Tomato spotted wilt virus (TSWV) Isolates Encoding Truncated NSs Proteins Are not Transmitted by its thrips vector Frankliniella occidentalis and Are Severely Impaired in Accumulation in Adults but not in 2nd-Instar Larvae

Tomato spotted wilt virus (TSWV) (Tospovirus, Bunyaviridae) is one of the most agronomically important viruses worldwide. The virus is transmitted between plants by insects in the genera Thrips and Frankliniella (Thysanoptera, Thripidae), in a propagative mode. Lack of reverse genetic systems hampers the functional analysis of TSWV genome, but thanks to the selective pressure of the Tsw resistance gene in pepper, we obtained a number of TSWV isolates, carrying a deletion in the NSs coding region. Such deletions caused a recovery phenotype on Capsicum spp. and Datura stramonium, linked to lack of suppression of silencing activity for the resulting truncated NSs, but in N. benthamiana some of the NSs recovery isolates accumulated to wild type level. We show that Frankliniella occidentalis, its most efficient vector, fails to transmit TSWV coding for truncated NSs protein. Quantitative RT-PCR and immuno-staining of individual insects detected the mutant virus in 2nd-instar larvae and adults, demonstrating that insects can acquire and accumulate NSs-defective mutants. Nevertheless adults carried a significantly lower viral load, showing for the first time a specific role of NSs in maintaining a high titer of virus in adult thrips. Sequence analyses of reassortant isolates revealed a strict association of lack of transmission to the mutation in the NSs-coding sequence.