Molecular identification and expression of papaya ringspot virus gene in tobacco (Nicotiana benthamiana Domin) plants

Abstract

Papaya ringspot virus (PRSV) belongs to Potyviridae family, genus Potyvirus and mainly transmitted by aphids (genera Macrosiphum and Myzus). Infected plants show mottling, blister-like patches and distortion of leaves as well as causes ring spot disease in papaya (Carica papaya L.) fruits. The HC-Pro (helper component proteinase), CI (cylindrical inclusion), NIb (nuclear inclusion body b) and Cp (coat protein) gene are involved in many other essential processes during the virus infection cycle as well as for transmission of potyvirus. Novel degenerate primers have been designed for amplification these genes. Phylogenetic analysis showed that the HC-Pro had the closest relationship with PRSV P isolate MP, CI with PRSV P isolate DEL and partial Nib plus CP with PRSV P isolate DEL as well as PRSV P isolate USA. Eluted PCR product of amplified gene was checked in 1 % agarose gel and cloned in binary vector with reporter gene (GUS). Agroinfiltration and GUS histochemical staining was performed for detecting the movement of a particular gene. The high sensitivity of GUS assays permitted temporal analysis of early expression of potyviral genes. Expression study proved that all genes are involved in long distance movement in host plants.