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Complete sequence, improved detection and functional analysis of Grapevine Leafroll-associated Virus 1(GLRaV-1) / by Alan Little.Little, Alan January 2004 (has links)
"April, 2004" / "List of figures" - inside back cover. / Bibliography: leaves 83-93 / 93, [8] leaves : ill., plates (some col.), photos ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / The specific objectives of this work include: 1. Completion of the GLRaV-1 genome sequence and bioinformatic analysis of the viral open reading frames ; 2. Production of an appropriate GLRaV-1 certification protocol addressing the shortcomings of the current tests for leafroll detection ; 3. Intracellular localisation of the GLRaV-1 gene products via generation of green fluorescent protein (GFP)-fusion constructs, in an attempt to further characterise the function of these proteins. / Thesis (Ph.D.)--University of Adelaide, School of Agriculture and Wine, Discipline of Wine and Horticulture, 2004
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Complete sequence, improved detection and functional analysis of Grapevine Leafroll-associated Virus 1(GLRaV-1) /Little, Alan. January 2004 (has links) (PDF)
Thesis (Ph.D.)--University of Adelaide, School of Agriculture and Wine, Discipline of Wine and Horticulture, 2004. / "April, 2004" "List of figures" - inside back cover. Bibliography: leaves 83-93.
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Generation of full-length cDNA clone and functional analysis of leader proteases of grapevine leafroll-associated virus-2 /Liu, Yu-Ping. January 1900 (has links)
Thesis (Ph. D.)--Oregon State University, 2009. / Printout. Includes bibliographical references (leaves 61-74). Also available on the World Wide Web.
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The construction of plant expression vectors for the introduction of leafroll disease resistance in grapevineVan Straten, Celene Debra 12 1900 (has links)
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.
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A pathogen-derived resistance strategy for the broad-spectrum control of grapevine leafroll-associated virus infectionFreeborough, Michael-John, 1971- 12 1900 (has links)
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.
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Evaluation of two pathogen-derived resistance strategies for Grapevine leafroll-associated virus 3Suidgeest, Faira 04 1900 (has links)
Thesis (MSc)--Stellenbosch University, 2015. / ENGLISH ABSTRACT: Grapevine leafroll disease (GLD), caused by the members of the family Closteroviridae, is
one of the most economic important viral diseases affecting grapevine. Grapevine leafroll
associated virus 3 (GLRaV-3), of the genus Ampelovirus, is the most widespread member of
the leafroll associated virus family. To prevent the spread of GLD, management strategies
such as rogueing and insect vector control are required to limit crop losses. Alternative control
strategies based on genetic modification of the grapevine genome, such as pathogen-derived
resistance (PDR), is proven to be effective in conferring resistance to several viruses.
Therefore, the focus of this study was to evaluate pathogen-derived resistance strategies
for GLRaV-3 using the following two approaches; 1) evaluation of transgenic plants
expressing a dysfunctional GLRaV-3 heat shock protein 70 homolog (HSP70h) in order to
confer resistance against GLRaV-3, and 2) the construction of artificial microRNAs
(amiRNAs) to use as a tool for silencing specific sequences of GLRaV-3 in the grapevine host
and the development of an amiRNA-mediated silencing validation system.
In the first part of this study, six transgenic plant lines (plant lines #1, #3, #9, #14, #15 and
#17) as well as a non-modified plant line, were inoculated with GLRaV-3 by grafting buds of
each onto GLRaV-3 infected plant material. After approximately five months, GLRaV-3 virus
titres of all grafted plants were quantified relative to two reference genes using RT-qPCR.
Results were evaluated by comparing the relative virus titre of each transgenic plant line to
that of the non-modified control plant line. Results showed that resistance levels of plant line
#3 was significantly enhanced (>99%) and remarkably, plant line #14, showed to be more
susceptible to the virus.
The second part of the study was the construction and validation of amiRNAs targeting
GLRaV-3 sequences. Two 21 nt regions of GLRaV-3 were successfully incorporated into
miRNA backbone vvi167b of grapevine. Moreover, target constructs were developed by
incorporating corresponding GLRaV-3 target sequences into the 3’ UTR of a green
fluorescence protein (GFP) gene. Subsequently, the target constructs were co-infiltrated with
the constructed amiRNA in Nicotiana benthamiana and GFP expression levels were
quantified to determine the silencing efficiency of the amiRNAs. Results showed that the
amiRNAs were successful in silencing the GFP target construct, however, they were not
specific in silencing exclusively their corresponding target. These amiRNA constructs are
ideal for further viral studies to determine the efficiency of silencing GLRaV-3 in GLD
infected grapevines. / AFRIKAANSE OPSOMMING: Wingerd rolblaar siekte (GLD), wat veroorsaak word deur die lede van die familie
Closteroviridae, is een van die ekonomies mees belangrike virus siektes van wingerd.
Grapevine leafroll-associated virus 3 (GLRaV-3), van die genus Ampelovirus, is die mees
wydverspreide lid van die rolblaar geassosieerde virus familie. Om die verspreiding van GLD
te voorkom, is bestuur strategieë, soos die verwydering van geïnfekteerde plante en
insekvektor beheer, ’n vereiste om oes verliese te beperk. Alternatiewe beheer strategieë
gebaseer op genetiese modifikasie van die wingerdgenoom, soos patogeen-afgeleide
weerstand (PDR), is bewys om effektief te wees in die verlening van weerstand teen verskeie
virusse. Daarom was die fokus van hierdie studie om patogeen-afgeleide weerstand strategieë
vir GLRaV-3 te evalueer met behulp van die volgende twee benaderings; 1) die evaluering
van transgeniese plante wat 'n disfunksionele GLRaV-3 hitte-skok proteïen 70 homoloog
(HSP70h) uitdruk, ten einde weerstand te verleen teen GLRaV-3, en 2) die konstruksie van
kunsmatige mikroRNAs (amiRNAs) om te gebruik as 'n instrument vir die ondrukking van
spesifieke genoomvolgordes van GLRaV-3 in die wingerd gasheer en die ontwikkeling van ’n
stelsel om amiRNA-bemiddelde onderdrukking te bevestig.
In die eerste deel van hierdie studie, is ses transgeniese plant lyne (plant lyne # 1, # 3, # 9, #
14, # 15 en # 17) sowel as 'n nie-gemodifiseerde gesonde plant lyn, geïnokuleer met GLRaV-
3 deur enting van ogies van elk op GLRaV-3 besmette plantmateriaal. Na ongeveer vyf
maande, is GLRaV-3 virus konsentrasies van alle ingeënte plante gekwantifiseer relatief tot
twee verwysing gene deur gebruik te maak van tru-transkripsie kwantitatiewe PCR (RTqPCR).
Resultate is geëvalueer deur die relatiewe virus konsentrasie van elke transgeniese
plant lyn te vergelyk met dié van die nie-gemodifiseerde kontrole lyn. Resultate het getoon
dat weerstand vlakke van plant lyn # 3 beduidend verbeter is (> 99%) en merkwaardig is plant
lyn # 14 bewys om meer vatbaar vir die virus te wees.
Die tweede deel van die studie was die konstruksie en bevestiging van kunsmatige
mikroRNAs (amiRNAs) wat GLRaV-3 genoomvolgordes teiken. Twee 21 nt streke van
GLRaV-3 is suksesvol geïnkorporeer in die ruggraat van wingerd mikroRNA vvi167b. Verder
is teiken konstrukte ontwikkel deur die inkorporering van ooreenstemmende GLRaV-3 teiken
genoomvolgordes in die 3'UTR (3’ ongetransleerde area) van 'n groen fluoressensie proteïen
(GFP) geen. Daarna is die teiken konstrukte gesamentlik geïnfiltreer met die gekonstrueerde
amiRNA in Nicotiana benthamiana en GFP uitdrukkingsvlakke is gekwantifiseer deur die
onderdrukkingsdoeltreffendheid van die amiRNAs te bepaal. Resultate het getoon dat die
amiRNAs suksesvol was in die onderdrukking van die GFP teiken konstruk, maar hulle was
egter nie-spesifiek in die eksklusiewe onderdrukking van die ooreenstemmende teiken.
Hierdie amiRNA konstrukte is ideaal vir verdere virus studies om die doeltreffendheid van
GLRaV-3 onderdrukking in GLD besmette wingerdstokke te bepaal.
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Identification and molecular characterization of three genetic variants of Grapevine leafroll-associated virus 3 (GLRaV-3) from South African vineyards and their spread in local vineyardsJooste, Anna Elizabeth Catharina 03 1900 (has links)
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.
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Identification and characterisation of grapevine leafroll-associated virus 3 genomic and subgenomic RNAsMaree, Hans Jacob 12 1900 (has links)
Thesis (PhD (Genetics))--University of Stellenbosch, 2010. / Includes bibliography. / Title page: Dept. of Genetics, Faculty of Science / ENGLISH ABSTRACT: Grapevine leafroll-associated virus 3 (GLRaV-3) is the type strain for the genus
Ampelovirus, family Closteroviridae. There has been only one report that claimed the
complete nucleotide sequence of GLRaV-3 (isolate NY-1, AF037268). Here we report the
complete sequence of the South African GLRaV-3, isolate GP18 (EU259806) and show a
significantly extended 5’ end. We used RLM-RACE to determine the 5’ end of GP18 and
found the 5’ UTR to be 737 nt compared to 158 nt in the NY-1 sequence. This extended
UTR was found in all other South African isolates of GLRaV-3 that were tested. In two
collaborative studies the existence of the extended 5’ UTR was confirmed and further
investigated. In the first study (Coetzee et al., 2010), metagenomic data generated by next
generation sequencing (Illumina Genome Analyzer II) was analysed for GLRaV-3 specific
sequences. Sequences similar to the GP18 isolate confirmed the sequence of the extended
5’ UTR. In the second study (Jooste et al., 2010), three genetic variants were identified and
their respective 5’ UTRs studied. Great diversity was observed between the 5’ UTRs of the
different genetic variants, however within a variant the 5’ UTR was found to be highly
conserved. Grapevine leafroll-associated virus 3 is a positive sense, single stranded RNA
virus that has been shown, like other closteroviruses, to produce subgenomic (sg) RNAs
during replication. These sgRNAs are deployed for the expression of the ORFs on the 3’
half of the genome. In this study a dsRNA blot confirmed the presence of three, 3’ coterminal
sgRNAs species [sgRNA(ORF3/4), sgRNA(ORF5) and sgRNA(ORF6)] in
GLRaV-3-infected plant material when using a probe directed at the coat protein gene. The
specific 5’ terminal nucleotides for these sgRNAs as well as four additional sgRNAs
[sgRNA(ORF7), sgRNA(ORF8), sgRNA(ORF9) and sgRNA(ORF10-12)] were
determined by RLM-RACE for GLRaV-3 isolate GP18. The construction of a GLRaV-3
mini-replicon, analogous to RNA1 of Lettuce infectious yellows virus, for the evaluation
of putative sg-promoters is also described. / AFRIKAANSE OPSOMMING: Grapevine leafroll-associated virus 3 (GLRaV-3) is ‘n lid van die Closteroviridae familie
en die hooflid vir die genus Ampelovirus. Tot dusver was daar net een studie wat die
volledige nukleïensuurvolgorde van GLRaV-3 gerapporteer het (isolaat NY-1, AF037268).
In hierdie studie rapporteer ons die volledige volgorde van ‘n Suid-Afrikaanse GLRaV-3,
isolaat nl. GP18 (EU259806) wat noemenswaardig langer is aan die 5’ kant. RLM-RACE
is gebruik om die 5’ eindpunt van GP18 te bepaal en daar is gevind dat die 5’
ongetransleerde streek (UTR) 737 nt lank is in vergelyking met die 158 nt van die NY-1
volgorde. Die verlengde 5’ UTR is gevind in alle Suid-Afrikaanse monsters wat getoets is.
Die verlengde 5’ UTR is bevestig en verder bestudeer tydens twee samewerkingsprojekte.
In die eerste studie (Coetzee et al., 2010), is metagenomiese data gegenereer deur
volgende-generasie volgordebepaling (Illumina Genome Analyzer II) en geanaliseer vir
GLRaV-3 spesifieke volgordes. Volgordes soortgelyk aan die GP18 isolaat het die
verlengde 5’ UTR volgorde bevestig. In die tweede studie (Jooste et al., 2010), is drie
genetiese variante van GLRaV-3 geidentifiseer en hulle onderskeie 5’ UTR volgordes
bepaal en bestudeer. Daar is groot diversiteit tussen die 5’ UTRs van die verskillende
genetiese variante gevind, maar tussen isolate van dieselfde variant is die volgordes
gekonserveerd. Grapevine leafroll-associated virus 3 is ‘n positiewe-sin, enkelstring RNA
virus wat al voorheen bewys is om, soos ander closterovirusse, subgenomiese (sg) RNAs te
produseer tydens replisering. Hierdie sgRNAs word ingespan vir die uitdrukking van die
ORFs op die 3’ helfte van die virusgenoom. In hierdie studie is ‘n dsRNA klad gebruik om
die voorkoms van 3’ ko-terminale sgRNAs [sgRNA(ORF3/4), sgRNA(ORF5) and
sgRNA(ORF6)] te bevestig in GLRaV-3 geinfekteerde plantmateriaal deur gebruik te
maak van ‘n peiler teen die kapsiedproteïengeen. Die spesifieke 5’ terminale nukleotiedes
vir hierdie sgRNAs sowel as vier additionele sgRNAs [sgRNA(ORF7), sgRNA(ORF8),
sgRNA(ORF9) and sgRNA(ORF10-12)] is bepaal deur gebruik te maak van RLM-RACE
op die GLRaV-3 isolaat GP18. Die konstruksie van ‘n GLRaV-3 mini-repliserings
konstruk, analoog aan die RNA1 van Lettuce infectious yellows virus, vir die evaluasie
van moontlike sg-promotors word ook beskryf.
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