The construction of an infectious clone of grapevine virus A (GV A)

[摘要] An infectious clone of a viral RNA genome is one that can be used, either as an in vitro transcript or ascDNA, to produce an infection in a susceptible plant. Infectious clones serve as a tool to study viralRNA genomes at a molecular level to gain deeper insight into genome organization, viral genefunction, presence of regulatory sequences and gene expression. In the Western Cape (and elsewhere) anew crippling grapevine disease, known as Shiraz disease, is emerging of which the aetiology andpathogenic agents involved are not yet fully understood. Grapevine virus A (GVA), genus Vitivirus,family Flexiviridae, is thought to be the associated with this disease. The aim of this study was toconstruct a full-length infectious cDNA clone of GVA, which will aid in the molecular study of theviral genome. This clone could ultimately be used to investigate GVA's involvement in Shiraz disease,which could lead to the unravelling of the aetiology and control of the disease. A full-length clone ofGVA, named GVA-IC2/T7-2972-3, was constructed in several steps using restriction digestion/ligationand primer overlap extension PCR. Grapevine virus A cDNA fragments were obtained from GVAinfectedNicotiana benthamiana and Vitis vinifera plants using three different techniques, of which theRapid direct-one-tube RT-PCR was most successful. A 5' T7 promoter and a 3' poly-A tail wereincorporated and the full-length clone was cloned into pBluescript II SK (+). Full-length sequencingof the clone, revealed two significant frameshift mutations. The first mutation was a single base pairinsertion (one G) in a slippery site of 6 G's at position 1380 – 1385 in open reading frame one (ORF 1)of the viral genome. This mutation was corrected by PCR-based site-directed mutagenesis, whichresulted in pSK-GVA-mutagen-3 and pSK-GVA-mutagen-4. The second mutation was a single basepair deletion (one G) at position 6959 in ORF4, which coded for the coat protein (CP). Severaltechniques were attempted to correct this mutation, but none were successful. Even though the secondmutation could not be corrected, in vitro transcriptions were performed on three clones followed bysubsequent infections of N. benthamiana plants. The three clones included pSK-GVA-mutagen-3, pSKGVA-mutagen-4 (both hosting the mutation at position 6959) and GVA-IC2/T7-2972-3 (hosting bothmutations). At 21 days post-inoculation no significant visual symptoms were observed in plantsinfected with in vitro RNA or in plants infected with wild type GVA. Rapid direct-one-tube RT-PCRresults revealed the presence of viral RNA in infected leaves and apical leaves of infected plants, andprovided preliminary evidence that the mutated clones were still capable of systemic infection and viralmovement. These results are still inconclusive, and several post-infection studies will have to beperformed to confirm these findings. Koch's postulates will also have to be proved in order to confirm the infectious nature of the clones. The effect of the two mutations in the constructed clones will beinvestigated further and post-infection analysis performed to deduce whether the viral progeny aredevoid of the mutations. Three full-length GVA cDNA clones (hosting mutations) seemingly capableof systemic infection in N. benthamiana plants were constructed in this study and have laid thefoundation for molecular and mutational analysis of the GVA genome. This could lead to the study ofpathogen-host interactions in order to unravel the aetiology of Shiraz disease in the future.

[发布机构] Stellenbosch University