Thesis (MScAgric)--Stellenbosch University, 2003. / ENGLISH ABSTRACT: The resistance of plants to infection by phytopathogenic microorganisms is the
result of multiple defence reactions comprising both constitutive and inducible
barriers. While disease is the exception, such exceptions can be costly and
even devastating. In particular, fungal diseases remain one of the major factors
limiting crop productivity worldwide, with huge losses that need to be weighed
up against massive cash inputs for pesticide treatments.
Part of the defence reactions of plants is the synthesis of
pathogenesis-related proteins, such as the plant hydrolases, glucanases and
chitinases. In recent years, attention has been paid to the implementation of
these proteins in plant transformation schemes. The rationale for this approach
was that these antimicrobial agents not only degrade the main cell wall
components of fungi, but also produce glucosidic fragments that act as elicitors
of the biosynthesis of defence metabolites by the host. Furthermore, since
these active antimicrobial agents are individually encoded by single genes,
these defence systems should and have been shown to be highly amenable to
manipulation by gene transfer.
In this study, yeast glucanases from Saccharomyces cerevisiae were
evaluated for their potential as antifungal proteins. The glucanases tested for
their antifungal activity against Botrytis cinerea were the yeast EXG1 and BGL2
genes, encoding an exoglucanase and an endoglucanase respectively. An in
vitro assay performed on these glucanases indicated that exoglucanase had a
more detrimental effect on B. cinerea hyphal development and growth than the
endoglucanase; the former caused typical disruption of the cells and leakage of
cell material. The yeast exoglucanase was subsequently subcloned into a plant
expression cassette containing the strong constitutive 358 promoter, yielding
plasm ids pEXG1 and pMJ-EXG1. The pMJ-EXG1 construct targeted the
exoglucanase to the apoplastic region with a signal peptide from an
antimicrobial peptide from Mirabilis jalapa, Mj-AMP2. The pEXG1 and
pMJ-EXG1 constructs were mobilised into Agrobacterium tumefaciens to
facilitate the subsequent tobacco transformation, which yielded transgenic tobacco lines designated E and MJE respectively. Transgene integration was
confirmed with southern blot and PCR analyses for both the E and MJE lines.
The expression and heterologous production of the EXG1-encoded
exoglucanase in the E-transgenic lines was shown with northern blots and
activity assays respectively. Moreover, the high level of expression of the yeast
exoglucanase led to a decrease in susceptibility of the E lines to B. cinerea
infection in comparison to the untransformed tobacco controls. An average
decrease in disease susceptibility of 40% was observed in an in planta
detached leaf assay. Crude protein extracts from the E lines were also
analysed in an in vitro quantitive fungal growth assay, inhibiting in vitro fungal
growth by average 20%, thus further confirming the antifungal nature of the
yeast exoglucanase.
Although integration of the MJ-EXG1 expression cassette was confirmed,
no mRNA levels could be detected with northern blot or RT-PCR analysis of the
MJE lines. These lines also did not show any in vitro antifungal activities or a
decrease in susceptibility to B. cinerea infection in the detached leaf assay. It is
suspected that this result is possibly linked to gene silencing, a phenomenon
quite frequently associated with heterologous and/or overexpression of
glucanases in plant hosts. It appears as if the targeted overexpression to the
apoplastic space triggered the gene silencing response, since the intracellularly
overexpressed product was produced and shown to display activity. The yeast
exoglucanase thus joins the list of silenced glucanases in overexpression
studies in plants.
Overall, this study confirmed the antifungal characteristics of the
Saccharomyces exoglucanase and provides valuable information of the
possibility of utilising yeast glucanases in a transgenic environment. A
decrease in the susceptibility of tobacco to B. cinerea infection, as shown by the
overexpressed EXG1-encoded exoglucanases, merits further investigation into
the use of this gene in the engineering of disease-resistant crops. / AFRIKAANSE OPSOMMING: Die weerstand van plante teen infeksie deur fitopatogeniese mikroórganismes is
die resultaat van verskeie meervoudige verdedigingsreaksies wat beide
konstitutiewe en induseerbare versperrings behels. Terwyl siekte die
uitsondering eerder as die reël is, kan sulke uitsonderinge duur en selfs
verwoestend wees. In die besonder is swamsiektes een van die vernaamste
faktore wat gewasproduksie wêreldwyd beperk, met enorme verliese wat teen
kontantinsette vir plaagdoders opgeweeg moet word.
Deel van die verdedigingsreaksie van plante is die sintese van
patogeen-verwante proteïene, soos die planthidrolases, -glukanases en
-chitinases. In die onlangse tyd is aandag geskenk aan die implementering van
hierdie proteïene in plant transformasieskemas. Die grondrede hiervoor was
dat hierdie antimikrobiese agente nie net die hoof selwandkomponente van
swamme kan afbreek nie, maar ook glukosidiese fragmente produseer wat as
ontlokkers van metabolietbiosintese vir die verdediging van die gasheer kan
optree. Aangesien hierdie aktiewe antimikrobiese agente individueel deur
enkele gene enkodeer word, blyk hierdie verdedigingsisteme om hoogs
ontvanklik vir manipulasie deur geenoordrag te wees.
In hierdie studie is die gisglukanase van Saccharomyces cerevisiae vir
hul potensiaal as antifungiese proteïene geëvalueer. Die glukanases wat vir hul
antifungiese aktiwiteit teen Botrytis cinerea getoets is, was die gis EXG1- en
-BGL2-gene, wat onderskeidelik vir "n eksoglukanase en 'n endoglukanase
enkodeer. "n In vitro toets wat op hierdie glukanases uitgevoer is, het aangedui
dat die eksoglukanase 'n meer skadelike effek op die hife-groei en
-ontwikkeling van B. cinerea as die endoglukanase gehad het; eersgenoemde
het die tipiese ontwrigting van die selle en die uitlek van selmateriaal tot gevolg
gehad. Die gis-eksoglukanase is gevolglik in 'n plant uitdrukkingskasset wat die
sterk konstitutiewe 35S promotor bevat, gesubkloneer, wat plamiede pEXG1 en
pMJ-EXG1 opgelewer het. Die pMJ-EXG1-konstruk het die eksoglukanase na
die apoplastiese gebied geteiken deur 'n seinpeptied vanaf "n antimikrobiese
peptied van Mirabilisjalaba, Mj-AMP2. Die pEXG1- en pMJ-EXG1-konstrukte is in Agrobacterium tumefaciens gemobiliseer, wat die gevolglike
tabaktransformasies gefasiliteer het wat die E en MJE transgeniese tabaklyne
onderskeikelik gelewer het. Transgeen-integrasie is deur suidelike klad- en
PKR-analises vir beide die E en MJE lyne bevestig. Die uitdrukking en
heteroloë produksie van die EXG1-enkodeerde eksoglukanase is in die
transgeniese E lyne deur noordelike klad en aktiwiteitstoetse onderskeidelik
aangetoon. Verder het die hoë uitdrukkingsvlak van die gis-eksoglukanase tot
'n vermindering in die vatbaarheid van die E lyne vir B. cinerea-infeksie relatief
tot die ongetransformeerde tabakkontroles gelei. 'n Gemiddelde vermindering
in siektevatbaarheid van 40% is in 'n in planta verwyderde-blaartoets
waargeneem. Ru proteïen-ekstrakte van die E lyne is ook in 'n in vitro
kwantitatiewe swamgroeitoets geanaliseer en het in vitro swamgroei met tot
gemiddeld 20% geïnhibeer, wat dus verder die antifungiese aard van die
gis-eksoglukanase bevestig het.
Alhoewel die integrasie van die pMJ-EXG1 uitdrukkingskasset bevestig
is, kon geen mRNA-vlakke met die noordelike klad- of RT-peR-analises van die
MJE-Iyne waargeneem word nie. Hierdie lyne het ook geen in vitro antifungiese
aktiwiteite of 'n vermindering in die vatbaarheid vir B. cinerea-infeksie getoon
nie, soos in die verwyderde-blaartoets uitgevoer is nie. Dit word vermoed dat
hierdie resultaat moontlik aan geenstilmaking gekoppel is, 'n verskynsel wat
gereeld met heteroloë- en/of ooruitdrukking van glukanases in plantgashere
gekoppel word. Dit blyk dat die ooruitdrukking wat tot die apoplastiese ruimte
geteiken is, tot die geenstilmaking-respons aanleiding gegee het, aangesien die
intrasellulêre ooruitgedrukte produk gemaak is en aktiwiteit getoon het. Die
gis-eksoglukanase word dus deel van die lys van stilgemaakte glukanases in
die ooruitdrukkingstudies van plante.
In die algemeen het hierdie studie dus die antifungiese kenmerke van die
Saccharomyces eksoglukanase bevestig en waardevolle inligting oor die
moontlike gebruik van gis-glukanases in 'n transgeniese omgewing verskaf. 'n
Afname in die vatbaarheid van tabak vir infeksie deur B. cinerea, soos deur die
ooruitdrukking van EXG1-enkodeerde eksoglukanase getoon is, verdien dus
verdere ondersoek van die gebruik van hierdie geen in die skepping van
siekteweerstandbiedende gewasse.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/53634 |
| Date | 12 1900 |
| Creators | Basson, Esmé Maree |
| Contributors | Vivier, M. A., Pretorius, I. S., Van Rensburg, P., Stellenbosch University. Faculty of AgriSciences. Dept. of Plant Pathology. |
| Publisher | Stellenbosch : Stellenbosch University |
| Source Sets | South African National ETD Portal |
| Language | en_ZA |
| Detected Language | English |
| Type | Thesis |
| Format | [xiv], 70 p. : ill. |
| Rights | Stellenbosch University |
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