Thesis (PhD)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: Grapevine fanleaf virus (GFLV) is one of the oldest, most widespread and devastating
viruses infecting grapevine, and occurs globally where Vitis vinifera is grown. In South
Africa (SA) GFLV is predominant in the Breede River Valley, one of the highest wine
producing regions in SA. To date, only three GFLV isolates have been completely
sequenced internationally, and limited sequence information is available for SA GFLV
isolates. In this study, the first full-length GFLV genome sequence from a South African
isolate, GFLV-SAPCS3, was determined. Full-length sequences were used for
phylogenetic analysis and revealed that the SA isolates are separate from other
sequenced GFLV isolates. Full-length sequences were also used to investigate putative
intra- and interspecies recombination events involving GFLV-SAPCS3 RNA1 and RNA2
between GFLV and Arabis mosaic virus (ArMV) isolates. Using two different
recombination analysis software packages, the most notable of the putative
recombination events involving GFLV-SAPCS3 indicated that the GFLV-SAPCS3 RNA2
5’ 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 collaborative
study (see Chapter 4 for details), more than a 100 GFLV- infected grapevine plants
were screened for satRNAs. SatRNAs were present in only two plants, containing
isolates GFLV-SACH44 and GFLV-SACH47. The full-length nucleotide sequences of
the GFLV-SACH44 genomic RNAs 1 and 2, and the associated satRNA were
determined. No significant sequence variation could be detected between the GFLV
isolates that had the presence of a satRNA and those that had not. The GFLV-SACH44
RNA2 5’ UTR also had the same conserved sequence that was found in GFLVSAPCS3,
which suggests that GFLV-SACH44, like GFLV-SAPCS3, may have arisen
from a common ancestor, which may have originated from an interspecies
recombination event. The GFLV-SACH44 satRNA was found to be more closely related
to the ArMV large satRNA than to the satRNA associated with GFLV-F13. A full-length
cDNA clone of GFLV-SACH44 satRNA was constructed and its replication and systemic spread in herbaceous hosts, when mechanically co-inoculated with two GFLV isolates
as helper viruses, was demonstrated. Replication of the GFLV-SACH44 satRNA cDNA
clone was however abolished when co-inoculated with an ArMV helper virus, even
though it is phylogenetically more closely related to ArMV satRNAs. The full-length
satRNA clones were modified to be used as vectors for expression and/or silencing of
foreign genes, by inserting the green fluorescence protein (GFP) full-length or partial
sequences downstream of the open reading frame of the satRNA. These constructs
were cloned into a binary vector to allow for agro-infiltration into plants. Full-length
cDNA clones of GFLV-SAPCS3 RNA1 and RNA2 were constructed to be used in
conjunction with modified GFLV-SACH44 satRNA full-length clones. The full length
GFLV-SAPCS3 RNA1 and RNA2 clones were however not infectious in Nicotiana
benthamiana after agro-infiltration and therefore the evaluation of the modified satRNA
expression and silencing constructs had to be aborted. Attempts to understand this
failure revealed that, among other point mutations, four frameshifts had occurred in the
RNA1 full-length clone, rendering the transcripts untranslatable, and hence noninfectious.
Strategies to correct the mutations are discussed. Once these mutations
have been corrected this study can continue in evaluating the use of the satRNA
component for expression and silencing analysis. / AFRIKAANSE OPSOMMING: Grapevine fanleaf virus (GFLV) is een van die oudste, mees wydverspreide en mees
verwoestende virusse wat wingerd affekteer en word wêreldwyd waar Vitis vinifera
verbou word, gevind. In Suid Afrika (SA) kom GFLV veral in die Breederivier vallei, een
van die mees produktiewe wyn-produserende areas in SA, voor. Tot dusver is daar net
drie GFLV isolate waarvan die volledige nukleïensuurvolgorde internasionaal bepaal is.
Die nukleïensuurvolgorde informasie vir SA GFLV isolate is redelik beperk. In hierdie
studie was die eerste volledige nukleïensuurvolgorde van ‘n SA GFLV isolaat, GFLVSAPCS3,
bepaal. Die volledige nukleïensuurvolgordes was vir filogenetiese analise
gebruik, en vermeende intra- en interspesie rekombinasie gebeurtenisse, wat GFLVSAPCS3
RNA1 en RNA2 betrek, tussen GFLV en Arabis mosaic virus (ArMV) isolate
was ondersoek. Twee verskillende rekombinasie-analise sagteware programme was
gebruik en die noemenswaardigste van die vermeende rekombinasie gebeurtenisse,
met betrekking tot GFLV-SAPCS3, het aangedui dat die GFLV-SAPCS3 RNA2 5’
ontransleerde streek (UTR) waarskynlik van ‘n interspesie rekombinasie gebeurtenis
tussen ‘n GFLV-F13-tipe en ‘n ArMV-Ta-tipe isolaat ontwikkel het. Die teenwoordigheid
van satelliet RNAs (satRNAs), wat met SA GFLV isolate geassosieer is, was ook
ondersoek. Meer as ‘n 100 GFLV ge-infekteerde wingerd plante was in ‘n
samewerkingsprojek (sien Hoofstuk 4 vir besonderhede) getoets vir die
teenwoordigheid van satRNAs. SatRNAs was net in twee plante teenwoordig, in isolate
GFLV-SACH44 en GFLV-SACH47. Die vollengte nukleïensuurvolgordes van GFLVSACH44
RNA1, RNA2 en geassosieerde satRNA was bepaal. Geen beduidende
volgorde variasie tussen die GFLV isolate wat satRNAs bevat het, en die GFLV isolate
sonder satRNA was waargeneem nie. Die GFLV-SACH44 RNA2 5’ UTR het ook die
gekonserveerde volgorde, wat in GFLV-SAPCS3 teenwoordig was, gehad en dit dui
daarop dat GFLV-SACH44, soos GFLV-SAPCS3, van dieselfde stamvader, wat tydens
‘n vorige rekombinasie gebeurtenis ontstaan het, mag ontwikkel het. Die GFLVSACH44
satRNA was meer naverwant aan die ArMV satRNAs as aan die satRNA, wat
met GFLV-F13. ‘n Vollengte cDNA kloon van die GFLV-SACH44 satRNA was ontwikkel en die replisering en sistemiese verspreiding in sagte plante, nadat dit met twee GFLV
isolate as helper virusse saam ge-inokuleer was, was gedemonstreer. Replisering van
die GFLV-SACH44 satRNA cDNA kloon was egter ontwrig toe dit saam met ‘n ArMV
helper virus saam ge-inokuleer was, al is dit filogeneties meer verwant aan ArMV
satRNAs. Die vol-lengte satRNA klone was gemodifiseer om as vektore vir uitdrukking
en/of uitdowing van transgene te dien, deur om vol-lengte of gedeeltelike groen
fluoressensie proteïen (GFP) nukleïensuurvolgordes aan die einde van die satRNA
leesraam te koppel. Hierdie konstrukte was in ‘n binêre vektor gekloon om agroinfiltrasie
in plante toe te laat. Vol-lengte cDNA klone van GFLV-SAPCS3 RNA1 en
RNA2 was ontwikkel om in samewerking met die gemodifiseerde GFLV-SACH44
satRNA konstrukte gebruik te word. Die vol-lengte GFLV-SAPCS3 RNA1 en RNA2
klone het egter nie in Nicotiana benthamiana gerepliseer na agro-infiltrasie nie, daarom
was die evaluasie van die gemodifiseerde satRNA konstrukte gestaak. Pogings om die
mislukking te verstaan, het daarop gewys dat, behalwe punt mutasies, vier leesraam
versteurings in die RNA1 vollengte kloon voorgekom het, wat ontransleerbare
transkripte, en dus nie-repliserende konstrukte tot gevolg gehad het. Strategieë om die
mutasies te korrigeer is bespreek. Sodra die mutasies gekorrigeer is, kan die studie
voortgaan om te evalueer of die satRNA komponent vir uitdrukking en uitdowing analise
gebruik kan word.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/85600 |
| Date | 12 1900 |
| Creators | Lamprecht, Renate Luise |
| Contributors | Burger, J. T., Stephan, D., Stellenbosch University. Faculty of AgriSciences. Dept. of Genetics. |
| Publisher | Stellenbosch : Stellenbosch University |
| Source Sets | South African National ETD Portal |
| Language | en_ZA |
| Detected Language | English |
| Type | Thesis |
| Format | xv, 152 p. : ill., maps. |
| Rights | Stellenbosch University |
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