Expression of hairpin RNA (hpRNA) targeting the three CTV-silencing suppressor genes confers sweet orange with stem-pitting CTV tolerance
2017
Cheng, Chunzhen | Zhang, Yongyan | Yang, Jiawei | Zhong, Yun
Silencing the silencing suppressor genes of viruses by RNA interference (RNAi) has been demonstrated to be an efficient way to combat viral diseases in plants. Citrus tristeza virus (CTV) has three silencing suppressor genes, p20, p23, and p25. To silence them, the splicing by overlap extension by PCR (SOE-PCR) was applied in this study for construction of two RNAi vectors, pCAMBIA2301G-hp2520 (to target p25 and p20) and pCAMBIA2301G-hp252023 (to target p25, p20 and p23). Following Agrobacterium-mediated transformation, seven and one independent sweet orange transgenic lines were obtained for expressing the fusion transgenes, hp2520 (hairpin RNA of p25 and p20) and hp252023 (hairpin RNA of p25, p20 and p23), respectively. Challenging the transgenic plants by graft-inoculation with severe stem-pitting CTV strain CT14A showed that no transgenic line was immune to the strain. However, lower to much lower CTV titer was detected in two lines of hp2520 and the only line of hp252023. Vegetative progenies of the hp252023-expressing line showed no stem-pitting symptoms 2 years post CT14A inoculation, although a low level of CT14A was still detectable in their leaves. Our results show that expressing the hpRNAs of CTV silencing suppressor genes conferred sweet orange with high tolerance to stem-pitting CTV.
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