Polygalacturonase-inhibiting proteins (PGIPs) are cell wall-associated plant proteins that inhibit endopolygalacturonases (endo-PGs) from phytopathogenic fungi. For proteins to confer resistance to invading plant pathogens, it is preferred that they are either associated with the plant cell wall or secreted into the intercellular spaces where they can act almost immediately upon pathogen attack. The bactericidal efficacy of the Hen Egg White Lysozyme (HEWL) has previously been unequivocally demonstrated in transgenic plants; however, most of the protein remains intracellular. It was hypothesized that bean PGIP1, that has previously been expressed correctly in transgenic tomato plants and was found to inhibit the endopolygalacturonase activity of Stenocarpella maydis in a reducing sugar assay, would deliver the HEWL protein to the intercellular spaces due to its inherent translocation to the plant cell wall by means of a translational fusion between bean pgip1 and hewl genes. In this study, the efficacy of such a translational fusion was determined. The bean pgip1-hewl fusion was inserted into the binary vector pCAMBIA2300 and transformed into Nicotiana tabacum cv. LA Burley 21 plants by Agrobacterium-mediated transformation. Phenotypically normal transgenic plants were produced. Stable transgene insertion into the transgenic N. tabacum genomes was verified by PCR and Southern blot analyses. To demonstrate the efficacy of the bean PGIP1-HEWL fusion, independent homogenate and intercellular fluid protein extracts were prepared from transgenic N. tabacum leaf material. Protein extracts prepared so as to enrich for PGIP activity were tested in vitro for inhibition of S. maydis endo-PGs whereas protein extracts for HEWL activity were tested for lysis of Micrococcus luteus cells. Biochemical assays showed that bean PGIP1-HEWL inhibited S. maydis endo-PGs and cleaved M. luteus cell walls sufficiently to suggest that the PGIP1- HEWL fusion was structurally and functionally stable. Total protein extracts from the PGIP-HEWL and HEWL transgenic plants showed similar levels of HEWL specific activity, whereas intercellular fluid samples from PGIP-HEWL transgenic plants showed high activity in contrast to HEWL plants. With the success of showing protein activity in vitro of HEWL in intercellular spaces, bean PGIP1 can be recommended as a vehicle for delivery of other proteins to cell surfaces. Copyright 2006, University of Pretoria. All rights reserved. The copyright in this work vests in the University of Pretoria. No part of this work may be reproduced or transmitted in any form or by any means, without the prior written permission of the University of Pretoria. Please cite as follows: Feltman, NR 2006, Development of the polygalacturonase inhibiting protein (PGIP) for delivery of foreign proteins to the surfaces of plant cells, MSc dissertation, University of Pretoria, Pretoria, viewed yymmdd < http://upetd.up.ac.za/thesis/available/etd-07152008-111131 / > E416/gm / Dissertation (MSc)--University of Pretoria, 2012. / Plant Science / unrestricted

Thesis (MScAgric (Viticulture and Oenology. Wine Biotechnology))--University of Stellenbosch, 2010. / ENGLISH ABSTRACT: Necrotrophic fungi infect many economically important crop plants. This results in great losses in the agricultural sector world-wide. Understanding the nature by which plants respond to pathogens is imperative for genetically enhancing disease resistance in plants. Research tools have significantly contributed to our understanding of how the plant responds to pathogen attack, identifying an array of defence mechanisms used by plants upon attack. Many fungal pathogens secrete endopolygalacturonases (endoPGs) when infecting plants. These hydrolytic enzymes are inhibited by polygalacturonase-inhibiting proteins (PGIPs) associated with plant cell walls. PGIPs are well characterised and their current known functions are all linked to endoPG inhibition and the subsequent upregulation of plant defence pathways. Work on grapevine PGIPs have shown that apart from being efficient antifungal proteins, leading to protection of the plant against Botrytis cinerea when overexpressed, PGIPs might also have additional functions linked to cell wall strengthening. This working hypothesis formed the motivation of this study where a cinnamyl alcohol dehydrogenase (CAD) (1.1.1.195) gene was targeted for functional analysis in tobacco (Nicotiana tabacum). Some previous work and genetic resources obtained is relevant to this study, specifically previously characterized transgenic tobacco lines overexpressing the Vitis vinifera pgip1 (Vvpgip1) gene. These lines have confirmed PGIP-specific resistance phenotypes against B. cinerea, as well as increased levels of CAD transcripts in healthy plants. Moreover, preliminary evaluations indicated increased lignin levels as well as differential expression of several other cell wall genes in these overexpressing lines (in the absence of infections). In this study we generated a transgenic tobacco population, overexpressing the native CAD14 gene, via Agrobacterium transformations. The transgene was overexpressed with the Cauliflower Mosaic Virus promoter (CaMV 35Sp). The CAD transgenic population was analyzed for transgene integration and expression and showed active transcription, even from leaves that normally don’t express CAD to high levels. These lines, together with the untransformed control, and a representative transgenic VvPGIP1 tobacco line previously characterized with elevated expression of CAD were used for all further analyses, specifically CAD activity assays of stems and leaves, as well as whole plant infections with B. cinerea. CAD enzyme activity assays were performed on healthy uninfected plant lines, without inducing native CAD expression or resistance phenotypes (i.e. without Botrytis infection). CAD activity was detected in leaves and stems, but a statistically sound separation between the CAD population and the untransformed control was only observed in the stems. The CAD assays also confirmed previous results that indicated that CAD transcription was upregulated in the PGIP line in the absence of infection. Overall, in all plant lines the stems exhibited 10-fold higher levels of CAD activity than the leaves, but the transgenic VvPGIP1 line showed a further 2-3-fold increase in CAD activity in the stems, when compared to the untransformed control and the majority of the CAD overexpressing lines. Disease assessment by whole plant infections with B. cinerea of the CAD transgenic plants revealed reduced disease susceptibility towards this pathogen. A reduction in disease susceptibility of 20 – 40% (based on lesion sizes) was observed for a homologous group of transgenic lines that was statistically clearly separated from the untransformed control plants following infection with Botrytis over an 11-day-period. The VvPGIP1 transgenic line displayed the strongest resistance phenotype, with reduction in susceptibility of 47%. The reduction in plant tissue maceration and lesion expansion was most pronounced in the VvPGIP1 line compared to the CAD transgenic plants, while the CAD transgenic plants showed more reduction than the untransformed control. In combination, the data confirms that CAD upregulation could lead to resistance phenotypes. Relating this data back to the previously observed upregulation of CAD in the VvPGIP1-overexpressing lines, the findings from this study corroborates that increased CAD activity contributes to the observed resistance phenotypes, possibility by strengthening the cell wall. In conclusion, this study yielded a characterized transgenic population overexpressing the CAD14 gene; this overexpression contributed to increased RNA transcription compared to the untransformed control plant, increased CAD activity (most notably in the stems) and a disease resistance phenotype against Botrytis. These findings corroborates the current working hypothesis in our group that PGIPs might have a role in preparing the plant cell for attack by contributing to specific cell wall changes. The exact mechanisms are still currently unknown and under investigation. The transgenic lines generated in this study will be invaluable in the subsequent analyses where these various phenotypes will be subjected to profiling and accurate cell wall analyses. / AFRIKAANSE OPSOMMING: Nekrotrofiese swamme infekteer en beskadig verskeie ekonomies belangrike gewasse. Dit lei wêreldwyd tot groot verliese vir die landbousektor. Dit is noodsaaklik om te verstaan hoe plante reageer teenoor patogene, sodat die siekteweerstand van plante verbeter kan word. Navorsingshulpbronne het beduidend bygedra tot die kennis van plantreaksies tydens patogeniese aanvalle, en het sodoende ‘n reeks van weerstandmeganismes, wat die plant inspan tydens ‘n aanval, geïdentifiseer. Verskeie patogeniese swamme skei endopoligalakturonases (endoPGs) af tydens plantinfeksie. Hierdie hidrolitiese ensieme word geïnhibeer deur poligalakturonase-inhiberende proteïene (PGIPs) wat met die plantselwand geassosieerd is. PGIPs is goed gekarakteriseerd en al hulle huidiglik bekende funksies is gekoppel aan endoPG inhibisie en die daaropvolgende opregulering van plant weerstandspaaie. Navorsing op wingerd PGIPs het gewys dat, afgesien van die feit dat PGIPs goeie antifungiese proteïene is wat lei tot beskerming van die plant teen Botrytis cinerea wanneer dit ooruitgedruk word, PGIPs ook moontlik addisionele funksies verrig wat verwant is aan selwandversterking. Hierdie werkshipotese vorm die motivering vir hierdie studie waarin ‘n sinnamiel alkohol dehidrogenase (SAD) (1.1.1.195) geen geteiken is vir funksionele analise in tabak (Nicotiana tabacum). Vorige navorsing en genetiese hulpbronne daardeur verkry is belangrik vir hierdie studie, spesifiek die gekarakteriseerde transgeniese tabaklyne wat die Vitis vinifera pgip1 (Vvpgip1) geen ooruitdruk. Hierdie lyne besit bevestigde PGIP-spesifieke weerstandsfenotipes teen B. cinerea, sowel as hoër vlakke van SAD transkripte in gesonde plante. Voorlopige analises het ook aangedui dat hierdie ooruitdrukkende lyne hoër lignien vlakke het, asook differensiële uitdrukking van verskeie ander selwandgene (in die afwesigheid van infeksie). In hierdie studie is ‘n transgeniese tabakpopulasie gegenereer wat die natuurlike tabak SAD14 geen ooruitdruk, deur middel van Agrobacterium transformasie. Die transgeen is ooruitgedruk deur die Blomkool Mosaïek Virus promoter (CaMV 35Sp). Die SAD transgeniese populasie is geanaliseer vir transgeen integrasie en uitdrukking en het aktiewe transkriptering getoon, selfs in blare waar daar normaalweg nie hoë vlakke van SAD uitgedruk word nie. Hierdie lyne, die ongetransformeerde wilde-tipe kontrole sowel as ’n verteenwoordigende transgeniese VvPGIP1 tabaklyn wat vooraf gekarakteriseerd was met hoë SAD uitdrukking, is gebruik vir alle verdere analises, spesifiek SAD aktiwiteitstoetse in stingels en blare, asook heelplantinfeksies met B. cinerea. Aktiwiteitsanalises van die SAD ensiem is gedoen op gesonde ongeinfekteerde plantlyne, sonder om natuurlike tabak SAD uitdrukking of weerstandsfenotipes te induseer (dus sonder Botrytis infeksie). SAD aktiwiteit is waargeneem in beide die blare en stingels, maar ‘n statisties betekenisvolle skeiding is slegs gevind tussen die SAD populasie en die ongetransformeerde kontrole in die stingels. Die SAD toetse het ook vorige resultate bevestig wat aangedui het dat SAD transkripsie opgereguleer word in die PGIP lyn in die afwesigheid van infeksie. Die stingels het oor die algemeen ‘n 10-voudige vermeerdering in SAD aktiwiteitsvlakke getoon in vergelyking met die blare, maar die transgeniese VvPGIP1 lyn het ‘n verdere 2-3-voudige verhoging in SAD aktiwiteit gehad in die stingels ,in vergelyking met die ongetransformeerde kontrole en die meerderheid van die SADooruitdrukkende lyne. Siekteweerstand ondersoeke deur middel van heelplantinfeksies met B. cinerea van die SAD transgeniese plante, het verminderde vatbaarheid aangedui ten opsigte van hierdie patogeen. ‘n Afname in siekte-vatbaarheid van 20 – 40% (gebaseer op wondgroottes) is waargeneem vir ‘n homoloë groep transgeniese lyne wat statisties betekenisvol geskei kon word van die ongetransformeerde kontrole plante na infeksie met Botrytis in ‘n infeksietoets wat 11 dae geduur het. Die VvPGIP1 transgeniese lyn het die mees weerstandbiedende fenotipe gehad, met ‘n afname in siekte-vatbaarheid van 47%. Die afname in plantweefselafbreking en wondgrootte was die duidelikste in die VvPGIP1 lyn in vergelyking met die SAD transgeniese plante, terwyl die SAD transgeniese plante ‘n groter afname aangedui het as die ongetransformeerde kontrole. In kombinasie het die data bevestig dat SAD opregulasie kan lei tot weerstandbiedende fenotipes. Hierdie data, in vergelyking met die vorige bevinding van opregulasie van SAD in die VvPGIP1-ooruitdrukkende lyne, bevestig dat hoër SAD aktiwiteit bydra tot die waargenome weerstandbiedende fenotipes, moontlik deur versterking van die plantselwand. Ter afsluiting, hierdie studie het ‘n gekarakteriseerde transgeniese populasie wat die SAD14 geen ooruitdruk gelewer; hierdie ooruitdrukking het bygedra tot hoër RNA transkripsie in vergelyking met die kontrole, verhoogde SAD aktiwiteit (veral in die stingels) en siekteweerstandbiedende fenotipes teenoor Botrytis. Hierdie bevindinge ondersteun die huidige werkshipotese in ons groep dat PGIPs moontlik ‘n rol speel in die voorbereiding van die plantsel teen infeksie deur spesifieke selwandveranderinge te veroorsaak. Die spesifieke meganismes is steeds onbekend en word verder ondersoek. Die transgeniese lyne wat tydens hierdie studie gegenereer is, sal baie belangrik wees in opvolgende analises waar hierdie verskillende fenotipes gebruik kan word om die profiel van selwandkomponente, maar ook die akkurate selwandsamestelling te bestudeer.