Identification of the minimal sequence required for vascular-specific activity of Tomato mottle Taino virus Replication-associated protein promoter in transgenic plants.

A 597 nt fragment from Tomato mottle Taino virus (ToMoTV) DNA-A, with 459 nt located upstream of the Replication-associated protein translation start codon, was tested for promoter activity in solanaceous plants. The promoter activity of this fragment (pRep(459::Rep)) was demonstrated when it was introduced upstream the uidA reporter gene into tobacco, potato and tomato plants by genetic transformation. It became active in 7-day-old transgenic tobacco seedlings as revealed by a vascular-specific pattern of gene expression which was maintained during the continued growth of the plant. Transformed potato and tomato plants also showed a vascular-specific pattern of expression. In comparative assays, pRep(459::Rep) showed an expression activity 10-40-fold less than the 35S promoter from Cauliflower mosaic virus. To delimit the minimal cis-acting elements necessary for vascular specificity of this promoter, a set of PCR deletion mutants of pRep(459::Rep) (pRep(459), pRep(324), pRep(203), pRep(145), pRep(132) and pRep(115)), were generated and used to transform tobacco plants. Transgenic tobacco plants belonging to all the pRep versions were blue stained in the vascular system except those from the pRep(115) version. The results described in this report demonstrate that the minimal sequences necessary for the pRep promoter activity are confined in a segment of 132 nts (located between the nts 2454 and 2585 of the ToMoTV DNA A) and that this promoter harbors those elements sufficient for vascular-specific expression.