RNAi-Mediated Transgenic Tospovirus Resistance Broken by IntraspeciesSilencing Suppressor Protein Complementation

Abstract

Extension of an inverted repeat transgene cassette, containing partial nucleoprotein (N) gene sequences from four different tomato-infecting Tospovirus spp. with a partial N gene sequence from the tomato strain of Tomato yellow ring virus (TYRV-t), renders transgenic Nicotiana benthamiana plants additionally resistant to this strain but not to the soybean strain of this Tospovirus sp. (TYRV-s), both strains exhibiting 14.4% nucleotide sequence divergence in their N genes. Surprisingly, coinoculation of the TYRV-t-resistant transgenic lines with both TYRV-t and TYRV-s resulted in rescue of the former. Mass-spectrometric analysis of the viral ribonucleocapsids accumulating in the transgenic plants showed the presence of the N proteins of both strains excluding hetero-encapsidation as rescue mechanism and indicating suppression of TYRV-t N gene transcript breakdown by RNA interference. Prior (Potato virus X [PVX]-vector-mediated) expression of the TYRV-s silencing suppressor (NSs) gene also allowed TYRV-t to break the resistance. This phenomenon was also observed when the homologous (TYRV-t) NSs gene was provided from a PVX replicon, demonstrating that TYRV can break RNA-mediated host resistance upon a priori expression of its NSs protein. Remarkably, mixed inoculation of TYRV-t with other Tospovirus spp. or nonrelated viruses did not result in resistance breaking, indicating that the rescuing activity of NSs—though based on suppressing RNA silencing—is species-dependent.