THE SEQUENCE IN THE POTATO SPINDLE TUBER VIROID REQUIRED FOR ITS cDNA TO BE INFECTIVE: A PUTATIVE PROCESSING SITE IN VIROID REPLICATION

Abstract

This paper describes the minimum length of the sequence required for the potato spindle tuber viroid (PSTV) cDNA cloned in plasmid or phage DNA vectors to be infective. To examine the infectivity, we cloned the BamHI fragments containing the full length of PSTV cDNA into the BamHI sites of pUC plasmids or double-stranded forms of M13mp phage DNA. When the BamHI monomeric unit was inserted into pUC8 or M13mp8 in a particular orientation, the 14 base pair sequence (GGATCCCCGGGGAA) of PSTV cDNA was directly duplicated at both of the insertion junctions. These recombinant DNAs were infectious to tomato plants at the same level as the recombinant containing a tandem dimer of cDNA. Insertion of the BamHI monomer into pUC12 or M13mp10 created the direct repetition of the 11 base pair sequence (GGATCCCCGGG) at both of the insertion junctions. Infectivities of both the pUC12 and the M13mp10 recombinants were tenfold lower than those of the pUC8 and the M13mp8 recombinants. On the other hand, when the plasmids or the phage DNAs carried the BamHI monomer in the opposite orientation so as to create the terminal repetition of the 7 base pair sequence (GGGATCC), the infectivity was scarcely detected. We also constructed a recombinant DNA having the AluI monomeric unit in either orientation, in which no terminal repetition was generated. This recombinant DNA showed no detectable infectivity. These results indicate that the cloned BamHI monomer having the direct repetition of the 14 base pair sequence at both the termini is able to express fully the infectivity, whereas the monomer having the shorter stretch of repetition shows only limited infectivity. Neither the sequences of vectors used nor the orientations of the cDNA sequence are crucial for infection by cloned cDNAs. Since the 14 nucleotide sequence is found in the middle of the regions which are conserved in many viroid sequences, this sequence is thought to be an essential site for viroid replication, probably a processing site of the oligomeric forms of viroid RNA found in infected plants.