Citrus tristeza virus-based induced gene silencing of phytoene desaturase is more efficient when antisense orientation is used
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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; ∼ 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.
KeywordsCitrus sinensis Phytoene desaturase Carotenoids Abscisic acid Virus-induced gene silencing Antisense
The authors acknowledge our CREC colleagues for their helpful discussions. We thank Shelley E. Jones for the technical assistance and Laina Lindsey and Floyd Butz for maintaining the trees in greenhouses. This project is funded by the grant no. 201500955-04 from SCRI-NIFA, USDA.
NK conceptualized the research; NK, YN, FH, SB, and SG provided resources and data; JC, NK, YN, FH, SB, and SG performed experiments, conducted data analysis, and curated data; NK, YN, FH, and SG wrote the original draft and were responsible for data visualization; NK, FH, and SG revised the manuscript; NK administered the project.
Conflict of interest
The authors declare no conflict of interest.
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