Thesis (Ph. D.)--University of Alberta, 2009. / Title from pdf file main screen (viewed on June 29, 2009). "A thesis submitted to the Faculty of Graduate Studies and Research in partial fulfillment of the requirements for the degree of Doctor of Philosophy Plant Science, Department of Agricultural, Food and Nutritional Science, University of Alberta." Includes bibliographical references.

Thesis (M.S.)--University of Wyoming, 2008. / Title from PDF title page (viewed on June 26, 2009). Includes bibliographical references (p. 86-111).

Thesis (Ph. D.)--University of Washington, 2004. / Vita. Includes bibliographical references (p. 84-109).

Thesis (Ph. D.)--University of Chicago, Committee on Genetics, March 2003. / Includes bibliographical references. Also available on the Internet.

M.Sc. / Field grown plants are constantly challenged with a variety of stressful factors, such as high temperatures, drought and pathogen infection that adversely affect crop production and quality. These stresses seldom occur as single entities in plants and in warm climates, heat stress is often a common dominator in combinatorial stress. The heat shock (HS) response in plants has priority over other stress responses, including the pathogen-induced stress response. Activation of the HS response prevents the normal plant defence strategy, leaving the plant vulnerable to pathogen attack. However, prior exposure to elevated temperatures confers protection from subsequent, otherwise lethal, temperatures (thermotolerance) and a variety of other stress conditions including heavy-metals, chilling injury and certain pathogens (cross tolerance). In general, litterature supports a central role for heat shock proteins (HSP), in particular the 70 kDa HSP (Hsp70), in thermotolerance. Incompatible host-pathogen interactions lead to the activation of an array of defence mechanisms, including the promotion of phenylpropanoid metabolism. Phenylalanine ammonia-lyase is a key regulator of this metabolic pathway, influencing the production of salicylic acid, lignin and phytoalexins among other essential defence products. In this study it was hypothesised that prior exposure to non-lethal HS confers protection from subsequent heat-related suppression of the phenylpropanoid pathway, induced as a defence mechanism during an incompatible plant-pathogen interaction. This hypothesis was verified by analysing the effect of thermotolerance on pathogen-related stimulation of PAL promoter activity, enzyme activity and lignin deposition. The tomato, Lycopersicon esculentum cultivar UC82B and Ralstonia solanacearum, the causative agent of bacterial wilt, were used as host-pathogen model. Specific objectives in the study were: (1) Development of PAL promoter-GUS reporter transformed Lycopersicon esculentum. (2) Establishment of a thermotolerance protocol that ensures optimal Hsp70 levels at subsequent HS. (3) Evaluation of the influence of prior heat treatment on phenylpropanoid metabolism after exposure to HS in combination with Ralstonia solanacearum. Results obtained support the hypothesis indicating that thermotolerance protects phenylpropanoid metabolism, in particular PAL promoter and enzyme activity, and to a certain extent lignin production, induced by avirulent Ralstonia solanacearum during a second severe HS. In contrast, HS without a prior heat treatment, suppressed phenylpropanoid metabolism. The protective potential of prior heat treatment during subsequent infection under hyperthermic conditions support the application of HSP in the development of novel plant protection strategies.

M.Sc. / Plants are in constant conflict with pathogens and have evolved intricate mechanisms to protect themselves against pathogens. The gene-for-gene response is regarded as the first line of defence when plant and pathogen meet. This interaction leads to the induction of defence proteins such as PR proteins that protect the plant from invading pathogens. A seemingly unrelated topic to plants and pathogens is heat shock proteins (HSP). HSP are a highly conserved group of defence proteins induced in all organisms in response to a variety of environmental stresses to provide protection from, and adaptation to cellular stress. HSP are in general not considered to be part of the defence response classically induced by avirulent pathogens and whether they are induced and play a role in plant-pathogen interactions is controversial. The protective chaperoning capacity of HSP makes them ideal proteins to exploit to target as endogenous defence proteins in the search for new strategies in the management of infectious diseases. In humans, HSP induction during infection is a complex phenomenon depending on the pathogen, whether the infection is acute or chronic, the host cell type and its differentiative state as well as environmental factors. In this investigation the expression of the inducible and constitutive isoforms of the 70kDa HSP (Hsp70/Hsc70) was investigated in tomato, Lycopersicon esculentum in response to virulent and avirulent strains of Ralstonia solanacearum, the causative agent of bacterial wilt. Expression of Hsp70 was studied in conjunction with the accumulation of PR-la and host cell viability. A quick, non-toxic, tetrazolium-based assay was developed from the Alamar Blue assay, commonly used in mammalian cells, and applied for the evaluation of host cell viability. The results shown suggest Hsp70/Hsc70 is significantly induced in tomato cell suspensions during an incompatible interaction 24h to 48 h following co-cultivation with the avirulent R. solanacearum strain compared to normal levels at this interval in cells exposed to the virulent strain. In both compatible and incompatible interactions Hsp70/Hsc70 levels eventually (72 h) accumulated correlating significantly with decreased viability. PR-la accumulation was significantly induced from 6 h to 18 h by the virulent as well as the avirulent R. solanacearum strains. In general, comparable results were obtained using leaf discs as an in vivo model. Based upon the differential induction of Hsp70/Hsc70 by virulent and avirulent pathogens it is proposed that HSP may play an important role in determining the outcome of the interaction between tomato and R. solanacearum. Successful defence may not only involve a limited number of defence genes but may result from a concerted action of a large number of defence genes.

Plants have evolved mechanisms to defend themselves against pathogen attack. These defense mechanisms consist of a series of inducible responses (including specific recognition of pathogen invasion, signal transduction and defense gene activation) that result in resistance. Plants responses to pathogen invasion also result in the suppression of various housekeeping activities of the cells, thus diverting the cellular resources to defense responses. Systemic acquired resistance (SAR), an inducible defense response enhanced as a result of initial infection with a necrotising pathogen, lead to long-term resistance in a plant. Differential gene expression of genes related to defense in cultured cotton cells and leaf disks that have been challenged with a purified elicitor from Verticillium dahliae, as well as a chemical inducer of defense responses, DL-b-amino-n-butyric acid, were investigated. The mRNA differential display reverse transcriptase polymerase chain reaction (DDRT-PCR) was used to identify differentially expressed genes 5 h after application of either 50 mg mL-1 Verticillium dahliae elicitor or 1 mM DL-b-amino-n-butyric acid to cotton cell suspension cultures and leaf disks. Identified cDNAs up- or down-regulated for this study were classified into seven groups: ‘Transcription factor’, ‘Ubiquitin and Proteasome’, ‘Mitochondria’, ‘Protein kinase/Receptor-like kinase’, ‘Defense/Resistance’, ‘Carbohydrate metabolism/Cell wall’ and ‘Other’. The identified cDNAs up-regulated after Verticillium dahliae elicitor treatment, classified in the ‘Transcription factor’ group, coded for a MYB family transcription factor, zinc finger protein and a RMA1 RING zinc finger protein. The identified cDNA classified in the ‘Mitochondria’ group coded for a cytochrome C oxidase subunit I and II and the cDNA classified in the ‘Protein kinase/Receptor-like kinase’ group coded for a serine/threonine protein kinase. The identified cDNA classified in the ‘Defense/Resistance’ group coded for a disease resistance protein family and the cDNAs classified in the ‘Carbohydrate metabolism/Cell wall’ group coded for a beta-1,4-Nacetylglucosaminyltransferase, a cellulose synthase-like protein, a 3-deoxy-D-manno-octulosonic acid transferase-like protein and a hydroxyproline-rich glycoprotein homolog. In addition, a cDNA classified in the ‘Other’ group, coded for a urea active transporter-like protein. The cDNA identified that was down-regulated after Verticillium dahliae elicitor treatment, classified in the ‘Carbohydrate metabolism/Cell wall’ group, coded for a proline-rich protein family and cDNAs classified in the ‘Other’ group coded for a thioredoxin reductase1 and ‘hookless1’ homologue. Among the identified cDNAs up-regulated after DL-b-amino-n-butyric acid treatment, classified in the ‘Ubiquitin and Proteasome’ group, were a 20S proteasome subunit alpha type 5 and an ubiquitin. The identified cDNA classified in the ‘Mitochondria’ group coded for a NADH dehydrogenase subunit 6, a mitochondrial DNA product. The identified cDNAs classified in the ‘Other’ group coded for an armadillo repeat containing protein and a phosphoinositide-specific phospholipase C. The cDNA identified that was down-regulated after DL-b-amino-n-butyric acid treatment, classified in the ‘Protein kinase/Receptor-like kinase’ group, coded for a casein kinase I like protein. The identified cDNA classified in the ‘Carbohydrate metabolism/Cell wall’ group, coded for a putative glycine rich protein. Also, the identified cDNA classified in the ‘Other’ group, coded for a NADH dehydrogenase subunit F that is coded for by chloroplast DNA. The differential expression of the cDNAs up-regulated after the Verticillium dahliae elicitor treatment was confirmed for seven of the nine cDNA clones with a Reverse Northern dot blot. Also, the differential expression of two cDNAs up-regulated after DL-b-amino-n-butyric acid treatment was confirmed and the induction kinetics was followed with a Reverse Northern dot blot. The mRNAs corresponding to C8B5, the gene encoding an ubiquitin, were detectable after 2.5 h and showed a significant increase in expression up to 7.5 h, after which the expression levels decreased to levels similar to those detected at 2.5 h. The mRNAs corresponding to L4B4, a homologue of an a-type subunit of 20S proteasome, were detectable after 2.5 h with an gradual increase in expression levels up to 7.5 h after which the expression levels decreased to levels similar to those detected at 2.5 h. This study facilitated a better understanding of differential gene regulation during triggering of defense responses in cotton following elicitation with the Verticillium dahliae elicitor and DL-b-aminon- butyric acid. / Prof. I.A. Dubery

M.Sc. / Plant disease can be naturally suppressed by plant growth promoting rhizobacteria and endophytic / endorhizosphere bacteria. Apart from direct antagonism against pathogenic organisms, these plant growth promoting bacteria and endophytes can induce a form of systemic resistance (ISR) in plants. The main bacterial inducing component has been suggested to be the outer membrane lipopolysaccharides (LPS), found in the cell walls of Gramnegative bacteria. Burkholderia cepacia (Pseudomonas cepacia) is a bacterial endophyte that has potential as a biocontrol agent. Although a few studies have indicated that LPS from, certain Pseudorrionads has a protective effect in plants against disease, a controlled investigation has not been attempted previously with a purified preparation of LPS. LPS was isolated from the bacterial cell wall, prepared and characterized by denaturing electrophoresis. Characterization of the LPS also included the determination of 2-keto-3-deoxyoctonate, carbohydrate —, as well as the protein content. The purified LPS was found to possess activity as an elicitor of plant defence responses in tobacco where the induction of pathogenesisrelated (PR) proteins were investigated and electrophoretically analysed. An optimum LPS concentration range of 50-150 14/m1 was determined by studying cell death using the Evans blue procedure. Time and concentration ranges for LPS induced responses were established in cell suspensions, leaf discs, whole leaves and whole plants. It was determined that the PR-protein response could be optimally induced after four days following elicitation with 100 fag/ml LPS. Systemic induction of resistance was tested by treatment of the lower leaves and following the response in the upper leaves; as well as bacterial inoculation of the plant roots followed by PR-protein extraction of the leaves. Treatment of tobacco plants with LPS protected the plants against subsequent infection by the pathogen Phytophthora nicotianae, thereby suggesting a role for LPS as activators of systemic acquired resistance (SAR). It can be concluded from this study that the lipopolysaccharides from Burkholderia cepacia, that were used in this study, are effective local as well as systemic inducers of the defense PR-proteins in Nicotianae tabacum cv Samsun NN. The fact that protection is associated with PR-protein induction distinguishes it from the protection induced by rhizobacteria.

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.