S.Y. Morozov

Probable reassortment of genomic elements among elongated RNA-containing plant viruses.

SummaryThe relationships of genome organization among elongated (rod-shaped and filamentous) plant viruses have been analyzed. Sequences in coding and noncoding regions of barley stripe mosaic virus (BSMV) RNAs 1, 2, and 3 were compared with those of the monopartite RNA genomes of potato virus X (PVX), white clover mosaic virus (WClMV), and tobacco mosaic virus, the bipartite genome of tobacco rattle virus (TRV), the quadripartite genome of beet necrotic yellow vein virus (BNYVV), and icosahedral tricornaviruses. These plant viruses belong to a supergroup having 5′-capped genomic RNAs. The results suggest that the genomic elements in each BSMV RNA are phylogenetically related to those of different plant RNA viruses. RNA 1 resembles the corresponding RNA 1 of tricornaviruses. The putative proteins encoded in BSMV RNA 2 are related to the products of BNYVV RNA 2, PVX RNA, and WClMV RNA. Amino acid sequence comparisons suggest that BSMV RNA 3 resembles TRV RNA 1. Also, it can be proposed that in the case of monopartite genomes, as a rule, every gene or block of genes retains phylogenetic relationships that are independent of adjacent genomic elements of the same RNA. Such differential evolution of individual elements of one and the same viral genome implies a prominent role for gene reassortment in the formation of viral genetic systems.

Potato virus X-related single- and double-stranded RNAs Characterization and identification of terminal structures

Six species of 3′‐coterminal poly(A) ‐containing RNAs of subgenomic (sg) size have been found in plants infected with potato virus X (PVX): two major (0.9 kb — the coat protein mRNA, and 2.1 kb) and four minor (1.4, 1.8, 3.0 and 3.6 kb). The 5′‐end of the shortest sgRNA is located 26 nucleotides upstream of the initiating codon of the coat protein gene (812 nucleotides from the 3′‐terminal poly(A) tract of the PVX genomic RNA). Double‐stranded analogues have been found for most sgRNAs. The genomic‐size double‐stranded RNA (the replicative form) is shown to carry a poly(A)‐poly(U) hybrid of a predominant length of 150–250 bp on one end, and an unpaired G residue on the other (the 3′‐end of the negative chain). In contrast to this(—) the chains of double‐stranded 0.9 and 2.1 kbp sgRNAs lack the unpaired G and both end in C.

Translation of plant virus messenger RNAs

Publisher Summary A study of the synthesis of virus-specific proteins is closely related to the regulatory aspect of gene expression. This chapter focuses on the peculiarities of the translation of plant virus mRNAs and some mechanisms controlling the process. General principles of mRNA replication and expression with respect to viruses containing virion plus-strand RNA, minus-strand RNA, double-stranded RNA (dsRNA), and DNA are discussed in the chapter. Functions encoded in the genome of RNA-containing plant viruses are performed by the corresponding gene products-virus-specific (i.e., virus-coded) proteins. Aside from the virus-specific proteins, virus multiplication may be either positively or negatively affected by the products (proteins or possibly RNAs) coded for by the host cell genome, synthesis of which is induced by virus infection (so-called virus-induced products). An example of a virus-induced protein in the virus-animal cell system is interferon. Plant virologists have realized that the coat proteins of many viruses are synthesized in excess with respect to other virus-coded products. Ion concentration in cell-free extracts is an important regulatory factor in the translation of RNAs of viruses. There is some evidence that ionic concentration may influence the process of plant virus RNA translation in vitro .

Movement protein-derived resistance to triple gene block- containing plant viruses

Two mutant potato virus X (PVX) movement protein (MP) genes (m 12K-Sal and m 12K-Kpn) were obtained by inserting specific linkers at the boundary between the N-terminal hydrophobic and putative transmembrane segment, and the central invariant hydrophilic region of the respective 12 kDa, 12K, triple gene block (TGB) protein. Several transgenic potato lines which expressed m 12K-Sal or m 12K-Kpn to different degrees were resistant to infection by PVX, potato aucuba mosaic potexvirus and the carlaviruses potato virus M and S over a wide range of inoculum concentrations (3-300 micrograms/ml). However, they were not resistant to potato virus Y, which lacks a TGB protein. We suggest that the resistance of m 12K-Sal and m 12K-Kpn transgenic potato lines is MP-derived and not RNA-mediated.

Genome sequences of poa semilatent and lychnis ringspot hordeiviruses

SummaryThe nucleotide sequences of the genomic RNAs of two hordeiviruses, poa semilatent virus (PSLV) and lychnis ringspot virus (LRSV), were determined. The genome organization of both viruses is similar to that of the type hordeivirus, barley stripe mosaic virus (BSMV). Comparisons of the amino acid sequences of the hordeivirus replicase components revealed a high level of sequence similarity to those of soil-borne wheat mosaic virus (SBWMV), peanut clump virus (PCV), and Indian peanut clump virus. The cysteine-rich γ b proteins of hordeiviruses are similar to those of SBWMV, PCV, and tobraviruses. Analysis of cis-acting elements in the genomes of the three hordeiviruses revealed conservation of putative core elements of subgenomic RNA promoters. Complex patterns of conservation were also found in the 5′-untranslated regions of hordeivirus genomic RNAs α, β and γ.

Translation arrest of potato virus X RNA in Krebs-2 cell-free system: RNase H cleavage promoted by complementary oligodeoxynucleotides

Translation arrest of genomic potato virus X (PVX) RNA promoted by complementary oligodeoxynucleotides in Krebs‐2 cell‐free system is described. 14–15 mer oligodeoxynucleotides complementary to the 5′‐proximal cistron of PVX RNA were shown to induce specific truncation of the major non‐structural polypeptide coded by PVX RNA. Evidence is presented that effective translational arrest of PVX RNA in the presence of complementary oligonucleotides restults from the site‐specific cleavage of RNA by endogenous RNase H intrinsic to the Krebs‐2 extract. No similar translational arrest was found in the rabbit reticulocyte lysate cell‐free system.

Novel miR390-Dependent Transacting siRNA Precursors in Plants Revealed by a PCR-Based Experimental Approach and Database Analysis

TAS loci in plant genomes encode transacting small interfering RNAs (ta-siRNAs) that regulate expression of a number of genes. The function of TAS3 precursor in Arabidopsis thaliana is controlled by two miR390 target sites flanking two ta-siARF sequences targeting mRNAs of ARF transcription factors. Cleavage of the 3′-miR390-site initiates ta-siRNAs biogenesis. Here we describe the new method for identification of plant ta-siRNA precursors based on PCR with oligodeoxyribonucleotide primers mimicking miR390. The method was found to be efficient for dicotiledonous plants, cycads, and mosses. Based on sequences of amplified loci and a database analysis, a novel type of miR390-dependent TAS sequences was identified in dicots. These TAS loci are characterized by a smaller distance between miR390 sites compared to TAS3, a single copy of ta-siARF, and a sequence conservation pattern pointing to the possibility that processing of novel TAS-like locus is initiated by cleavage of the 5′-terminal miR390 target site.

Subcellular localization and self-interaction of plant-specific Nt-4/1 protein

The Nicotiana tabacum Nt-4/1 protein is a plant-specific protein of unknown function. Analysis of bacterially expressed Nt-4/1 protein in vitro revealed that the protein secondary structure is mostly alpha-helical and suggested that it could consist of three structural domains. Earlier studies of At-4/1, the Arabidopsis thaliana-encoded ortholog of Nt-4/1, demonstrated that GFP-fused At-4/1 was capable of polar localization in plant cells, association with plasmodesmata, and cell-to-cell transport. Together with the At-4/1 ability to interact with a plant virus movement protein, these data supported the hypothesis of the At-4/1 protein involvement in viral transport through plasmodesmata. Studies of the Nt-4/1-GFP fusion protein reported in this paper revealed that the protein was localized to cytoplasmic bodies, which were co-aligned with actin filaments and capable of actin-dependent intracellular movement. The Nt-4/1-GFP bodies, being non-membrane structures, were found in association with the plasma membrane, the tubular endoplasmic reticulum and endosome-like structures. Bimolecular fluorescence complementation experiments and inhibition of nuclear export showed that the Nt-4/1 protein was capable of nuclear-cytoplasmic transport. The nuclear export signal (NES) was identified in the Nt-4/1 protein by site-directed mutagenesis. The Nt-4/1 NES mutant was localized to the nucleoplasm forming spherical bodies. Immunogold labeling and electron microscopy of cytoplasmic Nt-4/1-containing bodies and nuclear structures containing the Nt-4/1 NES mutant revealed differences in their fine structure. In mammalian cells, Nt-4/1-GFP formed cytoplasmic spherical bodies similar to those found for the Nt-4/1 NES mutant in plant cell nuclei. Using dynamic laser light scattering and electron microscopy, the Nt-4/1 protein was found to form multimeric complexes in vitro.

RNA-binding properties of the plant protein Nt-4/1.

The tobacco α-helical protein Nt-4/1 with unknown function forms ribonucleoprotein (RNP) complexes in vitro. Results obtained by retardation of RNP complexes in agarose gel were confirmed by Western-Northern hybridization. Several deletion and point mutants of Nt-4/1 were constructed, and the RNA-binding site was mapped in a positively charged region of the C-terminal domain of the protein. The results of this study and those described earlier support our hypothesis of the participation of Nt-4/1 protein in spreading RNA-containing pathogens in the plant.

TAS3 genes for small ta-siARF RNAs in plants belonging to subtribe senecioninae: Occurrence of prematurely terminated RNA precursors

The various classes of plant 21- to 24-nt siRNAs derive from long dsRNA precursors that are processed by the ribonuclease Dicer-like (DCL). The species of ta-siRNA were originally discovered in Arabidopsis thaliana. Four gene families have been identified in Arabidopsis that each produces a number of ta-siRNAs: TAS1, TAS2, TAS3 and TAS4. The TAS3 genes encode ta-siR-ARF species which target the mRNA of three Auxin Response Factor (ARF) genes (ARF2, ARF3/ETT and ARF4) for subsequent degradation. The function of TAS3 precursor RNA is controlled by two miR390 target sites flanking tandem of ta-siARF sequences. In this paper, we have studied the presence of ta-siARF RNA genes in the representatives of subtribe Senecioninae. Senecioninae is the largest tribe of Asteraceae, comprised of ca. 150 genera and 3000 species which include many common succulents of greenhouses. Approximately one-third of species are placed in genus Senecio, making it one of the largest genera of flowering plants. However, there was no information on the structure of TAS genes in these plants. We revealed that the TAS3 species (TAS3-Sen1) in Senecio representatives was actively transcribed, and its homologues are distributed among many Asteracea plants and found to be similar to Arabidopsis AtTAS3a gene. We revealed several prematurely terminated transcripts of TAS3-Sen1. Finding the alternative shortened transcripts of TAS3-Sen1 lacking the 3′-terminal site cleaved by miR390 and retaining the 5′-terminal miR390 non-cleaved site suggested their using as decoys for the modulation of miR390 activity to regulate synthesis of ta-siARF RNAs in different Senecioninae species.