Molecular etiology of grapevine leafroll disease: role of the viral silencing suppressors

Gouveia, Paulo Alexandre Oliveira

http://hdl.handle.net/10400.1/7812

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Authors

Gouveia, Paulo Alexandre Oliveira

Advisor(s)

Nolasco, Gustavo

Abstract(s)

Grapevine leafroll disease is one of the most economically important and widespread grapevine viral diseases. Symptoms are caused by a number of distinct Grapevine leafroll-associated viruses (GLRaVs, family Closteroviridae). The main goal of this research was to study the role of the viral suppressors of RNA silencing (VSRs) in the molecular etiology of the leafroll disease analysing their variability in relation to symptom production in a model plant. This research was focused on GLRaV-3, the most prevalent agent of leafroll and type member of the genus Ampelovirus; and GLRaV-2, the only leafroll-associated virus of the genus Closterovirus and also been associated with other symptoms. By analogy with the genomic location and molecular signatures of the VSRs previously described for closteroviruses, the GLRaV-3 p21, p19.6 and p19.7 proteins were screened for VSR activity. Only p19.7 revealed suppressing activity, demonstrated against diverse silencing inducing systems. It was found that this activity varies across the phylogenetic groups and some variants originated virus-like symptoms. Phylogenetic analysis based on viral coat protein (CP) gene of GLRaV-3 revealed the existence of five well-defined clusters. Based on this, a typing tool based on asymmetric PCR-ELISA (APET) was developed to assess the prevalence of each phylogenetic group among the infected grapevine varieties. The genetic diversity of GLRaV-2 was studied focusing the whole p24 gene, previously identified to express a VSR. The p24 sequences obtained in this work clustered into five phylogenetic groups. It was showed that variants of p24 acts differently among the different types of Agrobacterium-mediated transient expression assays. One of the variants, a “truncated mutant variant”, was unable to suppress RNA silencing. A long hairpin constructed with p24 (lhRNA-p24) was verified to partially inhibit the VSR activity triggered by p24, even when was jointly inoculated with p24 variants from distant groups.