Transgene silencing in Phalaenopsis expressing the coat protein of Cymbidium Mosaic Virus is a manifestation of RNA-mediated resistance

Abstract

Phalaenopsis orchid is grown extensively for commercial production as cut flowers and potted plants. Susceptibility to the cymbidium mosaic virus (CymMV) has drawn concern among orchid growers worldwide as its natural host remains confined to orchids. Hence it was reasoned to improve the resistance of Phalaenopsis to CymMV by expressing a virus-derived transgene. A DNA cassette containing a CymMV coat protein (CP) cDNA and a nos terminator placed downstream of a maize ubiquitin promoter was transformed into Phalaenopsis orchid by particle bombardment. Southern blot analysis of transgenic orchid plants was conducted to confirm the successful integration of multiple copies of the CP gene in the orchid genome. Although low levels of CP mRNA transcript signals were observed by northern blot analysis, CP was not detected by western blot analysis in transgenic orchid plants. However these transgenic orchid plants exhibited enhanced resistance to virus infection, as confirmed by RT-PCR and ELISA. T0 progeny lines were assayed for resistance to CymMV as it occurs in the field. Five among the 13 tested lines showed CymMV protection in more than 50% of their progeny. Nuclear run-on and small interfering RNA (siRNAs) analyses showed that CymMV resistance was RNA-mediated through a post-transcriptional gene silencing mechanism (PTGS) in the silenced transgenic orchid plants. The detection of siRNAs, and not CP mRNA, proved to be an indicator of their resistance to CymMV infection. Results from the current study indicate that Phalaenopsis expressing heterologous CymMV CP can effectively confer resistance to CymMV.