Promoter/leader deletion analysis and plant expression vectors with the figwort mosaic virus (FMV) full length transcript (FLt) promoter containing single or double enhancer domains INDU B. MAITI , SIDDARAME GOWDA , JENNIFER KIERNAN, SUDIP K. GHOSH and ROBERT J. SHEPHERD Department of Plant Pathology, University of Kentucky, Lexington, KY 40546-0091, USA (Fax: +1 606 323 1077) Received 30 October 1995; revised 7 February 1996; accepted 16 February 1996 The boundaries required for maximal expression from the promoter/leader region of the full length transcript of figwort mosaic virus (FLt promoter) coupled to reporter genes were defined by 59 and 39 deletion analyses. In transient expression assays using protoplasts of Nicotiana edwardsonii, a 314 bp FLt promoter fragment sequence ( 249 to +65 from the transcription start site) was sufficient for strong expression activity. Plant expression vectors developed with modified FLt promoters were tested with GUS or CAT as reporter genes in transgenic plants. The FLt promoter is a strong constitutive promoter, with strength comparable to or greater than that of the CaMV 35S promoter. The FLt promoter with its double enhancer domain linked to GUS or CAT reporter genes provides an average 4-fold greater activity than the FLt promoter with a single enhancer domain ( 55 to 249 bp upstream fragment) in tests with transgenic plants and in protoplast transient expression assays. Keywords: caulimovirus; transcriptional enhancer; expression vectors; figwort mosaic virus; 35S promoter; transgenic plants; transient expression Introduction For constructing plant genetic transformation vectors, a number of strong constitutive promoters have been derived from caulimoviruses, particularly cauliflower mosaic virus (CaMV), and from Agrobacterium tumefaciens. These promoters, namely the CaMV 35S and 19S promoters (Lawton et al., 1987), the figwort mosaic virus (FMV, strain DxS) promoter for the full length transcript (Gowda et al., 1989), the FMV strain M3 34S promoter (Sanger et al., 1990), the commelina yellow mottle virus promoter (Medberry et al., 1992), the nopaline synthase promoter (An et al., 1986) and the octopine synthase promoter (Ellis et al., 1987), are examples of useful promoters for directing heterologous gene expression in transgenic plants. The genomes of five caulimoviruses of known nucleotide sequence, CaMV (Gardner et al., 1981), FMV (Richins et al., 1987), carnation etched ring virus (CERV) (Hull et al., 1986), soya bean chlorotic mottle virus (soyCMV) (Hasegawa et al., 1989), and peanut chlorotic streak virus (PClSV) (Richins, 1993; Richins et al., 1994) generally harbour at least two active transcriptional promoters. FMV (Scholthof et al., 1992) and PClSV (Richins, 1993) have been shown to have transcripts similar to the 19S and 35S RNAs (Howell and Hull, 1978; Odell et al., 1981) found in CaMV infected plant cells. The transcriptional promoter for the major transcript of FMV, which we first described several years ago (Gowda et al., 1989), is the subject of this investigation. The full length transcript of FMV is equivalent to the 35S transcript of CaMV. The 35S promoter from cauliflower mosaic virus Transgenic Research 6, 143–156 (1997) 0962–8819 1997 Chapman & Hall To whom correspondence should be addressed. Present Address: Citrus Research and Education Center, University of Florida, 700 Experiment Station Road, Lake Alfred, FL 33850-2299, USA. Present Address: Dept of Tropical Public Health, Harvard School of Medicine, 665 Huntington Ave., Boston, MA 02115, USA.