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Fusarium oxysporum (Fo) is a soil-borne fungal pathogen that causes vascular wilt disease on a broad range of plants, including agricultural crops and the model plant Arabidopsis thaliana. There are non-pathogenic members of the Fo species complex that confer defense benefits against other pathogens to the host plant, however alteration to the host’s physiology through interaction with one of these strains, Fo47, have not been described. In this study, we aimed to establish the Fo47-A. thaliana interaction and determine if Fo47 reduces disease severity of a pathogenic Fo isolate, Fo5176. Additionally, we sought to use bioinformatics to mine transcriptomic data of the infection between Fo47 and A. thaliana for putative effectors from the non-pathogenic isolate using a pipeline that is validated by identifying known effectors in the interaction between Fo5176 and A. thaliana. Phenotypic characterization of A. thaliana plants inoculated with Fo47 or Fo5176 has revealed a significant increase in rosette biomass of Fo47 inoculated plants when compared to mock (sterile water) inoculated plants. As is observed in other systems, treatment of plants with Fo47 prior to challenging with pathogenic Fo significantly reduces the disease severity over time. The results of this study suggest that Fo47 is a possible biocontrol agent against Fo5176, and that inoculation with non-pathogenic Fo alters the physiology of A. thaliana such that it has a higher rosette biomass without alterations to the water status of the plant. Our pipeline for extracting putative effectors using transcriptomic data as a critical filter generated 13 candidate genes for further experimentation to determine their role in the Fo47-A. thaliana interaction. This research reports the first known observation that Fo47 increases the shoot biomass of the host plant it is interacting with, and that the model plant A. thaliana can be used as a host to examine the spectrum of interactions capable within the Fusarium oxysporum species complex.

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

Appressorium-mediated plant infection is a common strategy used by many plant pathogenic fungi. Understanding the underlying genetic network that controls cellular differentiation of appressorium is therefore pivotal to design durable resistance strategies for these devastating pathogens. This thesis describes four published studies, which investigate the role of septin GTPases in infection and the role of secretion during plant tissue invasion by the rice blast pathogen Magnaporthe oryzae. Appressorium development involves a series of morphogenetic changes that are tightly regulated by cell cycle checkpoints. Entry into mitosis allows differentiation of an appressorium, while penetration peg emergence appears to require progression through subsequent cell cycle checkpoints and cytokinesis. The studies presented here show that symmetry-breaking events that occur during appressorium differentiation are mediated by scaffold proteins, named septins. Septin GTPases recruit actomyosin ring components during septation and define the site of cytokinesis. They also recruit a toroidal cortical F-actin network to the appressorium pore that provides cortical rigidity to facilitate plant infection. Septins act as diffusion barriers for proteins that mediate membrane curvature necessary for penetration peg formation. Repolarization of the F-actin cytoskeleton at the appressorium pore is essential for plant penetration and is controlled by cell polarity regulators, such as Cdc42 and Chm1. Septin-mediated plant infection is regulated by NADPH oxidase (Nox) dependent generation of reactive oxygen species (ROS). The Nox2/NoxR complex is essential for septin organization at the appressorium pore. Septins are therefore key determinants of appressorium repolarization. I also report an investigation of fungal secretory processes during tissue invasion and present evidence that distinct pathways are involved in effector secretion by Magnaporthe oryzae. A BrefeldinA-sensitive pathway is necessary for secretion of apoplastic effectors, such as Bas4 and Slp1, while a BrefeldinA-insensitive pathway is necessary for secretion of effectors destined for delivery to rice cells.

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).

Low nutrition availability in the soil can be a major limitation of plant growth. To improve nutrient acquisition, the majority of land plants engage in symbiosis with arbuscular mycorrhizal (AM) fungi. The accommodation of fungal colonisation structures in the roots requires their radical reprogramming. This starts during pre-symbiotic communication, where signals are exchanged between the fungus and plant across the rhizosphere. The receptor D14-LIKE emerged as a vital component of this pre-symbiotic communication when it was found to be absolutely required for symbiosis in rice. However, the broader relevance of the receptor, both in terms of functional conservation across plant species and its relation to other pre-symbiotic plant signalling components, remained unclear. The aim of this thesis was to elucidate these two key points. To address the fragmented picture of fungal signals, plant receptors and signalling pathways, a large scale transcriptomic experiment in rice was conducted to tie D14L together with other distinct pre-symbiotic components. In the absence of D14L-mediated signalling, rice was found to be compromised in the perception of germinated spore exudates, as well as specific chitinaceous signals, meaning that normal transcriptional reprogramming could not be achieved in response to any of these treatments. In addition, the functional conservation of D14L signalling was explored using trans-species complementation experiments. It was found that the Arabidopsis homolog AtKAI2 could complement the developmental phenotype of the d14l rice mutant, but not symbiosis. Likewise, D14La from early diverging Marchantia polymorpha and Marachantia paleacea could rescue developmental phenotypes in d14l rice, but again failed to complement symbiosis. This demonstrated a functional separation between developmental and symbiotic signalling. The data generated during my PhD foster D14L as a central node for multiple inputs to pre-symbiotic reprogramming, and provides new insights into pre-symbiotic communication mechanisms which are required for the successful establishment of symbiosis.

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.

Xanthates, key compounds used in the mining industry for the separation of metals from ores, may have a significant impact on the ecosystems once discarded into the tailings. The aims of this study were to : characterize chemical and microbiological conditions in a tailings lagoon (Brown Creek gold mine); examine the feasability of using indigenous bacteria to degrade xanthate under laboratory conditions; and, optimize the biodegradation process by employing biofilm technology and plant-microbe associations. To achieve these, a simple but accurate HPLC method was developed. The chemical and biological constitution of the tailings lagoon was monitored over a 2 year period from March 1995 to February 1997. Xanthate degradative performance was improved by Phragmites australis and its root associated bacterial community. Cumulative amounts of xanthate degraded by this plant-microbe association were significantly higher than any other treatment, specifically glass-attached and free-living bacterial populations. An increased bacterial number in the extensively developed root, the presence of extracellular layer as revealed from scanning electron microscope and significance of plant removal via uptake were thought to be the possible explanations for the enhanced biodegradation activity observed. In view of the above, use of glass-attached bacteria and plant-microbe associations might provide a feasible solution to degrade xanthate in the tailings lagoon. The results obtained from this study suggest that the plant-microbe associations could provide a cost effective method to degrade/remove xanthate from the tailings lagoon runoff. This approach offers several ecological advantages. Firstly, it offers the advantage of a solar-powered treatment system that is aesthetically pleasing. Secondly, bioremediation can be done on site, thereby eliminating waste transportation costs. Finally, it has the added attraction of a higher potential for public acceptance / Doctor of Philosophy (PhD)

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

The effect of Ascophyllum nodosum extracts on the nodulation and growth of alfalfa was investigated. Plant growth assay revealed that alfalfa treated with 2 g L-1 ANE exhibited a significant increase in leaf area. Under salt stress, alfalfa treated with 0.5 g L-1 ANE exhibited a significant increase in total length compared to controls. A root hair deformation assay indicated that ANE 0.5 g L-1 stimulated the synthesis of Nod factors secreted by rhizobia thus accelerate root hair deformation of alfalfa. Similarly, ANE 0.5 g L-1 caused an increase in nodC gene expression suggesting that ANE may act similarly to flavonoids in the rhizobium-legume symbiosis. Under field conditions, ANE increased the total number of functional nodules, total root length and total leaf area. Taken together, the results suggest that ANE may contain compound(s) that promote specific metabolic pathway both in alfalfa and bacterium thus enhance the symbiotic relationship.