Dissertation (PhD)--University of Stellenbosch, 2007. / ENGLISH ABSTRACT: Plants have developed sophisticated means of combating plant diseases. The
events that prepare the plant for, and follow plant-pathogenic interactions, are
extremely complex and have been the topic of intensive investigation in recent
years. These interactions involve a plethora of genes and proteins, and intricate
regulation thereof; from the host and pathogen alike. Studying the contribution of
single genes and their encoded proteins to the molecular dialogue between plant
and pathogen has been a focus of plant molecular biologists.
To this end, a gene encoding a polygalacturonase-inhibiting protein (PGIP)
was recently cloned from Vitis vinifera. These proteins have the ability to inhibit
fungal endopolygalacturonases (ePGs), enzymes which have been shown to be
required for the full virulence of several fungi on their respective plant hosts. The
activity of PGIP in inhibiting fungal macerating enzymes is particularly attractive
for the improvement of disease tolerance of crop species. The VvPGIP-encoding
gene was subsequently transferred to Nicotiana tabacum for high-level expression
of VvPGIP. These transgenic plants were found to be less susceptible to infection
by Botrytis cinerea in an initial detached leaf assay. Also, it was shown that ePG
inhibition by protein extracts from these lines correlated to the observed decrease
in susceptibility to B. cinerea. This study expands on previous findings by
corroborating the antifungal nature of the introduced PGIP by whole-plant, timecourse
infection assays. Six transgenic tobacco lines and an untransformed wildtype
(WT) were infected and the lesions measured daily from day three to seven,
and again at day 15. The transgenic lines exhibited smaller lesions sizes from
three to seven days post-inoculation, although these differences only became
statistically significant following seven days of incubation. At this point, four of the
six lines exhibited significantly smaller lesions than the WT, with reductions in
disease susceptibility ranging between 46 and 69% as compared to the WT. Two
of the lines exhibited disease susceptibility comparable to the WT. In these
resistant plant lines, a correlation could be drawn between Vvpgip1 expression,
PGIP activity and ePG inhibition. These lines were therefore considered to be
PGIP-specific resistant lines, and provided ideal resources to further study the
possible in planta roles of PGIP in plant defense.
The current hypothesis regarding the role(s) of PGIP in plant defense is twofold.
Firstly, PGIPs have the ability to specifically and effectively inhibit fungal
ePGs. This direct inhibition results in reduced fungal pathogenicity. Alternatively,
unhindered action of these enzymes results in maceration of plant tissue and
ultimately, tissue necrosis. Subsequently, it could be shown that, in vitro, the
inhibition of ePGs prolongs the existence of oligogalacturonides, molecules with
the ability to activate plant defense responses. Thus, PGIPs limit tissue damage
by inhibition of ePG; this inhibition results in activation of plant defense responses
aimed at limiting pathogen ingress. Several publications reported reduced susceptibility to Botrytis in transgenic
plant lines overexpressing PGIP-encoding genes. However, none of these
publications could expand on the current hypotheses regarding the possible in
planta roles of PGIP in plant defense. In this study we used transgenic tobacco
lines overexpressing Vvpgip1 as resources to study the in planta roles for PGIP.
Transcriptomic and hormonal analyses were performed on these lines and a WT
line, both before and following inoculation with Botrytis cinerea.
Transcriptomic analysis was performed on uninfected as well as infected
tobacco leaf material utilizing a Solanum tuberosum microarray. From the analysis
with healthy, uninfected plant material, it became clear that genes involved in cell
wall metabolism were differentially expressed between the transgenic lines and
the WT. Under these conditions, it could be shown and confirmed that the gene
encoding tobacco xyloglucan endotransglycosylase (XET/XTH) was
downregulated in the transgenic lines. Additionally, genes involved in the lignin
biosynthetic pathway were affected in the individual transgenic lines. Biochemical
evidence corroborated the indication of increased lignin deposition in their cell
walls. Additionally, phytohormone profiling revealed an increased indole-acetic
acid content in the transgenic lines. These results show that constitutive levels of
PGIP may affect cell wall metabolism in the Vvpgip1-transgenic lines which may
have a positive impact on the observed reduced susceptibilities of these plants.
An additional role for PGIP in the contribution to plant defenses is therefore
proposed. PGIP may directly influence defense responses in the plant leading to
the strengthening of cell walls. This might occur by virtue of its structural features
or its integration in the cell wall. These reinforced cell walls are thus “primed”
before pathogen ingress and contribute to the decrease in disease susceptibility
observed in lines accumulating high levels of PGIP.
Transcriptional and hormonal analyses, at the localized response, were
performed on Botrytis-infected leaf tissue of the transgenic lines and a WT line.
Several Botrytis responsive genes were found to be upregulated in both the WT
and the transgenic lines. Although limited differential expression was observed
between the two genotypes, the analyses identified a gene which was
upregulated two-fold in the transgenic lines, as compared to WT. This was
confirmed by quantitative Real-Time PCR. This gene is involved in the
lipoxygenase pathway, specifically the 9-LOX branch, leading to the synthesis of
the divinyl ether oxylipins colneleic and colnelenic acid, which show inhibitory
effects on Botrytis spore germination. Phytohormone profiling revealed that the
transgenic lines accumulated more of the defense-related hormone pool of
jasmonates. These are formed via the 13-LOX pathway and have been shown to
be important for the restriction of Botrytis growth at the site of infection.
Collectively, the results from the infection analyses indicate that in these
transgenic lines, both branches of the lipoxygenase pathway are differentially
induced at the level of the localized response to Botrytis infection. Similarly, an increased induction of the synthesis of the defense-related hormone salicylic acid
could be observed, although this hormone did not accumulate to significantly
higher levels. These results are the first report of differential induction of a
defense-related pathway in pgip-overexpressing lines and substantiate the
proposal that following ePG inhibition by PGIP, signaling which activates plant
defense responses, takes place.
Taken together, these results significantly contribute to our understanding of
the in planta role of PGIP in plant defense responses. / AFRIKAANSE OPSOMMING: Plante het deur evolusie gesofistikeerde meganismes teen die aanslag van
plantsiektes ontwikkel. Die gebeure wat die plant voorberei, asook dié wat op
plant-patogeen interaksies volg, is uiters kompleks en vorm die kern van verskeie
navorsingstemas die afgelope paar jaar. Etlike plant- én patogeengene en
proteïene is by hierdie interaksies betrokke en aan komplekse
reguleringsprosesse onderworpe. Die bestudering van die bydrae van enkelgene
en hul gekodeerde proteïene tot die molekulêre interaksie tussen ‘n plant en
patogeen is ‘n sterk fokus van plant-molekulêre bioloë.
Met hierdie doel as fokus, is ‘n geen wat vir ‘n poligalakturonaseinhiberende
proteïen (PGIP) kodeer, van Vitis vinifera gekloneer. Hierdie
proteïene beskik oor die vermoë om fungiese endopoligalakturonases (ePG's),
ensieme wat benodig word vir die virulensie van verskeie fungi op hul
gasheerplante, te inhibeer. Die inhibisie van ePG's deur PGIP en die
gepaardgaande verminderde weefseldegradasie is ‘n baie belowende strategie vir
die verbetering van verboude gewasse se patogeentoleransie. Die VvPGIPenkoderende
geen is gevolglik na Nicotiana tabacum oorgedra vir hoëvlakuitdrukking
van VvPGIP. Daar is gevind dat hierdie transgeniese plante minder
vatbaar vir Botrytis cinerea-infeksies was in ‘n inisiële antifungiese toets wat
gebruik gemaak het van blaarweefsel wat van die moederplant verwyder is. Daar
is ook ‘n korrelasie gevind tussen B. cinerea-siekteweerstand en ePG-inhibisie
deur proteïenekstrakte van die transgeniese populasie. Die huidige studie bou
voort op en bevestig vorige bevindinge betreffende die antfungiese aard van die
heteroloë PGIP in die heelplant en oor tyd. Ses transgeniese tabaklyne en 'n
ongetransformeerde wilde-tipe (WT) is geïnfekteer en die lesies is vanaf dag drie
tot sewe, en weer op dag 15, gemeet. Die transgeniese lyne het in die tydperk
van drie tot sewe dae ná-inokulasie kleiner lesies as die WT getoon, alhoewel
hierdie verskille slegs statisties beduidend geword het na sewe dae van
inkubasie. Op daardie tydstip het vier van die ses lyne aansienlik kleiner lesies as
die WT getoon, en verlagings in siektevatbaarheid het, in vergelyking met die WT,
van 46% tot 69% gewissel. Twee van die lyne het siektevatbaarheid getoon wat
vergelykbaar was met dié van die WT. In die siekteweerstandbiedende plantlyne
was daar 'n verband tussen Vvpgip1-ekspressie, PGIP-aktiwiteit en ePG-inhibisie.
Hierdie plantlyne is dus as PGIP-spesifieke siekteweerstandslyne beskou en dien
dus as ideale eksperimentele bronne vir die ontleding van die moontlike in plantafunksies
van PGIP in plantsiekteweerstandbiedendheid.
Die huidige hipotese betreffende die funksie(s) van PGIP in
plantsiekteweerstand is tweeledig. Eerstens het PGIP die vermoë om fungusePG's
spesifiek en doeltreffend te inhibeer. Hierdie direkte inhibisie veroorsaak ‘n
vermindering in patogenisiteit van die fungus op die gasheer. Indien ePG's egter
hulle ensimatiese aksie onverstoord voortsit, sal weefseldegradasie en uiteindelik weefselnekrose die gevolg wees. Daar kon ook bewys word dat die in vitroinhibisie
van ePG's deur PGIP die leeftyd van oligogalakturoniede, molekules wat
die vermoë het om die plantweerstandsrespons aan te skakel, kan verleng. PGIP
het dus nie net die vermoë om ePG's, en dus weefseldegradasie, te inhibeer nie;
maar hierdie inhibisie lei ook daartoe dat plantweerstandsresponse aangeskakel
word met die oog op die vermindering van patogeenindringing.
Verskeie publikasies het reeds gerapporteer oor verminderde Botrytisvatbaarheid
in PGIP transgeniese plantlyne. Geeneen van hierdie publikasies kon
egter uitbrei op die huidige hipotese aangaande die moontlike in planta-funksie
van PGIP in plantsiekteweerstand nie. In hierdie studie is transgeniese tabaklyne
wat PGIP ooruitgedruk gebruik om hierdie moontlike in planta-funksies vir PGIP
uit te klaar. Transkriptoom- en hormonale analises is op hierdie plantlyne en ‘n
WT voor en ná inokulasie met die nekrotroof Botrytis cinerea uitgevoer,.
Transkriptoomanalises is uitgevoer op ongeïnfekteerde, sowel as
geïnfekteerde tabakblaarmateriaal deur gebruik te maak van ‘n Solanum
tuberosum-mikroraster. Die analises met gesonde, ongeïnfekteerde
plantmateriaal het daarop gewys dat gene betrokke by selwandmetabolisme
tussen die transgeniese lyne en die WT verskillend uitgedruk was. Dit kon bewys
word dat, sonder infeksiedruk, die geen wat xiloglukaan-endotransglikosilase
(XET) kodeer, in die transgeniese lyne afgereguleer was. Gene wat betrokke is in
die lignien-biosintetiese pad was ook in die individuele transgeniese lyne
beïnvloed. Biochemiese toetse het ook die aanduiding van verhoogde
ligniendeposisie in die transgeniese lyne se selwande bevestig. Addisionele
fitohormoonprofiele het getoon dat hierdie lyne ook beskik oor verhoogde vlakke
van indoolasynsuur (IAA). Hierdie resultate wys daarop dat konstitutiewe vlakke
van PGIP selwandmetabolisme in die Vvpgip1-transgeniese lyne moontlik kan
beïnvloed, wat plantsiekteweerstand in dié lyne positief kan beïnvloed. Dit wil dus
voorkom asof PGIP 'n bykomende funksie in plantsiekteweerstand het.
Plantweerstandsreponse kan direk deur PGIP beïnvloed word, wat tot die
versterking van plantselwande kan lei; dit kan geskied by wyse van die strukturele
eienskappe van die proteïen of die integrasie daarvan in die selwand. Hierdie
selwande is dus “voorberei” alvorens patogeenindringing plaasvind en kon bydra
tot die verminderde siektevatbaarheid wat waargeneem is in lyne wat hoë vlakke
van PGIP akkumuleer.
Transkriptoom- en hormonale analises is ook uitgevoer op Botrytisgeïnfekteerde
blaarmateriaal van beide die transgeniese lyne en ‘n WT. Verskeie
Botrytis-responsgene is in beide die transgeniese lyne en die WT opgereguleer.
Differensïele geenekspressie tussen die twee genotipes was taamlik beperk,
maar in die analises kon ‘n geen geïdentifiseer word wat tweevoudig in die
transgeniese lyne opgereguleer was in vergelyking met die WT. Hierdie resultaat
is ook bevestig met behulp van die “Real-Time” Polimerasekettingreaksie (PKR).
Hierdie geen is betrokke in die lipoksigenase (LOX) -pad (spesifiek die 9-LOXarm), wat tot die sintese van die diviniel-eter oksilipiene “colneleic-” en
“colnelenic”-suur lei. Daar is al bewys dat hierdie twee verbindings Botrytisspoorontkieming
kan inhibeer. Fitohormoonprofiele van die geïnfekteerde plante
het gewys dat die transgeniese lyne verhoogde vlakke van die poel van
jasmonate wat plantsiekteweerstands-hormone is, ná inokulasie akkumuleer.
Hierdie hormone word in die 13-LOX-arm van die lipoksigenase pad gevorm en is
belangrik vir die beperking van Botrytis by die infeksiesetel. Die resultate van die
analises wat op Botrytis-infeksie volg, dui daarop dat beide arms van die
lipoksigenasepad in die transgeniese lyne verskillend by die lokale respons
geïnduseer word. ‘n Verhoogde induksie van ‘n ander
plantsiekteweerstandshormoon, salisielsuur, kon ook opgemerk word, alhoewel
die totaal geakkumuleerde vlakke nie beduidend hoër was as dié van die WT nie.
Hierdie resultate is die eerste wat onderskeidende induksie van ‘n
siekteweerstandspad in enige van die pgip-ooruitgedrukte plantlyne rapporteer.
Daarmee ondersteun dit ook die hipotese dat, seintransduksie wat
plantweerstandsresponse aanskakel, ná inhibisie van ePG deur PGIP plaasvind.
Die resultate wat met hierdie studie verkry is, dra dus beduidend by tot die
huidige kennis van die in planta-funksie van PGIP in
plantsiekteweerstandsresponse.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/17476 |
| Date | 12 1900 |
| Creators | Becker, John van Wyk, 1975- |
| Contributors | Vivier, M.A., Denby, K.J., 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 | xv, 95 leaves : ill. |
| Rights | Stellenbosch University |
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