Endopolygalacturonases (PGs) are the first cell wall degrading enzymes that are produced when pathogenic fungi encounter the host cell wall (Albersheim and Anderson, 1971). The role that these enzymes play in pathogenicity has been investigated for numerous pathogenic fungi. Although the results are not conclusive, there is evidence for some fungi that these enzymes are significant for their pathogenecity. Furthermore, plants contain polygalacturonase inhibiting proteins (PGIPs) in their cell walls, which are able to inhibit PGs (De Lorenzo et al, 2001; 2002). Colletotrichum SHK2148 is a pathogenic fungus causing anthracnose of lupin plants in South Africa. The identity of the fungus has been described as Colletotrichum tortuosum (Koch, 1996). However, this was based on morphological evidence only. Thus, the classification of the South African lupin- associated Colletotrichum isolates was re-assessed by comparing Colletotrichum SHK2148 on a morphological and molecular level to the recently described Colletotrichum lupinispecies (Nirenberg et al, 2002) as well as previously described Colletotrichum acutatum lupin anthracnose isolates (Talhinas et al, 2002). Based on the culture morphology, ITS and <font face=”symbol”>b</font>â-tubulin sequence data, it was concluded that Colletotrichum SHK2148 groups with C. lupini, more specifically, C. lupini var. setosum. The fungus, renamed Colletotrichum lupini SHK2148, was evaluated for its PG activity in pectin media (pH 5) over a 12 day growth period by using an agarose diffusion assay. The specific PG activity reached its highest level after three days, whereafter it decreased. Previous studies performed at the ARC, revealed that the fungus produced PG activity and this crude activity was inhibited by a PGIP produced in apple. A study was launched to isolate and characterise the gene(s) responsible for PG production. PG gene sequences from Colletotrichum gloeosporioides f.sp. malvae and Colletotrichum lindemuthianum were compared and conserved regions were identified from which primers were designed to amplify a fragment of a PG gene from C. lupini SHK2148. Inverse PCR was used to resolve the 5’ and 3’ sequences of the PG gene whereafter a complete copy of the gene was isolated from the genome of the fungus and characterised. The isolated gene was approximately 1Kb, contained a single intron of 59 bp and was very similar to the PG gene from C. gloeosporioides f.sp. malvae (cmpgII) as well as one of the PG genes (clpg2) from C. lindemuthianum. Southern blot analyses revealed that the gene was present as a single copy in the genome of the fungus. The in vitro expression of the PG gene from C. lupini SHK2148, grown in pectin media (pH 5), was investigated via northern blot analyses as well as RT-PCR, which revealed that the gene was expressed in the same time period that the highest PG activity was observed. A full cDNA copy of the PG gene was isolated using mRNA harvested from mycelia that was grown for 4 days on pectin. The cDNA copy confirmed the predicted intron position of the previously isolated genomic PG gene. Due to the unavailability of a full cDNA copy of the C. lupini SHK2148 PG gene at the time when expression studies were initiated, a complete cDNA copy was constructed by swapping an internal cDNA PG fragment with its counterpart in the complete genomic PG gene copy. The resulting cDNA PG copy was used as a template from which PG constructs were prepared for expression in Pichia pastoris. Constructs containing the PG gene with its native signal peptide, the PG gene with the β-MF signal peptide factor as well as hybrid constructs where the N terminal part of the mature PG proteins of Fusarium moniliforme and C. lupini SHK 2148 were exchanged, were transformed into P. pastoris . No PG activity was observed with an agarose diffusion assay for any of the Pichia clones. SDS-PAGE analyses were used to evaluate total protein isolations from the P. pastoris clones. The supernatant and cells of the clones were subjected to western blot analyses using antibodies directed againstAspergillus niger PG as well as F. moniliforme PG. The only positive hybridisation signal was observed between the A. niger antibody and a protein in supernatant extracts of the P. pastoris clones. However, the size of the hybridising band was very large. This could be due to glycosylation of the C. lupini SHK 2148 PG in P. pastoris, although the size increase is unusually large. The results indicated that it is unlikely that the C. lupini SHK 2148 PG was expressed in P. pastoris transformed with any of these constructs. Copyright / Dissertation (MSc)--University of Pretoria, 2010. / Plant Science / unrestricted

Cell wall loosening and degradation are important processes in major stages of plant development including fruit ripening. Three main mechanisms have been proposed to contribute towards cell wall polysaccharide degradation in vivo: enzymic hydrolysis by endopolygalacturonase (EPG), enzymic elimination by pectate lyase (PL), and non-enzymic scission by hydroxyl radicals (•OH). However, little idea as to which of these three mechanisms predominates in homogalacturonan degradation especially during fruit ripening. This study presents an attempt to discover the respective contribution of those three mechanisms of attack. The strategy used to achieve the objective of this study was to identify and measure homogalacturonan molecules that exhibit symptoms of each mechanism of attack. A method that was developed in this study is a fluorescent labelling method mainly to study the •OH attack on pectic polysaccharides. This labelling method is based on the ability of 2-aminoacridone (2-AMAC) to reductively aminate oxo groups of sugar moieties followed by exhaustive digestion with Driselase. In a model in-vitro experiment, the developed novel fluorescent labelling method, when applied to homogalacturonan, that had been attacked by •OH (Fenton reagent), produced at least three fluorescent ‘fingerprint’ compounds, separable by high-voltage paper electrophoresis (HVPE) based on their charge/mass properties at pH 6.5 and also by high pressure liquid chromatography (HPLC) on a C18 column with a fluorescence detector at λem= 520 nm. These fingerprint compounds include: a monomer, 1A*; a dimer, 2A*; and an unidentified compound, X*. In-vivo application with alcoholinsoluble residue (AIR) of seven species of fruit (pear, mango, banana, apple, avocado, strawberry and strawberry tree fruit) at three stages of softening produced at least two fluorescent fingerprint compounds: a monomer, 1AF and a dimer, 2AF. XF, an interesting compound found in a few samples in in-vivo experiments, showed electrophoretic mobility similar to X*; however, the retention time of this compound on HPLC did not agree with that of X*. 2AF was suggested to be exclusive evidence for •OH attack in vivo while 1AF was suggested to be a useful evidence not only to reveal •OH attack but also to reveal EPG and PL attack on pectic polysaccharides during fruit softening. HVPE and HPLC results showed an increasing pattern of 2AF in mango, banana, avocado and strawberry tree fruit, which indicated progressive •OH attack on pectic polysaccharides during the softening process. There was no clear evidence of 2AF at any stage of softening in apple and strawberry, which may suggest that fruit softening in apple and strawberry was not associated with •OH attack. On the other hand, HVPE analysis of 1AF showed and increasing pattern in pear, mango, banana, avocado and strawberry tree fruit, which may indicate EPG, PL and/or •OH attack during fruit softening. Production of these fluorescent fingerprint compounds provides good evidence for •OH attack on pectic polysaccharides, and has the potential to give useful information for EPG and PL attack in vivo.

Thesis (MSc)--University of Stellenbosch, 2011. / ENGLISH ABSTRACT: Plants are constantly exposed to biotic and abiotic stress inducing factors that threaten their existence. Biotic factors such as pathogens are the cause of huge yield losses to crop plants worldwide with fungal pathogens debatably constituting the worst damage. Fungal pathogens such as Botrytis cinerea, which has a wide host range, release cell wall degrading enzymes called endopolygalacturonases (ePGs) during plant infection. These ePGs break down the pectin component of the cell wall, thus providing an entry route, as well as nutrients for the fungus. Plants have evolved mechanisms to counteract and suppress the action of the ePGs. This is achieved through the action of cell wall associated proteins called polygalacturonaseinhibiting proteins, PGIPs. PGIPs directly inhibit ePGs and their inhibitory action also prolongs the existence of longer chain oligogalacturonide residues which are believed to elicit a cascade of defence responses. In grapevine, a PGIP encoding gene, VvPGIP1, was previously isolated and characterised. VvPGIP1, as well as nine non-vinifera grapevine PGIPs have been expressed in tobacco and shown to be potent antifungal proteins that caused the transgenic tobacco to have strong resistance phenotypes against Botrytis in whole plant infection assays. Following on the tobacco study, two of the non-vinifera PGIPs were expressed in cultivars of the susceptible Vitis vinifera. Characterisation of the putative transgenic population showed that transgene integration was successful, the transgenes were being expressed and there were at least 29 transgenic lines with independent integration events. The transgenic lines were confirmed to have active PGIPs (transgene-derived) in their leaves. Crude protein extracts from 22 lines exhibited 100% inhibition against crude B. cinerea PGs (BcPGs). The plant lines with positive transgene integration, expression, independent integration events and exhibiting 100% transgene-derived PGIP activity were further selected for whole plant and detached leaf antifungal assays where they were challenged with B. cinerea. The whole plant infection assay showed that expression of the non-vinifera PGIPs in V. vinifera promotes susceptibility to B. cinerea, not resistance. This surprising result could perhaps be explained by a quicker and stronger recognition between the pathogen and the host and the stronger activation of defence responses in the host. A more active hypersensitive response in the host would benefit Botrytis being a necrotroph. The type of lesions and the onset and speed of lesion development observed on the transgenics lines versus the wild type support this possibility. Knowledge gaps with regards to the efficiency of the ePG inhibition by the nonvinifera PGIPs during infection of grapevine tissue; the potential changes that might be caused by expressing PGIPs in a grapevine host with a native PGIP with high homology to the transgenes (including potential gene silencing) and the potential impact on defence signalling and defence responses all provides further avenues of study to elucidate this very interesting phenotype further. Overall, this study provides a comprehensively characterised population of transgenic plants that provides useful resources for in vivo analysis of PGIP function in defence, where the host plant harbours a native copy of the PGIP encoding gene. / AFRIKAANSE OPSOMMING: Plante word voortdurend blootgestel aan biotiese en abiotiese faktore, wat stres veroorsaak en hul bestaan bedreig. Biotiese faktore, soos patogene, veroorsaak groot verliese in wêreldwye gewasopbrengste, met swampatogene wat moontlik die grootste skade veroorsaak. Swampatogene, soos Botrytis cinerea, wat ‘n wye reeks gasheerplante kan infekteer, stel selwand-afbrekende ensieme tydens plantinfeksie vry, wat as endo-poligalakturonases (ePG’s). bekend staan. Hierdie ePG’s breek die pektienkomponent van die selwand af, wat gevolglik as ‘n ingangspunt dien,asook voedingstowwe vir die swam verskaf. Plante het meganismes ontwikkel om die aktiwiteit van hierdie ePG’s te bekamp en te onderdruk. Die aktiwiteit van die selwand-geassosieërde proteïene, genaamd poligalakturonase-inhiberende proteïene (PGIP’s), speel hier ‘n rol. PGIP’s inhibeer ePG’s direk en hul inhiberende aktiwiteit verleng ook die bestaan van langketting oligogalakturoniedresidu’s, wat blykbaar ‘n kaskade van weerstandsreaksies kan inisieer. ‘n PGIP-koderende geen, VvPGIP1, is voorheen uit wingerd geïsoleer en gekarakteriseer. VvPGIP1, asook nege nie-vinifera wingerd-PGIP’s is voorheen in tabak uitgedruk en bevestig as proteïene met sterk anti-swamaktiwiteit, soos bevestig deur die bevinding dat die transgeniese tabak ‘n weerstandsfenotipe teen Botrytis in heelplant-infeksietoetse het. Ná die tabakstudie is twee van die nie-vinifera PGIP’s uitgedruk in vatbare V. vinifera-kultivars. Karakterisering van die vermeende transgeniese bevolking het getoon dat die transgeen-integrasie suksesvol was, dat die transgeen uitgedruk word en dat daar ten minste 29 transgeniese lyne met onafhanklike integrasie gebeurtenisse geskep is. Daar is verder bevestig dat die transgeniese lyne aktiewe PGIP’s (transgeen-afkomstig) in hul blare het. Ongesuiwerde proteïenekstrakte van 22 lyne het 100% inhibisie teen ‘n mengsel van ongesuiwerde B. cinerea PGs (BcPGs) getoon. Die plantlyne met positiewe transgeenintegrasie en -uitdrukking, asook onafhanklike integrasiegebeure en wat 100% transgeen-afkomstige PGIP-aktiwiteit getoon het, is verder aan heel-plant en verwyderde blaarswaminfeksies met B cinerea onderwerp. Die heelplantinfeksietoetse het getoon dat uitdrukking van nie-vinifera PGIP’s in V. vinifera ‘n toename, in plaas van ‘n afname, in vatbaarheid teen B. cinerea veroorsaak. Hierdie verbasende resultaat kan moontlik toegeskryf word aan ‘n vinniger en sterker herkenningsreaksie tussen patogeen en gasheer en die moontlike sterker stimulering van weerstandsreaksies in die gasheer. ‘n Meer aktiewe hipersensitiewe reaksie in die gasheer sal tot die voordeel van Botrytis, wat ‘n nektrotroof is, wees. Die tipe letsel, asook die aanvang en spoed van letselontwikkeling wat waargeneem is in transgeniese lyne teenoor die wilde-tipe ondersteun hierdie moontlikheid. Gapings in kennis ten opsigte van die doeltreffendheid van die ePG-inhibisie deur die nievinifera PGIP’s tydens infeksie van wingerdweefsel, die moontlike veranderinge (insluitend ‘n moontlike geenuitdowingseffek) wat veroorsaak kan word deur die uitdrukking van PGIP-gene in ‘n kultivar met ‘n inheemse en baie homoloë PGIP-geen, kon ‘n invloed op weerstandseine en weerstandsreaksies gehad het. Hierdie aspekte lewer verdere studiemoontlikhede om hierdie interessante fenotipe verder te verklaar.Algeheel lewer hierdie studie ‘n breedvoeriggekarakteriseerde bevolking trangeniese plante, wat dien as nuttige hulpbronne vir in vivoanalise van PGIP se funksie in siekteweerstandbiedendheid, veral waar die gasheerplant ‘n inheemse kopie van die PGIP-koderende geen huisves.

O maior desafio para a produção de bioenergia continua sendo a precisa desconstrução da parede celular de vegetal. Tendo em vista viabilizar a produção de etanol de segunda geração (2G) em larga escala, bem como otimizar o procedimento de produção do etanol de primeira geração (1G), estudos vêm sendo desenvolvidos com foco na produção de enzimas lignocelulósicas, principalmente as de origem fúngica. Esses estudos visam o aperfeiçoamento de coquetéis enzimáticos, compostos principalmente por enzimas que atacam celulose e hemiceluloses. No entanto, sabemos que as paredes celulares vegetais possuem também pectinas e recentemente foi demonstrado que pectinases podem melhorar sensivelmente o processo de hidrólise (até 30&#37;). Após o levantamento de dados referentes a diferentes enzimas pectinolíticas denominadas endopoligalacturonases (EPGs) já caracterizadas e catalogadas no banco de dados CAZy, foi possível verificar que aquelas produzidas por fungos filamentosos contabilizam mais de 65&#37;. A partir disso foi realizada uma análise filogenética de todas as EPGs caracterizadas produzidas por diferentes organismos. O resultado demonstrou que enzimas dessa família produzidas por bactérias também com interesse biotecnológico são mais próximas de plantas do que dos próprios fungos. Além disso, a proporção de enzimas caracterizadas de bactérias e plantas era significativamente menor que o observado para EPGs de fungos. Isso evidenciou uma lacuna no conhecimento sobre enzimas de plantas, ainda pouco exploradas no contexto de biotecnologia e bioenergia. Este trabalho apresenta a clonagem, expressão heteróloga e caracterização da EPG de cana-de-açúcar recombinante. O principal objetivo é obter informações a respeito de uma enzima com atividade chave na degradação de parede celular, haja vista ter sido ela isolada de um processo de ataque endógeno à pectina da própria cana-de-açúcar. Como resultado, a endopoligalacturonase de Saccharum sp. foi eficientemente expressa em sistema de expressão heterólogo com Pichia pastoris X33. O clone produtor selecionado gerou os maiores valores de proteínas totais no sobrenadante de cultivo com 96 horas de indução com metanol. A partir do extrato bruto do sobrenadante coleta, os melhores valores de atividade enzimática foram obtidos quando da incubação por 22 horas a 45 C em tampão acetato de sódio pH 5,0. Sais, detergentes e quelantes atuaram como inibidores da atividade enzimática e, por outro lado, o agente redutor ditiotreitol (DTT) foi responsável pelo incremento de atividade enzimática. Por fim, os parâmetros cinéticos obtidos para a enzima caracterizada indicam a possibilidade de utilizá-la como complemento de coquetéis enzimáticos, conhecidamente pobres em pectinases. / The biggest challenge for bioenergy production remains the disassembly of the extracellular matrix of some plant species. For the large-scale production of second generation ethanol (2G), and optimizing the first generation ethanol (1G) production process, studies have been developed focusing on the production of lignocellulosic enzymes, especially those of produced by fungi. These studies aim at the improvement of enzymatic cocktails, composed mainly of enzymes that are capable of attacking cellulose and hemicelluloses. However, it is a known fact that the plant cell walls also contain pectins and it has been recently demonstrated that pectinases could significantly improve the hydrolysis process (up to 30&#37;). After an analysis of different pectinolytic enzymes known as endopoligalacturonases (EPGs), already characterized and cataloged under the CAZy database, it was possible to verify that fungi enzymes account for more than 65&#37; of all of the characterized EPGs. The result has shown that enzymes from this family produced by bacteria - also with biotechnological interest - are closer to plants than fungi. Besides, the proportion of characterized enzymes from bacteria and plants was significantly lower than that observed for fungal EPGs. This evidenced a gap in knowledge about plant enzymes, still little explored in the context of biotechnology and bioenergy. This work presents the cloning, heterologous expression, and characterization of recombinant sugarcane EPG. The primary objective is to obtain information about an enzyme with key activity in cell wall degradation since it has been isolated from a process of an endogenous attack on sugarcane pectin itself. As a result, the endopoligalacturonase of Saccharum sp. was efficiently expressed through the heterologous expression system with Pichia pastoris X33. The selected clone generated the highest total protein values in the culture supernatant at 96 hours of methanol induction. From the crude extract of the supernatant collected, the best enzyme activity values were obtained upon incubation for 22 hours at 45&#176;C in pH 5.0 sodium acetate buffer. Salts, detergents, and chelators acted as inhibitors of the enzymatic activity and, on the other hand, the reducing agent dithiothreitol (DTT) was responsible for the increase of enzymatic activity. Finally, the kinetic parameters obtained for the enzyme characterized indicate that it can be used as a complement to enzymatic cocktails, which are known to be poor in pectinases.

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