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.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/53285 |
| Date | 12 1900 |
| Creators | Freeborough, Michael-John, 1971- |
| Contributors | Pretorius, I. S., Burger, J. T., Stellenbosch University. Faculty of AgriSciences. Dept. of Viticulture and Oenology. Institute for Wine Biotechnology. |
| Publisher | Stellenbosch : Stellenbosch University |
| Source Sets | South African National ETD Portal |
| Language | en_ZA |
| Detected Language | Unknown |
| Type | Thesis |
| Format | 119 p. : ill. |
| Rights | Stellenbosch University |
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