Virus-induced gene silencing (VIGS) in Lilium leichtlinii using the Cucumber mosaic virus vector

Abstract

Lilies (Lilium) are among the most important floriculture crops, and to accelerate research regarding lily genetics, the development of reverse-genetics tools is necessary. However, Agrobacterium-mediated transformation in Lilium is time-consuming, since the plants require several years to progress from acclimation to flowering. Thus, virus-induced gene silencing (VIGS) is an attractive method for assaying gene function. In the present study, we modified a lily-derived strain of Cucumber mosaic virus (CMV-HL) as a VIGS vector and evaluated its effectiveness for inducing gene silencing in Lilium leichtlinii by introducing L. leichtlinii phytoene desaturase (LlPDS) gene fragments into an intercistronic region between the 3a and 3b genes of the CMV-HL RNA3 genome. At 30 days after inoculation (dpi) with LlPDS-containing CMV-HL, photo-bleaching was observed in the upper leaves of L. leichtlinii, and at 57 dpi, we observed that the natural orange color in flower tepals had faded. Reduced LlPDS expression and the detection of small interfering LlPDS RNA indicated that the color changes were the result of LlPDS gene silencing. In addition, the leaves also exhibited a mild photo-bleaching phenotype in the following year. Therefore, our results indicate that CMV-HL spreads systemically in the leaves and flowers of Lilium during the first year of infection, as well as in new shoots during the following year, and that the vector system can be successfully applied to induce short-term endogenous gene silencing in lilies.

Keywords: Cucumber mosaic virus (CMV)-HL; lily; photo-bleaching; phytoene desaturase (PDS); small interfering RNA (siRNA).

Figure 1. Virus-induced gene silencing in Lilium leichtlinii (P33-1) inoculated with Cucumber mosaic virus (CMV) harboring a 33-nt LlPDS fragment. (A) Leaf phenotypes of healthy plant (H1), a plant inoculated with empty vector (E1), and a plant infected with CMV-HL harboring the 33-nt LlPDS fragment (P33-1) at 30 dpi. Leaf width was approx. 9 mm. (B) RT-PCR amplified the CMV RNA3 region including a cloning site to confirm insertion of the PDS fragment in upper leaves. Fragments amplified from the plasmid of pCHL3-SB without LlPDS fragment (Empty, 252 bp) and pCHL3-SB harboring 33-nt fragments of LlPDS (LlPDS33, 278 bp) are also shown. (C) Relative expression of LlPDS in upper leaves of H1, E1, and P33-1. Vertical bars indicate the standard error of three technical replicates. (D) End-point PCR amplification of siRNA (60-nt) in the upper leaves of the P33-1, H1 plants, and two plants inoculated with empty vector (E1 and E2). –RT: reverse transcriptase was not added at the pulsed RT reaction. U6: U6 small nuclear RNA (77 bp). (E) Phenotypes of flowers at 57 dpi. Orange color at the tip of both outer and inner tepals had faded in the P33-1 plant. Bar=1 cm. (F) Relative expression of LlPDS in the orange tepals of the H1 plant and color-faded (white) and orange tepal regions of the P33-1 plant. Vertical bars indicate the standard error of three technical replicates.

Figure 2. Transmission of LlPDS silencing in the year following inoculation. (A) Leaf phenotypes: Photo-bleaching appeared on the P33-1 leaf but not on the leaves of the P33-2, a plant inoculated with empty vector (E1), and a healthy plant (H1). Leaf width was approx. 9 mm. (B) RT-PCR amplified the CMV RNA3 region including a cloning site to confirm the possession of the LlPDS fragment in lower, middle, and upper leaves of the P33-1 plant, and the leaves of the P33-2, E1, E2, E3, H1, H2, and H3 plants. M: molecular weight marker. (C) Relative expression of LlPDS in leaves. Vertical bars indicate the standard error of three leaves.

Figure 3. Virus-induced gene silencing in Lilium leichtlinii (P52) inoculated with Cucumber mosaic virus (CMV) harboring a 52-nt LlPDS fragment in the year after inoculation. (A) Photo-bleaching phenotype in the P52 plant. (B) RT-PCR amplified the CMV RNA3 region, including a cloning site, to confirm the insertion of the LlPDS fragment in six randomly selected leaves from the P52 plant (#1–6) and leaves of three plants (E1, E2, and E3) inoculated with empty vector. M: molecular weight marker. (C) Relative expression of LlPDS in six photo-bleached leaves from the P52 plant and four leaves of E1 plant. Vertical bars indicate the standard error of six (P52) and four (E1) leaves. (D) Detection of siRNA (stem-loop PCR, 60-nt) in leaves of the P52 and E1 plants. U6: U6 small nuclear RNA (77 bp).