Citrus tristeza virus-based induced gene silencing of phytoene desaturase is more efficient when antisense orientation is used

In this study, phytoene desaturase (PDS) in citrus and green fluorescent protein (GFP) in Nicotiana benthamiana (line 16c) were silenced by virus-induced gene silencing (VIGS) with Citrus tristeza virus (CTV), using the sense and antisense orientations. The antisense orientation showed a stronger reduction of GFP expression in N. benthamiana compared with the sense orientation. Similarly, the antisense orientation of pds induced stronger leaf photobleaching than the sense orientation. The antisense orientation produced higher levels of pds- and gfp-specific short interfering RNAs (siRNAs; approximate to 21 nucleotides) than the sense orientation. In addition, the antisense orientation of pds resulted in lower expression of carotenoid biosynthetic genes and produced lower levels of carotenoids and chlorophylls compared to the sense orientation. Furthermore, the antisense insertion resulted in higher accumulation of the colorless carotenoid phytoene compared to the sense orientation. Our results indicate that the gene fragments inserted using the antisense orientation induced higher gene silencing than the sense orientation because more short interfering RNAs were produced. These siRNAs are complementary to the mRNAs of the target gene and consequently guide the degradation of target mRNA. Better understanding of gene silencing mechanisms in plants is necessary to enhance VIGS technology.