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.

M.Sc. (Botany and Plant Biotechnology) / Wheat (Triticum aestivum L.) is the one of the three most extensively cultivated cereal crops worldwide (Shewry, 2009). In South Africa, wheat is cultivated in both summer and winter rainfall regions as monocultures typical to modern industrialised agriculture. Monocultures provide uniform crop quality and allow processes such as planting and harvesting to be mechanised (Altieri et al., 2009). However, the genetically homogeneous nature of monocultures increases the vulnerability of the crop to both biotic and abiotic stresses (Faraji, 2011). Future food production is challenged by predicaments such as an increasing human population while the ratio of arable land to population is decreasing. Yield losses of wheat due to biotic factors alone were estimated as 29 % (2001-2003) (Oerke, 2006). The need to reduce the gap between attainable yield and actual yield is therefore crucial in order to maximise crop production for future food security (Duveiller et al., 2007). One of the most damaging pests to worldwide wheat production is the Russian wheat aphid (RWA), Diuraphis noxia (Kurdjumov) (Arzani et al., 2004). A native pest of central Asia, the RWA has spread to all cereal producing areas of the world with the exception of Australia (Burd et al., 2006). While feeding on susceptible hosts, the aphid injects an eliciting agent into the host, which causes the breakdown of the chloroplast and cellular membranes, leading to the appearance of symptoms typical of RWA feeding, including leaf rolling (Botha et al., 2005). Leaf rolling creates a sheltered environment for the aphid from insecticides and predators, and this together with their parthenogenic and viviparous reproductive nature makes their rapid increase in numbers extremely difficult to control (Goggin, 2007). Resistant wheat genotypes currently represent the most effective long term solution to control RWA infestations; however resistance breaking aphid biotypes are rapidly overcoming the incorporated resistance genes under field conditions (Burd et al., 2006; Jankielsohn, 2011). Understanding the molecular basis of plant resistance to the RWA is crucial in creating cultivars with durable resistance (Botha et al., 2005).

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

M.Sc. / in the current study the effect of the addition of methyl Jasmonate (MeJA), chitosan, a cell wall elicitor prepared from Phytophthora nicotlanae to tobacco cells and the subsequent defense responses elicited in these cells were Investigated. The defense responses investigated can be divided into three categories according to the time scale whereby resistance responses in plant cells are induced: early events which included the analysis of lipid peroxidation, the induction of lipoxygenase (L0)0 enzyme activity as well as the changes in phosphoprotein profiles; intermediate to later responses which included investigations of peroxidase (POD) activity, lignin content, phytoalexin content and phenolic content and also late responses which included studies of pathogenesis-related proteins (PR) and 13-1,3-giucanase activity. An approach also followed in this study was the addition of MeJA to tobacco cells for 24 h followed by the addition of either the cell wall elicitor or chitosan as a secondary elicitors, to investigate possible preconditioning or sensitisation by MeJA. Results obtained in this study revealed the time and concentration dependent accumulation of phytoalexins (secondary metabolites) when MeJA was added to tobacco cells and an optimal concentration of MeJA to use in further studies was determined as 1 mM. MeJA was the most effective inducer of lipid peroxidation (22 fold induction), a response observable after 2 h of exposure to MeJA. Conditioning with MeJA, followed by both chitosan (19 fold induction) and elicitor (25 fold induction) led to an earlier accumulation as well as significant increases in the levels of malondialdehyde, the product of lipid peroxidation. LOX enzyme activity was significantly increased by the addition of MeJA (6 fold Induction), chitosan (4 fold induction) and elicitor (3.8 fold induction). Conditioning with .MeJA, followed by both chitosan (3.3 fold induction) and elicitor (3.9 fold Induction) also led to noteworthy increases in enzyme activity. Analysis of the phosphoprotein profiles do not reveal the accumulation of phosphorylated proteins when MeJA was added to cells and very little accumulation of such proteins when chitosan was added. Phosphorylated proteins could be observed in cells treated with elicitor and In the cases where conditioning with MeJA, followed by secondary elicitation with either chitosan or elicitor, was studied, the differential induction of phosphorylated cellular proteins could also be observed. No significant induction of POD activity could be observed under any of the conditions, except for a possible slight increase in POD activity starting at 16 - 24 h after the elicitor had been added and a more definite increase after 24 h which was sustained up to 48 h after the addition of MeJA. PAGE of peroxidase, followed by activity staining revealed the presence of a slow migrating anionic peroxidase as well as a fast migrating peroxidase. Conditioning with MeJA, followed by secondary elicitation with both chitosan and elicitor revealed enhanced POD activity as well increased induction of a fast migrating anionic peroxidase on PAGE gels. MeJA was a more effective inducer of elevated levels of lignin content than the elicitor or chitosan and the addition of MeJA to tobacco cells led to a 2.2 fold increase in the lignin content, a response observed after 24 h and sustained up to 48 h. Chitosan as secondary elicitor did not lead to any increase in lignin content, but the cell wall elicitor as secondary agent significantly increased the lignin content after 40 - 48 h. Analysis of phenolic content did not show any significant increases In the total soluble phenolics when the agents were used on their own and only the phenolic content of the MeJA-conditioned cells, followed by the addition of chitosan showed a slight increase. In this case, the HPLC analysis of the phenolics also revealed a shift In the profiles for phenolics. SDS-PAGE of PR proteins revealed the induction of constitutive as well as new proteins when MeJA and elicitor, but not chitosan were used as elicitation agents. However, In the MeJA-pretreated cells addition of both chitosan and elicitor led to increased accumulation of PR proteins with molecular masses ranging from 6 - 70 kDa. Results from the i3-1,3-glucanase activity assay indicate a strong induction (4-5 fold) when MeJA and elicitor (4 fold), but not when chitosan was added to cells. Conditioning effects were revealed when both chitosan (3 fold induction) and elicitor (2.5 fold induction) were used as secondary elicitors. The increases in intensities of bands with molecular masses ranging from 31- 35 kDa observed on SOS-PAGE gels where chitosan and elicitor were added as secondary agents corresponded in a time dependent manner with the increased levels obtained in thep-1,3-glucanase activity assay.

In South Africa the genus, Eucalyptus plays an important role as a major economic component in the forest and mining sector. Unfortunately, this genus has problems due to damage by pests. The Eucalyptus snout beetle, Gonipterus scutellatus, feeds and defoliates the leaves of eucalypts. Plants produce secondary metabolites, which protects them against defoliation by insects and herbivores. This study focuses on the variations of total soluble phenolic content of nine Eucalyptus species between the species and within the species throughout the year. Total soluble phenols were quantified with the Folin-Ciocalteau reagent. There was a general increase in the concentration of total soluble phenols throughout the year. The content of total soluble phenols were generally higher compared to carbohydrates. This experiment needs to be done on a continuous basis so as to formulate a screening method for eucalypt species that are resistant to G. scutellatus. / Dissertation (M Inst Agrar (Sustainable Insect Management))--University of Pretoria, 2005. / Zoology and Entomology / unrestricted

In South Africa, the genus Eucalyptus plays an important role as a plantation tree and hence forms a major economic component in the forest sector. An insect pest of these Eucalyptus species, Gonipterus scutellatus, causes periodic defoliation in the plantations. Plants have extraordinary array of chemicals (secondary metabolites), which defend them from herbivores. This study reports on the seasonal variation of the tannin concentration and carbohydrate content of the leaves of nine Eucalyptus species. Soluble tannins were quantified using Hagerman test and the carbohydrate content with a refractometer. Generally high tannin concentrations were observed during spring and late summer with low concentrations during autumn and winter. During the months of February, April, July tannin concentration was usually found to be higher than the carbohydrate content. The higher concentration of tannin could be linked to the allocation of carbon for the production of tannin instead of carbohydrates for growth. / Dissertation (MInst Agrar)--University of Pretoria, 2006. / Zoology and Entomology / unrestricted

The sequence of 3553 nucleotides corresponding to the 3'-terminal region of potato virus S (PVS) has been determined from cloned cDNA. The sequence obtained contains six open reading frames with the potential to encode proteins of Mr 10,734, Mr 32,515, Mr 7,222, Mr 11,802, Mr 25,092 and at least Mr 41,052. The amino acid sequence of the 33K ORF has been confirmed to be that of the viral coat protein gene. The nucleotide sequence of this ORF was obtained from expression plasmids which were isolated by binding with a specific monoclonal antibody to PVS, and the expression of coat protein fusion products was verified by Western blots of bacterial cell lysates. The deduced amino acid sequence of a 70 amino acid portion from the central region of the PVS coat protein was 59% identical to the analogous region of potato virus X. In addition, the 7K, 12K and 25K ORF's displayed significant sequence homology with similar sized ORF's from a number of potexviruses. The partial 41K ORF was homologous with the C-terminal portion of the viral replicase proteins of potato virus X and white clover mosaic virus. While the biological functions of the 12K and 25K non-structural proteins coded for by PVS and members of the potexvirus group remain unknown, the 12K protein displays a hydropathicity profile consistent with a membrane associated protein and the 25K protein contains a conserved sequence motif found in a number of nucleoside triphosphate binding proteins. Members of the carlavirus group are distinguished from the potexviruses by the presence of a small [11K (PVS, potato virus M) - 16K (lily symptomless virus)] 3' terminal ORF which appears to contain a sequence motif similar to the 'zinc-finger' domain found in many nucleic acid binding proteins. The coat protein gene from potato virus S (PVS) was introduced into Nicotiana debneyii tobacco as well as a commercial potato cultivar, 'Russet Burbank', by leaf disc transformation using Agrobacterium tumefaciens. Transgenic plants expressing the viral coat protein were highly resistant to subsequent infection following mechanical inoculation with the Andean or ME strains of PVS as indicated by a lack of accumulation of virus in the upper leaves. The coat protein mediated protection afforded by these transgenic plants was sufficient to prevent the accumulation of virus in the tissues of non-transformed 'Russet Burbank' shoots which had been grafted onto transgenic plants inoculated with PVS, and in reciprocal grafts, transgenic shoots accumulated less than 2% (6 weeks after grafting) of the concentration of PVS found in non-transformed shoots similarly grafted onto plants systemically infected with PVS. These transgenic plants also displayed a measure of resistance to inoculation with a related carlavirus from potato, potato virus M. In agreement with previous reports for plants expressing PVX coat protein, plants expressing PVS coat protein were also protected from inoculation with PVS RNA. These results provide further evidence that coat protein mediated protection for these two groups of viruses, which share similar genome organizations, may involve inhibition of some early event in infection, other than, or in addition to, virus uncoating. Specific monoclonal antibodies were prepared against a C-terminal derived 18 kDa portion of the 25K protein of PVS expressed as an in-frame chimeric fusion protein with the glutathione S-transferase gene. The in vivo expression of this non-structural protein in virus infected tissue, as well as tissue from transgenic tobacco (var Xanthi-nc) engineered to contain the entire 25K gene, was verified by Western immunoblot labelling. / Medicine, Faculty of / Biochemistry and Molecular Biology, Department of / Graduate

This study involves a measure of the variability of descendants from a cross between Ustilago hordei race 7 and race 11, on two varieties of barley, Trebi and Odessa. Components of variability were defined, statistically described and compared. Biometrical analyses uncovered the action of significant additive and non-additive genetic effects. Differential interactions between treatments and varieties revealed the existence of at least one virulence gene. Specific polygenes and the virulence gene were found to produce significant interactions with different environmental conditions. Homogeneity of variance of the genetic components of the F2 from three randomly chosen F1 dikaryotic lines demonstrated the highly homozygous condition of the parental teliospores. Covariance - variance regression analysis was used to study the dominance and epistatic differences between treatment dikaryons. There is evidence for ambidirectional dominance. The number of effective factors operating against the varieties, Trebi and Odessa, were estimated to be between 4-6 and 1-2 respectively. / Science, Faculty of / Botany, Department of / Graduate

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