Summary auto-generated
Researchers cloned and sequenced a naturally occurring defective RNA (D2.3) from citrus tristeza virus (CTV), a destructive citrus pathogen. The D2.3 RNA is 2,379 nucleotides long and consists of fused 5' and 3' terminal segments from the CTV genome with extensive internal deletions. The scientists created an infectious cDNA clone by inserting the D2.3 sequence downstream of a cauliflower mosaic virus promoter and tagging it with a chimeric DNA fragment. This construct was delivered into CTV-infected plant tissue via particle bombardment. The recombinant D2.3 RNA was detected in systemically infected leaves 2.5 to 7 months post-inoculation through RT-PCR analysis, confirming replication and encapsidation into virus particles. The D2.3 RNA was the first CTV defective RNA identified with a putative continuous open reading frame. This work represents the first report of an infectious cDNA derived from a CTV defective RNA and demonstrates that defective RNAs require the helper virus for replication. The findings enable future studies of defective RNA accumulation and potentially allow functional analysis of CTV genes and expression of foreign genes.
Key findings
- Cloning and sequencing of a naturally occurring CTV defective RNA (D2.3) of 2,379 nucleotides, consisting of 5' and 3' terminal segments from the viral genome
- Construction of an infectious cDNA clone from the defective RNA that replicates when introduced into CTV-infected plants with a helper virus
- Detection of recombinant D2.3 RNA in systemically infected leaves for up to 7 months post-inoculation, confirming its encapsidation in CTV particles
- D2.3 is the first characterized CTV defective RNA containing a putative single continuous open reading frame
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Abstract
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.