Infection of Tobacco Mesophyll Protoplasts by Potato Virus X

SUMMARYBecause it contains the Nx gene, potato cv. 'Marls Piper' reacts hypersensitively to inoculation with group three strains of potato virus X (PVX). However, protoplasts prepared from aseptically grown shoot cultures of potato cv. 'Marls Piper' were infected reproducibly with either PVX or PVX RNA using inocula containing polyethylene glycol. The extent of infection of protoplasts was assessed by fluorescent antibody staining, and by assaying extracts for antigen by ELISA, for infectivity and for PVX RNA content by cDNA hybridization. Infection occurred in 43 to 65% of the protoplasts infected with PVX and in 57 to 70% of the protoplasts infected with PVX RNA. In comparison, when high quality protoplasts were obtained from leaves of potgrown plants 55 to 65% became infected when inoculated with PVX RNA. However, protoplasts obtained from leaves of whole plants varied greatly in both quantity and quality, whereas shoot cultures reproducibly gave high quality protoplast preparations in large numbers. In protoplasts from either source, yields of progeny virus were between 10 and 30 pg per infected protoplast. The multiplication of a group 3 strain of PVX in protoplasts containing the Nx gene did not induce necrosis and confirms that hypersensitivity is not always expressed in isolated protoplasts, Protoplasts isolated from the mesophyll tissue of leaves can be synchronously infected with, and support the replication of, numerous plant viruses. As a high efficiency of infection can be achieved, protoplast systems have been extensively used for studying virus replication (Harrison & Mayo, 1983;Takebe, 1983). However, the relative usefulness of different methods of preparing and inoculating mesophyll protoplasts depends on the combination of plant species and virus employed (Takebe, 1977). Potato (Solanum tuberosum L. ssp. tuberosum) protoplasts have been little used in such studies despite the value of the crop and its susceptibility to several economically important virus diseases. Protoplast preparations from potato plants grown under glasshouse conditions are highly variable in quantity and quality, and yields are influenced greatly by the age and physiological state of the plants and the position of the leaf used for protoplast isolation~ (O'Hara & Henshaw, 1982). We have found that these problems in consistently obtaining good yields of high quality protoplasts can be overcome by using aseptically grown shoot cultures as an alternative source of plant material. We report here a reliable and reproducible system for the infection of shoot culture-derived potato protoplasts with potato virus X (PVX).Potato cultivar 'Marls Piper', which carries PVX hypersensitivity genes Nx and Nb (Cockerham, 1955(Cockerham, , 1970, was used both as a source of mesophyll protoplasts from whole leaves and to obtain shoot cultures. Establishment and maintenance of the cultures was based on the methods described by Thomas (1981) and Nelson et al. (1983), Plants were grown from sprouted tubers in potting compost (John Innes No. 2)...

supporting

confidence: 81%

Infection of Protoplasts Derived from Potato Shoot Cultures with Potato Virus X

Adams

Jones²,

Coutts

1985

“…AD does not inhibit CPMV multiplication if added late in infection, and the same situation has been reported to occur with other viruses (Nassuth et al, 1983, Otsuki et al, 1974. We have shown, however, that at least in the case of CPMV multiplication in cowpea protoplasts, no conclusions can be drawn from experiments where AD is added late in infection.…”

Section: Effect Of Cordycepin Added Late In the Incubation Periodcontrasting

confidence: 46%

“…Actinomycin D (AD) inhibits DNA-directed RNA synthesis and it has been used to show that the replication of several RNA viruses depends upon continuing host gene expression (Alblas & Bol, 1977;Morris-Krsinich et al, 1979;Motoyoshi & Hull, 1974;Otsuki et al, 1974). Previously, it was reported (Rottier et al, 1979) that during the early stages of infection of cowpea protoplasts, the multiplication of cowpea mosaic virus (CPMV) was sensitive to AD.…”

Section: Introductionmentioning

confidence: 99%

A Reappraisal of the Effect of Actinomycin D and Cordycepin on the Multiplication of Cowpea Mosaic Virus in Cowpea Protoplasts

Varennes

Davies

Shaw

et al. 1985

SUMMARYActinomycin D (AD) administered to cowpea mosaic virus-infected cowpea protoplasts immediately after inoculation inhibited virus multiplication, whereas late in the incubation period neither virus multiplication nor host DNA transcription were affected. The data obtained suggested that neither virus uncoating nor encapsidation were inhibited by AD. The inhibition of virus multiplication was manifested as a decrease in the level of progeny viral nucleoproteins and (+) and (-) viral RNAs. Cordycepin strongly inhibited coat protein production and (+) and (-) viral RNA synthesis throughout the incubation period.

“…However, later work with various plant viruses has given conflicting and contradictory results. Thus, replication of TMV (Takebe & Otsuki, 1969), cowpea chlorotic mottle virus (Bancroft et al, 1975), cucumber mosaic virus (CMV) (Otsuki & Takebe, 1973), turnip yellow mosaic virus (TYMV) (Renaudila et al, 1975) or tobacco necrotic dwarf virus (Kubo & Takanami, 1979) in protoplasts was unaffected by Act D, whereas multiplication of TMV, cowpea mosaic and bean pod mottle viruses (Lockhart & Semancik, 1968Furusawa et al, 1970), potato virus X (PVX) or groundnut dwarf virus in leaf tissue (Kushnirenko et al, 1980), and of CMV (Takanami et al, 1977), TYMV (Renaudin & Bov6, 1977), PVX (Otsuki et al, 1974), alfalfa mosaic virus (Alblas & Bol, 1977), pea enation mosaic virus (Motoyoshi & Hull, 1974), turnip rosette virus (Morris-Krsinich et al, 1979) or cowpea mosaic virus (Rottier et al, 1979) in protoplasts could be inhibited by Act D. In many of these studies it was noted that when the addition of Act D to cultures was delayed for several hours after inoculation, little or no inhibition occurred, suggesting that Act D had affected an early step in virus multiplication (e.g. Dawson, 1978;Rottier et al, 1979).…”

Section: Discussionmentioning

confidence: 99%

Effects of Actinomycin D on the Infection of Tobacco Protoplasts by Four Viruses

Mayo¹,

Barker²

1983

SUMMARYThe addition of actinomycin D (25 Ixg/ml) or cordycepin (1 mi) to protoplast cultures immediately after inoculation with particles of tobacco mosaic, tobacco rattle, tobacco ringspot or potato leafroll viruses resulted in a decrease in the proportion of protoplasts becoming infected, as judged by staining with fluorescent antibody to virus particles. A delay of a few hours between the inoculation and the addition of either inhibitor largely or completely eliminated this effect. In contrast, infection was unaffected by the addition of actinomycin D when the protoplasts were inoculated with RNA preparations from tobacco mosaic or tobacco rattle viruses.