Instability of potato spindle tuber viroid (PSTVd) cDNA in Agrobacterium tumefaciens

Abstract

Potato spindle tuber viroid (PSTVd, severe strain) was isolated from tobacco plants transformed with a dimeric infectious expression construct 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 electrophoresis under native conditions. Electrophoretic analysis revealed an appearence of electrophoretically distinguishable heteroduplexes, suggesting an accumulation of mutated sequence variants of PSTVd had occurred in the plant transformants. TGGE analysis of PSTVd cDNA, re-cloned from the original expression vector pCB1413 to plasmid pUC18 confirmed the accumulation of mutations in the cDNA and the instability of this sequence in A. tumefaciens maintained at 4°C for 2.5 years. One of these point mutations was identified by sequencing the PSTVd cDNAs isolated from the individual E. coli colonies. This transition (GC→AT) was localized at the position of 81 in the PSTVd genome, causing the change of C to U in PSTVd plus RNA. Transformation of tobacco with the freshly prepared expression vector containing the dimeric sequence of PSTVd lethal KF440-2 lead to the propagation of PSTVd electrophoretically identical to that derived from the original sequence and maintained in the tomato by a conventional infection. © 1994 Institute of Experimental Botany.