M.Sc. (Biochemistry) / Plants respond to various stress stimuli by activating a broad-spectrum of defence responses that can be expressed locally at the site of pathogen infection (hypersensitive response-HR) as well as systemically in uninfected tissue (systemic acquired resistance-SAR). The ability to continuously respond to both abiotic and biotic stimuli leads to changes in the plants’ physiology, morphology and development. Therefore, there is a need to define and understand the mechanism of the plant defence system, including the mode of recognition, activation of signalling pathways and subsequent defence. In so doing, a long lasting and effective protection against various pathogens may be established. In the current study, the transcriptome status of cultured cells of Nicotiana tabacum was investigated using annealing control primer (ACP)-based differential display (DD) since it is an improved technology to compare patterns of gene expression in RNA samples, isolated from tissue / cells under different biological conditions, using a novel priming system. Here, ACP-DDRT-PCR was used in combination with a next-generation sequencing technology, namely 454 pyro-sequencing, which is the only technique that generates longer reads which are suitable for de novo assembly and annotation of non-model plants like tobacco of which the genome is not yet published in Genbank. SAR occurs following induction by biotrophic or necrotising pathogens. However, it can also be manifested artificially after chemical treatment. In this study, isonitrosoacetophenone (INAP), a novel compound that was originally isolated from extracts of citrus peel undergoing oxidative stress, was used as a chemical inducer and it was hypothesised that this compound induces defence-related responses in plants. In order to investigate this, tobacco cell suspensions were elicited with 1 mM INAP, followed by ACP-DDRT-PCR and subsequent identification of differentially expressed genes using pyro-sequencing.

Background: Heat-shock (HS) proteins (HSP) are induced or increasingly expressed to protect against lethal environmental stresses. Hsp70 in particular, provides protection against various stresses including oxidative stress, is implicated in thermotolerance and appears to have an anti-apoptotic function. Anti-inflammatory salicylates potentiate the induction of the 70 kDa HSP (Hsp70) in mammals in response to HS, enhance thermotolerance and induce apoptosis. In plants, salicylic acid (SA) is a natural signalling molecule, mediating resistance in response to avirulent pathogens. The effects of salicylic acid-mediated increases in Hsp70/Hsc70 expression and its relation to events associated with PCD/ apoptosis in plants are unknown. Hypothesis and Objectives: The hypothesis studied in this investigation was that SA influences Hsp70 expression similar to that found in mammalian cells and may influence the choice between survival or death, whether apoptosis or necrosis. In order to verify this hypothesis the effect of SA alone or in combination with HS on Hsp70/Hsc70 accumulation and events associated with apoptosis were investigated through three main objectives: 1) Determine whether SA in plants, as in mammalian cells, can potentiate heat-induced Hsp70/Hsc70 accumulation or induce Hsp70/Hsc70 by itself at elevated levels. This was done by investigating the effect of SA at various concentrations on Hsp70/Hsc70 expression at normal temperatures or following heat. 2) Establish flow cytometry as a rapid and quantitative alternative for the evaluation of Hsp70 accumulation in plant protoplasts to be evaluated in concert with various parameters indicative of cellular integrity. 3) Investigate whether Hsp70/Hsc70 expression modulated by SA influences cell death (apoptosis/necrosis) or associated events such as mitochondrial membrane permeability (MMP) or reactive oxygen species (ROS) in plant protoplasts using flow cytometry. Materials and Methods: The effect of SA alone or in combination with HS on Hsp70/Hsc70 levels in tomato cells was investigated using biometabolic labelling and Western blotting. A flow cytometric assay was developed to determine Hsp70/Hsc70 levels in tobacco protoplasts. MMP and ROS were monitored by the fluorescent probes DiIC1(5) and H2DCFDA respectively, phosphatidylserine externalisation by annexin V binding and DNA fragmentation by the TUNEL assay in protoplasts treated with SA and/or HS. Results: Results obtained in the attainment of the three main objectives were: 1) In plants, as in mammals, low concentrations of SA do not induce Hsp70/Hsc70 but significantly potentiate heat-induced Hsp70/Hsc70 levels while cytotoxic levels significantly induce Hsp70/Hsc70. In cell suspension cultures, this induction was preceded by increased membrane permeability. 2) Flow cytometry can be implemented as a rapid, quantitative alternative to detect intracellular Hsp70/Hsc70 accumulation in protoplasts. 3) In protoplasts exposed to low doses of SA at normal temperatures, PCD/apoptosis is increased as reflected by increased DNA fragmentation and phosphatidylserine externalisation, but not by increased MMP or ROS. High doses of SA were associated with increased levels of necrosis. Exposure of protoplasts to low doses of SA in combination with HS showed suppression of PCD/apoptosis (reflected by decreased DNA fragmentation and phosphatidylserine externalisation), accompanied by decreased levels of ROS and increased MMP. Discussion: These results suggest that SA-mediated increases in Hsp70/Hsc70 accumulation at normal temperatures are associated with cellular damage and protect cells against necrosis. On the other hand, low doses of SA that potentiate heat-induced Hsp70/Hsc70 accumulation abrogated the induction of apoptosis that was induced by low doses of SA at normal temperatures. The anti-apoptotic effects of Hsp70 could therefore influence plant resistance by interfering with the execution of PCD. These results could contribute to our understanding of heat-induced disease susceptibility, and the manipulation of SA-modulated Hsp70/Hsc70 should be carefully considered in the light of its ability to affect cell death, which may be advantageous or deleterious to the plant cell. / Prof. L. Bornman

The main objective of this thesis was the elucidation of the host-pathogen interaction between Pinus patula and Fusarium circinatum. This was accomplished by studying differential gene expression at the molecular level. Therefore, the first chapter reports the use of PCR-based methods in gene discovery and transcriptome analysis. The use of these techniques in the identification of novel transcripts in host-pathogen interactions addressed. These examples illustrate the differences and strong features of each technique. Chitinases are linked to defence responses in plants. In chapter tw0, the induction of chitinases in P. patula was assessed at both the protein and genetic level. Western blot analysis and enzyme activity assays indicate that chitinase enzyme is not detected a part of the defence response by P. patula after infection by F. circinatum. This was further confirmed by the lack of significant induction of two Pinus chitinase genes, LP6 and PSCHI4, as determined by RT-PCR analysis. Partial DNA sequence homologues for the LP6 and PSCH14 genes were determined and compared with a variety of plant chitinases. The low levels of detectable chitinase induction in P. patula might explain the high levels of susceptibility to the pitch canker fungus observed in seedlings of this tree. Pinus patula, the most widely planted species in South Africa, is highly susceptible to infection by F. circinatum. In chapter three, suppression subtractive hybridisation was used to elucidate the changes taking place at the molecular level early on in this interaction. Most of the identified transcripts shared homology to both biotic and abiotic stress in plants. The induction of one fragment, displaying homology to phytocyanin proteins, as followed through RT-PCR. Induction levels for this fragment differed significantly between less and more susceptible plants. Although most of the sequences isolated in this study can be Iiked to stress, most have not been linked with specific plant-pathogen interactions. This raises questions in regard to the function of these genes in host-pathogen interactions. Further research identify the function of these sequences in the defence response will be needed. These sequences can also be tested against a family of Pinus trees to ascertain if they will be useful in marker assisted selection. A molecular analysis of culture degeneration and pathogenicity of F. circinatum was attempted in chapter four. In this chapter, the differential induction of transcripts in F. circinatum was determined against several other Fusarium spp. Several of he identified fragments shared homology with stress related proteins. One transcript shared homology to a polyketide synthase, FUM5, that could be linked to fumonisin production in other Fusarium spp. ELISA detected no fumonisin production, although the FUM5 transcripts were detected. The identification of all the transcripts could provide a basis for more intensive gene discovery studies in F. circinatum and other Fusarium spp. The induction of these sequences in different isolates needs to be studied to prove their function in F. circinatum. This study also complements several other studies that studied the morphological characteristics of culture degeneration. Resistance gene analogues have been reported from a diverge set of plant species. In chapter five, degenerate PCR amplification was used to isolate TI-NBS-LRR like resistance gene analogues from a range of Pinus species. These sequences w re further characterised through comparative analysis with previously reported Pinus resistance gene analogues. Through motif analysis, several of the known conserved motifs found in NBS domains were identified and conservation with other plant NBS motifs is indicated. The P-Ioop and GLPL motifs displayed a high level of conservation on amino acid level with other plant NBS motifs. However, slight differences in several of the conserved regions were observed when the Pinus analogues were compared with Arabidopsis thaliana. The identification of differences between angiosperm and gymnosperm NBS sequences indicates that design of new degenerate probes and primers for the isolation of more ancient NBS sequences is needed. Further, phylogenetic and structural analyses of these sequences will also aid in understanding the relationship between angiosperm and gymnosperm NBS sequences. The knowledge gained from such a study will highlight the similarities between angiosperm and gymnosperm defence responses. This study represents the first detailed report on RGA in Pinus. / Thesis (PhD)--University of Pretoria, 2006. / Genetics / Unrestricted

Lipopolysaccharides from Gram-negative bacteria are amphipathic, tripartite molecules consisting of a hydrophobic lipid A portion, a core hetero-oligosaccharide and a repetitive hydrophilic O-antigen polysaccharide region. Through cell : cell interactions, plants can come into contact with LPS originating from root-associated rhizobacteria, bacterial endophytes as well as bacterial pathogens. Biologically active LPS molecules have been shown to act as determinants of bacterial virulence but also as determinants of induced systemic resistance (ISR) and activators of the phenotypically related systemic acquired resistance (SAR), characterised by accelerated and enhanced defence responses. LPS as a ¡¥pathogen associated molecular pattern, PAMP¡¦ molecule, has the ability to activate the innate mammalian immunity system and to act as an immunomodulator of immune ¡V and inflammatory systems via the conserved lipid A region. It is thus believed that LPS is able to promote plant disease resistance through activation of ISR and/or SAR; however here, the O-antigen region is also implicated to play a pivotal role in the signal perception and transduction in response to elicitation by this bio-active lipoglycan. LPS was isolated from the cell walls of the endophyte, Burkholderia cepacia, characterised by denaturing electrophoresis and compared to the equivalent from the pathogen Ralstonia solanacearum. When dissolved in the presence of Ca2+ and Mg2+, the LPS attained its biologically active micellar state through complex formation. The former LPS strongly induced the activation of two MAPKs following treatment of Nicotiana tabacum cv Samsun leaves, while comparative inductions with the R. solanacearum counterpart were extremely weak and might be ascribed to it lacking an extensive O-antigen region. No previous reports on LPS-responsive MAP kinases in plant tissues exist in the literature. The time- and dose dependent activation of the two kinases were therefore investigated and their physico-chemical properties compared. A novel 32 kDa MAP kinase was transiently activated in response to exposure to LPS with optimal activation at 7 min post-elicitation with 100 ƒÝg.ml-1 LPS. Its identity as an ERK (extracellular signal-related) MAPK was confirmed by immunodetection with a pTEpY-specific (anti-active) MAPK antibody, tyrosine-phosphorylated association of activation and inhibition of activation by U0126, an inhibitor of upstream MAPKKs. The kinase did not utilise casein, histone or myelin basic protein as substrates and no endogenous substrate could be identified. The activated MAP kinase exhibited a pI of 6.3, but two charge isomers of 32 kDa respectively were found upon two-dimensional electrophoresis. Although loss of the dual-phosphorylated epitope during purification attempts prevented extensive purification, 30% ammonium sulphate fractionation significantly (33 fold) enriched the MAPK. A second, distinct, 30 kDa MAP kinase was transiently activated in response to 125 ƒÝg.ml-1 LPS at 40 min post-elicitation, and its identity as a p38 MAPK, to date not yet found in plants, was confirmed by immunodetection with a pTGpY-specific (anti-active) MAPK antibody, tyrosine-phosphorylation associated with activation and inhibition of activation by SB203580, a direct inhibitor of p38 MAPKs. The kinase did not utilise casein, histone or MBP as substrates and no endogenous substrate could be identified. The kinase displayed a pI of 6.0, but two charge isomers of 30 kDa respectively were found following two-dimensional electrophoresis. Loss of the dual-phosphorylated epitope again prevented significant purification, but the protein was found to be significantly (83 fold) enriched by 30% ammonium sulphate fractionation. Although LPS has been reported to be capable of altering Ca2+ permeability and perturbation of Ca2+ homeostasis across plasma membranes, Ca2+ did not appear to potentiate or reduce the activation of either the 30 or the 32 kDa kinases. To date other MAP kinases have been shown to act either independently or upstream from reactive oxygen intermediates (ROI) produced during the oxidative burst. It was found that peroxide and concomitant ROI is either not generated in leaf tissue in response to LPS elicitation, or if generated, do not trigger the activation of the two kinases. The identification and partial characterisation of these two novel tobacco MAPKs in the signal perception and transduction response to LPS, significantly contributes to understanding the biochemical basis of the mechanism of action of LPS as a ¡¥resistance elicitor¡¦ involved in the triggering of effective plant defence responses and contributes towards relating the activation of mammalian innate immunity to similar responses in plants. / Prof. I.A. Dubery

Plants respond to pathogen attack by inducing a coordinated resistance strategy, which results in the expression of defense gene products. When a plant-pathogen interaction results in disease establishment, parasite colonization is caused by a delayed plant defense response, not due the absence of any response. Thus, the speed and intensity of the plant response and intracellular signalling determines the outcome of a plant-pathogen interaction. The acceleration of plant responses by the application of resistance inducers could provide a commercially, biologically and environmentally feasible alternative to existing pathogen control methods. Lipopolysaccharides are amphipathic lipoglycans that are attached to the outer bacterial membrane by a lipidic entity inserted into the bacterial phospholipid monolayer, with the saccharidic part oriented towards the exterior. The general structure of this compound is comprised of an anchor named lipid A associated with a core polysaccharide, which bears an O-antigen domain. LPS has been described as one of the pathogen-associated molecular patterns (PAMPs) capable of eliciting the activation of the plant innate immune system. LPS present in the outer membranes of plant growth-promoting rhizobacteria (PBPR) are major determinants of induced systemic resistance (ISR). In addition, LPS may function as an activator of systemic acquired resistance (SAR), providing non-specific immunization against later infection. Evidence suggests that LPS may advance plant disease resistance using the mechanism of ISR or SAR through its application to plants as a sensitizing agent, priming them to respond more effectively to subsequent pathogen attack. Phosphorylation plays a major role during the plant defense response, exemplified by its phosphorylation of transcription factors, required for the expression of defense-related genes. One of the most extensively documented phosphorylation responses is that of MAP kinase activation by phosphorylation in response to elicitation by race-specific and non-racespecific elicitors in various plant species.Proteins that undergo differential phosphorylation as a result of elicitation could be components of signal transduction pathways which connect pathogen perception with defense responses. Thus the identification of protein kinases, protein phosphatases and their substrates is essential in the elucidation of plant defense responses. The hypothesis behind this dissertation is that LPS elicitation results in alterations in the phosphorylation profile of Arabidopsis thaliana proteins. In this study, LPS was extracted from the cell walls of Burkholderia cepacia, a bacterial endophyte, and characterized by SDS-PAGE. The exposure of Arabidopsis callus culture cells to LPS resulted in distinctive changes in the phosphoprotein profile of the cells. Radioactive phosphorous labelling of proteins provided evidence that phosphorylation occurs in Arabidopsis following LPS perception, as part of a defense response related to LPS elicitation. Further investigation of differential protein phosphorylation via immunoblotting with antiphosphotyrosine antibodies revealed that tyrosine phosphorylation of Arabidopsis proteins occurs in response to LPS. One of the tyrosine-phosphorylated proteins was found to be a 42 kDa kinase, activated in response to LPS elicitation. The identity of the kinase as a mitogen-activated protein (MAP) kinase was confirmed by immunoblotting with anti-active MAP kinase antibodies. In addition, an assay of MAP kinase activity demonstrated the ability of the LPS-responsive MAP kinase to phosphorylate the ERK-MAP kinase substrate Elk1. In terms of the global phosphoproteome of Arabidopsis in response to LPS, phosphopeptides were purified from a crude protein digest by immobilized metal affinity chromatography and analyzed by liquid chromatography-tandem mass spectrometry (LCMS/ MS). While LC indicated both quantitative and qualitative differences resulting from LPS elicitation, no peptides could be positively identified as phosphopeptides by MS analysis. This work can however be repeated with further precautions to prevent the loss of phosphate groups prior to analysis. The results obtained in this study indicate that LPS causes specific alterations in Arabidopsis protein phosphorylation as a post-translational modification in response to the perception of LPS during a plant-pathogen interaction, proving the original hypothesis. / Prof. I.A. Dubery

M.Sc. / Plant cells have evolved the ability to detect pathogen ingress and subsequently activate defense-related functions as part of the plant pathogen response. One or more poorly defined signal transduction pathways, initiated upon recognition of the pathogen by the plant host, regulate expression of plant defense genes. Acquired resistance (AR) is an inducible defense mechanism exhibited by many plants that provides protection against a broad range of pathogens.Many chemical and environmental cues can elicit the same defenses or subsets therein. Cytochrome P450 enzymes are heme-dependent, mixed function oxidase systems that utilize dioxygen to produce a functionalized organic substrate and a molecule of water. They play important biosynthetic and detoxicative roles. They have been identified as being involved in the activation (e.g. allene oxide synthase) and execution of plant defense responses. To investigate the involvement of cytochromes P450 in the plant defense response - mainly through the activation of allene oxide synthases in the jamonate signaling pathway — Phaseolus vulgaris L.cv. Contender leaves were treated with chemical elicitors to mimic the plant-pathogen interaction and thereby activate plant defense responses. Through the use of differential display reverse transcription polymerase chain reaction and denaturing polyacrylamide gel electrophoresis, differentially expressed cDNA bands were isolated, cloned and sequenced. One of the cloned cDNA fragment proved to be a previously unreported cytochrome P450 cDNA, and was named CYP98A5. Dot blot analysis of bean leaves treated with various chemicals showed an enhanced expression of CYP98A5 in leaves treated with 3,5- dichlorosalicylic acid. Northern blot analysis of a time dependent induction study of CYP98A5 in bean leaves treated with this chemical compound indicated that 3,5-dichlorosalicylic acid induces CYP98A5 transcripts earlier than it is induced in control leaves. This might be indicative of a possible conditioning and sensitizing effect of 3,5- dichlorosalicylic acid on bean leaves to a more rapid and effective response with defense reactions once attacked by pathogens. CYP98A5, however, did not appear to be an allene oxide synthase when sequence comparison with other allene oxide synthases was performed; isolation and comparison of the complete CYP98A5 sequence could prove this wrong. It is not possible to assign any function to CYP98A5 at this stage; elucidation of the function of this enzyme in plants would provide more insight into this study.

No description available.

Thesis (MScAgric (Plant Pathology))--University of Stellenbosch, 2010. / ENGLISH ABSTRACT: A survey was undertaken on apple and pear trees in the Western Cape Province to determine the aetiology of trunk diseases with reference to trunk diseases occurring on grapevine. Grapevine trunk diseases cause the gradual decline and dieback of vines resulting in a decrease in the vine’s capability to carry and ripen fruit. In recent years, viticulture has been expanding into several of the well established pome fruit growing areas. The presence of trunk pathogens in pome fruit orchards may affect the health of the pome fruit trees as well as cause a threat to young vineyards planted in close proximity to these potential sources of viable inoculum. Several genera containing species known to be involved in trunk disease on pome fruit and grapevine were found, including Diplodia, Neofusicoccum, Eutypa, Phaeoacremonium and Phomopsis. Diplodia seriata and D. pyricolum, were isolated along with N. australe and N. vitifusiforme. Four Phaeoacremonium species, P. aleophilum, P. iranianum, P. mortoniae and P. viticola, two Phomopsis species linked to clades identified in former studies as Phomopsis sp. 1 and Phomopsis sp. 7, and Eutypa lata were found. In addition, Paraconiothyrium brasiliense and Pa. variabile, and an unidentified Pyrenochaetalike species were found. Of these the Phaeoacremonium species have not been found on pear wood and it is a first report of P. aleophilum occurring on apple. This is also a first report of the Phomopsis species and Eutypa lata found occurring on pome trees in South Africa Two new coelomycetous fungi were also found including a Diplodia species, Diplodia pyricolum sp. nov., and a new genus, Pyrenochaetoides gen. nov. with the type species, Pyrenochaetoides mali sp. nov., were described from necrotic pear and apple wood. The combined ITS and EF1-α phylogeny supported the new Diplodia species, which is closely related to D. mutila and D. africana. The new species is characterised by conidia that become pigmented and 1-septate within the pycnidium, and that are intermediate in size between the latter two Diplodia species. Phylogenetic inference of the SSU of the unknown coelomycete provided bootstrap support (100%) for a monophyletic clade unrelated to known genera, and basal to Phoma and its relatives. Morphologically the new genus is characterised by pycnidial with elongated necks that lack setae, cylindrical conidiophores that are seldomly branched at the base, and Phoma-like conidia. The phylogenetic results combined with its dissimilarity from genera allied to Phoma, lead to the conclusion that this species represents a new genus. A pathogenicity trial was undertaken to examine the role of these species on apple, pear and grapevine shoots. N. australe caused the longest lesions on grapevine shoots, while Pyrenochaetoides mali, Pa. variabile, D. seriata and P. mortoniae caused lesions that were significantly longer than the control inoculations. On pears, D. pyricolum and N. australe caused the longest lesions, followed by D. seriata and E. lata. On apples, the longest lesions were caused by N. australe and P. iranianum. D. seriata, D. pyricolum, E. lata, N. vitifusiforme, Pa. brasiliense, P. aleophilum and P. mortoniae also caused lesions on apple that were significantly longer than the control. The study demonstrated that close cultivation of grapevine to apple and pear orchards may have inherent risks in terms of the free availability of viable inoculum of trunk disease pathogens. / No Afrikaans abstract available.

Thesis (MSc)--University of Stellenbosch, 2005. / ENGLISH ABSTRACT: In the field of agriculture, plant pathogens are a major concern because of the severe damage these organisms cause to crops yearly. Fundamental studies regarding plant pathogens and their modes of action made it possible for researchers in the field of molecular biology to investigate pathogens further on a molecular level. Botrytis cinerea, has been used to great effect as a model system to investigate various aspects regarding pathogenesis, also on a molecular level. Molecular research done on B. cinerea over the last few years has shown that the endopolygalacturonases (EPGs) of this fungus are key role players in pathogenesis. This hydrolytic enzyme family of six members, encoded by the Bcpg1-6 genes, are important in breaking down the complex cell wall polymers of host plants, enabling the fungus to penetrate its host sufficiently. It has been shown that both BcPG1 and 2 are crucial for virulence of B. cinerea. A leucine-rich repeat inhibitor protein situated in the cell wall of various plant species, the polygalacturonase-inhibiting protein (PGIP), has been proven to interact with and inhibit EPGs, and thus the necrotic actions of B. cinerea. From literature it was clear that specific data regarding individual interactions of fungal EPGs with PGIPs are lacking currently. Furthermore, most experiments regarding the effects of EPG as well as interaction and inhibition studies of EPGs and PGIPs, rely on in vitro methods, without the possibility to contextualize the results on an in vivo or in planta level. The scope of this study was to specifically address the issues of individual EPG:PGIP interactions and the use of possible in vivo methodology by using EPGs from a highly virulent South African strain of B. cinerea and the grapevine VvPGIP1 that has been previously isolated in our laboratory. This PGIP, originally isolated from Vitis vinifera cv Pinotage, has been shown to inhibit a crude EPG extract from this strain with great efficiency. The approach taken relied on heterologous over-expression of the individual Bcpg genes and the isolation of pure and active enzymes to evaluate the inhibition of the EPGs with VvPGIP1. The genes were all successfully over-expressed in Saccharomyces cerevisiae with a strong and inducible promoter, but active enzyme preparations have been obtained only for the encoding Bcpg2 gene, as measured with an agarose diffusion assay. The in vitro PGIP inhibition assay is also based on the agarose diffusion assay and relies on activity of the EPGs to visualize the inhibiting effect of the PGIP being tested. The active EPG2, however, was not inhibited by VvPGIP1 when tested with this assay. The EPG encoding genes from B. cinerea were transiently over-expressed also in Nicotiana benthamiana by using the Agrobacterium-infiltration technique. Transgene expression was confirmed by Northern blot analysis and EPG-related symptoms were observed five to eight days post-infiltration. Differential symptoms appeared with the various EPGs, providing some evidence that the symptoms were not random events due to the infiltration or a hypersensitive response. Moreover, the symptoms observed for EPG2 was similar to those that were reported recently by another group on the same host. In spite of the expression data and the clear symptoms that developed, active preparations, as measured with the agarose diffusion plate asay, could only be obtained for EPG2 again. In our search for a possible in vivo method to detect and quantify EPG activity and inhibition by PGIPs, we tested and evaluated a technique based on chlorophyll fluorescence to detect the effect of EPGs on the rate of photosynthesis. Our results showed that the over-expression of these genes reduced the rate of electrons flowing through photosystem II, indicating metabolic stress occurring in the plant. We used the same technique to evaluate possible interaction between VvPGIP1 respectively with BcPG1 and 2 and found that the co-expressing of the Vvpgip1 gene caused protection of the infiltrated tissue, indicating inhibition of EPG1 and 2 by VvPGIP1. For EPG2, the observed interaction and possible inhibition by VvPGIP1 is the first report to our knowledge of an interaction between this specific EPG2 and a PGIP. Moreover, to further elucidate the in planta interaction between VvPGIP1 and the EPGs from the South African B. cinerea strain, we tested for possible interactions by making use of a plant two-hybrid fusion assay, but the results are inconclusive at this stage. Previous studies in our laboratory have shown that several natural mutations exist between PGIP encoding genes from different V. vinifera cultivars. Based on this finding and the fact that these natural mutations could result in changes with regard to EPG inhibition and ultimately disease susceptibility, we isolated an additional 37 PGIP encoding genes from various grapevine genotypes, some of which are known for their resistance to pathogens. Combined, these results make a valuable contribution to understand plant pathogen interactions, specifically in this case by modeling the interactions of pathogen and plant derived proteins. The possibility to use in vivo methods such as chlorophyll fluorescence to follow these interactions on an in planta level, provides exciting possibilities to strenghten and contextualize in vitro results. / AFRIKAANSE OPSOMMING: Plantpatogene organismes veroorsaak jaarliks erge skade aan landbougewasse en word dus as ’n ernstige probleem in die landbousektor beskou. Diepgaande studies wat handel oor plantpatogene en hul metodes van infeksie het dit vir molekulêre bioloë moontlik gemaak om patogene nou ook op molekulêre vlak verder te bestudeer. Botrytis cinerea is baie effektief as modelsisteem gebruik om verskeie aspekte van patogenese verder te bestudeer, ook op ‘n molekulêre vlak. Molekulêre navorsing op B. cinerea, het getoon dat die endopoligalakturonases (EPGs) van dié swam kernrolbelangrik in patogenese is. Hierdie sesledige hidrolitiese ensiemfamilie word gekodeer deur die Bcpg1-6 gene en is belangrik vir die afbraak van die komplekse selwandpolimere van plantgashere, om suksesvolle gasheerpenetrasie te veroorsaak. Daar is aangetoon dat beide BcPG1 en 2 essensieël vir virulensie van die patogeen is. ’n Leusienryke-herhalings inhibitorproteïen wat in die selwand van verskeie plantspesies voorkom, die poligalakturonase-inhiberende proteïen (PGIP), het interaksie met en inhibeer EPGs en gevolglik ook die nekrotiserende aksies van B. cinerea. Uit die literatuur is dit duidelik dat spesifieke inligting aangaande individuele interaksies van fungiese EPGs met PGIPs tans nog ontbreek. Verder word daar op in vitro metodologie staatgemaak wannneer die effekte van EPGs asook die interaksie en inhibisie met PGIPs bestudeer word, sonder om die konteks van die in vivo- of in planta-omgewing in ag te neem. Die fokus van hierdie studie was om aspekte van individuele EPG:PGIP interaksies, asook die moontlike gebruik van in vivo metodologie te bestudeer deur EPGs, afkomstig van ’n hoogs virulente Suid-Afrikaanse ras van B. cinerea en die wingerd VvPGIP1, wat vroeër in ons laboratorium geïsoleer is, te gebrruik. Hierdie PGIP wat uit Vitis vinifera cv Pinotage geïsoleer is, inhibeer ’n kru EPG-ekstrak van bogenoemde ras baie effektief. Die benadering wat gevolg is het op die ooruitdrukking van die individuele Bcpg-gene in heteroloë sisteme staatgemaak en die gevolglike isolering van suiwer en aktiewe ensieme om EPG-inhibisie deur VvPGIP1 te beoordeel. Al die gene is suksesvol in Saccharomyces cerevisiae ooruitgedruk onder ’n sterk induseerbare promotor, maar volgens ’n agarose-diffundeerbare toets kon aktiewe ensiempreparate slegs vir die enkoderende Bcpg2 verkry word. Die in vitro PGIP-inhibisie toets is ook op die gemelde toets gebasseer en vereis EPG-aktiwiteit om die inhiberende effek van die PGIP, te visualiseer. Die aktiewe EPG2 is egter nie deur VvPGIP1 geïnhibeer met die aanleg van hierdie toets nie. Die EPG-enkoderende gene van B. cinerea is ook tydelik in Nicotiana benthamiana ooruitgedruk deur gebruik te maak van ’n Agrobacterium-infiltrasietegniek. Transgeenuitdrukking kon met die Noordelike kladtegniek bevestig word en EPG-verwante simptome is vyf tot agt dae na infiltrasie waargeneem. Verskillende simptome vir die verskillende EPGs is waargeneem, wat aanduidend is dat die simptome nie lukrake gevolge van die infiltrasies, of ’n hipersensitiewe respons is nie. Verder kon die simptome wat EPG2 vertoon het, gekorreleer word met dié wat onlangs deur ’n ander groep op dieselfde gasheer waargeneem is. Ten spyte van die ekspressiedata en die waargenome simptome, kon aktiewe ensiempreparate op die agarose-diffundeerbare toets, weereens slegs vir EPG2 waargeneem word. ’n Metode wat gebasseer is op chlorofilfluoressensie is getoets en geëvalueer as ’n moontlike in vivo metode om EPG aktiwiteit en inhibisie deur PGIPs waar te neem en te kwantifiseer. Die resultate het bevestig dat die ooruitdrukking van hierdie gene die elektronvloeitempo deur fotosisteem II verminder het wat ’n aanduiding is dat metaboliese stres in die plant heers. Dieselfde tegniek is gebruik om die moontlike interaksies tussen BcPG1 en 2 en VvPGIP1 te bestudeer en het aangetoon dat die mede-uitdrukking van die Vvpgip1-geen aanleiding gee tot ’n beskermende effek van die geinfiltreerde weefsel, wat aanduidend is van inhibisie van EPG1 en 2 deur VvPGIP1. In die geval van EPG2 is hierdie interaksie en moontlike inhibisie met ’n PGIP die eerste waarneming in die verband. In ’n verdere poging om die in planta-interaksie tussen VvPGIP1 en die EPGs van die Suid-Afrikaanse B. cinerea ras uit te klaar, is ’n plantgebasseerde twee-hibriede toets aangelê, maar geen klinkklare resultate kon verkry word nie. Vorige werk het bevestig dat verskeie natuurlike mutasies in PGIP-enkoderende gene, afkomstig van verskillende V. vinifera kultivars, voorkom. Hierdie resultaat en die feit dat hierdie mutasies verskille in EPG inhibisie en uiteindelik vatbaarheid vir siektes kan beïnvloed, het aanleiding gegee tot die isolering van ’n verdere 37 PGIP-enkoderende gene uit ‘n verskeidenheid druifplantgenotipes, sommige waarvan juis bekend vir hul weerstand teen patogene is. Die gekombineerde resultate wat in dié studie verkry is, maak ’n waardevolle bydrae tot die verstaan van plant-patogeeninteraksies, spesifiek met die modelering van interaksies van patogeen- en plantgebasseerde proteïene. Die moontlikheid om in vivo-metodes soos chlorofilfluoressensie te gebruik in in planta-analises, is besonder bemoedigend om in vitro-resultate te versterk en ook in konteks te plaas.

Thesis (M.A.)--Humboldt State University, 2009. / Includes bibliographical references (leaves 37-42). Also available via Humboldt Digital Scholar.