[Pectin methylesterase as a factor of plant transcriptome stability].

Pectin methylesterase (PME), cell wall enzyme, is known as a factor of plant growth and morphogenesis. Recently, PME participation in gene silencing, plant virus reproduction and transgenesis was revealed. Here, the role of PME in antivirus resistance was studied. It has been shown that increasing PME enzymatic activity induced by additional PME gene and even empty vector resulted in suppression of tobacco mosaic virus (TMV) reproduction including short- and long-distance virus movement in plant. We revealed increased PME activity in stably-transformed intact plants. For example, transgenic tobacco and N. benthamiana plants expressing TMV movement protein gene and GFP appeared increased PME activity. The tomato plants co-suppressing polygalacturonase gene have increased PME activity as well. Moreover, light-induced psbO gene activation was accompanied by transcription of PME gene. The introduction of foreign gene in plant cell or excess transcription of own genome resulted in increasing PME activity leading to transcriptome status quo returning.

[Gene silencing in plants].

Here, the role of small RNAs is described in (i) cytoplasmic viral RNA silencing; (ii) cellular mRNA silencing via miPHK production. The role of cellular factors such as Dicers, Argonautes, RNA-dependent RNA-polymerase, RNA-polymerase IV and pectin methylesterase are discussed in details. Moreover, silencing suppression by viral proteins/RNAs and silencing as an approach of biotechnology are reviewed.

Stability of plant mRNAs depends on the length of the 3'-untranslated region.

Eukaryotic mRNAs that prematurely terminate translation are recognized and degraded by nonsense mediated decay (NMD). This degradation pathway is well studied in animal and yeast cells. The data available imply that NMD also takes place in plants. However, the molecular mechanism of recognition and degradation of plant RNAs containing premature terminator codon (PTC) is not known. Here we report that in plant cells this mechanism involves the recognition of the sizes of the 3'-untranslated regions (3'UTR). Plant 3'UTRs longer than 300 nucleotides induce mRNA instability. Contrary to mammalian and yeast cells, this destabilization does not depend on the presence of any specific sequences downstream of the terminator codon. Unlike nuclear-produced mRNAs, plant virus vector long 3'UTR-containing RNAs, which are synthesized directly in the cytoplasm, are stable and translated efficiently. This shows that RNAs produced in the cytoplasm by viral RNA-dependent RNA polymerase are able to avoid the proposed mechanism.

[Epitope mapping of the recombinant movement protein of the tobacco mosaic virus using monoclonal antibodies].

The movement protein (MP) of the tobacco mosaic virus (TMV) provides the intercellular transport of the viral RNA through plasmodesmata. The MP fulfills its function while interacting with host cell factors over the whole path of its intracellular movement from the subcellular site of its synthesis to the plasmodesmata of cellular walls. The MP conformation during its intracellular movement and fulfillment of the transport function still remains unknown. In this study, we describe the preparation of murine monoclonal antibodies (MAs) to TMV MP and mapping of the MP epitopes. Stable hybridoma lines that produce MAs to the partially denatured recombinant MP (MPr) were obtained. MAs were tested by immunoblotting and ELISA with the use of deletion variants of MPr. The epitopes of TMV MPr that recognize specific MAs were determined.

[Cloning transport protein genes for two strains of tobacco mosaic virus and their expression in Escherichia coli cells]. / Klonirovanie genov transportnykh belkov dvukh shtammov virusa tabachnoi mozaiki i ikh ékspressiia v kletkakh Escherichia coli.

Recombinant constructs expressing fusion (His)6 movement proteins of two strains (Cruciferous and Vulgare) of tobacco mosaic virus were generated by cloning the PCR-amplified fragments into the pQE-9 vector. The movement proteins containing N-terminal (His)6 affinity tags were purified on a metal chelate adsorbent.

[Synthesis of the tobacco mosaic virus in blocked spreading of infection]. / Sintez virusa tabachnoi mozaiki v usloviiakh bloka transporta infektsii.

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.

[Analysis of polypeptides of virus-specific informosomes induced by tobacco mosaic virus and potato virus X]. / Analiz polipeptidov virusspetsificheskikh informosom, indutsiruemykh virusom tabachnoi mozaiki i X-virusom kartofelia.

Informosome-like virus-specific ribonucleoprotein (vRNP) of tobacco mosaic virus (TMV) comprise a set of four major polypeptides having molecular weights of 17 500, 31 000, 37 000 and 39 000. Of the minor polypeptides, those of apparent molecular weights 25 000, 55 000, 68 000 and 70 000 had electrophoretic mobilities of polypeptides found in a ribonucleoprotein preparation from uninoculated plants. Polypeptide with mol.wt. 175 000 is TMV coat protein so far as: a) vRNP was precipitated with immunoglobulins against TMV and TMV coat protein; b) it had electrophoretic mobility similar to mobility of TMV coat protein; c) the peptide map of polypeptides with mol.wts 31 000, 37 000 and 39 000 are probably virus-specific-products. This is supposed because they are not present in cell informosomes protein, and they are not revealed in vRNP induced in cells after infection with potato virus X (PVX). Electrophoresis of vRNP-PVX protein reveals polypeptides of 23 000 (PVX coat protein), 55 000, 70 000, 78 000, 95 000, 120 000 and 145 000.

[Virus-specific informosomes in tobacco cells infected with tobacco mosaic virus]. / Virusspetsificheskie informosomy v kletkakh tabaka, inifitsirovannykh virusom tabachnoi mozaiki.

We have previously detected in TMV-infected cells virus-specific informosome-like ribonucleoproteins (vRNP) that differed in the CsCl buoyant density from mature TMV particles. It is shown in the present work that [3H]uridine-labelled TMV-specific structures, when fractionated in Cs2SO4, produce three types of structures, i. e. with a buoyant density of 1.23 g/cm3 (the so-called 1.23 material), 1.29 g/gm3 (mature virus) and 1.34--1.49 g/cm3 (vRNP). The 1.23 material has been investigated. The incorporation of [3H]palmitic acid and the sensitivity of this material to 0.1% Na dodecyl sulphate was interpreted to mean the presence of membrane components. Treatment of the 1.23 material Na dodecyl sulphate induces the release of the mature virus, vRNP and free viral RNA. vRNP was shown to contain genome TMV RNA (mol. weight, 2.0 x 10(6)) and a considerable amount of subgenomic TMV RNA (mol. weight, 1.1--1.3 x 10(6) and 0.6--0.8 x 10(6)). It is demonstrated that RNA isolated from vRNP codes for TMV-specific proteins and is able to hybridize with recombinant plasmid containing DNA-copy of 3'-end RNA TMV fragment (about one half of genome).