Thesis (MSc)--University of Stellenbosch, 2002. / ENGLISH ABSTRACT: Seed germination is the most vulnerable time in a plant's life cycle, since the thick protective
seed coat ruptures and the moist and humid soil environment not only favours seed
germination, but also the growth and development of plant pathogens. Infection of plant seeds
during germination, however, is the exception rather than the rule. Plant seeds have
- - -developed a--cemplex preformed defense mechanism that includes anttfungal agents thatdiffuse
into the surrounding environment to form a protective layer around the seed. This
protective layer prevents fungal and bacterial pathogens from infecting the young seedling.
Over the last decade, scientists have studied the defense mechanisms of different
seeds in an effort to understand and ultimately to introduce and/or manipulate these
mechanisms in plants as part of the plant's endogenous disease resistance to pathogens.
Various chemical compounds, peptides and proteins that showed strong in vitro activities
against various fungi were isolated in these efforts. The mere demonstration of in vitro activity
alone, however, is not sufficient to assign a defense role to these antifungal agents. Typically,
mutant plants that have lost the ability to produce the antifungal agent, or mutants that are
overproducing the agent, have been used to correlate the mutant phenotype to either a
decline or increase in disease resistance respectively. Genetic transformation and the
subsequent development of transgenic plants have made an unprecedented impact in this
regard, specifically in understanding the role of specific defense-related proteins and their
interaction with plant pathogens.
In this study, the antifungal peptide, Hs-AFP1, from Heuchera sanguinea, a plant
defensin, was evaluated in a heterologous in planta environment as a defense protein with
potential for engineering disease resistant crops. The in vitro assays performed with Hs-AFP1
against Botrytis cinerea showed antifungal activities of 88% growth inhibition at a
concentration of 8 J,lg/ml of the purified peptide, while inducing a characteristic
hyperbranching effect on the Botrytis hyphae. Tobacco was subsequently transformed with a
construct, pFAJ3068, expressing Hs-AFP1 under the strong constitutive 35S promoter. The
peptide was targeted to the apoplastic region with the signal peptide from Mj-AMP2, an
antimicrobial peptide from Mirabilis jalapa. Due to reports of peptide instability in transgenic
plant systems, two additional constructs were prepared and transformed into tobacco to
anticipate possible Hs-AFP1 instability in the heterologous tobacco environment. A putative
peptide stabilization construct, pHs-EXG1, consisted of a fusion between Hs-AFP1 and the
antifungal exo-glucanase (encoded by EXG1) from Saccharomyces cerevisiae. A control
construct, pMj-EXG1, expressing EXG1 targeted to the apoplastic region with the Mj-AMP2
signal peptide, was also prepared and transformed into tobacco to normalize the background
antifungal activity as a result of the exoglucanase in the fusion construct lines. Tobacco was successfully transformed with pFAJ3068, pHs-EXG1 and pMj-EXG1,
resulting in transgenic tobacco lines designated THs, THE and TME respectively. Transgene
expression was confirmed for the THs and THE transgenic lines. The translation of these
transcripts into proteins was also confirmed with Western blot analysis. Moreover, the
heterologous production of Hs-AFP1 in tobacco led to an increase in disease resistance to
B. cinerea in the THs lines in comparison with the untransformed tobacco controls. An
increase of up to 42% in disease resistance was observed in an in planta detached leaf
assay. Crude protein extracts from the THs lines were also analyzed in an in vitro quantitative
fungal growth assay. This assay confirmed the results obtained with the disease resistance
assay, with crude protein extracts exhibiting up to 40% fungal growth inhibition. The
incubation of B. cinerea in the presence of crude protein extracts from THs lines resulted in
hyperbranching of the fungal hyphae, which is characteristic of Hs-AFP1 activity.
From these analyses it was clear that the heterologously expressed Hs-AFP1 was
quite stable in the transgenic environment. The fusion between Hs-AFP1 and EXG1 did not
increase the stability of Hs-AFP1, but rather led to a loss of the Hs-AFP1 activity. All the
analyses performed showed the THE lines to be reduced in their ability to inhibit fungal
infection in comparison to the THs line. Also, microscopic analysis of the effects of the crude
THE extracts on B. cinerea growth showed no hyperbranching activity, again confirming the
loss of peptide activity due to the fusion to EXG1. This is in agreement with previous work, in
which sarcotoxin 1A was fused to a reporter gene and also lost activity.
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 TME lines. These lines
also did not show any in vitro antifungal activities, probably indicating post-transcriptional
gene silencing. This silencing was overcome in the fusion constructs that were expressed in
the THE plant lines. These lines also showed EXG1 protein activity, as measured by
~-glucosidase assays. Although the THE lines did not serve the functions originally
envisaged, they fortuitously showed that a fusion strategy might stabilize glucanase
expression in a transgenic environment. A variety of glucanases have been shown to be
prone to gene silencing when overexpressed in a plant environment and the yeast glucanase
can now be added to that list if it is not present as a fusion protein.
Overall, this study confirmed that Hs-AFP1 is involved in plant defense systems and
provided valuable information on the stability of small peptides in a heterologous environment.
The positive results obtained with overexpressed Hs-AFP1 on fungal inhibition in this study
merits further investigations into the use of this peptide in the engineering of disease-resistant
crops. / AFRIKAANSE OPSOMMING: Saadontkieming is die mees vatbare tyd vir siekteontwikkeling gedurende 'n plant se
lewenssiklus. Die saadhuid bars en die vogtige grondkondisies bevoordeel nie net
saadontkieming nie, maar ook die groei en ontwikkeling van plantpatogene. Infeksie van
plantsade tydens ontkieming is egter die uitsondering eerder as die reël. Plantsade besit
komplekse -veraeaigingsfueganfsmes-reen moontlike - patoqeeninteksies. Die meqanismes
sluit die produksie van antifungiese agense, wat tydens saadontkieming na die omliggende
omgewing diffundeer om 'n beskermende sone om die ontkiemende saad te vorm, in. Die
gevolglike antifungiese sone beskerm die saad teen infeksie deur bakterieë en swamme.
Gedurende die laaste dekade het navorsers baie aandag aan die bestudering van
plantsaadverdedigingsmeganismes gegee. Dié kennis word gebruik om die verdedigingsmeganismes
beter te verstaan, asook om dié meganismes te manipuleer en/of oor te dra aan
plantspesies met inherente swak weerstandsmeganismes wat gereeld aan
plantpatogeeninfeksies onderhewig is. Navorsing op plantsade het tot die isolasie van
verskeie chemiese agense, peptiede en proteïene, wat sterk in vitro aktiwiteite teen 'n wye
reeks swampatogene vertoon, gelei. Die vermoë van dié agense om swamme in 'n in vitro
omgewing te inhibeer, is alleen egter nie 'n bewys dat hulle 'n rol in plantverdeging speel nie.
Studies waar mutante gebruik word, is gewens om addisionele bewys te lewer dat die
substanse 'n rol in plantverdediging vervul. Sodanige mutante sluit plantlyne, waarin die geen
van belang gemuteer is of ooruitgedruk word om so die rol van die geen in 'n in planta
omgewing te bepaal in. In hierdie toepassings het genetiese transformasie en die daarstelling
van transgeniese plante 'n ongeëwenaarde bydrae gelewer.
In dié studie is die antifungiese peptied, Hs-AFP1, wat aan die peptiedgroep van plant-
"defensins" behoort en van Heuchera sanguine a afkomstig is, in 'n heteroloë in planta
omgewing geëvalueer as 'n verdedigingspeptied met die potensiaal om in die generering van
transgeniese siektebestande gewasse gebruik te word. Die antifungiese aktiwiteit van
Hs-AFP1 is teen Botrytis cinerea in 'n in vitro reaksie geëvalueer, waar die toediening van
8 ,",g/mlgesuiwerde Hs-AFP1 peptied aanleiding gegee het tot 'n 88% afname in hifegroei van
B. cinerea. Hipervertakkings van swamhifes, 'n kenmerkende eienskap van Hs-AFP1
aktiwiteit, kon duidelik waargeneem word. Tabakplante is voorts getransformeer met 'n
konstruk, pFAJ3068, wat die koderende geen van Hs-AFP1 onder die sterk konstitutiewe
CaMV 35S promotor bevat het. Die peptied is met behulp van die seinpeptied wat afkomstig
is van die Mirabilis jalapa antimikrobiese peptied, Mj-AMP2, na die apoplastiese omgewing
geteiken. Voorheen is gerapporteer dat transgeniese peptiede in die heteroloë omgewing
soms onstabiel is. Dit het gelei tot die generering van twee addisionele konstrukte om die
moontlikheid van peptiedonstabiliteit te ondervang. 'n Stabiliseringskonstruk, pHs-EXG1, bestaande uit In versmelting tussen Hs-AFP1 en In antifungiese eksoglukanase van
Saccharomyces cerevisiae, gekodeer deur EXG1, is in tabakplante getransformeer. In
Kontrolekonstruk, pMj-EXG1, met die EXG1-geen saam met die Mj-AMP2-seinpeptied, is ook
voorberei en in tabakplante getransformeer. Dit is gebruik om die antifungiese aktiwiteit van
die eksoglukanase in die antifungiese aktiwiteitstoetse van die stabiliseringskonstruk te
kwantifiseer en te normaliseer.
Tabak is suksesvol met pFAJ3068, pHs-EXG1 en pMj-EXG1 getransformeer, wat
onderskeidelik gelei het tot die sogenaamde THs, THE en TME transgeniese tabaklyne.
Transgeentranskripsie en -translasie in die THs en THE tabaklyne is onderskeidelik deur
Noordelike- en Westelike-kladanalises bevestig. Die aktiewe uitdrukking van Hs-AFP1 het die
vermoë van tabakplante om B. cinerea infeksies te weerstaan, met tot 42% verhoog in
vergelyking met ongetransformeerde kontrole tabakplante tydens 'n in planta
siekteweerstandstoets. Totale proteïenekstrakte van THs tabaklyne is voorts ook in In in vitro
inhibisietoets geëvalueer, wat gelei het tot resultate wat goed met dié van die in planta toetse
ooreenstem. Die totale proteïenekstrakte het swamgroei met 40% geïnhibeer en die
kenmerkende hipervertakking van Hs-AFP1-aktiwiteit is ook mikroskopies waargeneem.
Resultate wat verkry is vanaf al die analises wat op die transgeniese THs tabaklyne
uitgevoer is, het aangedui dat Hs-AFP1 baie stabiel in die heteroloë tabakomgewing is en
peptiedstabiliteit was dus nie In probleem, soos verwag is nie. Die fusie tussen Hs-AFP1 en
EXG1 het dus nie die stabiliteit van die reeds stabiele Hs-AFP1 peptied verder verbeter nie,
maar het wel tot die verlies van Hs-AFP1 aktiwiteit gelei. Die antifungiese analises van die
THE tabaklyne het verder bevestig dat dié lyne selfs swakker inhibisie van B. cinereainfeksies
tot gevolg gehad het, as ongetransformeerde tabakplante. Mikroskopiese analises
van totale THE proteïenekstrakte het voorts ook geen kenmerkende hipervertakkings in die
swamhifes vertoon nie, wat alles daarop dui dat die Hs-AFP1-deel van die fusieproteïen as
gevolg van die fusie met EXG1 geïnaktiveer is. Dié resultaat is in lyn met vorige navorsing,
wat getoon het dat In ander peptied, sarcotoxin 1A, sy antifungiese aktiwiteit verloor indien dit
met In verklikkergeen versmelt word.
Alhoewel integrasie van die pMj-EXG1-konstruk in die TME-tabaklyne bevestig is, kon
geen mRNA met Noordelike-klad- of trutranskriptase-PKR (RT-PKR)-analises waargeneem
word nie. Die TME plant het ook geen antifungiese aktiwiteit in in vitro toetse getoon nie en dit
het geblyk dat die pMj-EXG1-konstruk aan geenafskakeling in die heteroloë tabakomgewing
onderworpe was. Dié afskakelingseffek is egter in die THE plante oorkom, aangesien
laasgenoemde sterk EXG1 proteïenaktiwiteit met J3-glukosidase aktiwiteitstoetse vertoon het.
Alhoewel die THE plante nie die stabiliteit van Hs-AFP1 verbeter het nie, het dit onwerwags
tot die stabilisering van EXG1 in In heteroloë omgewing gelei. Versmeltingstegnologie kan
dus moontlik gebruik word as 'n strategie om ander glukanases, wat bekend is vir
geenafskakeling in transgeniese omgewings, heteroloog uit te druk. In die geheel gesien, het dié studie getoon dat Hs-AFP1 'n onbetwiste rol in
plantverdedigingsmeganismes speel en daar is voorts ook meer kennis oor die stabiliteit van
peptiede in 'n heteraloë plantomgewing ingewin. Die positiewe resultate t.o.v. die verhoogde
siekteweerstand in die transgeniese THs plantlyne regverdig ook die verdere bestudering van
dié peptied om transgeniese siekteweerstand in gewasse te bewerkstellig.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/52983 |
| Date | 04 1900 |
| Creators | De Beer, Abre |
| Contributors | Vivier, M. A., Pretorius, I. S., Stellenbosch University. Faculty of AgriSciences. Dept. of Viticulture and Oenology. |
| Publisher | Stellenbosch : Stellenbosch University |
| Source Sets | South African National ETD Portal |
| Language | en_ZA |
| Detected Language | English |
| Type | Thesis |
| Format | 84 p. : ill. (some col.) |
| Rights | Stellenbosch University |
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