Submitted to: Turkish Journal of Agriculture and Forestry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 7/11/2005
Publication Date: 3/1/2006
Citation: Cevik, B., Lee, R.F., Niblett, C.L. 2006. Genetic Transformation of Citrus Paradisi with Antisense and untranslatable RNA-dependent RNA Polymerase Genes of Citrus Tristeza Closterovirus. Turkish Journal of Agriculture and Forestry, 30 (3), Pgs. 173-182. Interpretive Summary: The RNA-dependent RNA polymerase (RdRp) of Citrus tristeza virus (CTV) is thought to be expressed by a +1 translational frameshift towards the 3’ end of ORF 1 of the CTV genome; ORF encodes a 400 kDa polyprotein. Using antibodies made against the RdRp of CTV expressed as a fusion protein in E. coli, the expression of the RdRp of CTV was studies in vivo and in vitro. While the 56 kDa RdRp of CTV is thought to be expressed in infected plants by a +1 translational frameshift, Western blot analyses detected a 50-kDa protein in CTV-infected tissue extracts but not from healthy tissue extracts. The 50-kDa protein was located mostly in the membrane fractions where most plant RdRps have been reported. When a cloned CTV-RdRp gene encoding a 60 kDa fusion protein was studied in vitro, in addition to the 60 kDa protein, two smaller proteins of about 50 kDa and 10 kDa were produced in a rabbit reticulocyte lysate translation system. All three proteins were immunoprecipitated with anti-CTV-RdRp serum suggesting that all were fragments of the expressed 60 kDa CTV-RdRp fusion protein. The results suggest that the CTV RdRp may be cleaved in vitro in the rabbit reticulocyte lysate.
Technical Abstract: Expression of the RNA-dependent RNA polymerase (RdRp) of Citrus tristeza virus (CTV) was studied in vivo and in vitro using a polyclonal antiserum raised against the recombinant CTV-RdRp protein. Although 56 kDa CTV-RdRp is thought to be expressed by a +1 translational frameshift at the carboxyl terminus of a 400 kDa polyprotein, a 50-kDa protein was detected in CTV-infected but not in healthy citrus tissue by Western blot suggesting that RdRp was cleaved from the CTV polyprotein. This 50-kDa protein was present in the cytoplasmic and membrane fractions, and accumulated mainly in the membrane fraction where the most replication-associated proteins of RNA viruses are found. When the expression of a cloned CTV-RdRp gene encoding a 60-kDa fusion protein was studied in vitro, besides the expected 60-kDa protein, two smaller proteins of about 50 kDa and 10 kDa were produced in rabbit reticulocyte lysate system. All three proteins were immunoprecipitated with the anti-CTV-RdRp serum suggesting that the 50 kDa and the 10 kDa proteins were fragments of the 60 kDa CTV-RdRp fusion protein. When the expression of the RdRp was analyzed at different times during in vitro translation the 60 kDa and the 50 kDa proteins were detected at all time points, and a small amount of the 10-kDa protein was detected after 30 min of translation. These results suggested that the CTV-RdRp also may be cleaved in vitro in the rabbit reticulocyte lysate.