Infection of Triticum monococcum Protoplasts with Barley Yellow Dwarf Virus

Abstract

SUMMARYProtoplasts from a Triticum monococcum cell culture line were successfully infected with barley yellow dwarf virus. Both purified virions and extracted RNA were shown to be infectious using a polyethylene glycol inoculation procedure. Up to 209/00 of the protoplasts contained viral antigens as judged by immunofluorescence assay. ELISA analysis showed that virus antigen expression was both dose-and time-dependent. Synthesis of new viral RNAs was confirmed by Northern blot hybridization. Studies of the expression of this economically important aphid-borne, phloem-limited virus should be greatly facilitated using this graminaceous protoplast system. It is the first reproducible protoplast infection system for this virus which can not be transmitted mechanically at the whole plant level.Barley yellow dwarf virus (BYDV) is a member of the luteovirus group and can cause significant economic losses to cereals (Bumett, 1984;Waterhouse et al., 1988). The luteovirus group is characteristically aphid-transmitted in a persistent manner and phloem-limited in infected plants. BYDV has 25 nm icosahedral particles with a single-stranded RNA genome of approx. Mr 2 x 106. Unlike many other plant viruses, luteoviruses are not mechanically transmissible. BYDV is currently divided into five serotypes on the basis of the specificity of its aphid vector and serological properties of the virions. Although the sequence of the genome of the PAV serotype of BYDV has been recently completed (Miller et al., 1988) there is still little biochemical information available on the replication and host interactions of this virus. This is due in part to the difficulties associated with the low yields produced and with working with a phloem-limited virus in plants.Protoplast infection systems have been described for two luteoviruses which infect dicotyledonous plants but none has been developed for BYDV, which infects monocotyledons. Both tobacco necrotic dwarf virus (TNDV) and potato leafroU virus (PLRV) can infect a high percentage of isolated tobacco mesophyll protoplasts (Kubo & Takanami, 1979). In addition, isolated RNA from PLRV has been demonstrated to be infectious for tobacco protoplasts (Mayo et al., 1982).Attempts to develop a similar bioassay system for BYDV expression have resulted in only limited success. The work of Barnett et al. (1981) suggests a low level of infection of cereal protoplasts with the BYDV MAV serotype. These investigators concluded that the large variability and low level of infection which they observed made such an approach unsuitable as a useful bioassay for BYDV replication. The techniques described here establish for the first time an effective and routine method for infection of monocotyledonous protoplasts with BYDV virions or RNA.A Triticum monococcum cell culture system provided a convenient and reliable source of graminaceous protoplasts suitable for infection with BYDV. A suspension cell culture line of T. monococcum was obtained from H. Lorz (K61n, F.R.G.) and maintained in WtMI medium t Present a...

Preparation and Electroporation of Oat Protoplasts from Cell Suspension Culture

“…Oat protoplasts have been used for studying viruses such as barley yellow dwarf luteovirus (BYDV), barley stripe hordeivirus, and brome mosaic virus (Furusawa and Okuno, 1978;Barnett et al, 1981;Zheng and Edwards, 1990). The protocol described herein was developed in the authors' laboratory using oat cell suspension culture for studying BYDV and is based largely on the work of Dinesh-Kumar et al (1992), whose protocols were derived from Young et al (1989). The oat cell suspension culture (Avena sativa cv.…”

The Yd2 resistance to barley yellow dwarf virus is effective in barley plants but not in their leaf protoplasts

“…A low level of infection of oat and barley leaf protoplasts was achieved by Barnett, Hammond & Lister (1981). We have recently developed a system based on protoplasts from Triticum monococcum (wheat) suspension culture in which BYDV replicates efficiently and reproducibly (Young et al, 1989).…”

Gene expression strategies of barley yellow dwarf virus

“…This protruded readthrough domain may determine aphid specificity by interacting with the receptors in the aphid vector (Bahner et al, 1990). Such protrusions have been observed on the surface of PLRV and BYDV particles (Harrison 1984;Waterhouse et al, 1989).…”

“…However, luteoviruses can replicate efficiently in the protoplasts derived from mesophyll tissues or suspension cells Young et al, 1989Young et al, , 1991Veldt et al, 1992). Different strains of BYDV induce distinct cytopathological events (Gill and Chong, 1979).…”

“…Total nucleic acids from protoplasts were isolated by the method of Young et al (1989). For northern blot analysis, RNA was separated from DNA by LiCI precipitation of the RNA (Sambrook et a!., 1989).…”