Advances in Understanding Plant Viruses and Virus Diseases.

▪ Abstract  Plant viruses have had an impact on the science of virology and on plant pathology ever since the virus concept was discovered with Tobacco mosaic virus at the end of the nineteenth century. In this review, we highlight those discoveries. We have divided plant virus research into a "Classical Discovery Period" from 1883-1951 in which the findings were very descriptive; an "Early Molecular Era" from 1952 to about 1983, in which information was developed that described further properties of the viruses, aided by the development of a number of salient techniques; and the "Recent Period" from 1983 to the present, when techniques have been developed to modify plant virus genomes, to detect nonstructural gene products, to determine the functions of viral gene products, and to transform plants to elicit novel forms of resistance to viral diseases.

Transgene translatability increases effectiveness of replicase-mediated resistance to cucumber mosaic virus.

Transgenic tobacco plants expressing an altered form of the 2a replicase gene from the Fny strain of Cucumber mosaic virus (CMV) exhibit suppressed virus replication and restricted virus movement when inoculated mechanically or by aphid vectors. Additional transformants have been generated which contain replicase gene constructs designed to determine the role(s) of transgene mRNA and/or protein in resistance. Resistance to systemic disease caused by CMV, as well as delayed infection, was observed in several lines of transgenic plants which were capable of expressing either full-length or truncated replicase proteins.

Elucidation of the genome organization of tobacco mosaic virus.

Proteins unique to tobacco mosaic virus (TMV)-infected plants were detected in the 1970s by electrophoretic analyses of extracts of virus-infected tissues, comparing their proteins to those generated in extracts of uninfected tissues. The genome organization of TMV was deduced principally from studies involving in vitro translation of proteins from the genomic and subgenomic messenger RNAs. The ultimate analysis of the TMV genome came in 1982 when P.

Cucumber mosaic virus is restricted from entering minor veins in transgenic tobacco exhibiting replicase-mediated resistance.

Transgenic tobacco plants expressing an altered form of the 2a replicase gene from cucumber mosaic virus (CMV) strain Fny exhibited a suppression of viral replication and restricted viral movement when inoculated mechanically or by insect vectors. Resistant plants could be infected, however, through a graft-union with an infected nontransformed plant. The infectious entity moved quickly through intergrafts of resistant tissue, indicating that it could move without replicating in the vascular system.

Viral RNA trafficking is inhibited in replicase-mediated resistant transgenic tobacco plants.

Transgenic tobacco (Nicotiana tabacum cv. Turkish Samsun NN) plants expressing a truncated replicase gene sequence from RNA-2 of strain Fny of cucumber mosaic virus (CMV) are resistant to systemic CMV disease. This is due to suppression of virus replication and cell-to-cell movement in the inoculated leaves of these plants.

A single nucleotide change in the coat protein gene of tobacco mosaic virus is involved in the induction of severe chlorosis.

YSI/1 is a mutant of the common strain (U1) of tobacco mosaic virus (TMV) which induces a severe yellow mosaic in Nicotiana tabacum instead of the light green/dark green mosaic induced by its parental U1 virus. Although there was less coat protein (CP) in whole leaf extracts of YSI/1-infected leaves than in U1-infected leaves, severalfold more CP was found in the chloroplasts, most of which was associated with the thylakoids. Sequencing the CP genes of both viruses showed the presence of nucleotide differences at viral RNA positions 5770 and 6127, both of which result in amino acid replacements; YSI/1 has an Asp-->Val change at amino acid 19 and a Ser-->Phe change at amino acid 138.

Specificity of replicase-mediated resistance to cucumber mosaic virus.

Plants transformed with a nucleotide sequence coding for a truncated RNA 2 replicase gene of the subgroup I strain of cucumber mosaic virus, Fny-CMV, are resistant to cucumber mosaic disease. Two resistant lines representing independent transformations in the original study have been propagated, and their progeny have been examined. Resistance to Fny-CMV was genetically integrated and was retained in the R4 generation.

Replicase-mediated resistance to cucumber mosaic virus in transgenic plants involves suppression of both virus replication in the inoculated leaves and long-distance movement.

Tobacco plants transformed with a gene encoding a truncated cucumber mosaic virus (CMV) 2a replicase protein are resistant to systemic CMV disease. Experiments using protoplasts derived from plants of two R2-generation CMV-resistant transgenic plant lines (lines R2-2 and R2-5) showed that resistance operates at the single cell level. Low levels of CMV-specific RNAs were detected in CMV-inoculated protoplasts obtained from both R2-2 and R2-5 plants indicating that resistance is due at least in large part to a marked but incomplete suppression of virus replication.

Replicase-mediated resistance to potato virus Y in transgenic tobacco plants.

Nicotiana tabacum 'Turkish Samsun NN' plants were transformed with nuclear inclusion b (NIb) gene sequences of potato virus Y, O strain (PVYO). The full-length construct included an additional in-frame initiation codon contiguous to the putative N-terminal amino acid codon and a stop codon replacing the C-terminal amino acid codon. Of 13 independently transformed lines, four yielded 37 (out of 100) plants in the R1 generation that were resistant to PVYO infection.

A defective replicase gene induces resistance to cucumber mosaic virus in transgenic tobacco plants.

Nicotiana tabacum cv. Turkish Samsun NN plants were transformed with a modified and truncated replicase gene encoded by RNA-2 of cucumber mosaic virus strain Fny. The replicase gene had been modified by deleting a 94-base-pair region spanning nucleotides 1857-1950; the deletion also caused a shift in the open reading frame, resulting in a truncated translation product approximately 75% as large as the full-length protein.

Resistance to tobacco mosaic virus induced by the 54-kDa gene sequence requires expression of the 54-kDa protein.

Tobacco plants transformed with the sequence encoding the 54-kDa putative replicase protein of tobacco mosaic virus were resistant to systemic virus disease (D. B. Golemboski, G.

Alleged common antigenic determinant of tobacco mosaic virus coat protein and the host protein ribulose-1,5-bisphosphate carboxylase is an artifact of indirect ELISA and western blotting.

We have reported the detection of an antigenic determinant shared by the tobacco mosaic virus coat protein and the large subunit of ribulose-1,5-bisphosphate carboxylase, a host protein (R.G. Dietzgen and M.

Plants transformed with a tobacco mosaic virus nonstructural gene sequence are resistant to the virus.

Nicotiana tabacum cv. Xanthi nn plants were transformed with nucleotides 3472-4916 of tobacco mosaic virus (TMV) strain U1. This sequence contains all but the three 3 terminal nucleotides of the TMV 54-kDa gene, which encodes a putative component of the replicase complex.

Ubiquitinated conjugates are found in preparations of several plant viruses.

Recently D.D. Dunigan, R.

Capping of tobacco mosaic virus RNA. Analysis of viral-coded guanylyltransferase-like activity.

The 5' end of tobacco mosaic virus (TMV) genomic RNA is capped with 7-methylguanosine. A virus-coded polypeptide with guanylyltransferase activity has been investigated. This enzyme is responsible for forming the 5'----5' linkage of guanosine 5'-monophosphate to the 5'-diphosphate of an acceptor RNA, thereby forming the cap.

Tobacco mosaic virus RNA enters chloroplasts in vivo.

Several lines of evidence are presented to allow us to conclude that tobacco mosaic virus (TMV) RNA enters the chloroplast in vivo. Chloroplasts were prepared from either directly inoculated or systemically infected leaves of tobacco plants inoculated with one of several strains of the virus and from uninfected control plants. Intact chloroplasts were isolated on Percoll gradients and treated with pancreatic RNase and thermolysin to destroy potential TMV virions and RNA on the outside or bound to their surfaces.

Tobacco mosaic virus particles contain ubiquitinated coat protein subunits.

Virions of tobacco mosaic virus (TMV) are composed of a single strand of RNA, encapsidated in about 2130 copies of a coat protein of MW 17,500. Asselin and Zaitlin [Virology 91, 173-181 (1978)] demonstrated that virion preparations also contained small amounts of a second protein of MW 26,500, which they termed "H protein." H protein, detectable to an average frequency of one per virion, was thought to be a protein of host origin.

Immunogold localization of the intracellular sites of structural and nonstructural tobacco mosaic virus proteins.

Antibodies raised against the 126K nonstructural protein (replicase) encoded by tobacco mosaic virus (TMV) RNA or the viral coat protein have been used to localize these proteins within virus-infected tobacco leaf cells by an immunogold labeling technique. A protocol is given for low-temperature fixation to facilitate immunogold labeling. In cells of TMV-infected leaf tissue, the 126K protein immunogold label was found almost exclusively in "viroplasms" in the cytoplasm and in pockets of virus particles at the viroplasmic periphery.

Tobacco mosaic virus particles uncoat and express their RNA in Xenopus laevis oocytes: implications for early interactions between plant cells and viruses.

Xenopus laevis oocytes were microinjected with tobacco mosaic virus (TMV) particles or unencapsidated TMV RNA at equivalent RNA concentrations. After a 5-hr incubation, batches of 10 oocytes from each treatment were frozen and lysed, and the supernatants were subjected to SDS-PAGE and Western immunoblotting with antiserum raised against a TMV RNA-encoded polypeptide of mol wt 126,000 (126K), purified from TMV-infected tobacco. Cross-reaction with a polypeptide of 126K synthesized in vitro, in a rabbit reticulocyte cell-free system programmed with TMV RNA, confirmed the specificity of the antiserum.

In vitro translation products of mrnas derived from TMV-infected tobacco exhibiting a hypersensitive response.

Expression of the hypersensitive response (HSR) to tobacco mosaic virus (TMV) infection in Nicotiana tabacum L. cv. Xanthi-nc (genotype NN) is controlled by the single dominant "N" gene and is temperature sensitive.

Nucleotide sequence analysis of six satellite RNAs of cucumber mosaic virus: primary sequence and secondary structure alterations do not correlate with differences in pathogenicity.

The nucleotide sequences of six satellite RNAs of cucumber mosaic virus (CMV) differing in their pathological properties have been determined. Although these RNAs varied in length from 333 to 342 nucleotides, the extent of nucleotide sequence homology to each other and to four other satellite RNAs ranged from 84 to 97%, allowing satellite RNA subgrouping on the basis of nucleotide sequence. This subgrouping was further extended by an analysis of the nature of the differences in nucleotide sequence, with most of the variation occurring within nucleotides 129 to 190 and 318 to the 3'-end.

The nature and biological significance of linear potato spindle tuber viroid molecules.

"Naturally occurring" linear potato spindle tuber viroid (PSTVL) was shown to be as infectious as circular PSTV (PSTVc). The occurrence of PSTVL was shown not to be (a) an artifact of the extraction procedure per se; (b) due to the presence of metal ions in extraction buffers; or (c) related to the host species used for propagation, to a particular PSTV strain, or to the duration of infection. From labeling and blot-hybridization experiments, it was concluded that in infected tissue PSTVc appears first followed by PSTVL, which is produced as the result of cleavage of PSTVc at specific sites.

Tobacco mosaic virus replicase and replicative structures.

The RNA-dependent RNA polymerase (replicase) mediating the replication of tobacco mosaic virus (TMV) has been investigated in a number of laboratories over a period of 20 years. Cell-free enzyme preparations have been prepared which can continue the synthesis of nascent complementary RNA, initiated in vivo; however, the enzyme does not require, nor does it respond to, exogenous viral RNA as a template. The presence in plants of a virus-stimulated, host-encoded RNA-dependent RNA polymerase (RdRp) has added confusion to this field; it is now generally conceded, however, that this enzyme is not the TMV replicase.

In vivo encapsidation of potato spindle tuber viroid by velvet tobacco mottle virus particles.

When Nicotiana clevelandii was inoculated simultaneously with velvet tobacco mottle virus (VTMoV) and potato spindle tuber viroid (PSTV), replication of the viroid was greatly suppressed. However, both pathogens replicated if PSTV was inoculated several days before the VTMoV. Small amounts of PSTV were detected by dot-blot analysis of RNA in highly purified VTMoV isolated from coinfected plants indicating that the viroid had been encapsidated by the virus particles.