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Plants produce a large number of specialized or secondary compounds that aid in their reproduction and protection against biotic and abiotic stress. In this work I investigated the metabolism and function of terpenes, the largest class of specialized metabolites, in switchgrass and carrot. Switchgrass (Panicum virgatum L.), a perennial C4 grass of the Tallgrass Prairie, represents an important species in natural and anthropogenic grasslands of North America. Its natural resilience to abiotic and biotic stress has made switchgrass a preferred bioenergy crop. I have investigated the metabolism of terpenes in switchgrass leaves and roots in response to herbivory or defense hormone treatments and the application of drought. With a focus on volatile terpene metabolites, I functionally characterized over thirty genes (terpene synthases, TPSs), of which one third could be correlated with the production and release of volatile monoterpenes and sesquiterpenes that likely function in direct chemical defense or in the attraction of insect predators or parasitoids. Drought stress application caused switchgrass roots to accumulate a larger amount of oxygenated terpenes and presumably non-volatile terpenes, the function of which in direct or indirect drought stress protection requires further investigation. I also examined the metabolic dynamics and role of the monoterpene borneol, which accumulates at high concentrations in the roots of switchgrass and to a lower extent in the roots of the close relative Setaria viridis, in root microbe interactions. Although we demonstrated a successful RNAi based knock down of the borneol terpene synthase TPS04, we found no immediate evidence that borneol significantly modifies bacterial communities in the root. Further studies on Setaria and equivalent RNAi lines in switchgrass will provide more detailed and needed insight to decipher the role of monoterpene accumulation in grasses interactions with mutualists, pathogens, and pests. In an applied project, I investigated terpene specialized metabolism in carrot (Daucus carota L.) to identify genetic determinants of carrot aroma and flavor. To determine central enzymes which contribute to the terpene component of carrot volatile blends, we first analyzed tissue specific expression patterns of carrot terpene synthase genes (TPS) in the genomic model carrot (cv. DH1) and in roots of four aromatically unique colored carrot genotypes (orange-4943B, red-R6637, yellow-Y9244A and purple-P7262). We selected nineteen key biosynthetic enzymes involved in terpene formation and compared in vitro products from recombinant proteins with native volatile profiles obtained from DH1 and colored carrot genotypes. We biochemically characterized several highly expressed TPSs with direct correlations to major compounds of carrot flavor and aroma including germacrene-D (DcTPS11), (DcTPS30) and -terpinolene (DcTPS03). Random forest analysis of colored carrot volatiles revealed that nine terpene compounds are sufficient for distinguishing the flavor and aroma of raw colored carrots. Interestingly, accumulation of specific terpene compounds rather than chemical diversity is responsible for differences in sensory quality traits in colored genotypes. As accumulations of specific terpene compounds can contribute to the undesired flavor in carrot, our report provides a detailed roadmap for future breeding efforts to enhance carrot flavor and aroma. / Doctor of Philosophy / Plants produce a large number of chemicals that are important for growth, defense, flavor, and aroma. While chemical production has been studied in some major food crops (corn, tomato, rice), knowledge of the formation and function of chemicals in switchgrass and carrot is still limited. Switchgrass (Panicum virgatum L.), a grass of the Tallgrass Prairie, represents an important species grasslands of North America. Its natural resilience to stress has made switchgrass a preferred bioenergy crop. I found that switchgrass produces many compounds in the chemical class of terpenoids in roots and leaves that likely serve as a defense against damage from pests. In addition, I found that drought stress leads to the production of terpenoid compounds that may have roles in protection when water is limited. My research also demonstrates that roots of switchgrass and the related grass Setaria maintain substantial levels of the essential oil compound borneol. This terpenoid compound can act as a nutrient source for specific bacteria and/or an antimicrobial agent. Therefore, I proposed that switchgrass and Setaria roots produce borneol to establish a distinct root microbiome by recruitment of beneficial bacteria and deterrence of harmful microorganisms. To test this hypothesis, we genetically engineered plants to reduce borneol formation and accumulation in roots. Using these plants, we evaluated changes in the root microbiome in response to altered borneol levels. We found that interfering with borneol production in Setaria roots has limited influence on the microbiome inside roots. Although a similar approach was used for switchgrass, we were unable to significantly reduce borneol V formation in roots. Results from this study provide a better understanding of belowground plant-microbe interactions, and potential for enhancing resistance traits into other crop species. I also investigated the flavor and aroma compounds produced in carrots, which are considered a key supplemental vegetable due to high nutritional value and pleasant taste. Surprisingly, little has been known about the genetic factors that control flavor and aroma traits in colored carrot varieties. Therefore, I performed a robust characterization of the biosynthesis of terpenoids, which are the predominant aroma and flavor compounds in carrot. I identified several enzymes in carrot that can produce a diverse blend of terpenoids which are associated with sweet, spicy and bitter tastes. In addition, I discovered that carrot stems and leaves also maintain a rich chemistry of terpenoids similar to that in roots. Results from this work provide a baseline for engineering enhanced flavor in carrot and provide a deeper insight into essential oil formation in root crops.

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.

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.

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 molecular basis for the gene-for-gene relationship of Vm-resistance in apple to Venturia inaequalis was investigated. Incompatible reactions involved a hypersensitive response (HR), which was accompanied by the accumulation of dark brown pigments and autofluorescent materials in epidermal and mesophyll cells at the site of invasion. Cell-free culture filtrates of the avirulent isolate elicited an HR in the Vm host (h5) leaves, but not in the susceptible host (h1). The elicitor activity was resistant to boiling but was abolished by proteinase K digestion. Elicitation of HR was used to monitor purification of the avirulence factor, AVRVm, from liquid cultures of the avirulent isolate following ultrafiltration, acetone precipitation and ion-exchange chromatography. The purest fraction contained three major proteins all with low isoelectric points (pI 3.0-4.5). The fraction also elicited HR on the differential host h4, but not on other resistant hosts (h2, h3 and h6) tested. Three candidate AVRVm proteins were identified and amino acid sequences were obtained using Edman degradation and mass spectrometry. Nucleotide sequences corresponding to these proteins were found in databases of V. inaequalis expressed sequence tags. There were no polymorphisms evident between avirulent and virulent isolates (representing races 1 and 5 respectively) either at genomic DNA or cDNA level of the full open reading frames. RT-PCR revealed that all genes were expressed in both avirulent and virulent isolates during in vitro and in planta growth. All three genes showed similar levels of expression between avirulent and virulent isolates during their in vitro growth. However, preliminary RT-PCR experiments showed that two of these genes were likely to be expressed at lower levels in the virulent compared with the avirulent isolate during compatible infection. Implications of this difference in expression and the future experiments to identify the genuine AvrVm gene were discussed.

The molecular basis for the gene-for-gene relationship of Vm-resistance in apple to Venturia inaequalis was investigated. Incompatible reactions involved a hypersensitive response (HR), which was accompanied by the accumulation of dark brown pigments and autofluorescent materials in epidermal and mesophyll cells at the site of invasion. Cell-free culture filtrates of the avirulent isolate elicited an HR in the Vm host (h5) leaves, but not in the susceptible host (h1). The elicitor activity was resistant to boiling but was abolished by proteinase K digestion. Elicitation of HR was used to monitor purification of the avirulence factor, AVRVm, from liquid cultures of the avirulent isolate following ultrafiltration, acetone precipitation and ion-exchange chromatography. The purest fraction contained three major proteins all with low isoelectric points (pI 3.0-4.5). The fraction also elicited HR on the differential host h4, but not on other resistant hosts (h2, h3 and h6) tested. Three candidate AVRVm proteins were identified and amino acid sequences were obtained using Edman degradation and mass spectrometry. Nucleotide sequences corresponding to these proteins were found in databases of V. inaequalis expressed sequence tags. There were no polymorphisms evident between avirulent and virulent isolates (representing races 1 and 5 respectively) either at genomic DNA or cDNA level of the full open reading frames. RT-PCR revealed that all genes were expressed in both avirulent and virulent isolates during in vitro and in planta growth. All three genes showed similar levels of expression between avirulent and virulent isolates during their in vitro growth. However, preliminary RT-PCR experiments showed that two of these genes were likely to be expressed at lower levels in the virulent compared with the avirulent isolate during compatible infection. Implications of this difference in expression and the future experiments to identify the genuine AvrVm gene were discussed.

The molecular basis for the gene-for-gene relationship of Vm-resistance in apple to Venturia inaequalis was investigated. Incompatible reactions involved a hypersensitive response (HR), which was accompanied by the accumulation of dark brown pigments and autofluorescent materials in epidermal and mesophyll cells at the site of invasion. Cell-free culture filtrates of the avirulent isolate elicited an HR in the Vm host (h5) leaves, but not in the susceptible host (h1). The elicitor activity was resistant to boiling but was abolished by proteinase K digestion. Elicitation of HR was used to monitor purification of the avirulence factor, AVRVm, from liquid cultures of the avirulent isolate following ultrafiltration, acetone precipitation and ion-exchange chromatography. The purest fraction contained three major proteins all with low isoelectric points (pI 3.0-4.5). The fraction also elicited HR on the differential host h4, but not on other resistant hosts (h2, h3 and h6) tested. Three candidate AVRVm proteins were identified and amino acid sequences were obtained using Edman degradation and mass spectrometry. Nucleotide sequences corresponding to these proteins were found in databases of V. inaequalis expressed sequence tags. There were no polymorphisms evident between avirulent and virulent isolates (representing races 1 and 5 respectively) either at genomic DNA or cDNA level of the full open reading frames. RT-PCR revealed that all genes were expressed in both avirulent and virulent isolates during in vitro and in planta growth. All three genes showed similar levels of expression between avirulent and virulent isolates during their in vitro growth. However, preliminary RT-PCR experiments showed that two of these genes were likely to be expressed at lower levels in the virulent compared with the avirulent isolate during compatible infection. Implications of this difference in expression and the future experiments to identify the genuine AvrVm gene were discussed.

The molecular basis for the gene-for-gene relationship of Vm-resistance in apple to Venturia inaequalis was investigated. Incompatible reactions involved a hypersensitive response (HR), which was accompanied by the accumulation of dark brown pigments and autofluorescent materials in epidermal and mesophyll cells at the site of invasion. Cell-free culture filtrates of the avirulent isolate elicited an HR in the Vm host (h5) leaves, but not in the susceptible host (h1). The elicitor activity was resistant to boiling but was abolished by proteinase K digestion. Elicitation of HR was used to monitor purification of the avirulence factor, AVRVm, from liquid cultures of the avirulent isolate following ultrafiltration, acetone precipitation and ion-exchange chromatography. The purest fraction contained three major proteins all with low isoelectric points (pI 3.0-4.5). The fraction also elicited HR on the differential host h4, but not on other resistant hosts (h2, h3 and h6) tested. Three candidate AVRVm proteins were identified and amino acid sequences were obtained using Edman degradation and mass spectrometry. Nucleotide sequences corresponding to these proteins were found in databases of V. inaequalis expressed sequence tags. There were no polymorphisms evident between avirulent and virulent isolates (representing races 1 and 5 respectively) either at genomic DNA or cDNA level of the full open reading frames. RT-PCR revealed that all genes were expressed in both avirulent and virulent isolates during in vitro and in planta growth. All three genes showed similar levels of expression between avirulent and virulent isolates during their in vitro growth. However, preliminary RT-PCR experiments showed that two of these genes were likely to be expressed at lower levels in the virulent compared with the avirulent isolate during compatible infection. Implications of this difference in expression and the future experiments to identify the genuine AvrVm gene were discussed.