Crude extracts of bacterially-expressed dsRNA protect orchid plants against Cymbidium mosaic virus during transplantation from in vitro culture

Abstract

SummaryCymbidium mosaic virus (CymMV) is one of the most prevalent viruses infecting and causing damage to orchids. As tissue culture methods are widely used for commercial orchid production, CymMV infection commonly occurs during transplantation and pruning, so the protection of plants during these stages would be of value to the industry. Double-stranded RNA (dsRNA) has been shown to mediate gene silencing via mRNA degradation and the inactivation of both endogenous and virally-expressed genes in plants. In this study, crude bacterial extracts were prepared from an RNase-deficient strain of Escherichia coli, HT115 (DE3), that expressed sense or anti-sense RNA corresponding to the CymMV coat protein gene (CP). A mixture of sense and anti-sense CP RNAs were used to produce dsRNA. Exogenous sense or anti-sense RNA introduced into the leaves of Brassolaeliocattleya hybrida by mechanical inoculation reduced the symptoms of CymMV infection and reduced expression of the CymMV CP RNA in virus-infected plants three-fold (with sense ssRNA) and over seven-fold (with anti-sense ssRNA) compared to the levels in virus-infected control plants with no ssRNA treatment. Most orchid plants infected with CymMV and treated with CymMV CP dsRNA, showed no symptoms of CymMV infection. The levels of expression of the CymMV CP gene in CymMV CP dsRNA-treated plants were 40-fold lower than in virus-infected control plants and similar to the CP RNA levels detected by quantitative RT-PCR in uninfected control orchid plants. These results demonstrate that the application of a crude extract of bacterially-expressed ssRNA or dsRNA corresponding to the CymMV CP gene was able to reduce viral symptoms and levels of CymMV CP RNA in infected orchids. This approach could provide a relatively low-cost method to protect orchid plants during commercial transplantation from tissue culture.