Molecular characterization of variants of a new 'rule-breaking' tobacco rattle virus RNA2 in potatoes.

In potato tubers showing pronounced corky ringspot symptoms, two related 'rule-breaking" tobacco rattle virus (TRV) RNA2s, named Da-2 and Db-2, were identified. Their coat protein (cp) genes are preceded on the 5' side by an additional gene for a 35 kDa protein for which no relationships with previously described TRV genes or their expression products were found. With estimated 4296 and 4247 nucleotides (nts) the two RNAs are the longest TRV RNA2s described so far. The difference in size between Da-2 and Db-2 is due to a duplication of a stretch of 49 nts in the 5' untranslated region of Da-2. An alignment of TRV coat proteins (cp) revealed that up to about amino acid (aa) 176 they form two rather uniform groups. The much shorter C-terminal parts of the cps, however, differ considerably in size and composition. With 56 aa this C-terminal part is much longer in the Da and Db sequences than in all other TRV cps. It differs in 18 positions in the two strains whereas their N-terminal 184 aa differ only in two positions. - In young potato plants developing from bud-cuttings of TRV Db-infected potato tubers which had been planted in soil free of virus and nematodes a gradual degradation of Db-2 was observed. In the newly formed rootlets already five days after planting a deletion of 80 nts was observed in the putative 2b gene which in other TRV strains encodes a protein necessary for nematode transmission. Thirty three days after planting the entire 2b gene, 119 nts at the 3'end of the cp gene and a portion of the original 3'untranslated region of Db-2 had been lost in the newly formed roots, leaves and stolons. The gene for the 35kDa protein was the only one which was not affected by deletions which seems to emphasize its importance for the virus. Fifty days after planting only TRV RNA1, but no TRV RNA2 were detectable.

Complete nucleotide sequences of two closely related tobacco rattle virus RNA2s for which previously possibly only deletion/recombination mutants had been described.

The almost complete nucleotide (nt) sequences lacking only short primer-derived 5' and 3' terminal portions were determined for the RNA2s of two closely related tobacco rattle virus (TRV) isolates which were detected in recently infected roots of Nicotiana benthamiana bait plants (isolate HaB) or of field-grown potato plants (isolate HaW), respectively. The sequences of c. 1350 5' proximal nts in these RNA2s were almost identical to those in four previously described TRV isolates from potatoes in the Netherlands and Poland which had been propagated in tobacco leaves. The RNA2s of the earlier isolates, however, consist of only c. 2000 nts, whereas the HaW and HaB RNA2s comprise more than 4000 nts. In addition to the coat protein gene which is present in the RNA2s of all isolates, the HaW and HaB RNA2s contain two further open reading frames which in the previous isolates were apparently lost either entirely or of which small remnants only were left. The RNA2s of the previously described isolates and of our new ones have different RNA1-related 3' ends which apparently have been acquired from different supporting RNA1s. After transmission to tobacco leaves HaB RNA2 formed deletion/recombination mutants which--as in the previous isolates--consisted of only c. 2000 nts. They had acquired the 3' end from their supporting RNA1.