Molecular characterisation of South African isolates of grapevine fanleaf virus and a new, associated satellite RNA

[摘要] ENGLISH ABSTRACT: Grapevine fanleaf virus (GFLV) is one of the oldest, most widespread and devastatingviruses infecting grapevine, and occurs globally where Vitis vinifera is grown. In SouthAfrica (SA) GFLV is predominant in the Breede River Valley, one of the highest wineproducing regions in SA. To date, only three GFLV isolates have been completelysequenced internationally, and limited sequence information is available for SA GFLVisolates. In this study, the first full-length GFLV genome sequence from a South Africanisolate, GFLV-SAPCS3, was determined. Full-length sequences were used forphylogenetic analysis and revealed that the SA isolates are separate from othersequenced GFLV isolates. Full-length sequences were also used to investigate putativeintra- and interspecies recombination events involving GFLV-SAPCS3 RNA1 and RNA2between GFLV and Arabis mosaic virus (ArMV) isolates. Using two differentrecombination analysis software packages, the most notable of the putativerecombination events involving GFLV-SAPCS3 indicated that the GFLV-SAPCS3 RNA25' UTR might have evolved from an interspecies recombination event between GFLVF13-type and ArMV Ta-type isolates. The presence of satellite RNAs (satRNA)associated with South African GFLV isolates was also investigated. In a collaborativestudy (see Chapter 4 for details), more than a 100 GFLV- infected grapevine plantswere screened for satRNAs. SatRNAs were present in only two plants, containingisolates GFLV-SACH44 and GFLV-SACH47. The full-length nucleotide sequences ofthe GFLV-SACH44 genomic RNAs 1 and 2, and the associated satRNA weredetermined. No significant sequence variation could be detected between the GFLVisolates that had the presence of a satRNA and those that had not. The GFLV-SACH44RNA2 5' UTR also had the same conserved sequence that was found in GFLVSAPCS3,which suggests that GFLV-SACH44, like GFLV-SAPCS3, may have arisenfrom a common ancestor, which may have originated from an interspeciesrecombination event. The GFLV-SACH44 satRNA was found to be more closely relatedto the ArMV large satRNA than to the satRNA associated with GFLV-F13. A full-lengthcDNA clone of GFLV-SACH44 satRNA was constructed and its replication and systemic spread in herbaceous hosts, when mechanically co-inoculated with two GFLV isolatesas helper viruses, was demonstrated. Replication of the GFLV-SACH44 satRNA cDNAclone was however abolished when co-inoculated with an ArMV helper virus, eventhough it is phylogenetically more closely related to ArMV satRNAs. The full-lengthsatRNA clones were modified to be used as vectors for expression and/or silencing offoreign genes, by inserting the green fluorescence protein (GFP) full-length or partialsequences downstream of the open reading frame of the satRNA. These constructswere cloned into a binary vector to allow for agro-infiltration into plants. Full-lengthcDNA clones of GFLV-SAPCS3 RNA1 and RNA2 were constructed to be used inconjunction with modified GFLV-SACH44 satRNA full-length clones. The full lengthGFLV-SAPCS3 RNA1 and RNA2 clones were however not infectious in Nicotianabenthamiana after agro-infiltration and therefore the evaluation of the modified satRNAexpression and silencing constructs had to be aborted. Attempts to understand thisfailure revealed that, among other point mutations, four frameshifts had occurred in theRNA1 full-length clone, rendering the transcripts untranslatable, and hence noninfectious.Strategies to correct the mutations are discussed. Once these mutationshave been corrected this study can continue in evaluating the use of the satRNAcomponent for expression and silencing analysis.

[发布机构] Stellenbosch University