No description available.

Thesis (Ph. D.) -- Lincoln University, 2009. / Also available via the World Wide Web.

Thesis (Ph. D.)--Johns Hopkins University. / Includes bibliographical references.

No description available.

Crown gall disease of plants is caused by introduction of foreign DNA into susceptible plant cells by strains of Agrobacterium tumefaciens. The expression of bacterial virulence genes is triggered by chemicals present in plant wound exudates. The exudates contain a number of phenolic compounds which act as chemical signals inducing expression of a number of genes directing the DNA transfer process. These are the virulence or vir genes, and vir::lac reporter gene fusions have been widely used to assay vir gene induction in Agrobacterium tumefaciens strains. Using such strains to monitor vir gene expression, Stachel et al. (1985) isolated from Nicotiana tabacum two active acetophenones: 3,5-dimethoxy-4-hydroxyacetophenone, ("acetosyringone" or AS), and α-hydroxy-3,5-dimethoxy-4-hydroxy-acetophenone, ("hydroxyacetosyringone" or HO-AS). However, in vitro assay results suggested that other more common compounds also exhibited activity (Spencer and Towers, 1988). This analysis of structure-activity relationships of induced vir expression in A. tumefaciens was presented in a previous thesis (Paul Spencer, M.Sc. thesis). The results revealed that a variety of commonly occurring plant phenolic compounds were capable of activating vir genes. In addition to the acetophenones, a variety of benzoic and cinnamic acid derivatives, and even a few chalcones of appropriate ring substitution were active. This thesis reports the isolation and identification of a number of these compounds in plant wound exudates. Some Agrobacterium tumefaciens strains are restricted in host range to certain grapevine cultivars. Subsequent to the development of a convenient and sensitive plate-bioassay method, a strongly active component in grapevine wound exudates was purified. A newly described vir-inducing phenolic compound was isolated from a number of Vitis cultivars using gel filtration, thin layer and high pressure liquid chromatographies. This was identified as syringic acid methyl ester (3,5-dimethoxy-4-hydroxybenzoic acid, methyl ester), using mass spectrometry. However, the presence of this compound in grapevine wound exudates does not provide a simple explanation for host range limitation of grapevine strains since it induces vir gene expression in both limited and wide host range strains of A. tumefaciens. Interestingly, neither AS nor HO-AS were present in grapevine-derived extracts. A convenient polyamide column chromatographic method was subsequently developed to permit rapid purification of plant-derived vir gene inducing mixtures, which were detected using the newly developed plate bioassay. Derivatized polyamide fractions were then analysed by combined gas chromatography-mass spectrometry (GC-MS). GC-MS proved to be an ideal means for the identification of the phenolic components in partially purified extracts. Examination of wound exudates from a range of host and non-host species revealed that the production of the acetophenones is restricted to members of the Solanaceae. Some experiments focussed on the biosynthetic precursors of the acetophenones in Nicotiana species. Wound exudates of the majority of species belonging to other plant families contained benzaldehydes and/or benzoic and cinnamic acid derivatives. The induction of virE gene expression was examined in the related Agrobacterium species, A. rhizogenes. To do this, the virE::lacZ gene fusion plasmid pSM358cd was introduced into A. rhizogenes A4 by triparental mating and the strain "A4/pSM358cd" was used to analyze vir activation. Acetophenones, chalcones, benzaldehydes, and benzoic and cinnamic acid derivatives were found to activate vir genes in A. rhizogenes. / Science, Faculty of / Botany, Department of / Graduate

Ph.D. / Current models regarding plant : pathogen interactions assume that recognition of pathogen-associated molecular pattern (PAMP) molecules can occur through pattern recognition receptors (PRRs) on the surface of plant cells. Lipopolysaccharides (LPS) embedded in the cell wall of Gram-negative bacteria can trigger defence responses or prime the plant in order to respond more rapidly, following perception of bacterial pathogens. Limited data has been reported on signal transduction and the nature of the LPS receptors in plants since no receptors have been identified yet. Parallels have been shown to exist between self-incompatibility and pathogen recognition with regard to self / non-self recognition. The two processes were reviewed and conceptual and mechanistic links between microbial recognition and self-incompatibility were discussed herein. The role of S-domain receptor-like kinases (RLKs) in defence mechanisms has previously not been widely recognized or explored. It was reasoned that S-domain RLKs could be utilized to function as resistance (R) genes or as pattern recognition receptors in perception of PAMPs of a non-protein nature. It has been found that genes encoding receptors may be up-regulated in response to perception of its ligand. A putative receptor-like kinase was previously reported to be induced by LPS. This 153 bp differentially expressed transcript, HAP3-15 (GenBank accession number DR109311), might be an expressed sequence tag (EST) for a gene encoding a receptor for LPS. The experimental characterisation of this EST was reported herein. Gene-walking, reverse transcriptase polymerase chain reaction (RT-PCR), rapid amplification of cDNA ends (RACE), cloning, sequencing and bio-informatic analyses were used to identify the full gene. These results revealed that it encoded a receptor-like protein kinase with an extracellular S-domain recognition motif. The 2842 bp genomic sequence obtained, showed that the sequence had a defined promoter region and six major domains. The first five domains were encoded by the first exon. These domains included a B-lectin / agglutinin domain, an S-locus glycoprotein domain, an EGF-like repeat, a PAN domain, a transmembrane region and part of the 6th domain. The 6th domain was a kinase domain consisting of eleven sub-domains interspersed by three introns. The gene was therefore designated as the N. tabacum S-domain Receptor-like kinase (NS-RLK).

The microorganisms colonizing plants can have a significant effect on host phenotype, mediating such processes as pathogen resistance, stress tolerance, nutrient acquisition, growth, and reproduction. Research regarding plant-microbe interactions has focused almost exclusively on vascular plants, and we know comparatively little about how bryophytes -- including mosses, liverworts, and hornworts -- are influenced by their microbiomes. Ceratodon purpureus is a dioecious, cosmopolitan moss species that exhibits sex-specific fungal communities, yet we do not know whether these microbes have a differential effect on the growth and physiology of male and female genotypes. Using a common-garden design, we reared ten axenic genotypes of C. purpureus in a controlled environmental chamber. Clonal C. purpureus replicates, with and without the addition of a microbial inoculation, were used to test the effect of a mixed microbial community on vegetative growth, sex expression, photosynthetic efficiency (Fv/Fm and ETR), and chlorophyll content (CFR) for male and female mosses. We found that microbes had a negative impact on the growth and photosynthesis efficiency of C. purpureus, and this effect varied among genotypes of C. purpureus for ETR and growth. Microbes also had a positive, sex-specific effect on chlorophyll content in C. purpureus, with males exhibiting lower CFR values in the absence of microbes. C. purpureus sex expression was marginally negatively affected by microbe addition, but gametangia production was low overall in our experiment. We also conducted preliminary surveys using direct counts from moss ramets to assess the community composition of epiphytic algae associated with our microbe addition and control C. purpureus. These surveys identified three algal morphospecies in association with the microbe addition C. purpureus genotypes, as well as cyanobacteria, nematodes, rotifers, and testate amoeba. No algae, cyanobacteria, or micro-fauna were observed in the control plants. Transplantation of a mixed microbial community from field-to-laboratory conditions may be applied to other bryophyte species under varying environmental conditions to provide insight into how these diminutive yet important ecosystems will respond to environmental perturbation.

Thesis (M. Sc.) -- Lincoln University, 2009. / Also available via the World Wide Web.

As sessile organisms, plants evolved a plethora of defenses against their attackers. Given the role of plants as a primary food source for many organisms, plant defense has important implications for community ecology. Surprisingly, despite the potential to alter entire food webs and communities, the factors determining plant investment in defense are not well-understood, and are even less understood considering the numerous symbiotic interactions in the same plant. Legume-rhizobia symbioses engineer ecosystems by fixing nitrogen from the atmosphere in trade for plant photosynthates, yet connecting symbiotic resource exchange to food web interactions has yet to be established. Here I test how rhizobia influence plant defense and tritrophic interactions in lima bean (Fabaceae - Phaseolus lunatus L.): a model plant in chemical ecology research characterized by a broad range of different defenses. Examining suites of traits among lima bean genotypes, highly cyanogenic cultivars and wild type plants (high cyanotypes) produce more hook-shaped trichomes, as a putative combined approach of chemical and mechanical defenses, forming defense syndromes to protect against multiple feeding guilds (Chapter 2). Testing costs that may have contributed to forming tradeoffs among strategies, high cyanotypes show reduced fitness under plant-plant competition relative to low cyanotypes, but when challenged with herbivory, high cyanotypes fitness reductions are no longer evident (Chapter 3). Young leaves, not reproductive organs, are the most cyanogenic lima bean organ, and removal quantitatively decreases fitness, supporting assumptions that the most valuable tissues will be most highly defended (Chapter 4). Testing the degree to which nitrogen-fixing rhizobia contribute to cyanogenesis, high cyanotypes form more nodules than low cyanotypes. Quantitative relationships between nodule number and plant traits highlight the role symbiotic investment plays a role in plant defense and nutritive phenotype, while simultaneously, genotypically-determined levels of defense shape plant investment in symbiosis (Chapter 5). Interestingly, traits that trade off by cyanotype (i.e. high cyanogenesis but low indirect defense) reflect the patterns in plants with nitrogen-fixing rhizobia. Rhizobia-inoculated lima beans show reduced indirect defenses, recruiting fewer parasitoid wasps (Chapter 6) and predatory ants (Chapter 7). Examining plant-ant attraction in greater detail, ants prefer headspace regions above EFN droplets, corresponding with species-specific differences in suites of volatiles, indicating EFN, like floral nectar, can be scented to manipulate insect behavior (Chapter 8). Overall, understanding when investing in traits to recruit predators is more effective than investing in defensive chemistry, and how particular ecological contexts, such as symbioses can influence the outcome of defense allocation strategies remains a fascinating area of research. Determining the mechanisms underlying why rhizobia and other belowground microbial symbionts influence their host plants' above ground interactions, whether plants traits affected by symbiotic microbes are simply a function of the costs and benefits from resource exchange, or whether symbionts can influence the success of primarily direct versus indirectly defended plants is an important question for understanding complex trophic systems and connecting to agricultural implications for more effective biological pest control.

Symbiosis is the close and protracted interaction between organisms. The molecular interactions that occur during symbiosis are complex with multiple barriers that must be overcome. Many Gram-negative, host-associated bacteria use a type III secretion system to mediate associations with their eukaryotic hosts. This secretion system is a specialized apparatus for the injection of type III effector proteins directly into host cells, which in the case of plant pathogens, are collectively necessary to modulate host defense. The type III secretion system is not a mechanism exclusive to pathogens, however, as many strains of commensal Pseudomonas fluorescens and mutualistic rhizobia demonstrably require a type III secretion system to interact with their host plants. The work presented in this thesis describes genome-enabled approaches for characterizing type III effector genes across the range of plant symbiosis. Using high-throughput sequencing technology, draft genome sequences were generated for the plant pathogen, Xanthomonas hortorum pv. carotae M081, the plant commensal, Pseudomonas fluorescens WH6, and six strains from the plant mutualists Sinorhizobium fredii and Bradyrhizobium japonicum. Analyses of the draft genome sequences and publicly available finished sequences contributed insights into mechanisms of host-association and to increasing the inventory of type III effector sequences as well as developing methods directly applicable for agriculture. Finally, characterization of the genetic diversity of type III effectors from rhizobia shows that collections of type III effectors of mutualists are static, with little diversity in content and sequence variation. This represents the first comprehensive cataloging of type III effector from species of mutualistic bacteria and the first to provide evidence for purifying selection of this important class of genes. / Graduation date: 2012