SERIAL PASSAGE OF TOBACCO-RATTLE-VIRUS UNDER DIFFERENT SELECTION CONDITIONS RESULTS IN DELETION OF STRUCTURAL AND NONSTRUCTURAL GENES IN RNA-2

The RNA genome of tobacco rattle virus (TRV) Is bipartite. RNA 2 of th e nematode-transmissible TRV isolate PPK20 encodes the viral coat prot ein (cp) and proteins with molecular weights of 29,400 and 32,860 (29. 4 K and 32.8 K proteins). When this isolate was serially passaged in t obacco by using phenol-extracted RNA as the inoculum in each transfer, defective interfering (DI) RNAs rapidly accumulated. A number of thes e DI RNAs were cloned. Six DI RNAs had single internal deletions in RN A 2 that removed most of the cp gene, the 29.4 K gene, and the 5' half of the 32.8 K gene. The borders of the deletions in these DI RNAs wer e found to be flanked in the genomic RNA 2 by short nucleotide repeats or sequences resembling the 5' end of TRV genomic and subgenomic RNAs . Two DI RNAs were found to be recombinants containing a 5' sequence d erived from RNA 2 and a 3' sequence;derived from RNA 1. When serial pa ssage of TRV isolate PPK20 was carded out by using leaf homogenates as inocula in each transfer, accumulation of a DI RNA (designated D7) wi th a functional cp gene was observed. The deletion in D7 covered the 3 ' end of the cp gene, the 29.4 K gene, and the 5' half of the 32.8 K g ene. An infectious cDNA clone of D7 RNA was made. In mixed infections, D7 RNA rapidly outcompeted RNA 2 but did not compete with RNA 1. The deletion in D7 RNA abolished the nematode transmissibility of the PPK2 0 isolate. These results may explain the observation that many laborat ory isolates of tobraviruses have lost their nematode transmissibility and contain RNA 2 molecules of widely different lengths.