OLIVE MILD MOSAIC VIRUS CP AND P6 ARE SUPPRESSORS OF RNA SILENCING AND THEIR SILENCING CONFERS RESISTANCE AGAINST OMMV

Abstract

RNA silencing is an important defense mechanism in plants, yet several plant viruses encode proteins that suppress this mechanism. Here the genome of Olive mild mosaic virus (OMMV) was screened for silencing suppressors in the herbaceous host Nicotiana benthamiana 16C using a green fluorescent based transient suppression assay. The full OMMV cDNA and 5 different OMMV open reading frames (ORFs) were cloned into the Gateway binary destination vector pK7WG2, transformed into Agrobacterium tumefaciens C58C1 and agroinfiltrated into N. benthamiana 16C. Among all ORFs tested, CP and p6 showed suppressor activity, with CP showing a significant higher activity when compared to p6, yet lower than that of the full OMMV. This suggests that OMMV silencing suppression results from a complementary action of both CP and p6. This is the first time that a silencing suppressor was found in a necrovirus and that two independent proteins act as silencing suppressors in a member of Tombusviridae family. Such discovery led to the use of those viral suppressors in the development of OMMV resistant plants through pathogen- derived resistance (PDR) based on RNA silencing. Two hairpin constructs targeting each suppressor were agroinfiltrated in N. benthamiana plants which were then inoculated with OMMV RNA. When plants expressed both constructs, a highly significant reduction in viral accumulation and symptom attenuation was observed as compared to those of controls. These results indicate that the use of both OMMV viral suppressors as transgenes is a very efficient and promising approach to obtain olive and other hosts resistant to OMMV.