Mol Gen Genet (1988) 212: 536-542 OIGG © Springer-Verlag 1988 Expression of bacterial chitinase protein in tobacco leaves using two photosynthetic gene promoters Jonathan D.G. Jones, Caroline Dean, David Gidoni, David Gilbert, Diane Bond-Nutter, Rino Lee, John Bedbrook, and Pamela Dunsmuir Advanced Genetic Sciences Inc., 6701 San Pablo Ave., Oakland, CA 94608, USA Summary. A bacterial chitinase gene from Serratia marces- cens (chiA) was fused to (i) a promoter of the ribulose bis- phosphate carboxylase small subunit (rbcS) gene and (ii) two different chlorophyll a/b binding protein (cab) gene promoters from petunia. The resulting constructions were introduced into Agrobaeterium Ti plasmid-based plant cell transformation vectors and used to generate multiple inde- pendent transgenic tobacco plants. ChiA mRNA and pro- tein levels were measured in these plants. On average, the rbcS/ehiA fusion gave rise to threefold more ehiA mRNA than either cab/chiA fusion. We investigated the influence of sequences around the translational initiation ATG codon on the level of ChiA protein. The rbcS/chiA and cab/chiA fusions in which the sequence in the vicinity of the transla- tional initiation codon is ACC ATGGC gave rise to trans- formants with higher levels of ChiA protein than those car- rying a eab/chiA fusion with the sequence CAT ATGCG in the same region. This difference in translational efficiency is consistent with previous findings on preferred sequences in this region of the mRNA. In those transformants show- ing the highest level of ChiA expression, ChiA protein accu- mulated to about 0.25% of total soluble leaf protein. These plants contained significantly higher chitinase enzymatic ac- tivity than control plants. Key words: Agrobacterium - Small subunit of ribulose bis- phosphate carboxylase - Chlorophyll a/b binding protein - Promoter comparisons - Chitinase activity Introduction Successful plant genetic engineering for crop improvement will frequently require the capacity to express introduced genes at high levels. One approach to achieving this end has been to isolate the 5' flanking regions of strongly ex- pressed plant genes, particularly the nuclear genes which encode abundant leaf proteins such as the small subunit of ribulose bisphosphate carboxylase (rbeS) and the chloro- phyll a/b binding protein (cab) (Dunsmuir et al. 1983; Dunsmuir 1985; Dean et al. 1985a, b). Several reports de- scribe the use of these photosynthetic gene promoters to direct the expression of foreign coding regions in leaves of transgenic plants (Jones et al. 1985; Poulsen et al. 1986). Other promoters, such as the 35S promoter from cauliflow- Offprint requests to: J.D.G. Jones er mosaic virus or various T-DNA promoters from Agro- bacterium, have also been used to direct useful levels of expression of introduced genes in plants (Powell Abel et al. 1986; Turner et al. 1987; Vaeck et al. 1987). We report here on experiments in which promoters from several different petunia photosynthetic genes were fused to ehiA, a bacterial chitinase gene from Serratia marcescens (Jones et al. 1986). The promoters that we compared were derived from (i) the petunia ribulose bisphosphate carboxyl- ase small subunit (rbcS) gene SSU301 which encodes an mRNA that constitutes 1.5% of petunia leaf polyadeny- lated mRNA (Dean et al. 1985, 1986, 1987a) and (ii)the two chlorophyll a/b binding protein (cab) genes cab22L and eab22R (Dunsmuir et al. 1983; Dunsmuir 1985), each of which gives rise to about 0.2% of total petunia leaf mRNA (Gidoni et al. 1987). We investigated the capacity of these different photo- synthetic promoters to direct high levels of synthesis of chiA mRNA and protein in transgenic tobacco plants. We also examined the effect of different sequences in the vicin- ity of the translation initiation codon on the accumulation of ChiA protein in leaves. Previous studies using nopaline synthase promoter (nos) fusions to chiA have indicated the importance of sequences in this region for expression levels of ehiA mRNA and protein (Taylor et al. 1987). The S. marcescens chiA gene encodes a protein which has been shown to possess in vitro antifungal activity (Jones et al. 1986). We conducted our gene fusion experiments with the chiA gene in order to develop transformed plant lines which express the ChiA protein at high levels and then to investigate whether these lines have enhanced resistance to fungal disease. As a first step toward evaluating the feasi- bility of this approach, we investigated whether the ChiA protein produced in tobacco leaves is enzymatically active. Materials and methods DNA biochemistry and bacterial methods. Ligation, Eseheriehia coIi transformation with plasmid DNA and preparation of plasmid DNA were all carried out by stan- dard techniques using the E. coli strains HB101, JM83 and MVl193 (Maniatis et al. 1983; Vieira and Messing 1987) and the pUC family of plasmid vectors or the mpl8 and mpl9 M13 phage vectors (Yanisch-Perron eta1. 1985). DNA sequences were determined using dideoxy sequencing with [35S] dATP (Biggin et al. 1983). Binary vector plasmids