Functionally Homologous Host Components Recognize Potato Virus X in Gomphrena globosa and Potato.

All known isolates of potato virus X (PVX), with the exception of a South American isolate PVXHB, induce an extreme resistance response on potato carrying the Rx gene and elicit the production of necrotic lesions on Gomphrena globosa: PVXHB establishes systemic infection on Rx genotypes of potato and infects the inoculated leaf of G. globosa without lesion formation. Previously, we have shown that the Rx-mediated resistance is affected by a feature of the coat protein that depends on the presence of a threonine residue at position 121 in the coat protein of PVXCP4 and that the resistance is an induced response expressed in protoplasts of potato with the Rx genotype. In this study, we provide evidence, based on the analysis of PVXCP4/PVXHB hybrids, that the elicitation of lesions on G. globosa also requires the presence of a threonine residue at position 121 of the viral coat protein. The lesion-forming phenotype was not associated with the ability of the viral isolate to accumulate in the infected plant. We therefore propose that there is a homologous component of both potato carrying Rx and G. globosa that interacts with a feature of the PVX coat protein and, following the interaction, activates an induced response in the plant cell.

A Potato Virus X Resistance Gene Mediates an Induced, Nonspecific Resistance in Protoplasts.

The Rx locus in potato controls extreme resistance to most isolates of potato virus X (PVX). The resistance is expressed in whole plants and in protoplasts. Rx-mediated resistance in protoplasts causes reduced accumulation of all PVX RNA species, including the (-) strand RNA after a lag of 8 hr postinoculation. In work reported elsewhere, we have shown that the Rx-breaking property of PVXHB was associated with the coat protein gene of PVXUK3 and PVXCP4. Here, we describe how a frameshift mutation in the coat protein gene had no effect on Rx resistance breaking but compromised the Rx-mediated resistance to PVXCP4. We also describe how in coinoculation experiments, the Rx-mediated resistance could be induced to affect PVXHB or cucumber mosaic virus (CMV). In these experiments, PVXHB or CMV was coinoculated to protoplasts (Rx genotype) together with an isolate of PVX, which is affected by Rx. We interpret this data to indicate that Rx-mediated resistance is induced when the PVX coat protein is produced in the infected cells and that the induced resistance mechanism is effective against viruses unrelated to PVX.

Structure of tobraviral particles: a model suggested from sequence conservation in tobraviral and tobamoviral coat proteins.

Comparisons of the coat protein sequences of four tobraviruses with those of seven tobamoviruses indicate that these proteins share a common evolutionary origin. Numerous amino acids for which specific functions have been identified in the molecular structure of the tobacco mosaic virus vulgare protein have identical or closely similar counterparts among the tobraviral proteins. These include those with roles in the hydrophobic core of the protein, those that contribute to the RNA binding site and those involved in the control of virus assembly. We suggest a model for the structure of the tobraviral particle that not only offers an explanation for the greater diameter of the tobraviral particle but also confirms an early suggestion for RNA placement within this particle.

Analysis of immune system gene expression in small rheumatoid arthritis biopsies using a combination of subtractive hybridization and high-density cDNA arrays.

Subtractive hybridization of cDNAs generated from synovial RNA which had been isolated from patients with rheumatoid arthritis (RA) or normal controls was used in conjunction with high-density array hybridization to identify genes of immunological interest. The method was designed to detect gene expression in small needle biopsy specimens by means of a prior amplification of nanogram amounts of total RNA to full-length cDNA using PCR. The latter was cut with Rsa I, ligated with adapters, hybridized with unmodified driver cDNA, and subjected to suppression subtraction PCR. Differentially expressed products were cloned into E. coli and picked into 384 well plates. Inserts were obtained by PCR across the multiple cloning site, and the products arrayed at high density on nylon filters. The subtracted cDNAs were also labelled by random priming for use as probes for library screening. The libraries chosen were the subtracted one described above and a set of 45,000 ESTs from the I.M. A.G.E consortium. Clones showing positive hybridization were identified by sequence analysis and homology searching. The results showed that the subtracted hybridization approach could identify many gene fragments expressed at different levels, the most abundant being immunoglobulins and HLA-DR. The expression profile was characteristic of macrophage, B cell and plasma cell infiltration with evidence of interferon induction. In addition, a significant number of sequences without matches in the nucleotide databases were obtained, this demonstrates the utility of the method in finding novel gene fragments for further characterisation as potential members of the immune system. Although RA was studied here, the technology is applicable to any disease process even in cases where amounts of tissue may be limited.

A model for the generation of tobacco rattle virus (TRV) anomalous isolates: pea early browning virus RNA-2 acquires TRV sequences from both RNA-1 and RNA-2.

Comparison of the 5'-terminal sequences of several tobraviruses suggests that the RNA-2 molecule of the tobacco rattle virus (TRV) anomalous isolate TCM arose from pea early browning virus (PEBV) RNA-2 by acquisition of 3' and 5' sequences from TRV RNA-1 and RNA-2 molecules, respectively. We have identified a region of homology in the RNA-2 molecules of PEBV, TRV and pepper ringspot virus which could have facilitated this recombination.

The complete nucleotide sequence of PEBV RNA2 reveals the presence of a novel open reading frame and provides insights into the structure of tobraviral subgenomic promoters.

The 3374 nucleotide sequence of RNA2 from the British PEBV strain SP5 has been determined. The RNA includes three open reading frames flanked by 5' and 3' noncoding regions of 509 and 480 nucleotides. The open reading frames specify coat protein, a 29.6K product homologous to the 29.1K product of TRV(TCM) RNA2 and a 23K product not homologous to any previously described protein. The homology demonstrated between the coat proteins of PRV, TRV and PEBV indicates a common evolutionary origin for these proteins. Upstream of each ORF are located sequences homologous to those with which subgenomic RNAs of other tobraviruses start. Subgenomic RNAs for the expression of the three ORFs may start at these points. On all five tobraviral RNA2 molecules sequenced to date, these sequences were found upstream of the coat protein ORF in association with a strongly-conserved potential secondary structural element. Similar potential structures were identified upstream of other tobraviral ORFs. These structures may contribute to the activity of the tobraviral subgenomic promoter.

A feature of the coat protein of potato virus X affects both induced virus resistance in potato and viral fitness.

The coat protein of PVX determines whether isolates of PVX are affected by Rx-mediated resistance in potato. Isolates with the coat protein of PVXHB are not affected by the resistance; those with the coat protein of PVXUK3 elicit an extreme resistance in the Rx potato that prevents virus accumulation, even on the inoculated leaf. In this paper we describe the analysis of a series of hybrid and mutant isolates of PVXHB and PVXCP4 which were inoculated to plants and protoplasts of Rx and rx cultivars of potato. From the virulence phenotypes of these isolates we conclude that elicitation of the resistance is affected by amino acids 121 and 127 of the viral coat protein, with codon 121 being the major determinant. PVXHB and hybrid or mutant isolates with lysine and arginine at positions 121 and 127 were able to overcome the resistance of Rx, whereas those with threonine and arginine were resistance sensitive both on plants and in protoplasts. The viral isolates with single mutations at either codon 121 or 127 were less infectious than the wild-type or double mutant isolates although, in protoplasts of the susceptible cultivar of potato, they accumulated as well as the wild-type virus. Taken together these data suggest that amino acids 121 and 127 affect a feature of the viral coat protein which may interact with cellular components involved in the spread of PVX and with the product of the Rx resistance gene.

Construction and analysis of infectious transcripts synthesized from full-length cDNA clones of both genomic RNAs of pea early browning virus.

Full-length cDNA clones of both RNAs of pea early browning virus have been constructed. Synthetic transcripts derived in vitro from these clones are infectious when inoculated onto plants. Electron microscopy revealed the presence of virions in transcript-inoculated plants, and both purified RNA and virions isolated from such plants could be used to infect other plants. Transcripts of RNA1 alone were able to replicate and spread systemically which is a characteristic of members of the tobravirus group of plant viruses.