The molecular and biological characterisation of ORF5 of three South African variants of Grapevine Vitivirus A

[摘要] Grapevine Vitivirus A (GVA), genus Vitivirus, family Flexiviridae is a wellcharacterised single-stranded RNA virus that has been implicated in the grapevinediseases, Kober stem grooving and Shiraz disease. The virus infects both itshost, Vitis vinifera and the experimental model plant, Nicotiana spp.. Biologicalstudies performed on the virus in its herbaceous host, Nicotiana benthami-ana, revealed that many divergent variants of the virus exists in South Africaand can induce di erent symptoms in the model plant. Further molecularanalysis divided the variants into three molecular groups based on molecularheterogeneity and nucleotide identity. The establishment of an infectiousfull-length cDNA clone of GVA contributed towards the elucidation of genefunctions for 4 of the 5 open reading frames (ORF's), and indicated ORF5as the pathogenicity determinant within the genome. Further studies alsoshowed that ORF5 encodes for a nucleic acid binding protein that exhibitssuppression activity of a plants' natural virus silencing mechanism. Many proteinsthat have previously been identi ed as the pathogenicity determinantwithin a viral genome have been found to encode for suppression activity.Although suppression activity has been elucidated within the ORF5 of theItalian cDNA clone of GVA, IS 151, no such study has yet been performed onthe divergent South African variants of GVA. Three variants, GTR1-1, GTR1-2 and GTG11-1, which represent each of the molecular groups (Group III, IIand I), were selected for this study. The aim of this study was to visuallyelucidate suppression activity of RNA transgene silencing by the ORF5's ofGTR1-1, GTR1-2 and GTG11-1 in a transient expression assays in transgenicN. benthamiana (line 16c). Pathogenicity studies for these variants were alsoperformed. The ORF5 of the infectious full-length clone, GVA118, which canalso serve as an expression vector, was deleted and provided with restrictionenzyme sites into which the respective ORF5s and the marker genes, GFP andGUS could be cloned directionally. Infectivity, symptom development and systemicmovement were compared between the di erent full length clones afterco-in ltration in N. benthamiana. Preliminary results obtained in this studyfailed to visually indicate any suppression activity encoded by the ORF5 ofGTR1-1, GTR1-2 and GTG11-1. The deletion of ORF5 within GVA118 wassuccessful and rendered the infectious full length clone asymptomatic. Directionalcloning of the ORF5 of GTR1-1 into the unique restriction enzymesprovided previously, resulted in much milder symptoms than those observe forGTR1-2 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 inthe herbaceous host, N. benthamiana. This construct can be further characterisedas a possible expression vector of foreign proteins in herbaceous hostsand grapevine.

[发布机构] Stellenbosch University