Thesis (MSc)--University of Stellenbosch, 2011. / ENGLISH ABSTRACT: Plants are constantly exposed to biotic and abiotic stress inducing factors that threaten their
existence. Biotic factors such as pathogens are the cause of huge yield losses to crop plants
worldwide with fungal pathogens debatably constituting the worst damage. Fungal pathogens
such as Botrytis cinerea, which has a wide host range, release cell wall degrading enzymes
called endopolygalacturonases (ePGs) during plant infection. These ePGs break down the
pectin component of the cell wall, thus providing an entry route, as well as nutrients for the
fungus.
Plants have evolved mechanisms to counteract and suppress the action of the ePGs.
This is achieved through the action of cell wall associated proteins called polygalacturonaseinhibiting
proteins, PGIPs. PGIPs directly inhibit ePGs and their inhibitory action also prolongs
the existence of longer chain oligogalacturonide residues which are believed to elicit a cascade
of defence responses. In grapevine, a PGIP encoding gene, VvPGIP1, was previously isolated
and characterised. VvPGIP1, as well as nine non-vinifera grapevine PGIPs have been
expressed in tobacco and shown to be potent antifungal proteins that caused the transgenic
tobacco to have strong resistance phenotypes against Botrytis in whole plant infection assays.
Following on the tobacco study, two of the non-vinifera PGIPs were expressed in cultivars of the
susceptible Vitis vinifera. Characterisation of the putative transgenic population showed that
transgene integration was successful, the transgenes were being expressed and there were at
least 29 transgenic lines with independent integration events. The transgenic lines were
confirmed to have active PGIPs (transgene-derived) in their leaves. Crude protein extracts from
22 lines exhibited 100% inhibition against crude B. cinerea PGs (BcPGs).
The plant lines with positive transgene integration, expression, independent integration
events and exhibiting 100% transgene-derived PGIP activity were further selected for whole
plant and detached leaf antifungal assays where they were challenged with B. cinerea. The
whole plant infection assay showed that expression of the non-vinifera PGIPs in V. vinifera
promotes susceptibility to B. cinerea, not resistance. This surprising result could perhaps be
explained by a quicker and stronger recognition between the pathogen and the host and the
stronger activation of defence responses in the host. A more active hypersensitive response in
the host would benefit Botrytis being a necrotroph. The type of lesions and the onset and speed
of lesion development observed on the transgenics lines versus the wild type support this
possibility. Knowledge gaps with regards to the efficiency of the ePG inhibition by the nonvinifera
PGIPs during infection of grapevine tissue; the potential changes that might be caused
by expressing PGIPs in a grapevine host with a native PGIP with high homology to the
transgenes (including potential gene silencing) and the potential impact on defence signalling
and defence responses all provides further avenues of study to elucidate this very interesting
phenotype further. Overall, this study provides a comprehensively characterised population of
transgenic plants that provides useful resources for in vivo analysis of PGIP function in defence,
where the host plant harbours a native copy of the PGIP encoding gene. / AFRIKAANSE OPSOMMING: Plante word voortdurend blootgestel aan biotiese en abiotiese faktore, wat stres veroorsaak en
hul bestaan bedreig. Biotiese faktore, soos patogene, veroorsaak groot verliese in wêreldwye
gewasopbrengste, met swampatogene wat moontlik die grootste skade veroorsaak.
Swampatogene, soos Botrytis cinerea, wat ‘n wye reeks gasheerplante kan infekteer, stel
selwand-afbrekende ensieme tydens plantinfeksie vry, wat as endo-poligalakturonases (ePG’s).
bekend staan. Hierdie ePG’s breek die pektienkomponent van die selwand af, wat gevolglik as
‘n ingangspunt dien,asook voedingstowwe vir die swam verskaf.
Plante het meganismes ontwikkel om die aktiwiteit van hierdie ePG’s te bekamp en te
onderdruk. Die aktiwiteit van die selwand-geassosieërde proteïene, genaamd
poligalakturonase-inhiberende proteïene (PGIP’s), speel hier ‘n rol. PGIP’s inhibeer ePG’s direk
en hul inhiberende aktiwiteit verleng ook die bestaan van langketting oligogalakturoniedresidu’s,
wat blykbaar ‘n kaskade van weerstandsreaksies kan inisieer. ‘n PGIP-koderende
geen, VvPGIP1, is voorheen uit wingerd geïsoleer en gekarakteriseer. VvPGIP1, asook nege
nie-vinifera wingerd-PGIP’s is voorheen in tabak uitgedruk en bevestig as proteïene met sterk
anti-swamaktiwiteit, soos bevestig deur die bevinding dat die transgeniese tabak ‘n
weerstandsfenotipe teen Botrytis in heelplant-infeksietoetse het. Ná die tabakstudie is twee van
die nie-vinifera PGIP’s uitgedruk in vatbare V. vinifera-kultivars. Karakterisering van die
vermeende transgeniese bevolking het getoon dat die transgeen-integrasie suksesvol was, dat
die transgeen uitgedruk word en dat daar ten minste 29 transgeniese lyne met onafhanklike
integrasie gebeurtenisse geskep is. Daar is verder bevestig dat die transgeniese lyne aktiewe
PGIP’s (transgeen-afkomstig) in hul blare het. Ongesuiwerde proteïenekstrakte van 22 lyne het
100% inhibisie teen ‘n mengsel van ongesuiwerde B. cinerea PGs (BcPGs) getoon.
Die plantlyne met positiewe transgeenintegrasie en -uitdrukking, asook onafhanklike
integrasiegebeure en wat 100% transgeen-afkomstige PGIP-aktiwiteit getoon het, is verder aan
heel-plant en verwyderde blaarswaminfeksies met B cinerea onderwerp. Die heelplantinfeksietoetse
het getoon dat uitdrukking van nie-vinifera PGIP’s in V. vinifera ‘n toename, in
plaas van ‘n afname, in vatbaarheid teen B. cinerea veroorsaak. Hierdie verbasende resultaat
kan moontlik toegeskryf word aan ‘n vinniger en sterker herkenningsreaksie tussen patogeen en
gasheer en die moontlike sterker stimulering van weerstandsreaksies in die gasheer. ‘n Meer
aktiewe hipersensitiewe reaksie in die gasheer sal tot die voordeel van Botrytis, wat ‘n
nektrotroof is, wees. Die tipe letsel, asook die aanvang en spoed van letselontwikkeling wat
waargeneem is in transgeniese lyne teenoor die wilde-tipe ondersteun hierdie moontlikheid.
Gapings in kennis ten opsigte van die doeltreffendheid van die ePG-inhibisie deur die nievinifera
PGIP’s tydens infeksie van wingerdweefsel, die moontlike veranderinge (insluitend ‘n
moontlike geenuitdowingseffek) wat veroorsaak kan word deur die uitdrukking van PGIP-gene
in ‘n kultivar met ‘n inheemse en baie homoloë PGIP-geen, kon ‘n invloed op weerstandseine
en weerstandsreaksies gehad het. Hierdie aspekte lewer verdere studiemoontlikhede om
hierdie interessante fenotipe verder te verklaar.Algeheel lewer hierdie studie ‘n breedvoeriggekarakteriseerde
bevolking trangeniese plante, wat dien as nuttige hulpbronne vir in vivoanalise
van PGIP se funksie in siekteweerstandbiedendheid, veral waar die gasheerplant ‘n
inheemse kopie van die PGIP-koderende geen huisves.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/6825 |
| Date | 03 1900 |
| Creators | Moyo, Mukani |
| Contributors | Vivier, M. A., University of Stellenbosch. Faculty of AgriSciences. Dept. of Viticulture and Oenology. Institute for Wine Biotechnology. |
| Publisher | Stellenbosch : University of Stellenbosch |
| Source Sets | South African National ETD Portal |
| Language | en_ZA |
| Detected Language | Unknown |
| Type | Thesis |
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