Abstract
Lithospermum erythrorhizon is a medicinal plant that produces shikonin, a red lipophilic naphthoquinone derivative that accumulates exclusively in roots. The biosynthetic steps required to complete the naphthalene ring of shikonin and its mechanism of secretion remain unclear. Multiple omics studies identified several candidate genes involved in shikonin production. The functions of these genes can be evaluated using virus-induced gene silencing (VIGS) systems, which have been shown advantageous in introducing iRNA genes into non-model plants. This study describes the development of a VIGS system using an apple latent spherical virus (ALSV) vector and a target gene, phytoene desaturase (LePDS1). Virus particles packaged in Nicotiana benthamiana were inoculated into L. erythrorhizon seedlings, yielding new leaves with albino phenotype but without disease symptoms. The levels of LePDS1 mRNAs were significantly lower in the albino plants than in mock control or escape plants. Virus-derived mRNA was detected in infected plants but not in escape and mock plants. Quantitative PCR and deep sequencing analysis indicated that transcription of another hypothetical PDS gene (LePDS2) also decreased in the defective leaves. Virus infection, however, had no effect on shikonin production. These results suggest that virus-based genetic transformation and the VIGS system silence target genes in soil-grown L. erythrorhizon.
Highlights
To identify candidate genes involved in these later steps of shikonin biosynthesis, we performed large-scale transcriptome and comparative proteome analyses, based on results showing that shikonin production can be strictly regulated by many chemical and physical factors[9]
To determine if apple latent spherical virus (ALSV) can infect L. erythrorhizon, we assessed its ability to knock down expression of the phytoene desaturase (PDS) gene in this plant species
The read number of LePDS1 was much smaller than that of LePDS2 (n = 7, p = 8.19 × 10–12) (Fig. 4D) and the LePDS1:LePDS2 ratio in infected (n = 7) plant leaves differed significantly from the ratios in escape (n = 14, p = 7.17 × 10–11) and mock infected (n = 14, p = 5.19 × 10–9) plant leaves (Fig. 4D). These results suggested that the designed sequence in this virus-induced gene silencing (VIGS) experiment preferentially knocked down LePDS1 rather than LePDS2, the expression of LePDS2 was negatively regulated to a lesser extent. qRT-PCR measurements of individual and combined expression of LePDS1 and LePDS2 and of the LePDS1: LePDS2 ratio in albino L5–L8 leaves expected by amplicon sequencing (Fig. 4E) showed that LePDS1 expression was significantly lower in leaves of infected than of escape (n = 4, p = 1.17 × 10–6) and mock infected (n = 4, p = 7.00 × 10–5) plants, LePDS2 expression, was significantly lower in leaves of infected than of escape (n = 4, p = 1.22 × 10–6) and mock treated (n = 4, p = 1.68 × 10–3) plants
Summary
Shikonin and its stereo-isomer alkannin have been reported to have various biological activities, including antibacterial, anti-inflammatory, anti-oxidant, antitumor, anti-angiogenic[1], and anti-topoisomerase[2] activities These compounds have been found to enhance granulation and glucose u ptake[3] and to reduce a diposity[4]. Shikonin production is tissue specific, being produced solely in the epidermis, but not in other root tissues, such as inside the central cylinder[6]. These characteristics enabled the identification of more than 10 putative candidate genes involved in shikonin biosynthesis[9] and more than 16 putative candidate genes involved in shikonin secretion from cells[11]
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