INSTABILITY OF POTATO SPINDLE TUBER VIROID (PSTVD) CDNA IN AGROBACTERIUM-TUMEFACIENS

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.