Infection of cowpea protoplasts with sonchus yellow net virus and festuca leaf streak virus

N.A.M. van Beek

Research output: Thesisinternal PhD, WU


The advantages of protoplast systems for plant virus research have been frequently reviewed (Zaitlin & Beachy, 1974; Takebe, 1975; Muhlbach, 1982; Sander & Mertens, 1984). Relatively little attention has been given to the limitations of such a system.Protoplasts do not exist under natural conditions. They lack a rigid cell wall and cell-to-cell connections are absent. Protoplasts are maintained in media that differ from the milieu in plant tissue with respect to nutrient composition, hormone balance, and, most importantly, tonicity. Several authors have documented the effects of osmotic stress in protoplasts of various sources (Lazar et al., 1973; Prevecz et al., 1978; Fleck et al. , 1982; Meyer & Aspart, 1983). These effects include altered nucleic acid and protein synthesis. Lazar et al. (1973) reported a more than ten-fold increase in RNase level upon protoplasts isolation. Although no reports have appeared that document effects of the altered physiological state of protoplasts on virus multiplication, extrapolation from the level of protoplast to plant must be done with caution.Our studies on Sonchus yellow net virus (SYNV) and Festuca leaf streak virus (FLSV) have been carried out with the aid of protoplasts, derived from the cowpea plant, a non-host for these viruses. Thus, our studies certainly are subject to the above stated limitations.In Chapter II we report on the infection of cowpea protoplasts with SYNV. The infection is mediated by polyethylene glycol, a compound that induces membrane fusion. Viral replication was demonstrated by the results of a biological assay, and it was shown that over 90% of the living protoplasts could be infected. Those SYNV particles, from which the envelope was removed, were much less efficiently introduced in protoplasts, indicating that fusion of viral envelope and protoplast membrane was an important mechanism for introduction. Conditions during the inoculation procedure were optimized. We showed that infection was blocked when a divalent cation chelating compound was included in the inoculation medium.A description at the ultrastructural level of the subsequent stages in the infection of cowpea protoplasts by SYNV is presented in Chapter III. The model for the budding process given by Francki (1973) is disputed. We observed virus particles in intermediate stages of budding that are highly suggestive for a simultaneous occurrence of coiling and budding as predicted by Peters & Schulz (1975). Budding of precoiled nucleoprotein strands as hypothesized by Francki (1973) was never observed.Budding of virus particles was prevented by tunicamycin. This led to the accumulation of massive amounts of coiled nucleoprotein strands and granular material in the nucleus. These structures were not seen in the cytoplasm, providing evidence that the inner nuclear membrane is the only site of assembly of SYNVAt later stages in infection, coiled nucleoprotein strands were observed free in the cytoplasm. They were shown to originate from mature particles as a consequence of fusion of their envelope with the endoplasmic reticulum membrane, followed by the release of the coiled nucleoprotein strand in the cytoplasm. The significance of these structures with respect to the decrease in infectivity of plant tissue and protoplasts in later stages of infection, and with respect to spread of the virus from cell-to-cell, is also discussed.In Chapter IV we show morphological stages of FLSV particles in cowpea protoplasts. Replication cannot be demonstrated formally since this virus has not been transmitted mechanically and a vector has not yet been identified. Thus, the infection of cowpea protoplasts with FLSV, until now only found in the monocotyledon Festuca gigantea has merits of its own,FLSV assembles at intracytoplasmic membranes. Striking similarities were noticed between the processes occurring in the cytoplasm of FLSV-infected protoplasts and those occurring in the nucleus of SYNV-infected protoplasts.A study on the protein synthesis of SYNV is presented in Chapter V. Four out of five structural proteins of SYNV (G, N, M 1 and M 2 ) were detected in infected protoplasts by gel electrophoresis of immunoprecipitates. The L protein could not be identified. The M 1 protein was shown to be phosphorylated and a polypeptide with a molecular weight of 38,000 was presumed to represent a less phosphorylated form of the M 1 protein. The G protein was proven to be glycosylated by N-glycosidical linked residues. The addition of 10 μg tunicamycin per ml incubation medium prevented glycosylation without markedly affecting protein synthesis. A nonglycosylated form of the G protein was not detected.
Original languageEnglish
QualificationDoctor of Philosophy
Awarding Institution
  • van der Want, J.P.H., Promotor, External person
  • Dijkstra, J.P.H., Promotor, External person
Award date7 Feb 1986
Place of PublicationWageningen
Publication statusPublished - 7 Feb 1986


  • cells
  • cowpeas
  • plant diseases
  • plant viruses
  • rhabdoviridae
  • vigna
  • viruses


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