected plants; b) a TMV preparation; or c) a mixture of TMV and vRNP. Multiplication of TMV in upper leaves was observed in neither of the variants; nevertheless in the vascular tissue and/or probably in adjoining parenchymal cells, two kinds of RNA were synthesized: of mol. w. (1.1--1.3) X 10(6) and (0.6--0.8) X 10(6). These RNA were not found in healthy plants in the presence of actinomycin D. The synthesis of genomic TMV RNA is suppressed under these conditions. Thus, some kind of abortive TMV infection takes place under the condition of experimental inoculation of plants through a cut stem. Molecular hybridization with the DNA of recombinant plasmid containing a nucleotide sequence complementary to the 3'-portion of genomic TMV RNA proves that short RNAs synthesized under the abortive infection conditions are TMV-specific. The experiments with differential temperature treatment of N-gene-containing plants under abortive infection conditions suggest that necrotization is not necessarily induced by genomic TMV RNA synthesis.     

Biological activities of human interferon and 2′–5′ oligoadenylates in plants

Exogenous human interferon 2 (IFN) and 2–5 oligoadenylates (2–5A) have been shown to cause at least a dual physiological effect in tobacco and wheat: (i) increased cytokinin activity and (ii) induced synthesis of numerous proteins, among which members of two groups of stress proteins have been identified, namely pathogenesis-related (PR) and heat shock (HS) proteins. These effects were observed only by low concentrations of these substances: IFN at 0.1–1 u/ml and 2–5A at 1–10 nM.     

Site-specific enzymatic cleavage of TMV RNA directed by deoxyribo- and chimeric (deoxyribo-ribo)oligonucleotides

The TMV RNA molecule can be cleaved at a single site by RNase H directed by chimeric oligo(deoxyribo-ribo)nucleotide with an internucleotide pyrophosphate bond.     

New phytoviral vector for superexpression of target proteins in plants

Previously, we obtained a new viral vector based on the Alternanthera mosaic virus strain MU (AltMV-MU). The gene of interest was placed under control of two subgenomic promoters (sgp). Viral vector provided the superexpression of target proteins in Nicotiana benthamiana. In the present work, to increase the level of protein expression, this viral vector was modified and the coat protein gene was placed under control of three sgp. The new viral vector provided superexpression of the protein of interest in plants, and its amount was more than 50% of the total soluble protein in cells. The efficiency of the target protein expression in the plants transformed with viral vectors containing two and three sgp was compared.     

Interaction of Plum Pox Virus with Specific Colloidal Gold-Labeled Antibodies and Development of Immunochromatographic Assay of the Virus

Two monoclonal antibodies (mABs) raised against plum pox virus (PPV) were shown to recognize its D, M, and C strains. Conjugates of the antibodies with colloidal gold (CG) nanoparticles averaging 26 nm in diameter were synthesized. The binding constants of PPV with both the native and conjugated mABs were determined using a Biacore X device. The complexes between the CG-mAB conjugates and plum pox virions were examined by means of transmission electron and atomic force microscopy. Using the conjugates with optimal component ratio, an express immunochromatographic assay of PPV was developed with a detection limit of 3 ng/ml and duration of 10 min. The assay was tested for PPV detection in sam- ples of stone fruit tree leaves and demonstrated a good compatibility with the data obtained by “sandwich”-ELISA. The developed assay can be used in the field and applied for monitoring viral infection and for quarantine purposes.     

Increase of histidine content in Brassica rapa subsp. oleifera by over-expression of histidine-rich fusion proteins

An approach that enables the increase of the quantity of a specific amino acid in crop plants is reported. Oleosin gene from Arabidopsis thaliana or 30K movement protein gene of Tobacco mosaic virus (TMV; genus Tobamovirus) were cloned under the control of napin or hybrid promoters, and in fusion to synthetic poly-histidine (poly-His) sequences for transformation into spring turnip rape (Brassica rapa subsp. oleifera; synonym to B. campestris). The most stable expression cassettes for the poly-His production prior to the plant transformation were selected by analyzing the protein expression in in vitro translation and in transient plant expression systems using GFP as marker. Expression of the poly-His-constructs in transgenic Brassica rapa plants was analyzed using dot and western blotting and PCR. The constructs were stably expressed in the third generation of the transgenic plant lines. Histidine content was measured from the seeds of the transgenic plants, and some plant lines had more than 20% increase in histidine content compared to wild type. The methodology may be widely applicable to increase the content of any amino acid in crop plants including those encoded by rare codons.     

Role of the leader sequence in tobacco pectin methylesterase secretion

We report that unprocessed tobacco pectin methylesterase (PME) contains N-terminal pro-sequence including the transmembrane (TM) domain and spacer segment preceding the mature PME. The mature portion of PME was replaced by green fluorescent protein (GFP) gene and various deletion mutants of pro-sequence fused to GFP were cloned into binary vectors and agroinjected in Nicotiana benthamiana leaves. The PME pro-sequence delivered GFP to the cell wall (CW). We showed that a transient binding of PME TM domain to endoplasmic reticulum membranes occurs upon its transport to CW. The CW targeting was abolished by various deletions in the TM domain, i.e., anchor domain was essential for secretion of GFP to CW. By contrast, even entire deletion of the spacer segment had no influence on GFP targeting.