Electrotransfection of turnip yellow mosaic virus RNA into Brassica leaf protoplasts and detection of viral RNA products with a non-radioactive probe.
We describe here a convenient and efficient system for studying turnip yellow mosaic virus (TYMV) replication in leaf protoplasts. Inoculation of rapeseed (Brassica napus) or Chinese cabbage (B. sinensis) protoplasts was achieved via electroporation, and sensitive detection of viral RNA products was performed by Northern blot analyses using a non-radioactive digoxigenin-labelled cDNA probe. Virus replication was detected when 1.5 x 10(6) rapeseed protoplasts were inoculated with 20 ng of TYMV RNA. Electrotransfection of TYMV RNA was more efficient in rapeseed than in Chinese cabbage protoplasts, and gave somewhat higher signals than those of TYMV virions. TYMV RNA appeared to replicate equally well whether the protoplasts were incubated in the dark or under constant light.
Institut Jacques Monod, Tour, Paris, France.
J C
JC
Boyer
B
B
Zaccomer
A L
AL
Haenni
D015152:Blotting, Northern; D001937:Brassica / Q000382:microbiology*; D004558:Electric Stimulation; D009029:Mosaic Viruses / Q000378:metabolism / Q000502:physiology*; D011523:Protoplasts / Q000382:microbiology; D012367:RNA, Viral / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D014162:Transfection; D014779:Virus Replication
D012367:RNA, Viral