H. Barker

Breeding virus resistant potatoes (Solanum tuberosum): a review of traditional and molecular approaches

Tetraploid cultivated potato (Solanum tuberosum) is the World’s fourth most important crop and has been subjected to much breeding effort, including the incorporation of resistance to viruses. Several new approaches, ideas and technologies have emerged recently that could affect the future direction of virus resistance breeding. Thus, there are new opportunities to harness molecular techniques in the form of linked molecular markers to speed up and simplify selection of host resistance genes. The practical application of pathogen-derived transgenic resistance has arrived with the first release of GM potatoes engineered for virus resistance in the USA. Recently, a cloned host virus resistance gene from potato has been shown to be effective when inserted into a potato cultivar lacking the gene. These and other developments offer great opportunities for improving virus resistance, and it is timely to consider these advances and consider the future direction of resistance breeding in potato. We review the sources of available resistance, conventional breeding methods, marker-assisted selection, somaclonal variation, pathogen-derived and other transgenic resistance, and transformation with cloned host genes. The relative merits of the different methods are discussed, and the likely direction of future developments is considered.

Invasion of non-phloem tissue in Nicotiana clevelandii by potato leafroll luteovirus is enhanced in plants also infected with potato Y potyvirus

Summary Nicotiana clevelandii plants doubly infected with potato leafroll luteovirus (PLRV) and potato Y potyvirus (PVY) developed more severe symptoms than plants infected with either virus alone. Compared with singly infected plants, the PLRV concentration was increased up to eightfold by the double infection and the proportion of leaf parenchyma protoplasts that could be stained with fluorescent antibody to PLRV was increased from 0.2 to 1.4%. The concentration of PVY was unaffected by the double infection. In potato plants, in contrast, double infection with PVY did not increase PLRV concentration and none of the parenchyma protoplasts obtained from singly or doubly infected leaves could be stained with fluorescent antibody to PLRV. PLRV seems restricted to phloem tissue in potato but also invades a few parenchyma cells in N. clevelandii, a process that is accentuated in plants also infected with PVY.

A review of host major-gene resistance to potato viruses X, Y, A and V in potato: genes, genetics and mapped locations

In view of modern developments in the technologies available for breeding potatoes for resistance to virus diseases, it is timely to review the host major genes that confer resistance, in Solanum species, to potato viruses X, Y, A and V (the viruses for which the resistance genes have been most extensively studied). Over the course of 60 years, many such genes in Solanum species have been characterized: a comprehensive list is presented. Inheritance studies are reviewed, including linkage studies and molecular mapping, and the positions of resistance genes mapped so far are listed. It is apparent from recent research that disease resistance genes are often clustered in particular regions of the chromosomes; the significance of these resistance gene clusters is discussed. The information presented will be useful for potato breeding, and for genetic and mapping studies and gene cloning.

Evidence that Potato Leafroll Virus RNA is Positive-stranded, is Linked to a Small Protein and Does Not Contain Polyadenylate

Summary RNA extracted from particles of potato leafroll virus (PLRV) infected tobacco mesophyll protoplasts. Treating the RNA with proteinase K did not abolish its infectivity. In messenger-dependent rabbit reticulocyte lysate, PLRV RNA induced the synthesis of specific polypeptides: a major product of mol. wt. 71000 but no product the size of coat protein. PLRV RNA is therefore positive-stranded. A genome-linked protein (apparent mol. wt. 7000) was detected in preparations of PLRV RNA but no polyadenylate sequence was found. These features may prove to be characteristic of luteoviruses.

Increase in Plasmodesmatal Permeability during Cell-to-Cell Spread of Tobacco Rattle Virus from Individually Inoculated Cells.

A microinjection technique was devised for inoculation of single Nicotiana clevelandii leaf trichome cells with virus particles. By removing inoculated trichomes at various times after microinjection, it was shown that at least 4 hr were required for tobacco rattle virus (TRV; tobravirus group) to move out of primarily inoculated cells. Effects of the early stages of TRV infection on plasmodesmatal permeability were examined by microinjection of fluorochrome-labeled molecules. Fluorescein-labeled insulin A chain (Mr, 2921) and fluorescein-labeled dextran (Mr, 4400) were observed to pass out of individual N. clevelandii trichome cells that had been inoculated with TRV by microinjection 5 hr previously. By contrast, Lucifer Yellow CH-labeled dextran (Mr, 10,000) was restricted to the inoculated cell. None of these fluorescent probes were able to move out of uninoculated cells or out of cells that had been inoculated with TRV only 2 hr previously. The movement of macromolecules through plasmodesmata, therefore, coincided with and probably resulted from cell-to-cell movement of TRV. The results are discussed with reference to the interaction of viruses and plasmodesmata and mechanisms of intercellular virus movement.

Polyadenylate in the RNA of Five Nepoviruses

Summary RNA extracted from particles of five nepoviruses [raspberry ringspot (RRV), strawberry latent ringspot, tobacco ringspot (TRSV), tomato black ring (TBRV) and tomato ringspot viruses] was bound to oligo(dT)-cellulose in buffers of high ionic strength (HS) whereas RNA from particles of tobacco mosaic virus or tobacco rattle virus was not. This suggests that nepovirus RNA molecules contain polyadenylate [poly(A)]. At least 97% of the infective RNA molecules of TRSV and TBRV were bound in HS buffer and eluted in buffer of low ionic strength. The two species of genome RNA of RRV and TBRV were bound equally to oligo(dT)-cellulose, but the satellite RNA (RNA-3) of TBRV was bound less avidly and to a smaller extent than the genome RNA. When 3H-borohydride-labelled TRSV RNA was digested with ribonucleases A + T1 about 29% of the radioactivity bound to oligo(dT)-cellulose, presumably as polyadenylate. Adenosine trialcohol was the only nucleoside trialcohol detected in alkali digests of borohydride-labelled RNA. Thus polyadenylate is probably located at the 3′-termini of the RNA molecules.

Replication of RNA-1 of tomato black ring virus independently of RNA-2.

Summary In hybridization experiments, using complementary DNA (cDNA) copies of the two genome parts of tomato black ring virus (TBRV RNA-1 and RNA-2), no sequence homology between the two RNA species was detected. When tobacco mesophyll protoplasts were inoculated with purified middle component particles, which contain only RNA-2, no replication of TBRV RNA could be detected. However, when they were inoculated with purified bottom component particles, which contain only RNA-1, extracts made 24 or 48 h later contained RNA that had the same mobility as RNA-1 in polyacrylamide-agarose gels, and that hybridized to cDNA copies of RNA-1. Thus RNA-1 can replicate in protoplasts that do not contain RNA-2. Moreover, this RNA-1 was capable, when mixed with nucleoprotein particles containing RNA-2, of inducing the formation of local lesions in Chenopodium amaranticolor leaves, and therefore was intact and attached to the genome-linked protein. The genome-linked protein of nepoviruses is probably virus-coded, and its production in protoplasts inoculated with bottom component particles therefore suggests that RNA-1 contains the gene for this protein.

Detection of potato virus Y in potato tubers: a comparison of polymerase chain reaction and enzyme-linked immunosorbent assay

SummaryA method was devised by which specific sequences of potato virus Y (PVY) RNA could be detected in total tuber RNA extracts by reverse transcription into cDNA and amplification by polymerase chain reaction (PCR). This method of PVY detection was tested and compared with the antibody-trapped antigen form of enzyme-linked immunosorbent assay (ATA-ELISA) using monoclonal antibodies. Both PCR and ATA-ELISA could detect PVY reliably in progeny tubers taken from growing plants of cv. Record, or tubers stored for 3 weeks after harvest. The ability to detect PVY decreased substantially after tubers had been stored for 20 weeks at 10°C. ATA-ELISA detected virus in only half the tubers from infected plants. However, PCR detected PVY very inefficiently in infected tubers after 20 weeks storage.

Potato mop-top virus RNA can move long distance in the absence of coat protein: evidence from resistant, transgenic plants.

Transgenic expression of a translatable version of the Potato mop-top virus (PMTV) coat protein (CP) gene (encoded by RNA 3) in Nicotiana benthamiana prevented production of symptoms and infective virus particles. RNAs 1 and 2 accumulated in inoculated and systemic leaves but, apart from small amounts of CP transgene RNA transcript, no genomic-length RNA 3 was found. Crude leaf extracts from inoculated plants were not infective. However, when RNA extracts from such transgenic plants were inoculated to nontransgenic N. benthamiana and N. clevelandii, RNA 1 and RNA 2 replicated in systemic leaves of both species in the absence of RNA 3 and virus particles, but symptoms did not develop. We suggest that the triple-gene block proteins of PMTV (encoded by RNA 2) represent a class of long-distance RNA movement factors.

Rapid production of single-chain Fv fragments in plants using a potato virus X episomal vector

We have used a plant virus episomal vector, based on potato virus X (PVX) to transiently express a single-chain Fv (scFv) and its diabody derivative in plants. The scFv was directed against a continuous epitope (cryptotope) on the coat protein of potato virus V. A cloned, full-length PVX vector sequence, containing the scFv gene, was used to direct in vitro transcription and the resulting RNA was used to inoculate Nicotiana clevelandii plants. Within a few days, plants developed characteristic symptoms and immunoblot analysis showed that accumulation of scFv protein coincided with accumulation of PVX. Targeting of the scFv to the apoplast greatly increased protein accumulation compared with cytosolic scFv and produced more severe symptoms on infected plants. ELISA demonstrated that the scFv and diabody extracted from infected plants showed the same antigen-binding specificity as that of the parental monoclonal antibody. The PVX vector is a convenient, rapid, low-cost in planta expression system that can also be used for assessment of scFv production and function prior to stable plant transformation.