Plant-infecting viruses, particularly the Pararetroviruses, have been used for many years as versatile genetic resources to design efficient plant expression vectors. The Pararetroviruses (members of the Caulimoviridae) typically contain two transcriptional promoters (the sub-genomic transcript promoter and the full-length transcript promoter) and 6-7 overlapping open reading frames (ORFs) with a genome size of 7-9 kB. Their promoter elements have been extensively exploited during the last two decades to construct effective gene expression systems. At the same time, the caulimoviral promoters have also been genetically manipulated with different molecular approaches to develop synthetic "chimeras" exhibiting precise functionality. Native and "tailor-made" synthetic promoters of Pararetroviruses are particularly attractive for formulating unique gene expression cassettes that perform extremely well in gene-stacking and gene-pyramiding in plant cells. In this chapter, we will mainly discuss important protocols associated with identifying novel/unique pararetroviral promoters that have optimal lengths with appropriate activities for developing efficient plant gene expression systems.
Keywords: Caulimoviral promoter; Electroporation; Infiltration; TSS; Transient expression.