Expression of an entire bacterial operon in plants

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.

Figure 1.

Analysis for the presence of prn genes in treated plants. A, DNA was extracted from systemic leaves and subjected to PCR using different sets of primers (Supplemental Table S1). M, Size markers. Amplification of: a prnA fragment (lane 1), TYLCV-IR (lane 2), a prnB and prnC-spanning fragment (lane 3), and a prnD fragment (lane 4). B, Presence of prnA in various tomato organs. Amplification with prnA primers of DNA extracted from roots (lane 1), leaves (lane 2), fruits (lane 3), leaves of an untreated plant (lane 5), and without template (lane 6). Lanes 4 and 7, Empty. M, Size markers.

Figure 2.

RT-PCR analysis for transcription of the prn operon. The amplified sequence is a segment of prnD. RT-PCR of the same plant RNAs amplifying a fragment of tomato actin is depicted at the bottom. M, Size markers. Lanes 1 to 8, Template RNA extracted from various prn-carrying plants. Lane 9, Control; RNA was extracted from untreated plant.

Figure 3.

HPLC and LC-MS analyses of the metabolites produced in PRN-expressing tomato plants. A, HPLC elution profiles. I, Synthetic PRN (positive control); II, root extract of PRN-expressing plants; III, leaf extract of PRN-expressing plants; IV, negative control. Root extract of untreated plants. Positions of PRN elution are indicated by arrows. B, Biological activity of the various fractions of AII. Each plate contains a disk of agar with R. solani mycelium and addition of (left to right) fractions eluted between 1 and 3 min, fractions eluted between 3 and 5 min, fractions eluted between 5 and 25 min, fractions eluted between 30 and 32 min, and synthetic PRN (0.2 μg/plate). C, LC-MS analysis of tomato extracts. The two top panels present extracts from plants not carrying IR-PRN. The two bottom panels present extracts from IR-PRN-harboring plants. LC patterns (total [negative] ion chromatogram [TIC]) are shown in the first and third panels from the top. Searches for PRN by mass (EIC, extracted ion chromatogram) are shown in the second and fourth panels.

Figure 4.

Expression of GFP in tomato plants harboring IR-PRN-GFP. Plant samples were taken 2 weeks after administration of the construct and examined under a confocal microscope (see “Materials and Methods”). A, Picture taken after excitation for GFP fluorescence. B, Picture taken with a filter that masks GFP fluorescence (chlorophyll autofluorescence is in red). C, Picture taken without excitation. D, Superposition of the two top panels.

Figure 5.

HPLC analysis of PRN in control tomato plants. I, Positive control (synthetic PRN). II, positive control. Extract of roots from plants carrying IR-PRN (same as in Fig. 3AII). III, Extract of roots carrying IR-PRN-GFP. IV, Extract of fruits from a tomato plant carrying IR-PRN. V, Extract of plants carrying IR-GUS (GUS replacing PRN). VI, Extract of TYLCV-infected tomato. VII, Extract of plants carrying IR-PRN with a deletion in prnB. Black arrows mark the position of PRN elution. Dashed arrows mark the expected positions of the eluted PRN.

Figure 6.

PRN-expressing plants are resistant to damping-off disease. Left: IR-PRN was introduced into tomato seedlings which were then planted in noninfested soil. The plants were transferred to R. solani-infested soil 1 week after administration of IR-PRN. The picture was taken 2 weeks after R. solani infestation. A PRN-treated plant is shown on the left and a nontreated plant on the right. Right: IR-PRN was administered to tomato seedlings which were immediately planted in R. solani-infested soil. The group of plants on the right was PRN treated, and the group on the left consists of untreated plants. The picture was taken 5 d after planting.

Figure 7

. IR-PRN is expressed as a long transcript. Lanes 1 and 2, Northern-blot analyses of RNA from IR-PRN-harboring plant (lane 1) and untreated plant (lane 2). A segment of prnA served as a probe. Lane 3, Long-distance PCR of cDNA reverse transcribed from ribosome-bound RNA. Primers for RT and PCR were from both ends of the prn operon (Supplemental Figs. S1 and 2 and Supplemental Table S1). M, Size markers.