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The functional analysis of Vitaceae polygalacturonase-inhibiting protein (PGIP) encoding genes overexpressed in tobaccoVenter, Alida 03 1900 (has links)
Thesis (MScAgric (Viticulture and Oenology. Wine Biotechnology))--University of Stellenbosch, 2010. / ENGLISH ABSTRACT: Agriculture worldwide is under great pressure to produce enough food in order to sustain the
ever-growing world population. Among the many challenges faced by food producers, crop
losses and damage caused by fungal plant pathogens is a major problem. The study of fungal
pathogens and the interaction between plants and fungi is therefore essential, and has been
carried out for many years. Much has been learned in this time, but the full mechanisms of the
various modes of fungal attack and plant defence have still not been elucidated.
Many fungi rely on the action of cell-wall degrading enzymes (CWDEs) to breach the
plant cell wall and facilitate access to the nutrients within. CWDEs are among the very first
enzymes to be secreted at the start of fungal attack, and many of them are considered to be
essential pathogenesis factors. Endopolygalacturonases (ePGs) are CWDEs that cleave the
homogalacturonan stretches of the plant cell wall and are vital virulence factors for a number of
fungi, including Botrytis cinerea. An important defence mechanism of plants involves the
inhibition of CWDEs in order to halt or slow down the fungal attack. Plant polygalacturonaseinhibiting
proteins (PGIPs) are cell wall associated CWDE-inhibiting proteins that specifically act
on fungal ePGs. Many different PGIPs from a number of diverse plant species have been
described to date. They are known to have differential inhibition capabilities that often result
from only a few key amino acid changes within the leucine-rich repeat (LRR) active domains.
Previously, the first grapevine PGIP was isolated and characterised from Vitis vinifera
cultivar Pinotage (Vvpgip1). This Vvpgip1 gene was overexpressed in the tobacco species
Nicotiana tabacum, and was shown to be very effective in reducing the susceptibility of tobacco
towards B. cinerea. The combined results confirmed transgene overexpression, increased PGIP
activity and a strong resistance response against Botrytis, leading to the characterisation of
these lines as having PGIP-specific resistance phenotypes. In a subsequent transcriptomic
analysis of these lines it was found that they display differential expression of cell wall
metabolism genes and biochemical characteristics that might indicate possible cell wall
strengthening compared to wild-type tobacco under uninfecting conditions.
The V. vinifera cultivars are all very susceptible to fungal attack, whereas other
grapevine species, specifically the North American Vitis species, are known for their strong
resistance and even immunity against many fungal pathogens. Thirty seven PGIPs have
previously been isolated from these more resistant species. The amino acid sequences of the
active domains of these PGIPs were previously aligned with that of VvPGIP1, and the proteins
were found to be highly homologous with each other and with VvPGIP1. The different nonvinifera
PGIPs separated into 14 subgroups based on their active domain sequences. For this
study, one PGIP from each group was selected for functional analysis in tobacco.
The selected PGIP-encoding genes were transformed into tobacco by means of
Agrobacterium tumefaciens. Analyses of the putatively transformed plantlets were performed to
test for transgene presence, transgene expression, and PGIP activity: final transgenic tobacco
populations consisting of three to twelve individually transformed lines of nine different nonvinifera
PGIPs were obtained. A subset of the resultant transgenic lines was infected with B.
cinerea in two independent whole plant infections over 11-14 days in order to investigate the
disease resistance afforded by the various PGIPs towards this fungus. A line from the
previously characterised VvPGIP1 population was included as reference; all the infections were
contrasted to the WT tobacco. All the infected lines overexpressing the non-vinifera PGIPs
displayed very strong disease reduction in comparison to the WT control: after initial primary
lesion formation, the spread of fungal infection was contained and halted in these lines, while
wild-type tobacco plants were severely affected. Although the VvPGIP1 line displayed the characteristic PGIP-defense response, the non-vinifera PGIP plants displayed smaller lesions,
indicating very strong resistance phenotypes.
The characterised non-vinifera PGIP overexpressing lines, together with the VvPGIP1
line and the WT control were also used to further evaluate the previous observation that
overexpression might lead to changes in expression of cell wall genes. Analysis of the
expression of a xyloglucan endotransglycosylase (xth) gene in the transgenic population
showed that this gene was down-regulated in healthy uninfected tissue from all the transgenic
lines tested. This confirmed previous results and have confirmed in all grapevine PGIP
overexpressing lines tested so far that this gene is downregulated. XTH is typically involved in
cell wall metabolism and specifically in controlling the strength and elasticity of the plant cell
wall. From previous work it is known that downregulation of this gene leads to strengthening of
the wall.
The results obtained in this study showed that the PGIP-specific resistance phenotype
seen for VvPGIP1-overexpressing tobacco could be confirmed in transgenic tobacco
overexpressing non-vinifera PGIPs from more resistant grapevine species as well. The fact that
these PGIPs lines all performed even better than the VvPGIP1 lines in conferring resistance
towards B. cinerea provides an interesting angle for further investigation into the structural
differences between the non-vinifera PGIPs and VvPGIP1. The transgenic lines are also
excellent material to study the in vivo functions of PGIPs further in the context of plant-pathogen
interactions. / AFRIKAANSE OPSOMMING: Die landboubedryf is wêreldwyd onder groot druk om genoeg voedsel te produseer vir die
groeiende wêreldbevolking. Een van die grootste probleme wat die bedryf ondervind, is die
groot skade wat aan gewasse aangerig word deur patogeniese swamme. Dit is dus noodsaaklik
om swamme en die interaksie tussen plante en swamme te bestudeer, en dit word al vir jare
gedoen. Hoewel daar al baie geleer is in hierdie tydperk, is die volle meganismes van die
verskeie maniere hoe swamme aanval en hoe plante hulleself verdedig, nog nie bekend nie.
Verskeie swamme maak staat op die aktiwiteit van selwand-afbrekende ensieme
(SWAEe) om deur die plantselwand te breek en sodoende toegang tot voedingstowwe in die
plantsel te fasiliteer. SWAEe is van die eerste ensieme wat tydens die begin van patogeniese
aanval deur swamme afgeskei word en verskeie SWAEe word as noodsaaklike patogeniese
faktore beskou. Endopoligalakturonases (ePGs) is SWAEe wat die homogalakturoniese dele
van die plantselwand verteer en is noodsaaklike virulensie faktore vir ‘n aantal swamme, onder
andere Botrytis cinerea. ‘n Belangrike weerstandsmeganisme van plante behels die inhibering
van swam SWAEe om sodoende die patogeen-aanval te stop of te vertraag. Die
poligalakturonase-inhiberende proteïne (PGIPs) van plante is selwand-geassosieerde SWAEinhiberende
proteïne wat spesifiek teen swam ePGs optree. Verskeie verskillende PGIPs vanuit
verskillende plantspesies is tot dusver beskryf. Dit is bekend dat hulle differensiële inhiberende
vermoëns het wat dikwels toegeskryf kan word aan slegs ‘n paar belangrike
aminosuurvolgordeverskille in die leusien-ryke herhalende (LRH) aktiewe areas.
Die eerste wingerd PGIP is vantevore geïsoleer vanuit Vitis vinifera kultivar Pinotage
(Vvpgip1) en gekarakteriseer. Hierdie Vvpgip1 geen is ooruitgedruk in die tabakspesie
Nicotiana tabacum en was baie effektief om die weerstand van tabak teen die swam Botrytis
cinerea te verhoog. Die ooruitdrukking van die transgeen, verhoogde PGIP aktiwiteit en goeie
weerstand teen Botrytis cinerea is bevestig, en het gelei daartoe dat die transgeniese VvPGIP1
plantlyne geklassifiseer is as lyne met PGIP-spesifieke weerstandsfenotipes. ‘n
Daaropvolgende transkriptomiese analise van die plantlyne het gewys dat hulle differensiële
uitdrukking van selwand-geassosieerde gene het, asook biochemiese eienskappe, wat ‘n
moontlike selwandversterking aandui in vergelyking met wilde-tipe tabak in die afwesigheid van
infeksie.
Die V. vinifera kultivars is hoogs vatbaar vir swamme, terwyl ander wingerdspesies,
spesifiek die Noord-Amerikaanse spesies, bekend is vir hoë weerstand en selfs immuniteit
teenoor verskeie patogeniese swamme. Sewe-en-dertig PGIPs is vantevore geïsoleer vanuit
hierdie meer weerstandbiedende spesies. Die aminosuurvolgordes van die aktiewe areas van
hierdie PGIPs is vantevore vergelyk met die van VvPGIP1 en dit is gevind dat hierdie proteïne
hoogs homoloog is aan mekaar, sowel as aan VvPGIP1. Die verskillende nie-vinifera PGIPs het
in 14 groepe verdeel na aanleiding van die homologie van hulle aktiewe areas. Vir hierdie studie
is een PGIP vanuit elkeen van hierdie groepe gekies vir verdere funksionele analise in tabak.
Die 14 nie-vinifera PGIP-koderende gene is stabiel oorgedra na tabak deur middel van
Agrobacterium tumefaciens. Die vermeende transgeniese plante is geanaliseer vir die
teenwoordigheid van die transgeen, die uitdrukking daarvan en PGIP aktiwiteit: bevestigde
transgeniese tabak populasies wat wissel van drie tot 12 individuele getransformeerde lyne kon
verkry word vir nege van die verskillende nie-vinifera PGIPs. ‘n Aantal van die transgeniese lyne
is geïnfekteer met B. cinerea in twee onafhanklike heelplantinfeksies vir 11-14 dae om die
siekteweerstand van hierdie PGIPs teenoor die swam te evalueer. ‘n Plantlyn van die
VvPGIP1-populasie is as ‘n verwysing ingesluit en al die infeksies is vergelyk met die wilde-tipe
tabak. Al die geïnfekteerde lyne wat die nie-vinifera PGIPs ooruitdruk het ‘n baie sterk afname in siektesimptome getoon in vergelyking met die wilde-tipe kontrole: na aanvanklikle primêre
lesies gevorm het, is die verspreiding van die infeksie ingeperk en gestop in hierdie lyne, terwyl
die wilde-tipe plante baie erg geaffekteer is. Terwyl die VvPGIP1 lyn ook die tipiese PGIPweerstandsrespons
getoon het, het die nie-vinifera PGIPe kleiner lesies ontwikkel, wat dui op
baie sterk weerstandsfenotipes.
Die gekarakteriseerde nie-vinifera PGIP ooruitdrukkende lyne, asook die VvPGIP1 lyn
en die wilde-tipe kontrole, is gebruik om die vorige waarneming dat die ooruitdrukking kan lei tot
veranderinge in selwandgeen-uitdrukking verder te ondersoek. Analise van die uitdrukking van
‘n xiloglukaan-endotransglikosilase (xth) geen in die transgeniese populasie het getoon dat
hierdie geen afgereguleer is in gesonde, oninfekteerde weefsel van al die transgeniese lyne wat
getoets is. Dit het vorige resultate bevestig en het ook bevestig dat hierdie geen afgereguleer is
in alle wingerd PGIP-ooruitdrukkende lyne wat tot dusver getoets is. XTH is tipies betrokke by
selwandmetabolisme, spesifiek by die beheer van selwandsterkte en selwandelastisiteit. Dit is
uit vorige werk bekend dat die afregulering van hierdie geen lei tot versterking van die
plantselwand.
Die resultate verkry tydens hierdie studie het gewys dat die PGIP-spesifieke weerstand
fenotipe van VvPGIP1-ooruitdrukkende tabak ook bevestig kon word in transgeniese tabak wat
nie-vinifera PGIPs vanuit meer weerstandbiedende wingerdspesies ooruitdruk. Die feit dat
hierdie PGIP lyne almal selfs beter weerstand teen B. cinerea bied as VvPGIP1 lyne is ‘n
interessante invalshoek vir opvolgende ondersoeke na die belang van strukturele verskille
tussen die nie-vinifera PGIPs en VvPGIP1. Hierdie transgeniese lyne is ook uitstekende
hulpbronne om die in vivo funksies van PGIPs verder te bestudeer in die konteks van plantpatogeen
interaksies.
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Evaluation of the role of PGIPs in plant defense responsesBecker, John van Wyk, 1975- 12 1900 (has links)
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.
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Plant defence genes expressed in tobacco and yeastBecker, John van Wyk 03 1900 (has links)
Thesis (MSc (Viticulture and Oenology. Wine Biotechnology))--University of Stellenbosch, 2002. / Pathogen devastation of food products has been the topic of extensive research efforts
worldwide. Fungal infections are foremost amongst these pests, contributing not only to
losses in product yield, but also significantly affecting the quality thereof. It is not surprising
then that producers of these foodstuffs and their derived products continually strive
towards the highest possible product quality. Therefore, it remains imperative that
satisfactory methods are implemented to control these fungal pathogens. The current
strategies are all hampered by drawbacks, and severe crop losses are still experienced.
New technologies are being explored; one such technology is the genetic
transformation of plant species. This method has enabled scientists to introduce foreign
genes, with known functions and predictable outcomes, into plants. Genes identified to be
involved in disease resistance have become the focus of numerous research efforts
concerned with the improvement of the plant's innate defence response. This study aimed
to enhance disease resistance to fungal pathogens by means of the genetic transformation
of two genes previously shown to be involved in disease resistance. These genes encode
polygalacturonase-inhibiting proteins (PGIPs) from Phaseolus vulgaris and resveratrol
synthase from Vitis vinifera. PGIPs specifically inhibit the action of fungal
polygalacturonases (PGs), which are enzymes responsible for the hydrolytic breakdown of
plant cell walls. These enzymes were also found to be the first enzymes that are secreted
by fungal pathogens during infection of the host plant. Additionally, PGIP-PG interaction
results in the existence of molecules involved in the activation of plant defence responses.
Resveratrol, the product of resveratrol synthase, exerts its antifungal action by destruction
of the microbial cellular membranes. These mentioned genes were transformed alone, and
in combination, into Nicotiana tabacum and the resultant transgenic lines were evaluated
for enhanced disease resistance and for possible synergistic effects between the
transgenes.
Several independent transgenic lines were regenerated with genes integrated into the
tobacco genome. Almost all the plants harbouring only pgip or vst1 genes also expressed
these genes at a high frequency. Some non-expressing lines were identified from the
transgenic plants that had integrated both genes, but several lines were obtained
expressing both transgenes. Good correlations were observed between transgene product
activity and enhanced resistance to the fungus Botrytis cinerea in an antifungal in planta
assay. Lines showing the highest PGIP activity and resveratrollevels were more resistant
to the pathogen, leading to disease resistance of up to 80% seven days after inoculation in
comparison to an untransformed control. These lines maintained their strong inhibition,
even three weeks post-inoculation, showing a complete halt in disease development and
fungal growth. These results provide good indications of the efficacy of these transgenes
in the upregulation of plant defence. However, the study will have to be expanded to include even more transgenic lines to elucidate the possible synergistic effects of these
genes.
In an additional pilot study, genes encoding for precursors and for the formation of
resveratrol were introduced into the yeast Saccharomyces cerevisiae. The resultant
recombinant yeast strains were evaluated for their ability to produce the phenolic
substance, resveratrol. This compound has been implicated in beneficial aspects relating
to human health, including positive effects on atherosclerosis and platelet aggregation as a
direct result of its antioxidant and anti-inflammatory activities.
Recombinant yeast strains were constructed that expressed genes coding for
coenzyme A ligase and resveratrol synthase. These strains were shown to be able to
produce the phenolic compound resveratrol from the precursors present in the yeast as
well as from the products introduced with the transformation. The resveratrol was
complexed with an added glucose moiety. These results are extremely positive,
considering the possibility of manipulating wine yeasts to produce resveratrol during the
wine fermentation, thereby adding to the health aspects of both red and white wine. This is
the first report of the production of this compound by the introduction of genes necessary
for its biosynthesis in a foreign host.
This study has confirmed the importance of PGIPs and resveratrol in the effort to
enhance disease resistance in plants through genetic transformation technology. It has
also shown that the health benefits of resveratrol could be exploited more optimally in the
wine industry, by producing wine yeasts with the ability to synthesise this important
antioxidant.
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