Thesis (MScAgric) -- University of Stellenbosch, 2002. / ENGLISH ABSTRACT: Fungi are an extremely diverse group of organisms and, by acting as pathogens, they can
colonise various other organisms, including humans, plants and animals. The effect of this is
usually detrimental, not only to agricultural crops and livestock, but also to human well-being.
The extensive farming of crops and livestock requires persistent control of fungal populations,
commonly through the use of chemical fungicides. However, the exclusive use of fungicides is
no longer a sustainable practice, as a result of serious problems, such as increasing fungicide
resistance in pathogen strains, the high costs of fungicides, as well as concern about the
environment. The search by producers and scientists for alternative control measures is an
ongoing process.
The fungal cell wall consists of polysaccharides that not only playa role in protection of the
fungi, but also in relaying signals for the invasion and infection of susceptible hosts. Chitin, a
polysaccharide composed of N-acteylglucosamine (GleNAc) residues linked by P-1,4 glucosidic
linkages, is one of the major components of the fungal cell wall, where it plays an important role
in the apical growth of the vegetative hyphae.
Chitinases (EC 3.2.1.14) are abundant proteins produced by a variety of microorganisms
and plants and are necessary for the hydrolysis of the chitin polymer. During the invasion of
many plant species by a pathogen, the production of a specific group of proteins, designated
pathogenesis-related (PR) proteins that include chitinases, is induced as part of their defence
response. Due to the facts that pathogenic fungi contain chitin in their cell walls and that plant
chitinases are induced upon pathogen attack, chitinases have been confirmed as an integral and
crucial part of the plant's natural defence response. Chitinases have increasingly been targeted
to upregulate plants' endogenous disease resistance mechanisms through transgenic
overexpression in a variety of hosts.
Several species of fungi, including various Trichoderma spp., are potent biocontrol agents
of plant pathogenic fungi and insects. The antagonistic activities of these biological control
agents towards phytopathogens are based on the secretion of extracellular hydrolytic enzymes,
such as cell wall-degrading chitinase enzymes. However, biological control is not restricted to
naturally occurring biocontrol agents. Through the process of genetic transformation, other
fungal or yeast species can be enhanced to produce their own chitinases or other antimicrobial
substances more effectively in order to yield potent biocontrol agents.
Various types of chitinases have been applied in the production of fungal resistant plants
and some research has been done on the application of chitinases, from a variety of
microorganisms, as biological control agents. In contrast, very little is known about the
antifungal activity of the Saccharomyces cerevisiae chitinase enzyme, encoded by the CTS1-2
gene. The CTS1-2 gene was utilised in this study as a candidate for overexpression in both
yeast and plant expression systems to analyse the ability of the encoding chitinase to inhibit
fungal growth.
The first objective of this study involved the high level expression and optimisation of the
secretion of the CTS1-2 gene in S. cerevisiae to render recombinant yeast with enhanced
antifungal abilities and with possible applications as a biocontrol agent to control plant pathogenic fungi. It was hypothesised that high-level expression and efficient secretion would
be prerequisites in a biocontrol yeast strain. To this end, two strong promoters and terminators
were included in the study and the secretion of the chitinase gene was evaluated by testing
three different secretion signals. The secretion signals included: the native CTS1-2 secretion
signal, the S. cerevisiae mating pheromone a-factor (MFa1) secretion signal, as well as the
Trichoderma reesei f3-xylanase 2 (XYN2) secretion signal. The phosphoglycerate kinase 1
(PGK1) and alcohol dehydrogenase 2 (ADH2) promoters and terminators were employed to
achieve high-level expression.
The results obtained from the analysis of the recombinant yeasts showed that the PGK1
promoter-terminator constructs yielded high level CTS1-2-expressing and chitinase-producing
strains of S. cerevisiae PRY488. The ability of the different secretion signals to efficiently
secrete the overexpressed chitinase was analysed and it was found that the non-native
secretion signals delivered significantly more protein to the extracellular environment. It was
thus evident that the performance of the MFa1 and XYN2 secretion signals was superior to that
of the native secretion signal. The antifungal activities of the recombinant chitinases produced
by these constructs were tested in in vitro assays against Botrytis cinerea. The enzymes led to
a significant reduction in hyphal development, caused by extreme structural damage to the
hyphal tips, the hyphal cell walls as well as the ability of the fungus to form reproductive and
survival structures, thereby confirming the antifungal abilities of this enzyme. The ADH2
promoter-terminator constructs yielded CTS1-2 transcripts, but no chitinase activity could be
detected with any of these strains. The reasons for this still remain unclear.
The second objective of this study was to assess the potential of the yeast chitinase gene to
upregulate defence against fungal infection in planta. In order to elucidate this, the CTS1-2
gene was constitutively overexpressed in tobacco plants, targeting the chitinase both to the
intra- and the extracellular environment. The results obtained showed that the transgenic
tobacco lines regenerated in this study stably integrated the transgene, exhibiting transgene
expression as well as the production of a biologically active yeast chitinase enzyme. The F,
progeny were rigorously tested for resistance to B. cinerea, and both in vitro and in planta
assays confirmed that the yeast chitinase increased the plant's tolerance to fungal infection;
some of the lines showed disease resistance of 65 and 70%. The plants expressing an
extracellularly targeted chitinase gene are still under evaluation. Interesting results are expected
relating to the effect of the chitinase on the plant surface with regards to disease resistance to
fungal pathogens.
In conclusion, the combined set of results from both the yeast and plant overexpression
studies has confirmed the strong antifungal effect of yeast chitinases. The yeast CTS1-2
chitinase could be instrumental in the development of a new generation of yeast strains with
improved antifungal capabilities. This enzyme could also play an important role in genetic
transformation technologies aimed at enhanced disease resistance. / AFRIKAANSE OPSOMMING: Swamme omsluit 'n uiterste diverse groep organismes wat mense, plante en diere deur
patogeniese aksie kan koloniseer. Die uitkoms hiervan op landbougewasse, die veebedryf en
menslike gesondheid is gewoonlik skadelik. Uitgebreide gewas- en veeboerderye benodig
voortdurende beheer van fungiese populasies, tipies deur van chemiese swamdoders gebruik te
maak. Die uitsluitlike gebruik van swamdoders is egter nie meer 'n lewensvatbare praktyk nie,
hoofsaaklik as gevolg van probleme soos die opbou van weerstand van patogeniese rasse teen
swamdoders, die hoë kostes van die middels, asook besorgheid oor die omgewing. Die soektog
na alternatiewe beheermaatreëls deur produsente en wetenskaplikes bly 'n aaneenlopende
proses.
Die swamselwand bestaan uit polisakkariede wat nie net In rol in die beskerming van die
swam speel nie, maar ook betrokke is in die oordrag van aanvals- en infeksieverwante seine in
'n vatbare gasheer. Chitien, 'n polisakkaried bestaande uit N-asetielglukosamien (GlcNAc)
residu's gekoppel deur 13-1,4glukosidiese bindings, is een van die hoofkomponente van die
swamselwand, waar dit 'n belangrike rol in die apikale groei van vegetatiewe hifes speel.
Chitinases (EC 3.2.1.14) is proteïene wat oorvloedig deur 'n verskeidenheid van
mikroërganismes en plante geproduseer word, waar hulle vir die hidrolise van die chitien
polimeer noodsaaklik is. Tydens die infeksie van verskeie plantspesies deur In patogeen, word
die produksie van 'n spesifieke groep proteïene, die sogenaamde patogeen-verwante (PR)
proteïene wat chitinases insluit, as deel van die plant se verdedigingsreaksie geïnduseer. Die
feit dat patogeniese swamselwande chitien bevat en dat plantchitinases tydens infeksie
geïnduseer word, het daartoe gelei dat dit bevestig is dat chitinases In integrale en kritiese deel
van die plant se natuurlike verdedigingsreaksie uitmaak. Chitinases word toenemend geteiken
in pogings om die plant se intrinsieke siekteweerstandsmeganismes te verbeter deur
transgeniese ooruitdrukking daarvan in 'n verskeidenheid van gashere.
Verskeie swamspesies, insluitend verskillende Trichodenna-spesies, is kragtige
bio-antagoniste van plantpatogeniese swamme. Die antagonistiese aksies van hierdie
biologiese beheeragente teenoor fitopatogene is gebaseer op die uitskeiding van ekstrasellulêre
hidrolitiese ensieme, soos die selwandverterende chitinase ensieme. Nietemin is biologiese
beheer nie net tot bio-antagoniste wat natuurlik voorkom beperk nie. Deur die proses van
genetiese transformasie kan ander swam- of gisspesies verbeter word om hul eie chitinases of
ander antimikrobiese substanse meer effektief te produseer, wat aanleiding sal gee tot kragtige
bio-antagoniste.
Verskeie tipes chitinases is al in die produksie van swambestande plante ingespan en
uitgebreide navorsing is gedoen op die toepassing van 'n reeks chitinases, afkomstig van 'n
verskeidenheid van mikroërganismes, as biologiese beheeragente. In teenstelling is baie min
bekend oor die antifungiese aktiwiteite van die Saccharomyces cerevisiae chitinase ensiem, wat
deur die CTS1-2 geen ge-enkodeer word. Die CTS1-2-geen is in hierdie studie gebruik vir
ooruitdrukking in beide gis- en plantuitdrukkingsisteme om die chitinase se vermoë om
swamgroei te inhibeer, te ondersoek. Die eerste oorkoepelende oogmerk van hierdie studie het hoë-vlak uitdrukking en
optimalisering van sekresie van die CTS1-2-geen in S. cerevisiae behels, met die toekomstige
doelwit om 'n rekombinante gis met verbeterde antifungiese eienskappe en met moontlike
toepassings as 'n bio-antagonis teen plantpatogeniese swamme te ontwikkel. Die hipotese was
dat hoë-vlak uitdrukking en voldoende sekresie voorvereistes vir 'n bio-antagonisras is. Omdié
rede is twee sterk promotors en termineerders by hierdie studie ingesluit en is die sekresie van
die chitinase-geen geëvalueer deur drie verskillende sekresieseine te toets. Die sekresieseine
sluit in: die wilde-tipe CTS1-2 sekresiesein, die S. cerevisiae paringsferomoon a-faktor (MFa1)
sekresiesein, en die Trichoderma reesei p-xilanase (XYN2) sekresiesein. Die fosfogliseraat
kinase 1 (PGK1) en alkohol dehidrogenase 2 (ADH2) promotors en termineerders is gebruik om
hoë-vlak uitdrukking te dryf.
Die resultate wat vanaf die analises van die rekombinante giste verkry is, het getoon dat die
PGK1 promotor-termineerder konstrukte hoë-vlak CTS1-2-uitdrukkende en
chitinase-produserende S. cerevisiae PRY488 rasse opgelewer het. Die vermoë van die
verskillende sekresieseine om die ooruitgedrukte chitinase voldoende uit te skei, is geanaliseer,
en daar is gevind dat die heteroloë sekresieseine aansienlik meer proteïene na die
ekstrasellulêre omgewing geloods het. Dit was dus duidelik dat die MFa1 en XYN2
sekresieseine beter as die wilde-tipe sekresiesein presteer het. Die antifungiese aktiwiteit van
die rekombinante chitinases wat deur hierdie konstrukte geproduseer is, is ook in in vitrotoetse
teen Botryits cinerea getoets. Die teenwoordigheid van die ensieme het gelei tot 'n aansienlike
afname in hife-ontwikkeling, veroorsaak deur ekstreme strukturele skade aan die hifepunte, die
hifeselwande, asook die vermoë van die swam om voortplanting- en oorlewingstrukture te vorm.
Die ADH2 promotor-termineerderkonstrukte het CTS1-2 transkripte vertoon, maar geen
chitinase-aktiwiteite kon in hierdie konstrukte waargeneem word nie. Die redes hiervoor is tot op
hede onbekend.
Die tweede oogmerk van hierdie studie was om die potensiaal van die gischitinase om
swaminfeksie in planta teë te werk, te ondersoek. Die CTS1-2-geen is konstitutief ooruitgedruk
in tabakplante, waarin die chitinase na beide die intra- en ekstrasellulêre omgewing geteiken is.
Resultate het getoon dat die geregenereerde transgeniese tabaklyne die transgeen stabiel
geïntegreer het, transgeenuitdrukking vertoon en dat 'n biologies aktiewe chitinase-ensiem
geproduseer is. 'n F1-generasie is aan strawwe toetse onderwerp om weerstand teen B. cinerea
te ondersoek. Beide die in vitro en in planta toetse het bevestig dat die gischitinase die plant se
verdraagsaamheid teenoor swaminfeksie verhoog het; sommige lyne het siekteweerstand van
tussen 65 en 70% getoon. Die plante wat 'n ekstrasellulêre chitinase produseer, word steeds
geëvalueer. Interessante resultate word verwag aangaande die effek van die chitinase op die
plant se oppervlak met betrekking tot siekteweerstand teen swampatogene.
Ten slotte, die gekombineerde stel resultate wat vanaf beide die gis- en
plantuitdrukkingstudies verkry is, het die sterk antifungiese effek van gischitinases bevestig. Die
gis CTS1-2 kan instrumenteel wees in die ontwikkeling van 'n nuwe generasie gisrasse met
verbeterde antifungiese eienskappe. Die ensiem kan ook 'n belangrike rol in genetiese
transformasietegnologieë, wat op verbeterde siekteweerstand gemik is, speel.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/53169 |
| Date | 04 1900 |
| Creators | Carstens, Maryke,1976- |
| Contributors | Vivier, M. A., Pretorius, I. S., Van Rensburg, P., 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 | English |
| Type | Thesis |
| Format | 101 p. : ill. |
| Rights | Stellenbosch University |
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