Thesis (Ph. D.)--Oregon State University, 2009. / Printout. Includes bibliographical references (leaves 61-74). Also available on the World Wide Web.

Thesis (MSc)--University of Stellenbosch, 2000. / ENGLISH ABSTRACT: Grapevine leafroll is one of the most damaging viral diseases that affect many viticultural regions of the world. Numerous reports over the last few years have associated closterovirus-like particles with leafroll disease. To date, eight serologically distinct closteroviruses have been isolated from leafroll infected vines, of which grapevine leafroll associated closterovirus-3 (GLRaV-3) is the best characterized. Virus resistance in transgenic plants based on the expression of a virusderived gene is known as pathogen-derived resistance. The viral coat protein (CP) gene, which expresses a structural protein responsible for coating the virus particles, was used in the first demonstration of virus-derived resistance. Coat protein-mediated resistance is currently the most feasible and most widely used method to obtain virus resistance in crop plants. The CP gene of a South African isolate of GLRaV-3 infected grapevine was isolated, cloned and sequenced. Double stranded RNA (dsRNA) was extracted from GLRaV-3 infected material and a high molecular weight band, of -18 kb was identified from infected vines. The dsRNA was used as a template in a reverse transcription PCR together with GLRaV-3 CP gene specific primers for the amplification of the GLRaV-3 CP gene (975 bp). The GLRaV-3 CP gene was cloned into the pGem®-T Easy vector. Clones hosting the CP gene in the sense (pLR3CP+) and antisense (pLR3CP-) orientations respectively were obtained. The sequence obtained from these two clones showed 99.26 % similarity to the only other GLRaV-3 CP nucleotide sequence available. The GLRaV-3 CP gene was excised from pLR3CP+ and pLR3CP- and subcloned into a plant expression vector, pCAMBIA 3301 in the sense (pCamBLR3CP+) and antisense (pCamBLR3CP-) orientations respectively, therefore enabling sense and antisense gene expression in transgenic plants. The GLRaV-3 CP gene was also subcloned from pCamBLR3CP+ into another plant expression vector, pCAMBIA 2301 in the sense orientation and designated as pCVSLR3CP+. These three constructs were given to Dr. M. Vivier (Institute for Wine Biotechnology, Stellenbosch) for grapevine transformation experiments. Two of these constructs, pCamBLR3CP+ and pCamBLR3CP- as well as pCAMBIA 3301 were used to transform Nicotiana tabacum by Agrobacterium tumefaciens-mediated transformation. Plants were selected for their ability to withstand the herbicide, Basta. This resistance is due to the presence of a plant selectable marker gene on each of these constructs, known as the bar gene. PCR with GLRaV-3 CP gene specific primers showed no amplification of the GLRaV-3 CP gene in the plants transformed with pCamBLR3CP+ and pCamBLR3CP-. Southern blot analysis with the GLRaV-3 CP gene as hybridization probe showed no signal for these plants, thus confirming the PCR results. PCR with bar gene specific primers showed no amplification of the bar gene in the plants infected with pCAMBIA 3301. The plants transformed with pCamBLR3CP+ and pCamBLR3CP- were also screened for the presence of the bar gene. Three of the eight plants tested showed amplification of the -560 bp bar gene. This result suggests that these plants were transformed with pCAMBIA 3301 (vector without the ligated GLRaV-3 CP gene) and not pCamBLR3CP+ or pCamBLR3CP- as had been expected. This project provides preliminary work for the subsequent transformation of grapevine with the GLRaV-3 CP gene, in an attempt to impart virus resistance. / AFRIKAANSE OPSOMMING: Wingerd rolblaar is een van die mees beskadigende virale siektes wat baie wingerd areas in die wêreld aantas. In Aantal verslae oor die afgelope jare het closterovirus partikels met wingerd rolblaar geassosieer. Tot hede, is agt serologiese onderskeibare closterovirusse geïsoleer vanuit geaffekteerde wingerde, waarvan wingerd rolblaar geassosieerde closterovirus-3 (GLRaV-3) die beste gekarakteriseerd is. Virus bestandheid in transgeniese plante gebaseer op die uitdrukking van gene afkomstig vanaf virusse, staan bekend as patogeen-afgeleide weerstand. Die virale kapsule protein (CP) geen vervaardig In strukturele protein wat verantwoordelik is vir die bedekking van die virus partikel. Dié geen was gebruik in die eerste demonstrasie van patogeen-afgeleide weerstand. Kapsuul protein-bemiddelde weerstand is tans die mees praktiese en algemene gebruikte metode om virus weerstand in plant gewasse te verkry. Die CP geen van In Suid Afrikaanse isolaat van GLRaV-3 geïnfekteerde wingerde is geïsoleer, gekloneer en die volgorde is bepaal. Dubbelstring RNA (dsRNA) was uit GLRaV-3 geïnfekteerde materiaal geëkstraheer en In hoë molekulêre gewig band van -18 kb is geïdentifiseer. Die dsRNA is gebruik as In templaat vir In omgekeerde transkripsie PKR saam met GLRaV-3 CP geen spesifieke inleiers vir die amplifikasie van die GLRaV-3 CP geen (975 bp). Die GLRaV-3 CP geen is gekloneer in die pGem®-T Easy vektor. Klone met die CP geen in die sin (pLR3CP+) en teensin (pLR3CP-) oriëntasies respektiewelik is verkry. Die volgorde wat verkry is vanuit hierdie twee klone dui op In 99.26 % ooreenstemming met die enigste ander GLRaV-3 CP geen volgorde wat beskikbaar is. Die GLRaV-3 CP geen is uit pLR3CP+ en pLR3CP- gesny en is gesubkloneer in In plant ekspressie vektor, pCAMBIA 3301 in die sin (pCamBLR3CP+) en teensin (pCamBLR3CP-) oriëntasies respektiewelik, wat die sin en teensin geen ekspressie in transgeniese plante in staat stel. Die GLRaV-3 CP geen was ook gesubkloneer vanaf pCamBLR3CP+ in In ander plant ekspressie vektor, pCAMBIA 2301 in die sin orientasie en is as pCVSLR3CP+ benoem. Hierdie drie konstruksies is aan Dr. M. Vivier (Instituut vir Wyn Biotegnologie, Stellenbosch) gegee vir wingerd transformasie eksperimente. Twee van hierdie konstruksies, pCamBLR3CP+ en pCamBLR3CP- asook pCAMBIA 3301 is gebruik om Nicotiana tabacum deur middel van Agrobacterium tumefaciens-bemiddelde transformasie te transformeer. Plante is geselekteer vir hul vermoë om die onkruiddoder, Basta, te weerstaan. Die teenwoordigheid van die plant selekteerbare merker geen, bar, op elke konstruksie lui tot dié weerstand. Die plante wat getransformeer is met pCamBLR3CP+ en pCamBLR3CP- is deur PKR saam met die GLRaV-3 CP geen spesifieke inleiers getoets, en geen amplifikasie van die GLRaV-3 CP geen is getoon nie. Southern blot analise met die GLRaV-3 CP geen as hibridisasie peiler het geen sein gewys vir hierdie plante nie, wat die PKR resultate bevestig. Die plante wat getransformeer is met pCAMBIA 3301 is deur PKR saam met die bar geen spesifieke inleiers getoets, en geen amplifikasie van die bar geen is getoon nie. Die plante wat getransformeer is met pCamBLR3CP+ en pCamBLR3CP- is ook getoets vir die teenwoordigheid vir die bar geen. Drie van die agt plante wat getoets is, het amplifikasie van die -560 bp bar geen getoon. Hierdie onverwagte resultate stel voor dat dié plante met pCAMBIA 3301 (vektor sonder die geligeerde GLRaV-3 CP geen) en nie met pCamBLR3CP+ en pCamBLR3CPgetransformeer is nie. Hierdie projek verskaf voorlopige werk vir die daaropvolgende transformasie van wingerd met die GLRaV-3 CP geen in 'n poging om virus bestandheid te verskaf.

Thesis (MSc)--Stellenbosch University, 2015. / ENGLISH ABSTRACT: Vitis vinifera is the woody crop most susceptible to intracellular pathogens. Currently 70 pathogens infect grapevine, of which 63 are of viral origin. Grapevine leafroll-associated virus 3 (GLRaV-3) is the type species of the genus Ampelovirus, family Closteroviridae. It is considered to be the primary causative agent of Grapevine leafroll disease (GLD) globally; however, the etiology of GLD is not completely understood. Here we report on the viral populations present in GLD symptomatic grapevines across the Western Cape province, South Africa. A widespread survey was performed to screen 315 grapevines for 11 grapevine-infecting viruses using RT-PCR. Additionally, GLRaV-3 variant groups were distinguished with high-resolution melt (HRM) curve analysis used in conjunction with real-time RT-PCR. Members of the family Closteroviridae were detected with the highest frequency, particularly GLRaV-3 that was detected in 87% of tested plants. Nextgeneration sequencing (NGS) is capable of detecting known and novel viruses without prior knowledge of viral sequences and when used in a metagenomic approach is able to detected viral populations within diseased vines. A total of 17 grapevine samples were subjected to NGS using either an Illumina MiSeq or HiSeq 2500 instrument to determine the virome within GLD vines. Collectively, more than 190 million reads were generated through NGS. Read datasets were trimmed and filtered for quality and subjected to both read-mapping and de novo assembly. Contigs assembled de novo were analyzed with BLAST (Basic Local Alignment Search Tool) against the NCBI (National Centre for Biotechnology Information) database and it was determined that GLRaV-3 was the best-represented virus, comprising 97.5% of the assembled contigs. Grapevine virus F (GVF) was detected for the first time in South African vineyards through de novo assemblies and the complete genome sequence validated through direct Sanger sequencing. The complete genome of GVF isolate V5 spans 7 539 nucleotides and shares 89.11% nucleotide identity to existing GVF genomes. The data generated through this study will assist in further understanding the etiology of GLD, support the current hypothesis of GLRaV-3 as the primary contributor to GLD, aid in understanding virus associations in diseased vines and potentially develop systems in which to control disease spread and symptom severity. / AFRIKAANSE OPSOMMING: Vitis vinifera is die houtagtige oes wat die mees vatbaarste is vir intrasellulêre patogene. Tans word wingerde deur 70 patogene geïnfekteer, waarvan 63 van virale oorsprong is. Grapevine leafrollassociated virus 3 (GLRaV-3) is die tipe spesie van die genus Ampelovirus, familie Closteroviridae. Dit word globaal beskou as die primêre oorsaak van Wingerd krulblaar-siekte (GLD), alhoewel die etiologie van GLD nie heeltemal begryp word nie. In hierdie verslag word die virale populasies teenwoordig in GLD simptomatiese wingerde oor die Wes-Kaap provinsie in Suid-Afrika gerapporteer. ‘n Wydverspreide opname was uitgevoer om 315 wingerde met 11 wingerdinfekterende virusse te ondersoek, deur gebruik te maak van tru-transkripsie polimerase ketting reaksie (PKR). Verder is variantgroepe van GLRaV-3 onderskei met hoë-resolusie smeltingskurweanalise, tesame met die gebruik van in-tyd tru-transkripsie PKR. Die hoogste frekwensie was van die lede van die familie Closteroviridae, veral GLRaV-3 wat in 87% van die ondersoekte plante gevind is. Nuwe-generasie volgorderbepaling (NGS) beskik oor die vermoë om bekende en nuwe virusse te herken in virale populasies in geaffekteerde wingerde sonder vorige kennis van virale volgorderbepalings en wanneer dit in ‘n metagenomiese benadering gebruik word kan die virale bevolkings binne siek wingerde ontdek. ‘n Totaal van 17 wingerd-steekproewe was blootgestel aan NGS deur die gebruik van of ‘n Illumina MiSeq of ‘n HiSeq 2500 instrument om die virome te bepaal van GLD wingerde. In totaal is meer 190 miljoen lesings gegenereer deur NGS. Hierdie data lesings was verwerk en gefilter vir kwaliteit om onderwerp te word vir beide kartering en de novo samestellings. Contigs verkry deur de novo samestellings was geanaliseer met BLAST (Basic Local Alignment Search Tool) teenoor die NCBI (National Centre for Biotechnology Information) databasis en dit was vasgestel dat GLRaV-3 was die mees-verteenwoordigende virus, bestaande uit 97.5% van die saamgestelde contigs. Grapevine virus F (GVF) was vir die eerste keer in Suid- Afrikaanse wingerde waargeneem deur de novo samestellings en die volledige genoom volgordger is geverifieer deur middel van direkte Sanger volgorderbepaling. Die volledige genoom van GVF isoleer V5 spanwydte van 7539 nukleotiedes en deel 89.11% nukleotied identiteite van bestaande GVF genome. Die gegenereerde data van hierdie studie sal bykomende begrip van die etiologie van GLD bystaan, die huidige hipotese van GLRaV-3 as die primêre bydraer tot GLD ondersteun, verhoogde begrip van virus-assosiasies in wingerdsiektes verseker en potensiële sisteme ontwikkel om siektes en simptome te beheer.

Complementary DNA (cDNA) clones representing approximately 5800 nucleotides of potato leafroll virus (PLRV) genomic RNA were generated, restriction-mapped, and partially sequenced. Within one of the cDNA clones an open reading frame (ORF) that could encode a 23 kDa protein was identified and further characterized. Comparison of the deduced amino acid sequence with the coat protein amino acid sequence of the PAV strain of barley yellow dwarf luteovirus (BYDV-PAV) showed significant similarity. This observation together with its size and internal location within the genome suggested that this gene encoded the PLRV coat protein. Other similarities were observed between PLRV and BYDV sequences in this region of their genomes, including a 17 kDa ORF within the ORF encoding the 23 kDa coat protein, and termination of the latter with an amber codon which is immediately followed by a large ORF in the same reading frame. Three PLRV coat protein gene sequences were used to transform tobacco and the potato cultivars 'Desiree' and 'Russet Burbank' via Agrobacterium tumefaciens mediated gene transfers. One construct possessed 12 nucleotides of the untranslated leader sequence 5' to the putative coat protein gene start codon. The other construct, which was also inserted in the reverse orientation to produce negative-sense RNA, had 192 nucleotides from this leader sequence. When these sequences were introduced as chimaeric genes under the control of a duplicated cauliflower mosaic virus 35S (CaMV) promoter, transcription levels were high. Both positive-sense transcripts produced potato leafroll coat protein which accumulated to maximum levels of approximately 0.5% and 0.01% of total leaf protein in tobacco and potato, respectively. Results show that significant levels of inoculum concentration-independent sustained resistance were obtained with each construct, resulting in PLRV titres as low as 1% of the level observed in untransformed plants, as determined by enzyme-linked immunosorbent assays. Virus transmission from PLRV-inoculated transgenic 'Russet Burbank' was reduced substantially and was correlated with virus titre. The pattern and level of protection were the same for constructs producing positive- and negative-sense RNA, suggesting a similar mechanism of resistance. Virus levels were negatively correlated to transcript levels within the transgenic plants. This resistance will have practical applications for the control, of PLRV. Elucidation of the mechanism of resistance may also help understand the mechanisms of virus infection. / Land and Food Systems, Faculty of / Graduate

Grapevine leafroll-associated virus type 3 (GLRaV-3) is the major causative agent of grapevine leafroll disease. The disease has a major negative impact on grape production for wineries, and can cause up to 62.8% loss in production. Despite the negative impact of GLRaV-3 on the grapevine industry worldwide, knowledge on the variability of the virus, which is essential for developing effective control measure of the virus in vineyards, is surprisingly scarce. To test this, six primers sets used in a one tube, one step polymerase chain reaction (PCR) protocol, together with ELISA were used to detect GLRaV-3 virus in 135 plant samples collected from a single vineyard. As expected the more sensitive PCR detected more infected samples than ELISA. However, some samples yielded positive results with the ELISA, but negative results using PCR. This might suggest that strain variants exist. Amongst PCR results of the different primer sets, anomalous results occurred, as often a plant will yield an amplicon with one primer set, but not with another primer set. Using the entire set of 7 PCR results per sample, each plant was assigned a PCR ‘fingerprint’. This yielded 24 different fingerprints in the vineyard. Mapping the spatial distribution of given fingerprints supported the possibility that strain variants exist. However, sequencing areas incorporating the primer binding sites showed no nucleotide sequence differences, indicating that the anomalous PCR results were not due to variants, but rather to protocol error. The PCR protocol used initially was adapted to obtain more optimal detection of virus. Different extraction methods and PCR protocols were tested. It was found that using the two step RT-PCR and using a less dilute plant macerate in ELISA extraction buffer (1:5), yielded amplicon of the expected size from all known infected plant samples. The protocol was further optimized with regards the RT step and subsequent PCR. Since the modified protocol did detect all known infected plant samples it can be concluded that in the 135 plant samples tested no significant sequence variation in strains at the primer binding sites occurred and that the anomalous PCR results initially obtained were due to a sub-optimal extraction method. / Dissertation (MSc (Microbiology))--University of Pretoria, 2008. / Microbiology and Plant Pathology / unrestricted

Grapevine Leafroll disease (GLD), one of the most destructive diseases of grapevines, has been found in every country where grapevines are grown. Grapevine Leafroll associated virus type 3 (GLRaV-3), one of several viruses associated with GLD globally, is the most prevalent virus in South African grapevines and therefore control of GLRaV-3 takes high priority in any strategy aimed at control of GLD. GLD can be controlled through the use of an integrated strategy which includes using certified plant material, controlling insect vectors through use of systemic insecticides and the removal of infected vines by roguing. Infected individuals are identified each autumn, using either symptom display (in red cultivars, where infected individuals display interveinal reddening and downward rolling of leaves) or ELISA (in symptomless white cultivars). ELISA is laborious, time consuming and relatively insensitivity compared to molecular techniques and a simpler, more rapid and more sensitive means of indentifying GLRaV-3 infected vines is required. A simple RNA extraction procedure combined with a single-tube reverse transcriptase loop-mediated amplification (RT-LAMP) has been developed which allows for the rapid, simple detection of GLRaV-3. Using RT-LAMP, a viral target can be amplified in 2 hours under isothermal conditions. This GLRaV-3 specific RTLAMP uses hydroxy napthol blue (HNB), a colourimetric indicator that changes from violet to sky blue only where a positive RT-LAMP reaction has occurred, making results quick and easy to interpret. The sensitivity of this technique was compared to ELISA and nested PCR by pooling samples at varying ratios of healthy to infected plants. Using nested PCR and RT-LAMP 1 infected sample could be detected amongst 50 healthy individuals while ELISA could only detect 1 amongst 30 infected making RT-LAMP more sensitive than ELISA. Further RT-LAMP could be performed in 2 hours compared to nested PCR and ELISA’s 8 and 48 hours respectively. Based on these results, RT-LAMP is viable alternative for ELISA for the detection of GLRaV-3 in the field. RT-LAMP was also tested for its ability to detect GLRaV-3 in grapevine rootstocks where, due to low viral titres and erratic distribution, it is notoriously difficult to detect. The rootstocks which were used for testing of GLRaV-3 had been tested in a previous study and it was found that only 28% of samples tested positive after 33 months (post inoculation). Using RT-LAMP, 78% of samples tested positive for GLRaV-3. Although further testing must be done, RT-LAMP may also be a viable alternative for testing grapevine rootstocks for GLRaV-3 infection. / Dissertation (MSc)--University of Pretoria, 2013. / gm2014 / Microbiology and Plant Pathology / unrestricted

Thesis (PhD (Genetics))--University of Stellenbosch, 2011. / Includes bibliography / ENGLISH ABSTRACT: Grapevine diseases, in particular virus and virus-like diseases, are threatening grapevine industries worldwide; also in South Africa. Grapevine leafroll (GLR) is one of the most important diseases of grapevines, occurring in all grape-producing countries worldwide. Grapevine leafroll-associated virus 3 (GLRaV-3) is known to be closely associated with GLR disease and occurs commonly in South African vineyards. In this study three genetic variants of GLRaV-3 were identified in vineyards of the Western Cape, South Africaby single strand conformation polymorphism (SSCP) profiles generated from a region amplified in ORF5. A specific SSCP profile could be assigned to each variant group and these wereconfirmed by sequencing of the ORF5 regions.These results demonstrated that SSCP analysis on this region in ORF5 provides a fast and reliable indication of the GLRaV-3 variant status of a plant, which in many instances showed mixed infections. The full genome sequence of one representative of each variant group i.e. isolates 621 (group I), 623 (group II) and PL-20 (group III), was determined by sequencing overlapping cloned fragments of these isolates. The sequences of genomic 5’ ends of these isolates were determined by RLM-RACE. Sequence alignment of the 5’UTRs indicated significant sequence and length variation in this region, between the three South African variant groups. Nucleotide sequence alignment of the Hsp70h and CP gene regions of these isolates with those of isolates from elsewhere in the world, followed by phylogenetic analysis, further supported the presence of three GLRaV-3 variants in South Africa, and that two or three additional variant groups occurs elsewhere in the world. We further investigated the prevalence of these three GLRaV-3 variants in mother blocksof different cultivars and from different vine growing regions, using SSCP analysis. The majority of the plants studied, were infected with the group II variant, similar to isolates 623 and GP18. The distribution of the three GLRaV-3 variants within a spatio-temporally recorded cluster of diseased plants was studied by means of SSCP profile analysis. We showed that different GLRaV-3 variants are transmitted to adjacent plants in an infection cluster. Results showed that, in some leafroll disease clusters, the variant that was present in the original GLRaV-3 infected plant of a cluster was transmitted to adjacent plants in a row and across rows. Some plants in the cluster were also infected with variants not present in the original plant. These infections could have been caused by mealybug vectors feeding on plants from surrounding areas and then infecting these plants. The scientific information generated on GLRaV-3 variants in this project contributed to the advancement of our knowledge of genetic variability and provides a basis of further epidemiology and vector-virus studies. The study showed for the first time that different GLRaV-3 variants were transmitted to adjacent plants in a row and across rows in a GLR disease cluster. The diversity detected in the 5’UTR between variants from the three genetic groups provides a platform for the further study of the biological characteristics of GLRaV-3 variants. / AFRIKAANSE OPSOMMING: Wingerdsiektes, veral virus siektes, bedreig wingerd industrieë wêreldwyd, asook die Suid Afrikaanse wingerdbedryf. Rolbladsiekte is een van die belangrikste siektes op wingerd en kom wêreldwyd voor. Die virus, grapevine leafroll-associated virus 3 (GLRaV-3), word sterk geassosieer met Rolbladsiekte en kom wydverspreid voor in Suid Afrikaanse wingerde. Tydens hierdie studie is drie genetiese variante van GLRaV-3 geïdentifiseer in wingerd moederblokke in die Wes-Kaap. Die GLRaV-3 variante is geïdentifiseer met ‘n tegniek wat ‘single-strand conformation polymorphism (SSCP)’ genoem word. Die SSCP profiele was gegenereer vanaf PKR produkte van die ORF5 area op die genoom van GLRaV-3. Die geamplifiseerde produk van die ORF5 gebied is gebruik om die SSCP profiele te verkry en DNA-volgorde data in die gebied het die drie SSCP profiele gestaaf. Hierdie metode om virus variasie te bestudeer in plante is vinnig en betroubare resultate is verkry. Gemengde infeksies, wat gereeld in wingerd voorkom, kon ook met die tegniek opgespoor word. Die volledige nukleotied-volgorde van elkeen van die drie GLRaV-3 genome is volledig bepaal. Die isolate wat die drie variant groepe verteenwoordig is isolaat 621 (groep I), 623 (groep II) en PL-20 (groep III). Die nukleotiedvolgorde in die 5’UTR is bepaal met die RLM-RACE tegniek. Wanneer die 5’UTRs van die drie variante vergelyk is, het dit getoon dat daar verskille is in die volgordes en lengtes voorgekom het. Ander dele van die genoom, o.a. die dopproteïen (CP) en Hsp70 areas, is filogeneties vergelyk met isolate van regoor die wêreld. In die filogenetiese analise is bevind dat die drie GLRaV-3 variante saamgegroepeer het met ander isolate in die wêreld en dat daar elders ook twee to drie addisionele variant groepe van GLRaV-3 voorkom. Die verspreiding van die drie GLRaV-3 variante in wingerde is bestudeer in verskillende kultivars en in verskillende verbouingsgebiede. Die meerderheid van die plante in die studie was geïnfekteer met die groep II variant wat dieselfde is as isolate 623 en GP18. Die voorkoms van die drie variante in ‘n siekte cluster is bestudeer d.m.v SSCP. Die studie het gewys dat verskillende GLRaV-3 variante versprei word na aangrensende plante in ‘n ry en tussen rye. In sommige gevalle is die variant wat in die oorspronklik geïnfekteerde plant voorkom, oorgedra na naasliggende plante. Sommige van die plante in the infeksie area was ook met ander GLRaV-3 variante geïnfekteer wat moontlik deur wolluise oorgedra is vanaf naburige geïnfekteerde plante. Die wetenskaplike inligting wat tydens hierdie studie beskryf word aangaande die identifikasie van GLRaV-3 variante, dra by tot die molekulêre kennis van GLRaV-3 en verskaf ‘n basis vir verdure epidemiologiese -en insek oordragingstudies. Die studie het vir die eerste keer bewys dat verskillende GLRaV-3 variante na aanliggende plante in ‘n ry asook oor rye oorgedra word. Die diversiteit tussen die GLRaV-3 variant groepe in die 5’UTR moet verder ondersoek word en die deel van die genoom kan ‘n belangrike rol speel in die biologiese eienskappe van die variante.

Thesis (PhD)--University of Stellenbosch, 2003. / ENGLISH ABSTRACT: Grapevine leafroll-associated virus-3 is one of ten members of the C/osteroviridae that are known to infect grapevine. Nine of these viruses are associated with grapevine leafroll disease, of which GLRaV-1 and GLRaV-3 are the most important and widespread. Members of the C/osteroviridae are unique amongst the viruses, as it is the only known family whose members encode a heat shock protein 70 kOa homolog (Hsp70h). The Hsp70h is a movement protein (MP) that is required for the active translocation of the virion structure through the plasmodesmata into adjacent cells. Broad-spectrum resistance to unrelated viruses can be obtained by a pathogen-derived resistance (POR) strategy that is based on the expression of a dysfunctional MP in plants. The Hsp70h has two distinct domains. The N-terminal two thirds of the protein is an ATPase domain and shares high homology with the ATPase domains of all Hsp70h proteins from the C/osteroviridae and Hsp70 proteins from the prokaryote and eukaryote kingdoms. Conserved amino acids are found in the ATPase domain and are required for the positioning of the ATP at the catalytic site for ATP hydrolysis. The C-terminal domain is variable and the function of this domain in the Closteroviridae is not known. In prokaryote and eukaryote Hsp70 proteins, the C-terminal domain is required for protein-protein interactions. The American NY-1 isolate of GLRaV-3 has been sequenced and POR strategies have been attempted with the coat protein, divergent coat protein and replicase genes, but not with a dysfunctional form of the hsp70h gene. In this study, double-stranded RNA was isolated from a commercial vineyard with unknown virus status, but with distinct grapevine leafroll symptoms, and from two grapevine sources of known virus status, one with mild and one with severe symptoms. The GLRaV-3 hsp70h gene was amplified by RT-PCR from the dsRNA and the gene sequence was analysed. The hsp70h gene from the three virus sources contained more than 94% nucleotide sequence homology to the NY-1 isolate and the conserved amino acids required for ATPase activity were present. The hsp70h gene isolated from GLRaV-3 from a commercial Stellenbosch vineyard showing clear leafroll symptoms was selected for further work and was subjected to site-directed mutagenesis to engineer four point mutations in the gene. These four mutations resulted in the substitution of Asn for Asp", Gly for Thr1O, Lys for Glu 174 and Asn for Asp 197. The wild type (WT) and mutated (Mut) forms of the hsp 70h genes were cloned into a bacterial expression vector. Expression of both the WT- and Mut-Hsp proteins was achieved, and the protein was expressed in the insoluble inclusion bodies. All attempts to refold and isolate active proteins from the inclusion bodies were unsuccessful. Attempts to increase the concentration of soluble protein within the expressing bacteria were unsuccessful. Due to the lack of active protein, biochemical tests on the ATPase activity of the WT- and Mut-Hsp proteins could not be conducted. The wt- and mut-hsp genes were cloned into a plant expression vector for transformation into tobacco plants. These transformations were successful and gave rise to 22 Km' and 18 Km' plants from the WT- and Mut-Hsp constructs respectively. Two plant lines, M5 and M10, transformed with the mut-hsp transgene construct, appeared to have a high level of resistance to the challenging potato X potexvirus, whereas all the other tested plants were susceptible to the challenging virus. It was thus shown that a dysfunctional form of the GLRaV-3 Hsp70h could provide resistance to an unrelated virus in tobacco. / AFRIKAANSE OPSOMMING: Wingerdrolblaar-geassosieerde virus 3 (GLRaV-3) is een van 10 lede van die Closteroviridae wat wingerd kan infekteer. Nege van die virusse is met wingerdrolblaar geassosieer. Die GLRaV-1 en GLRaV-3 is die belangrikste en mees wyd verspreide lede van die rolblaar-geassosieerde Closteroviridae. Lede van die Closteroviridae is uniek in die opsig dat die virusse vir 'n 70 kDa-homoloë hitteresponsproteïen (Hsp70h) kodeer. Die Hsp70 is 'n bewegingsproteïen (MP) wat belangrik is vir die translokasie van die virus deur die plasmodesmata na die naasliggende sel. Breë-spektrum weerstand teen onverwante virusse kan behaal word deur 'n patogeen-afgeleide weerstandstrategie (POR), wat op die uitdrukking van 'n disfunksionele MP wat in plante uitgedruk word, gebaseer is. Die Hsp70hproteïen het twee gebiede. Die N-terminale gebied is In ATPase-gebied en toon hoë homologie met ander ATPase-gebiede van Hsp70h-proteïene van die Closteroviridae, asook die prokariotiese en eukariotiese koninkryke. Gekonserveerde aminosure wat belangrik is vir die posisionering van ATP in die katalitiese domein vir ATP-hidrolise is in die ATPase-gebied gevind. Die C-terminale gebied is variërend en die funksie van die gebied in die Closteroviridae is onbekend. In prokariotiese en eukariotiese Hsp70h-proteïene is die C-terminale gebied belangrik vir proteïenproteïen interaksies. Die nukleotiedvolgorde van die Amerikaanse NY-1-isolaat van GLRaV-3 is al bepaal en POR-strategieë is ook op die kapsiedproteïen, uiteenlopende kapsiedproteïen en die replikasie-proteïen uitgevoer, maar nog nie op 'n disfunksionele vorm van die Hsp70h-geen nie. In hierdie studie is dubbelstring-RNA (dsRNA) van 'n kommersiële wingerd met onbekende virusstatus wat rolblaarsimptome toon, geïsoleer, asook van twee wingerde met 'n bekende virusstatus, een met ligte en een met strawwe simptome. Die GLRaV-3 hsp70h-geen is met hulp van die polimerasekettingreaksie-metode (PKR) vanaf die dsRNA geamplifiseer en die geen se nukleotiedvolgorde is bepaal. Die hsp 70-gene van drie verskillende wingerde het meer as 94% homologie met die NY-1-isolaat getoon. Die gekonserveerde aminosure wat vir ATPase-aktiwiteit belangrik is, was teenwoordig. Die hsp70h-geen van GLRaV-3, wat uit 'n kommersiële wingerd met duidelike rolblaarsimptome in die Stellenbosch-gebied geïsoleer is, is vir verdere navorsing gekies en dit is aan setel-gerigte mutagenese blootgestelom vier mutasies van die geen te bewerkstellig. Die gevolg van hierdie vier mutasies was die verandering van Asn na Asp", Gly na Thr1o, Lys na Glu174 en Asn na Asp197. Die wilde (WT) en veranderde (Mut) vorms van die hsp-gene is in 'n bakteriese uitdrukkingsvektor gekloneer. Uitdrukking van beide die WT- en die Mut-Hspproteïene is behaal, maar die proteïene was in die onoplosbare fraksie geleë. Pogings om die onoplosbare proteïene te isoleer en in 'n aktiewe oplosbare vorm te verkry, was onsuksesvol. Verdere pogings om die proteïene in die oplosbare fraksie van die bakteriese ekspressiesisteem uit te druk, was ook onsuksesvol. As gevolg van die gebrek aan aktiewe proteïen kon biochemiese toetse nie op die ATPaseaktiwiteit van die WT- en Mut-Hsp proteïne gedoen word nie. Die wt- en mut-hsp-gene is ook in In plantekspressievektor gekloneer vir transformasie in tabakplante. Hierdie transformasies was suksesvol en het aanleiding gegee tot 22 kanamisienbestande (Km') en 18 Km' plante vanaf die WT- en Mut-Hspkonstrukte onderskeidelik. Twee plantlyne, M5 en M10, wat met die mut-hsptransgene getransformeer is, het 'n hoë vlak van weerstand teen die infekterende aartappelvirus X getoon in vergelyking met ander plante wat met die virus geïnfekteer is. Daar is dus bewys gelewer dat 'n disfunksionele vorm van die GLRaV-3 Hsp70h weerstand kan bied teen 'n onverwante virus in tabak.

Potato leafroll virus (PLRV) causes yield and quality losses in potato. PLRV is identified by plant symptoms and serological tests such as an enzyme-linked immunosorbent assay (ELISA). A similar serological test, direct tissue blotting assay (DTBA), was used to detect and monitor PLRV movement in field-inoculated Russet Burbank plants and plant tissues from Russet Burbank and Russet Norkotah seed tubers submitted by growers for winter certification tests. DTBA was as accurate as ELISA and easier to use for detecting tuber-perpetuated PLRV in stems and petioles of plants grown from grower-submitted seed tubers. ELISA detected twice as many PLRV positives as DTBA in leaflet tests. DTBA detected PLRV in tuber tissue but results matched ELISA in only 74% or less of the samples. Results of DTBA tuber tests were sometimes difficult to interpret while stem and petiole results were distinct and unambiguous. As inoculations were delayed later in the season and as plants matured, PLRV infection levels decreased sharply, most often within a two week period in early July. In same-age plants inoculated 43 days after planting but 18 days apart, early inoculation produced higher PLRV levels. Conversely, when same-age plants were inoculated 62 days after planting but 19 days apart, late inoculation produced higher PLRV levels. This discrepancy is not fully understood, but larger tuber size at the later inoculation probably produced a stronger sink for source-to-sink translocation of nutrients and phloem-limited viruses. Results of DTBA winter grow-out tests of summer-infected tubers approximated those of ELISA and visual inspections. Indirect DTBA testing of tubers utilizing stem and petiole tissues from winter growout plants detected more PLRV than directly testing tuber tissue 21 days post inoculation in summer. DTBA detected current season (primary) PLRV less reliably than secondary (tuber-borne) PLRV, similar to reported ELISA results. PLRV infection increased tuber numbers but decreased size. Size reduction was most evident in plants infected early in the season. Average tuber size in healthy plots was always larger than the average tuber size in infected plots. Within an infected plant, small tubers tended to be infected less often than large tubers. / Graduation date: 1993

Bibliography: leaves 119-127 / viii, 138, 10 leaves, 8, [11] leaves of plates : ill. (some col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, Dept. of Plant Pathology, 1985