The molecular and biological characterisation of ORF5 of three South African variants of Grapevine Vitivirus A
Date
2009-03
Authors
Blignaut, Marguerite
Journal Title
Journal ISSN
Volume Title
Publisher
Stellenbosch : University of Stellenbosch
Abstract
Grapevine Vitivirus A (GVA), genus Vitivirus, family Flexiviridae is a well
characterised single-stranded RNA virus that has been implicated in the grapevine
diseases, Kober stem grooving and Shiraz disease. The virus infects both its
host, Vitis vinifera and the experimental model plant, Nicotiana spp.. Biological
studies performed on the virus in its herbaceous host, Nicotiana benthami-
ana, revealed that many divergent variants of the virus exists in South Africa
and can induce di erent symptoms in the model plant. Further molecular
analysis divided the variants into three molecular groups based on molecular
heterogeneity and nucleotide identity. The establishment of an infectious
full-length cDNA clone of GVA contributed towards the elucidation of gene
functions for 4 of the 5 open reading frames (ORF's), and indicated ORF5
as the pathogenicity determinant within the genome. Further studies also
showed that ORF5 encodes for a nucleic acid binding protein that exhibits
suppression activity of a plants' natural virus silencing mechanism. Many proteins
that have previously been identi ed as the pathogenicity determinant
within a viral genome have been found to encode for suppression activity.
Although suppression activity has been elucidated within the ORF5 of the
Italian cDNA clone of GVA, IS 151, no such study has yet been performed on
the divergent South African variants of GVA. Three variants, GTR1-1, GTR1-
2 and GTG11-1, which represent each of the molecular groups (Group III, II
and I), were selected for this study. The aim of this study was to visually
elucidate suppression activity of RNA transgene silencing by the ORF5's of
GTR1-1, GTR1-2 and GTG11-1 in a transient expression assays in transgenic
N. benthamiana (line 16c). Pathogenicity studies for these variants were also
performed. The ORF5 of the infectious full-length clone, GVA118, which can
also serve as an expression vector, was deleted and provided with restriction
enzyme sites into which the respective ORF5s and the marker genes, GFP and
GUS could be cloned directionally. Infectivity, symptom development and systemic
movement were compared between the di erent full length clones after
co-in ltration in N. benthamiana. Preliminary results obtained in this study
failed to visually indicate any suppression activity encoded by the ORF5 of
GTR1-1, GTR1-2 and GTG11-1. The deletion of ORF5 within GVA118 was
successful and rendered the infectious full length clone asymptomatic. Directional
cloning of the ORF5 of GTR1-1 into the unique restriction enzymes
provided previously, resulted in much milder symptoms than those observe for
GTR1-2 and GTG11-1. No GFP and GUS accumulation could be detected.
This study has established an infectious full-length cDNA clone, pBINSN-e35SGVA118
ORF5-1-1-pA, that can possibly induce much milder symptoms in
the herbaceous host, N. benthamiana. This construct can be further characterised
as a possible expression vector of foreign proteins in herbaceous hosts
and grapevine.
Description
Thesis (MSc (Genetics))--University of Stellenbosch, 2009.
Keywords
Pathogenicity, Dissertations -- Genetics, Theses -- Genetics, Grapes -- Diseases and pests -- Genetic aspects, Virus diseases of plants