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.

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.

D.Phil. (Biochemistry) / Plants are able to recognise and respond to signals from the environment through a complex array of biochemical pathways, which enables them to deter pathogenic micro-organisms and herbivores. Thousands of different structures of low-molecular weight organic compounds / natural products can be produced through an inducible chemical defence system; that can be manipulated for biotechnological purposes. The importance of natural products in medicine, agriculture and industry has led to numerous studies such as this, to understand the biosynthesis and biological activity of these substances...

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

Victoria blight, caused by fungus Cochliobolus victoriae, is a disease originally described on oats and recapitulated on Arabidopsis. Victoria blight is used as a model plant disease that conforms to an inverse gene-for-gene interaction. C. victoriae virulence is dependent upon its production of victorin, a host-specific toxin that induces programmed cell death in sensitive plants. In oats, victorin sensitivity and disease susceptibility is conferred by the Vb gene, which is genetically inseparable from the Pc-2 crown rust resistance gene. In Arabidopsis, victorin sensitivity and disease susceptibility is conferred by the LOCUS ORCHESTRATING VICTORIN EFFECTS 1 (LOV1) gene which encodes a NB-LRR protein, a type of protein commonly associated with disease resistance. LOV1-mediated cell death occurs when victorin binds Thioredoxin-h5 (TRX-h5) and LOV1 appears to "guards" TRX-h5. Together, these results suggest C. victoriae causes disease by inducing a resistance response. The work presented here aimed to determine if the response mediated by LOV1 is functionally related to a resistance response. We genetically characterized the response mediated by LOV1 with virus-induced gene silencing. We determined SUPPRESSOR OF THE G2 ALLELE OF SKP1 (SGT1), a gene required for the function of many resistance genes, is required for victorin sensitivity and involved in LOV1 protein accumulation. We screened a normalized library and identified six genes that suppressed victorin-mediated cell death and cell death induced by expression of the RESISTANCE TO PERONOSPORA PARASITICA PROTEIN 8 (RPP8) resistance gene: a mitochondrial phosphate transporter, glycolate oxidase, glutamine synthetase, glyceraldehyde 3-phosphate dehydrogenase and the P- and T-protein of the glycine decarboxylase complex. Silencing the latter four also inhibited cell death induced by the expression of an autoactive form of the resistance gene PTO, and reduced PTO-mediated resistance to Pseudomonas syringae pv. tabaci. These results provide evidence that victorin-mediated cell death is functionally similar to a resistance response, further supporting the hypothesis that a resistance response is exploited by C. victoriae for pathogenesis in Victoria blight. Resistance function of LOV1 was evaluated by observing Pseudomonas syringae pv. tomato virulence upon LOV1 activation. The LOV1 response pathway in Arabidopsis was adapted to activate upon infection with Pseudomonas syringae pv. tomato expressing the type III-dependent effector protein AvrRpt2, a well-characterized protease. We developed a construct to express a beta-glucuronidase (GUS) and TRX-h5 fusion protein separated by an AvrRpt2 proteolytic cleavage site, in which GUS sterically inhibits TRX-h5 function in LOV1-mediated cell death. The fusion is cleaved upon infection by P. syringae pv. tomato expressing avrRpt2, thereby leading to TRX-h5-mediated activation of LOV1 in the presence of victorin. However, when this strain was inoculated with victorin into transgenic LOV1 trx-h5 plants expressing the GUS/TRX-h5 fusion protein, no decrease in pathogen virulence was observed. Technical shortcomings likely prevented observable LOV1 resistance function. ��� / Graduation date: 2013 / Access restricted to the OSU Community at author's request from Oct. 9, 2012 - Oct. 9, 2013

[No abstract available] / xi, 176 leaves : ill. (chiefly col.) ; 29 cm

The nucleotide sequence of a cloned RAPD DNA marker (OPI08) linked to a disease resistance gene in L. sativa (lettuce) revealed homology with the conserved domain of the reverse transcriptase of Ty3/gypsy retrotransposons. To further characterize the presence of Ty3/gypsy-like sequences in plants, sets of degenerate primers deduced from archetype retrotransposons were used for PCR amplification of a sequence domain characteristic of the reverse transcriptase and the integrase of Ty3/gypsy retrotransposons. The nucleotide sequence of two cloned DNA fragments of Z. mays (maize) and A. thaliana proved to be homologous with the conserved domains of the reverse transcriptase and the integrase of Ty3/gypsy retrotransposons. Southern blot analysis also demonstrated homology of the Z. mays clone to Lycopersicon (tomato) and L. sativa. This is the first report of Ty3/gypsy-like sequences in A. thaliana, and L. sativa. This research brings to six the number of plant species where this type of element has been reported, in contrast to the large number of plant Ty1/copia transposable elements described. It is not known whether these elements are actively transposing in plant genomes.

No description available.

A three-year (1998-2000) field and laboratory study conducted in Fredericton, New Brunswick, Canada analyzed and quantified the dispersal of adult Colorado potato beetles within and between conventional and transgenic potato plots established according to the high-dose/refuge strategy. More specifically, the study addressed the following four predictions: (1) Adult Colorado potato beetle abundance or flight activity in transgenic potato fields is positively correlated to the abundance or flight activity in the immediately adjacent refuge field. (2) Colorado potato beetle intraspecific competition on potato plants will increase the flight take-off frequency of adult CPB; tolerating relatively high numbers of CPB egg masses or larvae or a high level of CPB defoliation on potato plants in the refuge could therefore be considered to increase the movement of beetles from the refuge to the transgenic field. (3) CPB flight take-off frequency will be higher on potato plants at the bloom than at the vegetative stage; planting of the non transgenic potato crop in the refuge earlier than the Bt transgenic potato crop in the main field could therefore be considered to increase movement of the Colorado potato beetles from the refuge to the transgenic field. (4) The aggregated distribution of CPB populations in the potato crop is caused by the presence of mating pairs; strategies changing the distribution of males and females in the refuge field could therefore be considered to increase dispersal from the refuge to the main crop field. / Population monitoring using plant counts, flight interception traps, flight landing traps and pitfall traps established that a transient population of adult CPB is present in the transgenic potato fields throughout the crop season and that the abundance of the beetle is higher than that required by the high dose/refuge strategy models. Furthermore results showed that the beetles invading the transgenic field population originate as much from the surrounding fields of conventional cultivars as from the adjacent refuges. It would therefore be possible to relax existing requirements for the refuge to be located immediately adjacent to the transgenic crop. / Like most insects, the adult CPB populations are aggregated and can be fitted to a negative binomial distribution over the crop season. This study revealed that the distribution results from the presence of mating pairs for the overwintered population and from the clumped pupation for the non breeding summer population. The activity of the males in search of females is at least partly responsible for the higher dispersal activity observed with the overwintered than with the summer populations. The comparatively low level of dispersal activity with the summer population could affect the efficacy of the high/dose refuge strategy during the later part of the crop season. / Results of flight chamber tests demonstrated that plant phenology and intraspecific competition have a positive effect on flight take-off frequency. These findings suggest that summer adult dispersal between the refuges and the transgenic crops could be stimulated by manipulating planting dates and the abundance of the different CPB life stages on the plants. / Together, the results of the thesis provide support for some of the premises of the high dose/refuge strategy and offer new information on the CPB dispersal that could be used to further improve its efficacy. Although the transgenic potato (NewLeaf) is not commercially available at this time, the threat of CPB resistance to new products or resistant cultivars under development makes it important to continue the research required by CPB resistance management plans.

No description available.