Potato spindle tuber viroid (PSTVd, severe strain) was isolated from t
obacco plants transformed with a dimeric infectious expression constru
ct which was maintained for a long time in Agrobacterium tumefaciens.
After in vitro hybridization of PSTVd to complete minus transcripts of
PSTVd (lethal), the resulting heteroduplexes were analyzed by electro
phoresis under native conditions. Electrophoretic analysis revealed an
appearence of electrophoretically distinguishable heteroduplexes, sug
gesting an accumulation of mutated sequence variants of PSTVd had occu
rred in the plant transformants. TGGE analysis of PSTVd cDNA, re-clone
d from the original expression vector pCB1413 to plasmid pUC18 confirm
ed the accumulation of mutations in the cDNA and the instability of th
is sequence in A. tumefaciens maintained at 4-degrees-C for 2.5 years.
One of these point mutations was identified by sequencing the PSTVd c
DNAs isolated from the individual E. coli colonies. This transition (G
C-->AT) was localized at the position of 81 in the PSTVd genome, causi
ng the change of C to U in PSTVd plus RNA. Transformation of tobacco w
ith the freshly prepared expression vector containing the dimeric sequ
ence of PSTVd lethal KF440-2 lead to the propagation of PSTVd electrop
horetically identical to that derived from the original sequence and m
aintained in the tomato by a conventional infection.