Thesis (MScAgric (Viticulture and Oenology. Wine Biotechnology))--University of Stellenbosch, 2010. / ENGLISH ABSTRACT: Necrotrophic fungi infect many economically important crop plants. This results in great losses
in the agricultural sector world-wide. Understanding the nature by which plants respond to
pathogens is imperative for genetically enhancing disease resistance in plants. Research tools
have significantly contributed to our understanding of how the plant responds to pathogen
attack, identifying an array of defence mechanisms used by plants upon attack.
Many fungal pathogens secrete endopolygalacturonases (endoPGs) when infecting
plants. These hydrolytic enzymes are inhibited by polygalacturonase-inhibiting proteins (PGIPs)
associated with plant cell walls. PGIPs are well characterised and their current known functions
are all linked to endoPG inhibition and the subsequent upregulation of plant defence pathways.
Work on grapevine PGIPs have shown that apart from being efficient antifungal proteins,
leading to protection of the plant against Botrytis cinerea when overexpressed, PGIPs might
also have additional functions linked to cell wall strengthening. This working hypothesis formed
the motivation of this study where a cinnamyl alcohol dehydrogenase (CAD) (1.1.1.195) gene
was targeted for functional analysis in tobacco (Nicotiana tabacum). Some previous work and
genetic resources obtained is relevant to this study, specifically previously characterized
transgenic tobacco lines overexpressing the Vitis vinifera pgip1 (Vvpgip1) gene. These lines
have confirmed PGIP-specific resistance phenotypes against B. cinerea, as well as increased
levels of CAD transcripts in healthy plants. Moreover, preliminary evaluations indicated
increased lignin levels as well as differential expression of several other cell wall genes in these
overexpressing lines (in the absence of infections).
In this study we generated a transgenic tobacco population, overexpressing the native
CAD14 gene, via Agrobacterium transformations. The transgene was overexpressed with the
Cauliflower Mosaic Virus promoter (CaMV 35Sp). The CAD transgenic population was analyzed
for transgene integration and expression and showed active transcription, even from leaves that
normally don’t express CAD to high levels. These lines, together with the untransformed control,
and a representative transgenic VvPGIP1 tobacco line previously characterized with elevated
expression of CAD were used for all further analyses, specifically CAD activity assays of stems
and leaves, as well as whole plant infections with B. cinerea. CAD enzyme activity assays were
performed on healthy uninfected plant lines, without inducing native CAD expression or
resistance phenotypes (i.e. without Botrytis infection). CAD activity was detected in leaves and
stems, but a statistically sound separation between the CAD population and the untransformed
control was only observed in the stems. The CAD assays also confirmed previous results that
indicated that CAD transcription was upregulated in the PGIP line in the absence of infection.
Overall, in all plant lines the stems exhibited 10-fold higher levels of CAD activity than the
leaves, but the transgenic VvPGIP1 line showed a further 2-3-fold increase in CAD activity in the stems, when compared to the untransformed control and the majority of the CAD
overexpressing lines.
Disease assessment by whole plant infections with B. cinerea of the CAD transgenic
plants revealed reduced disease susceptibility towards this pathogen. A reduction in disease
susceptibility of 20 – 40% (based on lesion sizes) was observed for a homologous group of
transgenic lines that was statistically clearly separated from the untransformed control plants
following infection with Botrytis over an 11-day-period. The VvPGIP1 transgenic line displayed
the strongest resistance phenotype, with reduction in susceptibility of 47%. The reduction in
plant tissue maceration and lesion expansion was most pronounced in the VvPGIP1 line
compared to the CAD transgenic plants, while the CAD transgenic plants showed more
reduction than the untransformed control. In combination, the data confirms that CAD
upregulation could lead to resistance phenotypes. Relating this data back to the previously
observed upregulation of CAD in the VvPGIP1-overexpressing lines, the findings from this study
corroborates that increased CAD activity contributes to the observed resistance phenotypes,
possibility by strengthening the cell wall.
In conclusion, this study yielded a characterized transgenic population overexpressing
the CAD14 gene; this overexpression contributed to increased RNA transcription compared to
the untransformed control plant, increased CAD activity (most notably in the stems) and a
disease resistance phenotype against Botrytis. These findings corroborates the current working
hypothesis in our group that PGIPs might have a role in preparing the plant cell for attack by
contributing to specific cell wall changes. The exact mechanisms are still currently unknown and
under investigation. The transgenic lines generated in this study will be invaluable in the
subsequent analyses where these various phenotypes will be subjected to profiling and
accurate cell wall analyses. / AFRIKAANSE OPSOMMING: Nekrotrofiese swamme infekteer en beskadig verskeie ekonomies belangrike gewasse. Dit lei
wêreldwyd tot groot verliese vir die landbousektor. Dit is noodsaaklik om te verstaan hoe plante
reageer teenoor patogene, sodat die siekteweerstand van plante verbeter kan word.
Navorsingshulpbronne het beduidend bygedra tot die kennis van plantreaksies tydens
patogeniese aanvalle, en het sodoende ‘n reeks van weerstandmeganismes, wat die plant
inspan tydens ‘n aanval, geïdentifiseer.
Verskeie patogeniese swamme skei endopoligalakturonases (endoPGs) af tydens plantinfeksie.
Hierdie hidrolitiese ensieme word geïnhibeer deur poligalakturonase-inhiberende
proteïene (PGIPs) wat met die plantselwand geassosieerd is. PGIPs is goed gekarakteriseerd
en al hulle huidiglik bekende funksies is gekoppel aan endoPG inhibisie en die daaropvolgende
opregulering van plant weerstandspaaie. Navorsing op wingerd PGIPs het gewys dat, afgesien
van die feit dat PGIPs goeie antifungiese proteïene is wat lei tot beskerming van die plant teen
Botrytis cinerea wanneer dit ooruitgedruk word, PGIPs ook moontlik addisionele funksies verrig
wat verwant is aan selwandversterking. Hierdie werkshipotese vorm die motivering vir hierdie
studie waarin ‘n sinnamiel alkohol dehidrogenase (SAD) (1.1.1.195) geen geteiken is vir
funksionele analise in tabak (Nicotiana tabacum). Vorige navorsing en genetiese hulpbronne
daardeur verkry is belangrik vir hierdie studie, spesifiek die gekarakteriseerde transgeniese
tabaklyne wat die Vitis vinifera pgip1 (Vvpgip1) geen ooruitdruk. Hierdie lyne besit bevestigde
PGIP-spesifieke weerstandsfenotipes teen B. cinerea, sowel as hoër vlakke van SAD
transkripte in gesonde plante. Voorlopige analises het ook aangedui dat hierdie ooruitdrukkende
lyne hoër lignien vlakke het, asook differensiële uitdrukking van verskeie ander selwandgene (in
die afwesigheid van infeksie).
In hierdie studie is ‘n transgeniese tabakpopulasie gegenereer wat die natuurlike tabak
SAD14 geen ooruitdruk, deur middel van Agrobacterium transformasie. Die transgeen is
ooruitgedruk deur die Blomkool Mosaïek Virus promoter (CaMV 35Sp). Die SAD transgeniese
populasie is geanaliseer vir transgeen integrasie en uitdrukking en het aktiewe transkriptering
getoon, selfs in blare waar daar normaalweg nie hoë vlakke van SAD uitgedruk word nie.
Hierdie lyne, die ongetransformeerde wilde-tipe kontrole sowel as ’n verteenwoordigende
transgeniese VvPGIP1 tabaklyn wat vooraf gekarakteriseerd was met hoë SAD uitdrukking, is
gebruik vir alle verdere analises, spesifiek SAD aktiwiteitstoetse in stingels en blare, asook
heelplantinfeksies met B. cinerea. Aktiwiteitsanalises van die SAD ensiem is gedoen op
gesonde ongeinfekteerde plantlyne, sonder om natuurlike tabak SAD uitdrukking of
weerstandsfenotipes te induseer (dus sonder Botrytis infeksie). SAD aktiwiteit is waargeneem in
beide die blare en stingels, maar ‘n statisties betekenisvolle skeiding is slegs gevind tussen die
SAD populasie en die ongetransformeerde kontrole in die stingels. Die SAD toetse het ook vorige resultate bevestig wat aangedui het dat SAD transkripsie opgereguleer word in die PGIP
lyn in die afwesigheid van infeksie. Die stingels het oor die algemeen ‘n 10-voudige
vermeerdering in SAD aktiwiteitsvlakke getoon in vergelyking met die blare, maar die
transgeniese VvPGIP1 lyn het ‘n verdere 2-3-voudige verhoging in SAD aktiwiteit gehad in die
stingels ,in vergelyking met die ongetransformeerde kontrole en die meerderheid van die SADooruitdrukkende
lyne.
Siekteweerstand ondersoeke deur middel van heelplantinfeksies met B. cinerea van die
SAD transgeniese plante, het verminderde vatbaarheid aangedui ten opsigte van hierdie
patogeen. ‘n Afname in siekte-vatbaarheid van 20 – 40% (gebaseer op wondgroottes) is
waargeneem vir ‘n homoloë groep transgeniese lyne wat statisties betekenisvol geskei kon
word van die ongetransformeerde kontrole plante na infeksie met Botrytis in ‘n infeksietoets wat
11 dae geduur het. Die VvPGIP1 transgeniese lyn het die mees weerstandbiedende fenotipe
gehad, met ‘n afname in siekte-vatbaarheid van 47%. Die afname in plantweefselafbreking en
wondgrootte was die duidelikste in die VvPGIP1 lyn in vergelyking met die SAD transgeniese
plante, terwyl die SAD transgeniese plante ‘n groter afname aangedui het as die
ongetransformeerde kontrole. In kombinasie het die data bevestig dat SAD opregulasie kan lei
tot weerstandbiedende fenotipes. Hierdie data, in vergelyking met die vorige bevinding van
opregulasie van SAD in die VvPGIP1-ooruitdrukkende lyne, bevestig dat hoër SAD aktiwiteit
bydra tot die waargenome weerstandbiedende fenotipes, moontlik deur versterking van die
plantselwand.
Ter afsluiting, hierdie studie het ‘n gekarakteriseerde transgeniese populasie wat die
SAD14 geen ooruitdruk gelewer; hierdie ooruitdrukking het bygedra tot hoër RNA transkripsie in
vergelyking met die kontrole, verhoogde SAD aktiwiteit (veral in die stingels) en siekteweerstandbiedende
fenotipes teenoor Botrytis. Hierdie bevindinge ondersteun die huidige
werkshipotese in ons groep dat PGIPs moontlik ‘n rol speel in die voorbereiding van die plantsel
teen infeksie deur spesifieke selwandveranderinge te veroorsaak. Die spesifieke meganismes is
steeds onbekend en word verder ondersoek. Die transgeniese lyne wat tydens hierdie studie
gegenereer is, sal baie belangrik wees in opvolgende analises waar hierdie verskillende
fenotipes gebruik kan word om die profiel van selwandkomponente, maar ook die akkurate
selwandsamestelling te bestudeer.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/4276 |
| Date | 03 1900 |
| Creators | Mbewana, Sandiswa |
| Contributors | Vivier, Melane, Joubert, Albert, 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 | English |
| Detected Language | English |
| Type | Thesis |
| Format | 54 p. : ill. |
| Rights | University of Stellenbosch |
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