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The ecological factors that maintain defensive chemical variation within and between plant species have intrigued ecologists for decades. While theory posits that polymorphisms may be maintained different forms of balancing selection, relatively few experimental studies have tested whether such balancing selection can maintain defensive chemical trait polymorphisms in nature. Further, evidence demonstrating balancing selection is rare for any trait. Here, I investigated a stereochemical trait polymorphism in Xanthium strumarium. This species is polymorphic with respect to the stereochemistry of the lactone ring junction of a prominent defensive compound class ? the sesquiterpene lactones. Individual plants typically produce only cis-fused or trans-fused lactones across their entire suite of compounds. Sesquiterpene lactones are known to influence feeding behavior and growth rates of various herbivores, but nothing is known about the ecological implications of variation in this stereochemical trait. I first examined whether sesquiterpene lactone stereochemical variation can influence folivore feeding behavior in the laboratory. Using pure sesquiterpene lactones in controlled feeding experiments, I found that laboratory-reared grasshoppers were less deterred by the cis-fused compounds than the trans-fused compounds. I then found that these patterns extended to the field: in common gardens, plants producing cis-fused lactones received more damage than plants producing trans-fused lactones. Additionally, folivore damage was negatively correlated with plant fitness. Taken together, these results indicate that herbivores can impose natural selection on this stereochemical trait polymorphism in nature. Finally, I found evidence that spatially variable selection leads to fitness patterns conducive to the maintenance of this polymorphism. Further, I found that the intensity of folivore damage across spatial scales predicted in which environments each morph outperformed the other, with plants producing cis-fused lactones achieving higher fitness than plants producing trans-fused lactones when herbivore pressure was low (and the reverse being true when herbivore pressure was high). This work demonstrates that relatively minor defensive chemical variation can have far-reaching impacts on the ecology and evolution of plant populations.

Iron is an essential component of various proteins and pigments for both plants and pathogenic fungi. However, redox cycling between the ferric and ferrous forms of iron can also catalyse the production of dangerous free radicals and iron homeostasis is therefore tightly regulated. During pathogen attack, plants quickly produce large amounts of reactive oxygen species at the site of attempted pathogen ingress. This so-called oxidative burst has received considerable attention, but no single enzyme has been shown to account for the phenomenon. Using inductively coupled plasma mass spectrometry and histochemistry, I show that iron is secreted to the apoplast of the diploid wheat <i>Triticum monococcum</i> during attack by the powdery mildew fungus <i>Blumeria graminis</i> f.sp. <i>tritici</i>. This iron accumulates at cell wall appositions synthesised de novo beneath sites of pathogen attack. I further show, using histochemistry and pharmaceutical inhibitors, that this apoplastic iron accumulation is required for production of H2O2 in the oxidative burst. To understand the impact of this massive change in iron homeostasis on gene transcription, I employ a 187 gene targeted macroarray platform and establish that iron overload induces the expression of iron homeostasis-related genes and defence-related genes through iron itself and iron-mediated H2O2 production, respectively. To illustrate how the plant is able to withstand the negative effects of its own oxidative defences, I characterise a novel quinone redox cycle, and show that simultaneous induction of a protective quinone reductase isoform and downregulation of reactive oxygen-producing quinone reductase isoform prevents the spread of reactive oxygen during pathogen attack. Finally, in an effort to understand the impact of iron on fungal pathogenicity, I investigate iron uptake in the head blight pathogen, <i>Fusarium graminearum</i>. Fungi use at least two separate systems to take up iron, one based on enzymatic iron reduction and the other based on the synthesis and secretion of small iron chelators termed siderophores. Using mutants disrupted in either of two modes of iron uptake, I establish that siderophore production is essential for full F. graminearum virulence on wheat. This thesis exposes iron as an important component of both plant defence and fungal virulence.

Iron is an essential component of various proteins and pigments for both plants and pathogenic fungi. However, redox cycling between the ferric and ferrous forms of iron can also catalyse the production of dangerous free radicals and iron homeostasis is therefore tightly regulated. During pathogen attack, plants quickly produce large amounts of reactive oxygen species at the site of attempted pathogen ingress. This so-called oxidative burst has received considerable attention, but no single enzyme has been shown to account for the phenomenon. Using inductively coupled plasma mass spectrometry and histochemistry, I show that iron is secreted to the apoplast of the diploid wheat <i>Triticum monococcum</i> during attack by the powdery mildew fungus <i>Blumeria graminis</i> f.sp. <i>tritici</i>. This iron accumulates at cell wall appositions synthesised de novo beneath sites of pathogen attack. I further show, using histochemistry and pharmaceutical inhibitors, that this apoplastic iron accumulation is required for production of H2O2 in the oxidative burst. To understand the impact of this massive change in iron homeostasis on gene transcription, I employ a 187 gene targeted macroarray platform and establish that iron overload induces the expression of iron homeostasis-related genes and defence-related genes through iron itself and iron-mediated H2O2 production, respectively. To illustrate how the plant is able to withstand the negative effects of its own oxidative defences, I characterise a novel quinone redox cycle, and show that simultaneous induction of a protective quinone reductase isoform and downregulation of reactive oxygen-producing quinone reductase isoform prevents the spread of reactive oxygen during pathogen attack. Finally, in an effort to understand the impact of iron on fungal pathogenicity, I investigate iron uptake in the head blight pathogen, <i>Fusarium graminearum</i>. Fungi use at least two separate systems to take up iron, one based on enzymatic iron reduction and the other based on the synthesis and secretion of small iron chelators termed siderophores. Using mutants disrupted in either of two modes of iron uptake, I establish that siderophore production is essential for full F. graminearum virulence on wheat. This thesis exposes iron as an important component of both plant defence and fungal virulence.

The molecular interaction between plants and insects is dynamic and multifaceted. We are interested in understanding the molecular mechanism that insects utilize to overcome plant defense proteins, as well as discovering novel plant insecticidal proteins. Three projects were developed. First, we evaluated the effects of soybean cysteine protease inhibitor (soyacystatin N, scN) on the growth and development in southern corn rootworm. Both subtractive suppressed hybridization (SSH) and cDNA microarray analyses were used to uncover the changes of gene expression profiles in southern corn rootworm under the scN challenge. The counterdefense-related genes were identified, suggesting that southern corn rootworm deployed several regulatory mechanisms to overcome the dietary scN. Second, to identify and confirm insecticidal properties of vegetative storage protein 2 in Arabidopsis (AtVSP2), the gene was cloned and expressed in E.coli. This protein showed acid phosphatase activity. Feeding assay indicated that AtVSP increased the mortality and delayed the development of two coleopteran and one dipteran insects. Third, to identify the molecular mechanism of this novel insecticidal protein, P element mutagenesis was utilized to generate AtVSP resistant mutants (VRs). Two balanced VR mutants and their revertants were generated, and can be used to further characterize the genetic loci of P element inserted in the mutants.

A suite of plants from the maize genus Zea L. (Poaceae) and the specialist herbivore Dalbulus maidis (DeLong and Wolcott, 1923) (Hemiptera: Cicadellidae) were used to test the hypotheses that anti-herbivore defenses are affected by plant life-history evolution and human intervention through domestication and breeding for high yield. The suite of plants included a commercial hybrid maize (Zea mays ssp. mays L.), a landrace maize, two populations of annual Balsas teosinte (Z. mays ssp. parviglumis Iltis & Doebley), and perennial teosinte (Z. diploperennis Iltis, Doebley & Guzman). Leaf toughness and pubescence, oviposition preference, and feeding and oviposition acceptance parameters were compared among the suite of host plants looking for effects of transitions in life history (perennial to annual teosinte), domestication (annual teosinte to landrace maize), and breeding (landrace maize to hybrid maize) on defenses against D. maidis. Observations on leaf toughness suggested that the life history and domestication transitions weakened the plant’s resistance to penetration by the herbivore’s mouthparts and ovipositor, as expected, while observations on pubescence suggested that the breeding transition led to stronger defense in hybrid maize compared to landrace maize, contrary to expectation. Observations on oviposition preference of D. maidis coincided with the expectations that life history and domestication transitions would lead to preference for Balsas teosinte over perennial teosinte, and of landrace maize over Balsas teosinte. A negative correlation suggested that oviposition preference is significantly influenced by leaf toughness. Observations on host plant feeding and iii oviposition acceptance under no-choice conditions suggested that D. maidis equally accepts all host plants considered in this study, thus these observations did not support the hypotheses associated with the life history, domestication, and breeding transitions evident in the herbivore’s host genus. Overall, the results of this study suggested that plant defenses against specialist herbivores are variably affected by plant life history evolution, domestication, and breeding. Additionally, the study’s results suggested that chemical defenses may play a role in Zea antiherbivore defense because the two physical defenses that were evaluated (i.e. leaf toughness and pubescence) only partially explained host preference of D. maidis

The parasitic angiosperm Orobanche aegyptiaca (Pers.) (Egyptian broomrape) is a root holoparasite that causes severe losses in yield and quality of many crops. Control of Orobanche is extremely challenging, in part because the parasite is hidden underground for most of its life cycle. However, the dependence of the parasite on the host suggests that broomrape-resistant hosts could be an ideal control method. Genetic engineering strategies may facilitate realization of this goal, but require an understanding of host defense responses to parasitism. Previous studies with tobacco indicated that broomrape parasitism induces host genes associated with jasmonic acid (JA)-mediated defenses such as wound responses and localized production of phenylpropanoid and isoprenoid phytoalexins. However, the gene for the pathogenesis-related (PR) protein, PR-1a, was not induced by parasitism in tobacco. Expression of PR-1a is correlated with the salicylic acid (SA)-mediated defense pathway that leads to systemic acquired resistance (SAR). The objective of this research was to extend the characterization of PR gene expression in order to define the scope of host defense response. Analyses of gene expression using RNA hybridization and RT-PCR in broomrape-parasitized Arabidopsis thaliana roots indicated that PR-1, PR-2, PR-5, as well as the JA-associated PDF1.2, were slightly induced by parasitism. Expression of PR-1, PR-5, and PDF1.2 in parasitized roots was not detectable by RNA hybridization analysis, but was demonstrated by RT-PCR. Interestingly, shoots of the parasitized plants showed greater PR gene expression levels than roots, indicating that O. aegyptiaca induced a response in the host that was systemic and amplified in shoots. Microarray analysis of parasitized Arabidopsis roots demonstrated a broad range of host gene expression changes including both defense- and non-defense-related genes. Genes induced were consistent with O. aegyptiaca preferentially stimulating JA-mediated responses. The failure of O. aegyptiaca to elicit SA-mediated defenses in host roots suggested that exogenous induction of this signaling pathway could enhance host resistance to parasitism. Treatment of O. aegyptiaca-inoculated tobacco with BTH, a SA analog that activates SAR, caused a 49% reduction in O. aegyptiaca numbers. Analysis of PR-1a using RNA hybridizations and protein immunoblots in treated plants showed the expected induction in shoots, but not in roots, confirming the organ-specific differences in defense response observed in Arabidopsis. Experiments using a strategy to engineer the hypersensitive response via the gene-for-gene interaction confirmed previous findings that parasite-specific activation of an R/Avr interaction in tobacco reduced parasitism by approximately 50%. This research suggests that approaches to stimulate SAR in susceptible host plants may be useful for reducing Orobanche parasitism / Master of Science

<p>Plants commonly produce multiple, seemingly redundant defenses, but the reasons for this are poorly understood. The specificity of defenses to particular herbivores could drive investment in multiple defenses. Alternatively, genetic correlations between defenses could lead to their joint expression, even if possessing both defenses is non-adaptive. Plants may produce multiple defenses if putative resistance traits do not reduce damage, forcing plants to rely on tolerance of damage instead. Furthermore, resource shortages caused by herbivore damage could lead to compensatory changes in expression and selection on non-defense traits, such as floral traits. Natural selection could favor producing multiple defenses if synergism between defenses increases the benefits or decrease the costs of producing multiple defenses. Non-linear relationships between the costs and benefits of defense trait investment could also favor multiple defenses.</p><p>Passiflora incarnata (`maypop') is a perennial vine native to the southeast United States that produces both direct, physical traits (leaf toughness and trichomes) and rewards thought to function in indirect defense (extrafloral nectar in a defense mutualism with ants), along with tolerance of herbivore damage. I performed two year-long common garden experiments with clonal replicates of plants originating from two populations. I measured plant fitness, herbivore damage, and defense traits. I ran a genotypic selection analysis to determine if manipulating herbivore damage through a pesticide exclusion treatment presence mediated selection on floral traits, and if herbivore damage led to plastic changes in floral trait expression. To evaluate the role of selection in maintaining multiple defenses, I estimated fitness surfaces for pairwise combinations of defense traits and evaluated where the fitness optima were on each surface. </p><p>I found that resistance traits did not reduce herbivore damage, but plants demonstrated specific tolerance to different classes of herbivore damage. Tolerance was negatively correlated with resistance, raising the possibility that tolerance of herbivore damage instead of resistance may be the key defense in this plant, and that production of the two type of defense is constrained by underlying genetic architecture. Plants with higher levels of generalist beetle damage flowered earlier and produced proportionally more male flowers. I found linear selection for both earlier flowering and a lower proportion of male flowers in the herbivore exclusion treatment. I found that selection favored investment in multiple resistance traits. However, for two tolerance traits or one resistance and one tolerance trait, investment in only one trait was favored. </p><p>These results highlight the possibility of several mechanisms selecting for the expression of multiple traits, including non-defense traits. Resistance traits may have a non-defensive primary function in this plant, and tolerance may instead be a key defense strategy. These results also emphasize the need to consider the type of trait--resistance or tolerance--when making broad predictions about their joint expression.</p> / Dissertation

Salicylic acid binding protein 2 (SABP2) that converts methyl salicylate to salicylic acid (SA) plays an obligatory role in the SA-mediated disease resistance pathway in plants. SABP2 interacts with SBIP24 in a yeast two-hybrid screening. SBIP24 belongs to the stearoyl-acyl carrier protein-desaturase protein family. To biochemically characterize the SBIP24, it was cloned from tobacco leaves using RT-PCR and expressed in E. coli. Recombinant SBIP24 was affinity purified using Ni-NTA chromatography. RT-PCR was performed to determine the role of SABP2 in modulating the expression SBIP24. TMV infected transgenic C3 (control tobacco plant containing empty silencing vector) and 1-2 (SABP2-silenced) transgenic tobacco plants were used. Preliminary results indicate that SABP2 may regulate the expression of SBIP24 in tobacco plants. Further studies are needed to confirm these preliminary results.

The fungal pathogen S. sclerotiorum (Sclerotinia sclerotiorum) impacts production and yield in one of Canada’s number one crops, canola (Brassica napus). Unfortunately, few cultivars show any tolerance to this devastating fungal pathogen. Thus, understanding how the plant responds to this aggressive fungus at the cellular level will facilitate the identification of genes and gene products responsible for improved plant performance. While our understanding of the host pathogen interaction is becoming clearer, there is remarkably little information available for Sclerotinia, especially its pathogenicity in canola. Moreover, we know nothing about how this interaction is specified at the cellular, physiological or molecular level directly at the site of infection in mature leaves following petal inoculation. Thus, we compared differences in plant structure, antioxidant response, and genes involved in the salicylic acid, jasmonic acid and ethylene defense pathways in a susceptible cultivar, Westar, and a previously described tolerant cultivar, Zhongyou821 (ZY821). Our data showed that at the cellular level, ZY821 was able to suppress the Sclerotinia penetration. The ascorbate-glutathione pathway and resistant signaling pathways were all associated with the canola defense response to S. sclerotiorum, while stronger antioxidant and signaling pathways responses were observed in ZY821 leaves at the site of infection. Also, transcriptional regulators not previously associated with plant defense in the Arabidopsis- S. sclerotiorum pathosystem were identified through bioinformatics approaches. By comparing plant susceptibility to S. sclerotiorum between Arabidopsis wild type and seven loss-of-function mutants, I found transcription factor JAM2 might be involved in plant tolerance to S. sclerotiorum. / October 2014