Abstract
Multigene expression is required for metabolic engineering, i.e. coregulated expression of all genes in a metabolic pathway for the production of a desired secondary metabolite. To that end, several transgenic approaches have been attempted with limited success. Better success has been achieved by transforming plastids with operons. IL-60 is a platform of constructs driven from the geminivirus Tomato yellow leaf curl virus. We demonstrate that IL-60 enables nontransgenic expression of an entire bacterial operon in tomato (Solanum lycopersicum) plants without the need for plastid (or any other) transformation. Delivery to the plant is simple, and the rate of expressing plants is close to 100%, eliminating the need for selectable markers. Using this platform, we show the expression of an entire metabolic pathway in plants and delivery of the end product secondary metabolite (pyrrolnitrin). Expression of this unique secondary metabolite resulted in the appearance of a unique plant phenotype disease resistance. Pyrrolnitrin production was already evident 2 d after application of the operon to plants and persisted throughout the plant's life span. Expression of entire metabolic pathways in plants is potentially beneficial for plant improvement, disease resistance, and biotechnological advances, such as commercial production of desired metabolites.
Highlights
Multigene expression is required for metabolic engineering, i.e. coregulated expression of all genes in a metabolic pathway for the production of a desired secondary metabolite
We present a case in which the universal DNA plant vector system, IL-60, mediated the introduction and expression of an entire bacterial operon in tomato (Solanum lycopersicum) plants
A plasmid was inserted into the replication-associated gene of Tomato yellow leaf curl virus (TYLCV), disabling rollingcircle replication but maintaining replication from doublestranded DNA to double-stranded DNA, which is directed solely by host factors
Summary
Replication, Expression, and Spread of the prn Operon in Tomato Plants. The components of the plant universal vector IL-60 employed throughout this study are described in Peretz et al (2007). Biological tests showed that the plant-extracted, HPLC-purified PRN was inhibitory to R. solani (Fig. 3B). We fused GFP (the 5#2untranslated region was deleted to remove predicted RBSs, and the first ATG was omitted to prevent possible translation initiation at the start of the GFP coding region) to prnD to produce IR-PRN-GFP (described in “Materials and Methods” and illustrated in Supplemental Fig. S1) and administered this construct to plants. Plants in which PRN had been replaced by an irrelevant gene (GUS) or TYLCV-infected plants did not produce PRN (Fig. 5). We isolated polyribosomes from PRNexpressing plants, prepared cDNA from the ribosomalbound RNA (as described in “Materials and Methods”), and amplified long cDNA (5–6 kb and p) by PCR with a PRN-specific primer (Fig. 7, lane 3). The polycistronic long transcript itself, rather than processed mature RNAs, served as the template for translation, as is the case in prokaryotes
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