A cDNA clone from a defective RNA of citrus tristeza virus is infective in the presence of the helper virus.

A naturally occurring defective RNA of 2379 nt (D2.3) from the VT strain of citrus tristeza closterovirus (CTV) was cloned and sequenced. The D2.3 RNA is a fusion of two regions of 1521 and 858 nt from the 5' and 3' ends of the CTV genome, respectively. A cDNA clone of D2.3 RNA was tagged by the insertion of a 0.47 kb chimeric DNA fragment and the recombinant cDNA was inserted downstream of the cauliflower mosaic virus 35S promoter. The resulting construct was bombarded into CTV-infected tissue, which was then grafted onto virus-free plants. The presence of recombinant RNA in systemically infected leaves was demonstrated by RT-PCR. Sequencing the RT-PCR products synthesized from double-stranded RNA confirmed the presence of the chimeric segment used for tagging. This is the first report of an infectious cDNA molecule derived from CTV D-RNA.

Multiple species of defective RNAs in plants infected with citrus tristeza virus.

Alemow (Citrus macrophylla) and sweet orange (C. sinensis) plants infected, respectively, with several Israeli and Florida isolates of the citrus tristeza virus (CTV) were found to contain multiple species of RNA molecules with features similar to defective-interfering RNAs. Northern blot hybridizations of dsRNAs extracted from serial passages of the Israeli VT isolate (CTV-VT) and from different plants infected with a single source of inoculum showed considerable variation both in the presence and in the relative abundance of the defective RNA (D-RNA) bands. The D-RNA molecules were found to be encapsidated in the CTV particles. Sequence analysis of two VT D-RNA molecules of 2.7 and 4.5 kb revealed that they were composed of two regions corresponding to 1818 and 4036 nucleotides from the 5' and 938 and 442 nucleotides from the 3' termini of the CTV-VT genomic RNA, respectively. A short (ca. 0.8 kb) nonencapsidated single-stranded positive-sense RNA species was also found in infected plants. This ssRNA, which copurified with dsRNAs, was shown by hybridization to encompass the 5'-terminal part of the CTV genome and might have an extensive secondary structure.