The functional analysis of Vitaceae polygalacturonase-inhibiting protein (PGIP) encoding genes overexpressed in tobacco

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