Agroinjection of tomato fruits. A tool for rapid functional analysis of transgenes directly in fruit

Abstract

Transient expression of foreign genes in plant tissues is a valuable tool for plant biotechnology. To shorten the time for gene functional analysis in fruits, we developed a transient methodology that could be applied to tomato (Solanum lycopersicum cv Micro Tom) fruits. It was found that injection of Agrobacterium cultures through the fruit stylar apex resulted in complete fruit infiltration. This infiltration method, named fruit agroinjection, rendered high levels of 35S Cauliflower mosaic virus-driven beta-glucuronidase and yellow fluorescence protein transient expression in the fruit, with higher expression levels around the placenta and moderate levels in the pericarp. Usefulness of fruit agroinjection was assayed in three case studies: (1) the heat shock regulation of an Arabidopsis (Arabidopsis thaliana) promoter, (2) the production of recombinant IgA antibodies as an example of molecular farming, and (3) the virus-induced gene silencing of the carotene biosynthesis pathway. In all three instances, this technology was shown to be efficient as a tool for fast transgene expression in fruits.

Figure 1.

Extent of agroinfiltration of tomato fruits using agroinjection. A, Fruit slices from tomatoes agroinjected with methylene blue-stained bacteria (left) and with an unstained culture (right). B, Close up from a tomato agroinjected with stained bacteria. Blue color reveals the tissues reached by the infiltrated culture.

Figure 2.

Plant promoter-driven expression of reporter genes in tomato fruits. A, pBIN-YFP/GUS-agroinfiltrated tomato at 4 dpi showing YFP fluorescence in the placental tissue under UV light. B, Confocal microscope image of a similar sample showing YFP fluorescence in cells from the placenta-locule transition zone. C, Histochemical GUS staining of a pBIN-YFP/GUS-agroinfiltrated tomato. D, Close up of the seed-placenta joining region decorated with GUS staining. E, Time course of glucuronidase activity in agroinfiltrated tomatoes. Bars show the average activity (pmol methylumbelliferone × g fresh weight⁻¹ × min⁻¹) of four tomatoes per time point ± sd. F, Heat shock induction of GUS activity directed by Arabidopsis HSP70 promoter. pHSP70∷GUS-agroinjected tomatoes at 4 dpi were separated in two halves and incubated for 6 h at 42°C and 25°C, respectively. Graph compares GUS-specific activity (pmol methylumbelliferone × μg protein⁻¹ × h⁻¹) of heat-shocked pieces (gray bars) with the negligible specific activity of control pieces (black bars).

Figure 3.

Expression of full IgA antibodies in tomato fruits. A, Schematic structure of pBINIgL and pBINIgH constructs. Black arrows represent cloning sites for phage display-derived variable sequences. B, Western analysis of tomato fruits agroinfiltrated with IgA antibody chains. Two phage display-derived clones (n8 and n10) were assayed. Tomatoes were infiltrated with cultures containing pBIN-IgL (L8 and L10 lanes), pBIN-IgH (H8 and H10 lanes), or coinfiltrated with a combination of HCs and LCs (A8 = L8 + H8; A10 = L10 + A10). Blots were decorated with anti-chicken IgH (alpha-specific) antibody (top section), anti-chicken LC (middle section), or anti-chicken IgY whole-molecule recognizing the native structure of chicken antibody (IgY and IgA share the same IgL in chicken).

Figure 4.

PDS silencing in tomato. A, Systemically (leaf-infiltrated) PDS-silenced plant showing photobleaching phenotype in leaves and fruits. B, Mature fruit from systemically (leaf-infiltrated) PDS-silenced plant showing red (LR) and yellow/orange (LO) sectors. C, Example of color evolution during ripening of Micro Tom fruits: G, green; B, breaker; O, yellow/orange; R, red; S, yellow/orange fruits showing different degrees of red pigmented sectors (ranging from S1 to S4). D, Fruits agroinjected with pTRV1/2-tPDS (S) or pTRV1 alone (R) showing drastic differences in red pigmentation at maturity. E, Longitudinal section of a mature tomato from a PDS-silenced plant showing internal red-yellow sectors. F, Close up of E showing viviparism in the yellow sector. G, Evolution of color in a group of 140 tomatoes agroinjected either with pTRV1/2-tPDS (left) or control pTRV1 (right) Agrobacterium cultures. Color was recorded for every tomato during 4 weeks (W1 to W4). Color categories were defined as in C. Number of tomatoes in every category is shown as a percentage of the total number of fruits. S category includes silenced fruits as well as a small number of nonsilenced fruits that were rapidly turning into red from the orange stage. H, Schematic representation of lycopene synthesis route in tomato.

Figure 5.

Effect of PDS silencing in tomato fruits. A, Relative abundance of PDS mRNA in pericarp from silenced tomatoes. Samples are defined as in Figure 4: LR, red sectors of systemically silenced tomatoes; LO, yellow/orange sectors of systemically silenced tomatoes; S, pericarp from pTRV1/2-tPDS-agroinjected tomatoes arrested at S stage. Relative mRNA levels were calculating using pricarp from TRV1-agroinjected red tomatoes (R) as a reference for the calculations. B, Carotene chromatographic profiles of the same samples as in A. C, Relative levels of lycopene (black bars) and the PDS substrate phytoene (white bars) in pericarp samples. Metabolite levels are given as a percentage of the total carotenoid content in every sample.