| Summary: | Thesis (PhD (Genetics))--University of Stellenbosch, 2010. === ENGLISH ABSTRACT: Grapevine (Vitis vinifera L.) is a very important agricultural commodity that needs to be
protected. To achieve this several in vivo tools are needed for the study of this crop and the
pathogens that infect it. Recently the grapevine genome has been sequenced and the next
important step will be gene annotation and function using these in vivo tools. In this study the
use of Grapevine virus A (GVA), genus Vitivirus, family Flexiviridae, as transient expression
and VIGS vector for heterologous protein expression and functional genomics in Nicotiana
benthamiana and V. vinifera were evaluated. Full-length genomic sequences of three South
African variants of the virus (GTR1-1, GTG11-1 and GTR1-2) were generated and used in a
molecular sequence comparison study. Results confirmed the separation of GVA variants into
three groups, with group III (mild variants) being the most distantly related. It showed the
high molecular heterogeneity of the virus and that ORF 2 was the most diverse. The GVA
variants GTG11-1, GTR1-2 and GTR1-1 were placed in molecular groups I, II and III
respectively. A collaboration study investigating the molecular divergence of GVA variants
linked to Shiraz disease (SD), described two interesting GVA variants of group II, namely
GTR1-2 and P163-M5 (Goszczynski et al., 2008). The group II variants were found to be
closely linked to the expression of SD. GTR1-2 was isolated from a susceptible grapevine
plant that never showed SD symptoms (Goszczynski 2007). The P163-M5 variant that
resulted in exceedingly severe symptoms in N. benthamiana and is that used as SD positive
control by the grapevine industry, was found to contain a 119 nt insert within the native
ORF2. Comparative analysis performed on the complete nt and aa sequences of group II GVA
variants suggested that the components in the GVA genome that cause pathogenicity in V.
vinifera are more complex (or different) to those that cause pathogenicity in N. benthamiana.
The three South African variants (GTR1-1, GTG11-1 and GTR1-2) were assembled into fulllength
cDNA clones under control of CaMV 35S promoters. After several strategies were
attempted, including a population cloning strategy for GTR1-2, none of the clones generated
were able to replicate in N. benthamiana plants. A single amino acid substitution at position
13 (Tyr/Y Cys/C) in ORF 5 of the GTR1-2 cDNA clone was shown to abolish or reduce
replication of the virus to below a detectable level. Two infectious clones of Israeli variants of
GVA (T7-GVA-GR5 and T7-GVA118, obtained from M. Mawassi) were brought under
control of a CaMV 35S promoter (35S-GVA-GR5 and 35S-GVA118). Both clones were
infectious, able to replicate, move systemically and induce typical GVA symptoms after
agroinfiltration in N. benthamiana. These Israeli clones served as backbone for further experiments in characterisation of transient expression and VIGS vectors. The use of GVA as
gene insertion vector (35S-GVA118) and gene exchange vector (35S-GVA-GR5-
ORF2+sgMP) in N. benthamiana and V. vinifera was compared. The gene insertion vector,
35S-GVA118 was based on the full-length GVA genome. The gene exchange vector, 35SGVA-
GR5- ORF2+sgMP, was constructed in this study by elimination of ORF 2 and
insertion of a sgMP and unique restriction sites to facilitate transgene insertion. In N.
benthamiana both vectors showed similar GUS expression levels and photobleaching
symptoms upon virus-induced NbPDS silencing. In V. vinifera limited GUS expression levels
and VIGS photobleaching symptoms were observed for the gene insertion vector, 35SGVA118.
No GUS expression was observed for the gene exchange vector 35S-GVA-GR5-
ORF2+sgMP in this host. As for silencing, one plant, agroinfiltrated with 35S-GVA-GR5-
ORF2-VvPDS+sgMP, developed photobleaching symptoms in 3 systemic infected leaves
after 4 months. This study showed that GVA can be used as gene insertion and gene exchange
vector for expression and VIGS in N. benthamiana, but in grapevine its use is limited to
expression and silencing of genes in the phloem tissue. It is also the first report that ORF 2 of
GVA is not needed for long distance movement in grapevine.
To investigate the possible role of the P163-M5 119 nt insertion and the GVA ORF 2 (of
unknown function), in expression of symptoms in plants, ORF 2 of a 35S-GVA-GR5 cDNA
clone was removed and subsequently substituted by the corresponding ORFs of four South
African GVA variants. Upon agro-infiltration into N. benthamiana leaves, all chimaeric GVA
constructs were able to move systemically through the plant. At this stage no correlation
could be found between severity of symptoms, the presence of the P163-M5 insert and the
specific GVA ORF 2 present in the chimaeras, indicating that other factors in the viral
genome or the host plant probably play a crucial role.
This study contributed to the pool of available in vivo tools for study and improvement of the
valuable grapevine crop. It also opened several exciting research avenues to pursue in the near
future. === AFRIKAANSE OPSOMMING: Wingerd (Vitis vinifera L.) is ‘n baie belangrike landboukundige gewas wat beskerm moet
word. Om die rede word verskeie in vivo gereedskap vir die bestudering van die
wingerdplant, en die patogene wat dit infekteer benodig. Die wingerd genoom se volgorde is
bepaal en dus is die volgende logiese stap om die gene te annoteer en funksie daaraan toe te
skryf. In hierdie studie is die gebruik van Grapevine virus A (GVA), genus Vitivirus, familie
Flexiviridae, as tydelike uitdrukking- en virus-geinduseerde geenuitdowingsvektor vir
heteroloë proteïen uitdrukking en funksionele genoomstudies in Nicotiana benthamiana en V.
Vinifera getoets. Vollengte genoomvolgordes van drie Suid-Afrikaanse variante van die virus
(GTR1-1, GTG11-1 en GTR1-2) is gegenereer en in ‘n molekulêre volgorde vergelyking
studie gebruik. Resultate het die verdeling van GVA variante in drie groepe, waar groep III
die verste verwant is, bevestig. Dit het ook gewys dat die virus ‘n baie hoë molekulêre
heterogeniteit het en dat oopleesraam 2 (ORF 2) die mees divers is. ‘n Samewerking studie
waar die molekulêre diversiteit van GVA variante, gekoppel aan Shiraz siekte (SD),
ondersoek is, is twee interessante variante van groep II beskryf, naamlik GTR1-2 en P163-M5
(Goszczynski et al., 2008). Groep II variante is vooraf gevind om nou verwant te wees aan die
ontwikkeling van SD in wingerd. Die GTR1-2 variant is uit ’n vatbare wingerd plant, wat
nooit SD-simptome vertoon het nie, geïsoleer (Goszczynski et al., 2007). In die ORF 2 van
die P163-M5 variant, wat simptome van die ergste graad in N. benthamiana geïnduseer het, en
ook deur die industrie as betroubare SD-positiewe kontrole gebruik word, is ’n 119 nt
invoeging gevind. Die vergelykende analise wat uitgevoer is, het daarop gedui dat die
determinante van patogenisiteit in die GVA genoom moontlik meer kompleks kan wees in V.
vinifera as in N. benthamiana. Die drie Suid-Afrikaanse variante (GTR1-1, GTG11-1 en
GTR1-2) is in afsonderlike vollengte cDNA klone, onder beheer van CaMV 35S promotors,
aanmekaargesit. Nadat verskeie kloneringstrategieë, insluitend ’n populasie kloneringstrategie
vir die GTR1-2 kloon, gebruik is, het geen een van die cDNA klone die vermoë besit om in
N. benthamiana te repliseer nie. ’n Enkele aminosuur substitusie in posisie 13
(Tyr/Y Cys/C) in ORF 5 van die GTR1-2 kloon, het die replisering van die virus tot laer as
’n opspoorbare vlak verlaag. Twee infektiewe klone van Israeliese GVA variante (T7-GVAGR5
en T7-GVA118, verkry van M. Mawassi) is onder beheer van ‘n CaMV 35S promotor
geplaas (35S-GVA-GR5 and 35S-GVA118). Beide klone het na agro-infiltrasie in N.
benthamiana plante gerepliseer, sistemies beweeg en tipiese GVA simptome geinduseer.
Hierdie twee klone het as raamwerk gedien vir verdere eksperimente in karakterisering van tydelike uitdrukkings- en VIGS vektore. Die gebruik van GVA as geen-insvoegingsvektor
(35S-GVA118) en geen-vervangingsvektor (35S-GVA-GR5- ORF2+sgMP) is in N.
benthamiana en V. vinifera vergelyk. Die geen-invoegingsvektor 35S-GVA118, was op die
vollengte GVA genoom gebasseer. Die geen-vervangingsvektor 35S-GVA-GR5-
ORF2+sgMP, was in hierdie studie gekonstrueer. Dit is gemaak eerstens deur eliminasie van
ORF 2 in die 35S-GVA-GR5 kloon, en tweedens deur die invoeging van ’n subgenomiese
promotor van die beweginsproteïen (sgMP) en unieke beperkings-ensiemsetels om klonering
van transgene te fasiliteer. Beide vektore het in N. benthamiana vergelykbare GUS
uitdrukkingsvlakke en fotobleikende simptome getoon na virus-geinduseerde NbPDS
uitdowing. In V. Vinifera is beperkte GUS uitdrukkingsvlakke en VIGS fotobleikende
simptome opgemerk met die geen-invoegingsvektor, 35S-GVA118. Geen GUS uitdrukking is
in hierdie gasheerplant met die geen-vervangingsvektor opgemerk nie. Slegs een wingerdplant
het fotobleikende simptome, na 4 maande in 3 sistemies geïnfekteerde blare gewys, na agroinfiltrasie
van die 35S-GVA-GR5- ORF2-VvPDS+sgMP konstruk. Hierdie studie het
bevestig dat GVA as geen-invoeging en geen-vervangingsvektor, vir heteroloë proteïenuitdrukking
en VIGS, in N. benthamiana gebruik kan word, maar dit blyk of die gebruik
daarvan in wingerd meer tot die floeëm weefsel beperk is. Hierdie studie wys vir die eerste
keer dat ORF 2 nie nodig is vir langafstand beweging van die virus in wingerd nie.
Om die moontlike rol van die P163-M5 119 nt invoeging en die GVA ORF 2 (met onbekende
funksie), in die uitdrukking van simptome in plante te ondersoek, is ORF 2 van die 35SGVA-
GR5 cDNA kloon verwyder en daaropvolgens vervang met die ooreenstemmende
ORFs van vier Suid-Afrikaanse GVA variante. Na agro-infiltrasie in N. benthamiana blare,
het al die chimeras die vermoë gehad om te repliseer, sistemies te beweeg en simptome te
induseer. Geen korrelasie kon gevind word tussen die graad van simptome, die
teenwoordigheid van die P163-M5 insersie en die spesifieke GVA ORF 2 teenwoordig in die
chimeras nie, wat dus daarop dui dat ander faktore in die virusgenoom of die gasheerplant `n
moontlike belangrike rol kan speel.
Hierdie studie het bygedrae tot die beskikbare poel van in vivo gereedskap vir die bestudering
en verbetering van die kosbare wingerdgewas. Dit het ook talle interessante
navorsingsgeleenthede oopgemaak om in die nabye toekoms te betree.
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