Prof. I.A. Dubery

M.Sc. / Plants produce volatile organic compounds in response to certain elicitors and environments. These compounds have a variety of functions, including the attraction of insects for pollination and seed dispersal, responses to both abiotic and biotic stresses and the priming or sensitizing of neighbouring plants for subsequent attack. The majority of the volatile blend is made up of terpenoid compounds and these compounds are formed through the action of an important class of enzymes termed Terpene Synthases. Lipopolysaccharides form part of the cell surface of Gram-negative bacteria and they are classed as “pathogen-associated molecular pattern molecules” and are thought to induce defence responses in plants by influencing different metabolic pathways that could ultimately result in the production of defence volatiles. LPS from Burkholderia cepacia that has been reported to induce the oxidative burst, the nitric oxide burst and changes in cytosolic calcium concentrations, was used in this study. In order to analyse the volatiles, Single-Drop Microextraction and Solid-Phase Microextraction were used as static headspace sampling techniques that allow the preconcentration of volatile analytes prior to analysis. Both these techniques are fast, simple and equilibrium based and both allow for minimal sample size and preparation. Luminometry was performed in order to test the efficacy of LPS and to determine if LPS is able to induce the oxidative burst in Arabidopsis thaliana. Histochemical staining of transgenic plants containing the PR1:GUS and PDF:GUS reporter gene constructs was performed in order to determine which signalling pathway LPS follows, either the jasmonic acid pathway or the salicylic acid pathway. SPME was then used to extract samples from both time and concentration studies. The time studies involved incubation times of 0 h, 2 h, 4 h and 6 h and 0 d, 1 d, 2 d and 3 d respectively, while the concentration studies involved using LPS concentrations of 0, 20 μg/ml, 40 μg/ml, 60 μg/ml, 80 μg/ml and 100 μg/ml. SPME was also used for the comparision of two A. thaliana ecotypes (Columbia and C24) as well as two A. thaliana knock-out lines (At5g44630 – multi-product sesquiterpene synthase and At5g23960 – (E)-β-caryophyllene synthase), and finally it was used for the sampling of A. thaliana leaf tissue. SDME was used to compare two solvents, namely octane and toluene and these results were compared to the SPME results. GC-MS was used only for the identification of volatiles with both SPME and SDME. Finally, GC-MS was used with SPME to identify volatiles that are produced by leaf tissue after priming.

M.Sc. / LPS, as a pathogen associated molecular pattern (PAMP) molecule can interact with eukaryotic host cells. Interaction occurs by either direct contact or due to the release of micelles containing LPS from bacterial cell surfaces. LPS activates innate host defence systems in both invertebrate and vertebrate animal/insect cells via analogous pathways, where the lipid A component,is responsible for the activities. LPS from several plant pathogens have been shown to activate a number of defence-related responses in plants. Initial concentration studies and cell viability assays were conducted to assess isonitrosoacetophenone (INAP) and LPS as elicitors of defensive responses in tobacco (Nicotiana tabacum cv. Samsun) cell suspensions. The effective concentrations were found to be 100vM INAP and 100μg/ml LPS. RNA was isolated, quantified and analysed to confirm the quality of the starting material for differential display analysis. The DDRT-PCR technique was successfully applied in order to obtain comparative "displays" of PCR amplicons derived from three sub-divided mRNA pools (i.e. each of the three different anchor primers, per treatment). Significant differences in the profiles of control, INAP and LPS treated cells were observed, indicating that the eliciting agents had prominent effects on cellular homeostasis, resulting in an altered gene expression profile. DDRT-PCR can be technically challenging at a number of steps. Modifications were incorporated to initially obtain differentially expressed transcripts (DETs), as well as reamplify the DETs. 223 Putative DETs were isolated from denaturing polyacrylamide sequencing gels. 172 Putative DETs were re-amplified, of which 126 appeared as good candidates for further analysis. Finally, 96 putative DETs were chosen for reverse Northern analysis. DDRT-PCR has been reported to be plagued by false positives. Reverse Northern analysis confirms the presence of the putative DET from the subdivided RNA pool, as well as affirming the differential expression, compared between the control and inducer blots. 26 DETs were selected for cloning, of which 16 were sequenced. Homologies between the DETs and known sequences were determined using BLASTN and BLASTX alignments, DNAssist software, as well as MIPS alignments to the Arabidopsis genome. Five of the DETs were assigned putative functions in plant signal perception, transduction and the defence response, based on their respective sequence homologies to sequences involved in innate immunity. It is proposed that the DET, HAP3-15, represents the plant equivalent of a component of the innate immunity pathway in mammals and Drosophila. It is further proposed that HAP3-15 represents a S-Receptor kinase protein (SRK), with a defensive role in distinguishing self from potential pathogens. Therefore, as a SRK, HAP3-15 would function as a transmembrane receptor able to conduct an external signal through the membrane to the cytoplasm as a form of signal perception. Subsequently HAP3-15 could ii play a role in phosphorylation cascades through the kinase domain and, consequently, be responsible for signal transduction. In addition, LPS would then represent the ligand creating the signal perceived by the SRK, HAP3-15, with oligosaccharide binding ability. HAP3-15 was also identified as a true positive by the INAP probe in reverse Northerns, implying that both the biological and chemical inducers used, activated the same receptor kinase. Whether the same signalling pathway was followed during the phosphorylation cascades has not been determined. Further analysis will require Northern blots in a time study to investigate the kinetics of induction. In addition, longer sequence information for each of the five DETs needs to be obtained to identify the corresponding genes in order to investigate their roles in innate immunity in plants.

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 water hyacinth (Eichhornia crassipes (Mart.) Solms-Laub (Pontederiaceae)) biological control programme makes use of tight plant-insect interactions to control the weed, by reestablishing the interactions between the plant and its natural enemies. Since the beginning of the water hyacinth biological control initiative, the impact of biological control agent herbivory on water hyacinth’s population growth and fitness have been well documented; however, very few investigations have been conducted to determine whether herbivory elicits insect-induced responses by water hyacinth. Studies were conducted to determine the presence and function of water hyacinth insectinduced responses, using the plant activator, BION®, in attempt to determine the plant hormone-mediated pathways regulating the final expressions of insect-induced defences in response to herbivory by the phloem-feeder, Eccritotarsus catarinensis (Carvalho) (Hemiptera: Miridae) and the leaf chewer, Neochetina bruchi Hustache (Coleoptera: Curculionidae). BION® (Syngenta, acibensolar-S-methyl (benzothiadiazole)) is a dissolvable, granular formulation that contains a chemical analogue of the plant hormone, salicylic acid (SA), which typically regulates defences against pathogens. The application of BION® results in the induction of the SA-mediated defence pathways in plants (activation of defences against pathogens), and consequently the inhibition of the jasmonic acid (JA)- mediated defence pathways (de-activation of defences against insect herbivores). To test for induced defence responses in water hyacinth, plants treated with BION® and then subjected to herbivory, were compared to un-treated plants that were also subjected to herbivory, BION®-only treated plants and control plants. The application of BION® did not confer resistance against the two insect herbivores, as herbivory, reductions in chlorophyll content and plant growth (leaf production and second petiole lengths) significantly increased in comparison to non-BION® treated plants. Furthermore, palatability indices significantly increased (>1.00) in BION® treated plants, reflecting increased weevil preferences for SAinduced water hyacinth plants. This concluded that SA-mediated defences are not effective against insect herbivory in water hyacinth plants, but are in fact palatable to insect herbivores, which reflects ecological and physiological costs of SA-mediated defences (pathogen defences) in water hyacinth. Biochemical analyses of leaves exhibited increases in nitrogen content in BION® treated plants. These elevated levels of nitrogenous compounds account for the increases in mirid and weevil preferences for BION® treated plants. The increases in nitrogenous compounds are probably structural proteins (e.g. peroxidises), because leaves treated with BION® increased in toughness, but only when exposed to herbivory. Regardless, insect herbivory was elevated on these leaves, probably because the nitrogenous compounds were nutritionally viable for the insects.

This dissertation focuses on the local defence-related industry as a beneficiary under the Department of Defence’s defence industrial participation (DIP) programme, managed by Armscor. Attention is given to the main construct of the development theory and how the DIP process in South Africa compares with the international reciprocal trade phenomena commonly referred to as ‘countertrade’. The author does an in-depth analysis of the Government’s policy regarding the defence-related industry (DRI) that forms part of the local defence industrial base (DIB), as well as the DIP policy, procedures and practices and their subsequent bearing on the local defence industry. The study is further substantiated with a comprehensive review of the consequences and outcomes resulting from the largest defence package deal (SDP), signed in December 1999, between the Department of Defence and several major foreign original equipment manufacturers (OEMs) and subsequently benchmarked against academic discourse on the subjects of international countertrade and development theories.

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.