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Evidence for similarity assisted recombination and predicted stem-loop structure determinant in potato virus X RNA recombination

¹ University of Goettingen;
² Institute of Sugar Beet Research

³ E-mail: varrelmann{at}ifz-goettingen.de

Virus RNA recombination, one of the main factors for genetic variability and evolution, is thought to be based on different mechanisms. Here, the recently described in vivo potato virus X (PVX) recombination assay (Draghici & Varrelmann, 2009), was applied to characterize structural parameters of recombination. The assay uses an Agrobacterium mediated expression system incorporating a PVX green fluorescent protein (GFP) labeled full-length clone. The clone contains a partial coat protein (CP) deletion that is defective in cell to cell movement, together with a functional CP + 3''-ntr transcript in Nicotiana benthamiana leaf tissue. The structural parameters assessed were the length of sequence overlap, the distance between mutations and the degree of sequence similarity. The effect on the observed frequency of reconstitution, and the composition of the recombination products were characterized. Application of four different type X intact PVX CP-genes with variable composition allowed the estimation of the junction sites of precise homologous recombination. Although one template switch would have been sufficient for functional reconstitution, between one to seven template switches were observed. Use of PVX GFP mutants with CP deletions of variable length resulted in a linear decrease of the reconstitution frequency. The critical length for homologous recombination observed was 20 to 50 nt. Reduction of the reconstitution frequency was obtained when a phylogeneticly distant PVX type Bi CP-gene was used. Finally, the prediction of CP and 3''-ntr RNA secondary structure demonstrated that recombination junction sites were mainly located in regions of stem-loop structures, allowing the categorization of the recombination observed as similarity assisted.

Received 30 June 2009; accepted 23 October 2009.