Novel TPLPV-mediated Virus-induced Gene Silencing System for Tea Plants (Camellia sinensis).

Abstract

In the post-genomic era of tea plants, the lack of an efficient genetic transformation system hinders the accurate identification of gene functions. Virus-induced gene silencing (VIGS) is a post-transcriptional silencing technology based on the plant's anti-virus mechanism, which does not rely on the stable transformation and regeneration system of plants. In this study, the tripartite tea plant line pattern virus (TPLPV) isolated from tea plants was developed into an infectious cDNA clone. The infectious cDNA clone of TPLPV, with an additional 30-bp poly(A) tail at the 3'-end of the TPLPV genome, and pCB301 as a binary expression vector were used to infect Nicotiana benthamiana and tea plants and caused line pattern symptoms. Furthermore, a VIGS vector was constructed for silencing phytoene desaturase (CsPDS) gene of tea plants. The copy number of TPLPV in tea plants was detected ranging from 14.0 to 1.40 × 108 by real-time quantitative reverse transcription PCR (qRT-PCR) analysis. The TPLPV-mediated VIGS system reduced CsPDS expression to 55.0% with a silencing efficiency of 40.0% and led to a chlorotic phenotype in systemic tea leaves at 45 days post-inoculation (dpi). The optimal concentration of Agrobacterium suspension containing TPLPV-VIGS vectors was OD600 = 0.6. The TPLPV-mediated VIGS system will serve as an effective tool for gene function analysis in tea plants.