GENOMIC CHARACTERIZATION AND MOLECULAR INVESTIGATIONS INTO NEGATIVE-STRANDED RNA VIRUSES OF PLANTS
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Tospoviruses are economically important viruses affecting a wide range of field and horticultural crops worldwide. Tospoviruses contain large (L) RNA, medium (M) RNA and small (S) RNA. Sequence analysis of the NSs gene in S RNA and the deduced protein sequences revealed two amino acid motifs that are conserved. Using Tomato spotted wilt virus (TSWV) as a model, the role of these motifs in suppressor activity of NSs was investigated. Using site-directed point mutations in two conserved motifs, glycine, lysine and valine/threonine (GKV/T) and tyrosine and leucine (YL), and an assay to measure the reversal of gene silencing in Nicotiana benthamiana line 16c, I showed that substitutions in these motifs abolished suppressor activity of the NSs protein, indicating that these two motifs are essential for the suppressor function of tospoviruses. RNA silencing-based approaches are successful for resistance using short regions of the viral genome. A new artificial micro RNA approach to confer resistance to was developed by Prof Mitter in collaboration with Prof Pappu. The candidate TSWV sequences (21nt in length) were introduced into an Arabidopsis amiRNA backbone. The effect of amiRNA constructs to impart resistance to TSWV was evaluated using transient assays in N. benthamiana as well as in transformed N. tabacum plants. In general, amiRNA constructs targeting the N gene were more effective against TSWV infection, while those specific to the NSs gene were not. This approach should be broadly applicable to other tospoviruses as well as other viruses that defy control due to lack of host plant resistance. EMDV is a rhabdovirus and consists of a large single-stranded RNA in negative sense. To obtain the complete RNA genome sequence from infected Agapanthus plants, deep-sequencing was carried out and the resulting sequences were assembled. De novo assembly of contigs, along with RACE to obtain the terminal sequences, showed that the viral genome is of 13,100-nt length and had 85.6% identity with the known EMDV genome from Greece. Sequence analysis showed five conserved motifs in the L gene, which may be useful in developing virus-specific as well as genus-specific detection tools for virus diagnosis and management.