Dissertations / Theses on the topic 'Inducible defense'

Plant-herbivore interactions are among the most common of ecological interactions. It is therefore not surprising that plants have evolved multiple mechanisms to defend themselves, using both constitutive chemical and physical barriers and by induced responses which are only expressed after herbivory has occurred. Herbivores, on the other hand, respond to these plant defenses by evolving counter-adaptations which makes defenses less effective or even useless. Adaptation can occur at different geographical scales, with varying coevolutionary interactions across a spatially heterogenous landscape. By looking at the underlying genes responsible for these defensive traits and herbivore related phenotypic traits, it is possible to investigate the coevolutionary history of these plant- herbivore interactions. Here I use molecular population genetic tools to investigate the evolutionary history of several inducible defense genes in European Aspen (Populus tremula) in Sweden. Two genes, belonging to the Polyphenol oxidase gene-family (PPO1 and PPO2), show skews in their site frequency spectrum together with patterns of diversity and divergence from an outgroup which correspond to signatures of adaptive evolution (Paper II). 71 single nucleotide polymorphisms (SNPs) from seven inducible defense genes (PPO1-PPO3, TI2-TI5) show elevated levels of population differentiation compared to control genes (genes not involved in plant defense), and 10 of these defense SNPs show strong signatures of natural selection (Paper III). These 71 defense SNPs also divides a sample of Swedish P. tremula trees into three distinct geographical groups, corresponding to a Southern, Central and Northern cluster, a patterns that is not present in control SNPs (Paper III). The same geographical pattern, with a distinct Northern cluster, is also observed in several phenotypic traits related to herbivory in our common garden in Sävar (Paper IV). These phenotypic traits show patterns of apparent local maladaptation of the herbivore community to the host population which could indicate the presence of “information coevolution” between plants and herbivores (Paper IV). 15 unique defense SNPs also show significant associations to eight phenotypic traits but the causal effects of these SNP associations may be confounded by the geographic structure found in both the underlying genes and in the phenotypic traits. The co-occurrence of population structure in both defense genes and herbivore community traits may be the result from historical events during the post-glacial recolonization of Sweden.
Interaktioner mellan växter och herbivorer är bland de vanligaste ekologiska interaktionerna och det är därför inte förvånande att växter har utvecklat flera olika mekanismer för att försvara sig. Dessa försvarsmekanismer består både av konstitutiva kemiska och fysiska barriärer så väl som inducerade försvar som bara är uttryckta efter att en växt har blivit skadad genom betning. Herbivorerna å sin sida svarar på dessa försvar genom att utveckla motanpassningar som gör växternas försvar mindre effektiva eller till och med verkningslösa. Dessa anpassningar kan ske över olika geografiska skalor beroende på om de samevolutionära interaktionerna varierar i ett rumsligt heterogent landskap. Genom att studera de underliggande gener som kontrollerar dessa försvarsegenskaper tillsammans med herbivorrelaterade fenotypiska egenskaper är det möjligt att undersöka den samevolutionära historien av interaktionerna mellan växter och herbivorer. Här använder jag mig av molekylärpopulationsgenetiska verktyg för att undersöka den evolutionära historien i flera inducerade försvarsgener hos asp (Populus tremula) i Sverige. Två gener, som tillhör genfamiljen Polyphenol-oxidaser (PPO1 och PPO2), uppvisar ett frekvensmönster som man förväntar sig vid positiv selektion. Detta mönster kan också ses i dessa geners diversitet samt i divergens från en utgrupp (Uppsats II). 71 ”single nucleotide polymorphisms” (SNPar) från 7 inducerade försvarsgener (PPO1-PPO3, TI2-TI5) visar förhöjda nivåer av populationsdifferentiering jämfört med kontrollgener (gener som inte är involverade i trädens försvar), och 10 av dessa försvars-SNPar visar även tecken på naturlig selektion (Uppsats III). Dessa 71 försvars-SNPar delar in ett urval av svenska aspar i tre distinkta geografiska grupper som beskriver ett sydligt, centralt och nordligt kluster som inte förekommer hos kontroll-SNPar (Uppsats III). Samma geografiska mönster, med ett distinkt nordligt kluster, återfinns däremot i ett antal fenotypiska egenskaper som är relaterade till herbivori i ett odlingsförsök utanför Sävar (Uppsats IV). Dessa fenotypiska egenskaper visar tecken på lokal felanpassning hos herbivorsamhället till den lokala värdpopulationen, vilket kan indikera förekomsten av ett ”samevolutionärt informationsutbyte” mellan växter och herbivorer (Uppsats IV). 15 unika försvars-SNPar påvisar också signifikanta associationer med 8 olika fenotypiska egenskaper, men om dessa har en verklig effekt eller inte är svårt att säga på grund av den geografiska strukturen som förekommer både hos de underliggande generna och hos de fenotypiska egenskaperna. Att denna populationsstruktur förekommer hos både försvarsgener och egenskaper som är förknippade med herbivorsamhället kan däremot vara ett resultat av historiska händelser som skett under aspens post-glaciala återkolonisation av Sverige.

My study was designed to determine how different thermal defense strategies and the intensity of high temperature challenges might affect demographic and physiological performance of limpets on rocky shores. Found together in the high intertidal zone, Lottia scabra employs a constitutive thermal defense strategy, whereas L. austrodigitalis has an inducible one. I measured loss and growth rates of both species as a function of average daily maximum temperature exposure in the field, and respiration rates for field-collected and lab-acclimated individuals under benign conditions in the lab before and after exposure to one of five peak temperatures (14, 24, 28, 32, or 36 °C) during a 4.5-hour simulated low tide. L. scabra was relatively unaffected by exposure to high temperatures, whereas L. austrodigitalis exhibited significant increases in loss rates from experimental plates, decreases in growth rates, and increases in oxygen consumption, consistent with activation of the heat shock response.

Infection by multiple parasites is a part of everyday life for many organisms. The host immune system may be a central mediator of the many ways parasites might influence one another (and their hosts). Immunity provides a means for the colonized to reduce the success of current and future colonizers and has evolved across the tree of life several times independently. Along the way, the immune systems of plants as well as many groups of animals has evolved perhaps an accidental vulnerability wherein defense against one parasite can increase susceptibility to others. This so-called immune 'cross-talk' is a conundrum worth investigating not only to understand the impact of parasites on focal organisms, but also to better predict how immunity itself influences the evolution and epidemiology of parasites whose spread we might like to curtail. For plants, co-infection often comes from insect herbivores and various bacteria that colonize the leaf interior. Both colonizers can reduce plant fitness directly or indirectly by potentiating future enemies via cross-talk in plant immunity. This phenomenon has largely been studied in laboratory model plants, leaving a substantial gap in our knowledge from native species that interact in the wild. This dissertation helps close this gap by investigating the ecology of co-infection of a native plant by its major insect herbivore and diverse leaf-colonizing bacteria. I revealed that leaf co-infection in the field by leaf-mining herbivores and leaf-colonizing ("phyllosphere") bacteria is substantially more common than single infection by either group and that bacterial infection can cause increased feeding by herbivores in the laboratory. Immune cross-talk can also shape the field-scale patterns of herbivory across a native plant population. Studying the main herbivore of this native plant in detail revealed that, in contrast to many specialist herbivores, our focal species avoids plant defenses likely because it does not possess a specialized means of avoiding their toxicity. Nonetheless, this species may depend on the very same defenses it avoids by being initially attracted to plants that produce them. This foraging strategy is unique among known specialists. Lastly, I moved beyond immune cross-talk to explore how co-occurring phyllosphere bacteria might directly impact one another through competition. In the lab, I found that different growth strategies underlie competitive ability for two major clades of bacteria within the genus Pseudomonas, and that toxin production and resistance may be important mediators of competition within the phyllosphere. However, competitively superior bacteria that produce toxins may indirectly facilitate the survival of inferior competitors through their being toxin resistant, which likely enhances co-existence of diverse bacteria in the phyllosphere. Together, this dissertation has revealed a variety of means by which co-infecting bacteria and insects might influence one another through plant defense cross-talk, as well as how the complex interplay of colonization and competition might affect the structure of leaf microbial communities in nature.

Phenotypic plasticity is an environmentally based change in phenotype and can be adaptive. Often, the change in an organism's phenotype is induced by the presence of a predator and serves as a defense against that predator. Defensive phenotypes are induced in freshwater physid snails in response to both crayfish and molluscivorous fish. Alternative morphologies are produced depending on which of these two predators snails are raised with, thus protecting them from each of these predators' unique mode of predation. Snails and other mollusks have been shown to produce thicker, differently shaped shells when found with predators relative to those found without predators. This production of thicker, differently shaped shells offers better protection against predators because of increased predator resistance. The first study in this thesis explores costs and limits to plasticity using the snailfish- crayfish system. I exposed juvenile physid snails (using a family structure) to either early or late shifts in predation regimes to assess whether developmental flexibility is equally possible early and late in development. Physid snails were observed to produce alternative defensive morphologies when raised in the presence of each of the two predators. All families responded similarly to the environment in which they were raised. Morphology was found to be heritable, but plasticity itself was not heritable. Morphology was found to become less flexible as snails progressed along their respective developmental pathways. In the second study, I raised physid snails with and without shell-crushing sunfish and examined the differences in shell thickness, shell mass, shell size and shell microstructural properties between the two treatment groups. Shells of snails raised with predators were found to be larger, thicker and more massive than those raised without predators, but differences in microstructure were found to be insignificant. I conclude that the observed shell thickening is accomplished by the snails' depositing more of the same material into their shells and not by producing a more complex shell composition.

Thesis (M.A.)--Georgia Southern University, 2008.
"A dissertation submitted to the Graduate Faculty of Georgia Southern University in partial fulfillment of the requirements for the degree Master of Arts." Under the direction of Joe Pellergino. ETD. Electronic version approved: May 2008. Includes bibliographical references (p. 83-86) and appendices.

The role of Ca²⁺ in the interaction between lucerne (Medicago sativa L.) and the wilt fungus, Verticillium albo-atrum R & B has been examined. Elicitor prepared from culture-filtrates of V.albo-antrum reduced the incorporation of ⁴⁵Ca²⁺ into protoplasts of lucerne. Elicitor and extracellular Ca²⁺ caused an efflux of ⁴⁵Ca²⁺ from protoplasts preloaded with ⁴⁵Ca²⁺, the elicitor causing the increase in efflux through the activity of an orthovanadate sensitive pump. Antagonists of Ca²⁺ ion channels were used to show that a constant cycling of Ca²⁺ occurs across the plasma membrane. Dibutyryl 3'5' cAMP and the calmodulin antagonist compound R24571 had no effect on the cycling of ⁴⁵Ca²⁺ suggesing cAMP and calmodulin are not directly involved in the mechanism of cycling. A Ca²⁺ dependent, calmodulin independent ATPase was identified which was sensitive to inhibition by sodium orthovanadate and the Ca²⁺ ion channel blocker verapamil, which could be responsible for the efflux of ⁴⁵Ca²⁺ across the plasma membrane. Tissues of lucerne were examined for the presence of receptor elements that could be components of a Ca²⁺ second messenger system. Calmodulin and a calmodulin dependent 3',5' cAMP phosphodiesterase were identified. The 3',5' cAMP phosphodiesterase associated with a column of immobilised calmodulin, was inhibited by compound R24571 and was stimulated by bovine calmodulin. This enzyme provides a link between the Ca²⁺ and the cAMP second messenger systems. Elicitor was shown to alter the pattern and increase the rate of phosphorylation of proteins from lucerne cell suspension cultures. A protein kinase activity that was stimulated by diacyl glycerol and phosphatidyl serine in the presence of Ca²⁺ was associated with a protein fraction prepared by phosphatidyl serine affinity chromatography. The preparation cross-reacted with monoclonal antibodies raised to mammalian Protein kinase C. Two Ca²⁺ dependent protein kinases and a calmodulin dependent protein kinase were identified in soluble extracts from lucerne. A Ca²⁺ dependent protein kinase activity was also identified in a plasma membrane preparation. All four kinase activities phosphorylated proteins of lucerne.

Francisella tularensis, the causative agent of tularemia, is a potent human and animal pathogen. Initially upon infection of the host, intramacrophage proliferation of F. tularensis occurs but after activation of the acquired host immunity, the phagocytes become activated to kill the bacterium. In my thesis, I focused on mechanisms utilized by F. tularensis to survive intracellularly and on host mechanisms responsible for macrophage-mediated killing and control of infection. The F. tularensis-specific protein IglC has been previously shown to be essential to the intramacrophage proliferation and virulence of the bacterium in mice. By electron microscopy of macrophages infected with either the live vaccine strain of F. tularensis or an isogenic mutant, denoted ∆iglC, expression of IglC was found to be necessary for the bacterium to escape from the phagosome. IFN-g-activated macrophages significantly inhibited the escape of the live vaccine strain of F. tularensis from the phagosome. iNOS and phox generate NO and O2-, respectively. These molecules and their reaction products possess both bactericidal and immunoregulatory properties. We investigated the capability of IFN-g-activated peritoneal exudate cells from gene deficient iNOS-/- or p47phox-/- mice to control an intracellular F. tularensis LVS infection. iNOS was found to contribute significantly to the IFN-g induced killing, while phox contributed only to a minor extent. Unexpectedly, bacteria were eradicated even in the absence of both a functional phox and an active iNOS. The eradication was found to depend on ONOO-, the reaction product of NO and O2-, because addition of a decomposition catalyst of ONOO- completely inhibited the killing. Studies on iNOS-/- or p47phox-/- mice infected with F. tularensis LVS showed phox to be important during the first days of infection, a stage when iNOS seemed dispensable. Eventually, iNOS-/- mice died of the infection, suggesting a role of iNOS later in the course of infection. iNOS-/- mice exhibited elevated IFN-g serum levels and severe liver damage suggesting that the outcome of infection was at least in part related to an uncontrolled immune response. Several pathogenic bacteria express Cu,Zn-SOD, which in combination with other enzymes detoxifies reactive oxygen species produced by the host. A deletion mutant of F. tularensis LVS lacking the gene encoding Cu,Zn-SOD was attenuated at least 100-fold compared to LVS in mice. In peritoneal exudate cells from mice, Cu,Zn-SOD was found to be required for effective intramacrophage proliferation and, in mice, important for bacterial replication at the very early phase of infection. In summary, the most conspicuous findings were a capability of IFN-g activated macrophages to retain F. tularensis LVS in the phagosome, an essential role of ONOO- in intracellular killing of F. tularensis, and an importance of Cu,Zn-SOD to the virulence of F. tularensis LVS.

Climate change is ecologically and socially complex, deemed the most important issue of our generation. Through this dissertation I have approached climate change research through an interdisciplinary perspective, investigating how this phenomenon will affect marine ecological systems, how we can better develop experimental systems to answer ecological questions, and how we can effectively educate about this issue. In Chapter 2, I provided accessible alternatives for researching the effects of climate change (elevated temperatures and pCO2) on marine ecosystems. I designed, built, and troubleshooted two accurate and inexpensive climate-controlled experimental systems capable of maintaining target conditions: a temperature-controlled system and an ocean acidification system. The temperature-controlled system was designed to manipulate experimental tank temperatures indirectly by controlling the temperature in a surrounding water bath, which buffered fluctuations and resulted in a high level of control. The ocean acidification experimental system was designed to elevate normally fluctuating pCO2 levels by a constant factor, which allowed pCO2 to fluctuate as expected in natural environments and made it more ecologically relevant than active pCO2-controlled systems. In Chapter 3, I experimentally tested the morphological responses of southern ribbed mussels Geukensia granosissima to two simultaneous stressors (elevated temperatures and the presence of water-borne predation cues from blue crab Callinectes sapidus) and if any effects of these treatments led to differences in handling times by predatory crabs. Bivalves may become more susceptible to predation as increased temperatures decrease the protection afforded by their shells, but few studies have tested the effects of elevated temperatures on inducible defenses in bivalves. Results showed that chronic heat stress can have detrimental morphological effects on intertidal mussels. Mussels reared in elevated temperatures manifested elongated shell shapes, exhibited a disruption of the predator effect on inducible defenses, and experienced decreased predator handling times. The observed responses to elevated temperatures could make southern ribbed mussels more vulnerable to predation. In Chapter 4, I experimentally tested the morphological responses of southern ribbed mussels to elevated pCO2 levels and the presence of water-borne predation cues from blue crabs, and if these effects led to differences in handling times by predatory crabs. Elevated pCO2 can have negative effects on bivalves’ morphology and physiology, but the consequences of these effects on predator-prey interactions are still unclear. I found that adult southern ribbed mussels’ inducible defenses were not affected by a medium-term exposure to elevated pCO2. Mussels grew more in shell length and width as a response to predation cues, independent of pCO2 conditions. However, and unexpectedly, mussels reared under elevated pCO2 exhibited greater growth in shell width independent of predator treatment, driving mussels reared in the presence of a predator under elevated pCO2 conditions to develop rounder shapes. On average, these effects on mussel morphometrics did not affect crab handling times, but mussels reared in the presence of a predator under elevated pCO2 conditions had highly variable handling times. It is important to consider the complexity of animal physiology, morphology, and interspecies relationships when making deductions on predator-prey relationships in a changing ocean. In Chapter 5, I analyzed the effectiveness of using an interdisciplinary approach to climate change education. Literature suggests that an interdisciplinary instructional framework in an outdoor setting, using tools from the experiential, active, and inquiry- and place-based learning approaches, as well as the socioscientific issues pedagogical framework, would be an excellent approach for climate change education. I found that students: increased their content knowledge on climate change causes and consequences, exhibited a deeper understanding of climate change through the words they used to describe it, and corrected common climate change misconceptions. This work can serve as an example for the development of effective climate change programs that uses already available instructional materials with intentional interdisciplinary goals. Our search to understand how marine ecosystems will cope with a changing climate has emphasized emerging issues in the way we gather data, the questions we seek to answer through research, and how we translate science of social importance to the public. Through this dissertation I strove to seek the answers to some of these questions and provide feasible solutions to some of the problems in climate change research and education through an interdisciplinary approach. As science continues to move towards answering questions of concern for both science and society, science research is moving towards more interdisciplinary approaches. This dissertation is an example of how this can be an efficient and comprehensive approach.

Orientador: Vera Nisaka Solferini
Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia
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Resumo: Populações espacialmente estruturadas tendem a apresentar alta diferenciação genética e forte evidência de processos micro-evolutivos. Indivíduos de Brugmansia suaveolens (Solanaceae) são restritos a manchas populacionais. Neste trabalho, a diversidade genética de nove populações de B. suaveolens foi estimada através de isoenzimas (capítulo 1). Nossos resultados demonstram alta variabilidade e estruturação genética entre as populações. O comportamento do polinizador e a dispersão hidrocórica parecem determinar a estruturação intrapopulacional. Existem evidências de que efeitos estocásticos (i.e.: fundação e migração) afetam o padrão de diferenciação interpopulacional. A maioria dos resultados pode ser esperada sob dinâmica de estabelecimento de metapopulações. Tais populações são bastante susceptíveis à diferenciação de caracteres quantitativos, seja devido à evolução fenotípica neutra (causada por deriva) ou ação de seleção (adaptação local). Para avaliar o papel da deriva e da seleção nessa diferenciação, a variação genética de caracteres pode ser comparada à variação neutra estimada por marcadores moleculares. Quatro caracteres foram avaliados em quatro das populações de B. suaveolens estudadas (capítulo 2), três deles relacionados à defesa química contra herbívoros (concentração de alcalóides). Nossos resultados mostram uma diferenciação genética ca. de três a quatro vezes maior que a estimada por marcadores neutros para três dos caracteres, embora essa diferença não seja significativa. A exceção ocorre para a razão de indução de alcalóides, que mostra sinais de seleção estabilizadora (ausência de diferenciação interpopulacional), contrariando o padrão esperado pela teoria
Abstract: Populations patchily distributed tend to show high genetic structure and strong micro-evolutionary process evidences. Brugmansia suaveolens (Solanaceae) individuals are restrict to population patches. In this work, the genetic diversity of nine B. suaveolens populations was estimate by means of allozyme electrophoresis (chapter 1). Our results indicate high variability and genetic structure among populations. The pollinator behaviour and the hydrochoric dispersal seem determine the intrapopulation structure. There are signals that stochastic effects (i.e.: migration and foundation) affect the differentiation among populations. The most of genetic patterns found can be created by a metapopulational establishment. These populations are very susceptible to the quantitative traits differentiation, due to neutral phenotypic evolution (generated by drift) or selection action (local adaptation). To evaluate the relative action of drift and selection on population differentiation, the genetic variation in quantitative traits can be compared to neutral variation estimated by molecular markers. Four traits were evaluated in four of the B. suaveolens populations studied (chapter 2), and three of them were related to chemical defense against herbivores (tropane alkaloids concentrations). Our results show a genetic differentiation ca. three to four times higher than the differentiation estimated by molecular markers for three of characters analyzed, although no significant. The exception is ¿alkaloids induction¿ trait, which show stabilizing selection signals (absence of interpopulation differentiation), instead of differentiation pattern expected by theory
Mestrado
Mestre em Ecologia

Inducible defenses alter the strength of interaction in food webs. Theoretical models that incorporate their effects are therefore critical for predicting community dynamics and stability. I examined ecological effects of an inducible morphological defense in a microbial model. I first investigated the effect of genotype. number of predators, and previous exposure to predators on the speed and maximum level of defense for eight clones in three species of the ciliate Euplotes. The effectiveness of defense depends on both of these aspects of defense induction: therefore these traits should evolve in concert. The speed and maximum level of induction varied among genotypes, showing that there is genetic variance for these traits and the potential for evolutionary change under selection. Higher predator densities led to more rapid induction and higher maximum levels of defense. but previous exposure to predators had no detectable effect on either of these traits. I then used a model selection approach to determine the shape of the functional response of clones that differed in their level of defense, and to estimate and compare the model parameters attack rate and handling time. Defense decreased the attack rate of Euploes on Chlorella vulgar-is algae in one highly defended clone, but did not affect the functional response in two less defended clones. My results demonstrate that Euploes ciliates can precisely and rapidly adjust their morphological defense to the magnitude of predation risk in a way that varies among genotypes. This variation will lead to diversity in prey vulnerability to predators under natural conditions and translates to genetically-based differences in the foraging impact on resources of Euplotes. These estimates of ecological effects of induced defense in this system allow their inclusion in the development and testing of dynamic models. This in turn will inform our understanding of the influence of induced defenses and related trait-mediated indirect effects on community dynamics and stability.

Plants are constantly attacked by pathogenic microbes. As a result, they have evolved a plethora of constitutive and inducible defense responses to defend against attempted pathogen infection. Although volatile organic compounds have been implicated in plant defense, direct evidence of their function in plant resistance is still lacking. I have examined the role of VOCs in Arabidopsis defense against the hemibiotrophic bacterial pathogen Pseudomonas syringae pv. maculicola. The obtained results show that the vegetative parts of Arabidopsis produces and emits the volatile phenylpropanoid MeSA and three kinds of terpenoids, (E,E)-4,8,12-trimethyltrideca-1,3,7,11-tetraene (TMTT), alpha-ionon and beta-farnesen, upon avirulent and virulent P. syringae inoculation. Whereas the most abundant volatiles, MeSA and TMTT, are already produced at early stages of infection in the compatible and incompatible interaction, enhanced emission of alpha-ionon and beta-farnesen can only be detected in later stages of the compatible interaction. It was revealed that pathogen-induced synthesis of TMTT in Arabidopsis requires the JA signaling pathway but occurs independently of SA defense signaling. Similarly, the production of MeSA is dependent on JA signaling but not on the SA defense signaling pathway. Furthermore, production of MeSA is dependent on the function of ISOCHORISMATE SYNTHASE1, which produces its precursor SA. Upon inoculation with avirulent P. syringae, endogenously produced JA activates the JA signalling pathway to mediate MeSA and TMTT synthesis. By contrast, in the compatible Arabidopsis-Psm interaction, production of MeSA predominantly depends on the P. syringea the virulence factor coronatine, which activates JA downstream signaling. To learn more about the role of inducible VOCs in plant defense responses, I have identified an Arabidopsis T-DNA insertions line with a defect in the TERPENE SYNTHASE4 (TPS4) gene. Emission profiles from this mutant revealed that the induced production of TMTT but not of alpha-ionone, beta-farnesene or MeSA are abolished, demonstrating that TPS4 specifically regulates the P. syringae-induced synthesis of TMTT in Arabidopsis. The lack of TMTT in tps4 mutants, however, does not affect plant defense responses and resistance induction against P. syringae. This excludes a role of the terpenoid as an effective phytoalexin in Arabidopsis leaves against the bacterial pathogen. Moreover, tps4 mutant plants are still able to mount a SAR response, excluding a signaling function of TMTT during SAR. An important aim of our studies was to address the defensive role of MeSA, the major VOC emitted from P. syringae-inoculated Arabidopsis leaves. MeSA has been recently proposed as a critical long distance signal in the development of SAR. I found that two independent T-DNA insertions lines with defects in expression of the pathogen-inducible SA methyl transferase gene BSMT1 are completely devoid of pathogen-induced production of MeSA. However, bsmt1 mutant plants are capable to increase the level of SA in systemic, non-infected leaves of Arabodopsis and develop SAR like wild-type plants upon local P. syringae-inoculation. Thus, MeSA does not function as a critical SAR signal in Arabidopsis. Further experiments showed that SA accumulation in distant leaves occurs due to de novo synthesis through isochorismate synthase. In addition, we also ruled out a critical defensive role of MeSA at inoculation sites, because bsmt1 mutants are able to build up SA-dependent defense responses and local resistance in a wild-type-like manner. The conversion of SA to MeSA and subsequently emission of MeSA from the plant might help the plant to detoxify an excess of SA. This process is regulated by the JA pathway and might be one means to mediate negative crosstalk between JA and SA signaling. Moreover, the COR-triggered conversion of SA to MeSA and emission of the volatile methyl ester could be a way by which virulent P. syringae is able to attenuate the SA-defense pathway
Pflanzen sind einer ständigen Bedrohung durch phytopathogene Mikroorganismen ausgesetzt und haben deshalb eine Vielzahl von konstitutiven und induzierbaren Abwehrstrategien entwickelt. Die Phytohormone Salicylsäure (SA), Jasmonsäure (JA) und Ethylen sind zum Beispiel entscheidende Regulatoren von induzierten Abwehrmechanismen. Eine Antwort der Pflanze auf mikrobielle Angriffe beinhaltet auch die Emission volatiler organischer Verbindungen (volatile organic compounds - VOCs). Antimikrobielle Wirkungen von VOCs wurden bisher jedoch nur in in-vitro-Assay beobachtet. Ein direkter Beweis für eine mögliche Rolle der VOCs in der Pflanzenabwehr wurde bisher nicht erbracht. Die Rolle pflanzlicher VOCs und deren Bedeutung für die Pathogenabwehr im Modellsystems Arabidopsis thaliana – Pseudomonas syringae ist das zentrale Element dieser Arbeit. Zunächst wurden Terpenoide, die die größte Gruppe der VOCs bilden, untersucht. Vegetative Teile von Arabidopsis emittieren nach Inokulation mit virulenten und avirulenten Stämmen von P. syringae pv. maculicola (Psm) vor allem drei Terpene: das Homoterpen (E,E)-4,8,12-Trimethyl-1,3,7,11-tridecatetraen (TMTT), alpha-Ionon und beta-Farnesen, welches zur Gruppe der Sesquiterpene gehört. Als Hauptkomponente des pathogen-induzierten VOC-Profils wurde das Phenylpropansäurederivat Methylsalicylsäure (MeSA) identifiziert. Um einen besseren Einblick in die Rolle der VOCs in der Pflanzenabwehr zu erhalten, wurden Arabidopsis T-DNA-Insertionslinien des Terpensynthase-gens TPS4 isoliert. Die Emissionsmuster zeigten, dass die induzierbare Freisetzung von TMTT, aber nicht von alpha-Ionon und beta-Farnesen oder MeSA reduziert war. Dies zeigt, dass TPS4 spezifisch die Psm-induzierte TMTT-Synthese in A. thaliana reguliert. Die verringerte Menge TMTT in den tps4-Mutanten hat jedoch keinen Einfluss auf die pflanzlichen Abwehrreaktionen und die Resistenzinduktion gegen P. syringae, was eine Rolle von TMTT als effektives Phytoalexin in A. thaliana gegen bakterielle Pathogene ausschließt. Ebenso hat TMTT keine Signalfunktion bei der Ausbildung der Systemisch erworbenen Resistenz (SAR), da tps4-Mutanten weiterhin in der Lage sind eine SAR-Antwort zu induzieren (Attaran et al. 2008). Als weiteres Teilprojekt wurde die Regulation von pathogen-induzierten VOCs in A. thaliana untersucht. Viele induzierte Abwehrmechanismen beinhalten Signaltransduktionsnetzwerke an denen Salicyl- oder Jasmonsäure beteiligt sind. Mit A. thaliana-Mutanten, die in der SA- oder JA- Synthese oder den jeweiligen Signalwegen beeinträchtigt sind, konnte gezeigt werden, dass die pathogen-induzierte TMTT-Produktion in A. thaliana über den JA-Signalweg, aber unabhängig von Salicylsäure verläuft. Auch die MeSA-Produktion ist JA-abhängig. Für die Biosynthese von SA, genauso wie für deren Derivat MeSA, wird ISOCHORISMAT SYNTHASE1 benötigt, die den MeSA-Vorläufer SA bildet. Im Rahmen einer inkompatiblen Interaktion wird die Bildung von MeSA in Abhängigkeit von der JA-Biosynthese gesteuert. Im Gegensatz dazu ist in der kompatiblen Interaktion die MeSA-Produktion vom bakteriellen Virulenzfaktor Coronatin abhängig. Coronatin-defiziente Stämme von P. syringae sind nicht fähig, eine MeSA-Emission zu induzieren (Attaran et al., 2009). Desweiteren wurde in der vorliegenden Arbeit die Rolle von MeSA in der Pflanzenabwehr untersucht. MeSA ist das VOC, welches von P. syringae-inokulierten A. thaliana-Blättern vorwiegend abgegeben wird. Kürzlich wurde für MeSA eine Signaleigenschaft als Langstreckensignal in der Etablierung der SAR postuliert (Park et al., 2007). Wir konnten zeigen, dass T-DNA Insertionslinien, bei denen keine Expression der pathogeninduzierten SA-Methyltransferase BSMT1 nachgewiesen werden konnte und die somit keine pathogen-induzierte MeSA-Produktion aufwiesen, auch in systemischen, nicht infizierten Blättern nach P. syringae-Inokulation einen erhöhten SA-Spiegel, eine verstärte Expression von Abwehrgenen und eine erhöhte Pathogenresistenz aufwiesen. Diese Mutantenlinien können also die SAR genauso und in demselben Maß wie Wildtyp-Pflanzen entwickeln. Damit konnte gezeigt werden, dass MeSA nicht als zentrales Signal für die Ausbildung der SAR in Arabidopsis wirken kann. Weitere Experimente machten deutlich, dass die SA-Akkumulation in distalen Blättern auf eine de-novo-Synthese durch die Isochorismat-Synthase zurückzuführen ist. Schließlich konnte auch eine wichtige Rolle von MeSA in der Pflanzenabwehr an den Infektionsstellen ausgeschlossen werden, da bsmt1-Mutanten SA-abhängige Abwehrreaktionen und lokale Resistenzantworten in gleicher Weise wie Wildtyp-Pflanzen zeigen (Attaran et al., 2009). Produktion und anschließende Emission von MeSA könnte daher in der Pflanze dazu beitragen, einen toxischen Überschuss an SA abzubauen. Reguliert wird dieser Prozess durch den JA-Signalweg, der dadurch einen negativen Einfluss auf den SAHaushalt der Pflanze innehat. Die Auslösung der MeSA-Produktion von dem bakteriellen Virulenzfaktor COR in der kompatiblen Wechselwirkung könnte eine Strategie von P. syringae sein, die Effizienz der SA-basierenden Abwehr zu verzögern

In order to analyze the inducible defense response of hybrid poplar (Populus trichocarpa x P. deltoides), macroarrays were used to profile transcript patterns elicited by wounding and by regurgitant from forest tent caterpillar (FTC; Malacosoma disstria), a poplar defoliator. FTC regurgitant proved to be a potent elicitor of defense gene expression and was shown to contain the insect-derived elicitor volicitin. Comparison of inducible defense responses elicited by FTC regurgitant and wounding with pliers revealed qualitatively similar responses in terms of transcript accumulation. Extensive overlap was also observed in the sets of induced genes from locally- and systemically-induced leaves. Systemic responses were further investigated and shown to also be inducible in roots, which implies shoot-root systemic signaling. Comparative macroarray analysis showed similarities between inducible responses in leaves and roots, including genes that encode previously identified leaf herbivore defense genes. The macroarray analysis also established a suite of marker genes for future studies of herbivore defense in poplar, many of which may play key roles in the defense response and are candidates for further study. Among these genes were several inducible Kunitz trypsin inhibitors (KTIs), which were investigated further with biochemical analyses. The sequenced poplar genome was used to select KTI genes that represent the diversity of this family. Recombinant proteins were generated and showed that the poplar KTI genes encode functional proteinase inhibitors and that they are functionally distinct, i.e. they have specific proteinase substrate preferences. Moreover, wounding increases accumulation of KTI proteins, as well as protease inhibitor activity in leaves, supporting a defensive role for this protein family. These proteins were therefore tested for their ability to inhibit insect digestive proteases from FTC and bertha armyworm. The poplar KTIs tested all inhibited at least some protease activity from FTC midgut extracts. The strongest inhibitor of FTC proteases, TI3. was further tested in bioassays and shown to reduce larval growth of FTC when incorporated into insect diet. confirming that this KTI functions as an anti-herbivore protein. In addition, analysis of FTC midguts from T13 feeding experiments showed that larvae responded to TI3 by producing more gut proteases. This hyperproduction of proteases may exacerbate the antinutritive effects of TI3 by reducing pools of essential amino acids.

Sinice nitkowate z rodzaju Phormidium są zdolne do obrony indukowanej w obecności kilku gatunków orzęsków wyspecjalizowanych w ich zjadaniu. Obrona przyjmuje różne formy w zależności od gatunku sinicy i presji orzęsków. Może ona polegać na tworzeniu przezroczystych, sztywnych otoczek (pochewek) wokół trychomów, wewnątrz których komórki sinic są dla orzęsków niedostępne. Inną formą jest wzmożona produkcja bezpostaciowych egzopolisacharydów otaczających maty sinic, wewnątrz których trychomy także są dla orzęsków niedostępne. W przypadku silnej presji ze strony orzęsków obserwowano bardzo szybką reakcję zwinięcia maty w kulę, również otoczoną egzopolisacharydami, która stanowi doskonałą ochronę dla pojedynczych trychomów. W prezentowanej pracy badano, czy istnieje sygnał chemiczny uruchamiający mechanizm obrony indukowanej u sinicy. Sprawdzano też, czy sygnał taki wydzielany jest jedynie przez orzęski wyspecjalizowane w odżywianiu się sinicami nitkowatymi, czy może pochodzić także od innych organizmów dla których sinice są pokarmem alternatywnym. Badano również, czy obrona sinicy może być indukowana przez sam czynnik mechaniczny. Wykazano, że sinica reaguje na obecność orzęsków oddzielonych od niej filtrem ograniczeniem rozprzestrzeniania, co wskazuje na istnienie rozpuszczalnego w wodzie sygnału chemicznego pochodzącego od orzęsków. Bezpośrednie obserwacje sinic atakowanych przez orzęski pokazały, że trychomy, które wcześniej poddane były pośredniemu wpływowi orzęsków oddzielonych filtrem, znacznie częściej wycofywały się w sztywnej pochewce niż te, na które orzęski wcześniej nie oddziaływały. Wynik ten wskazuje, że sygnał chemiczny stymuluje wytwarzanie pochewek. Jednocześnie fakt, że dwie godziny po tych obserwacjach udział procentowy orzęsków najedzonych był taki sam w studzienkach gdzie na sinice działał sygnał chemiczny jak i w kontroli, a następnie gwałtownie zmniejszył się w ciągu kolejnych 24 godzin w wyniku obrony sinicy sugeruje, że aby obrona ta mogła w pełni zadziałać konieczny jest łączny wpływ sygnału chemicznego i bezpośredniego kontaktu z orzęskami. Stymulujący wpływ sygnału chemicznego został także potwierdzony w eksperymencie w którym badano reakcję trychomów sinicy na bodziec mechaniczny. Sinice reagują na ten bodziec ucieczką, przy czym w zabiegu, w którym wcześniej poddane były wpływowi orzęsków oddzielonych filtrem, znacznie częściej jest to ucieczka wewnątrz sztywnej pochewki niż wtedy gdy nie były poddawane wpływowi orzęsków. Eksperyment, w którym badano specyficzność sygnału chemicznego pokazał, że tylko orzęski wyspecjalizowane w odżywianiu sinicami nitkowatymi mogą ograniczać dyspersję sinicy za pośrednictwem sygnału chemicznego. Inne organizmy, takie jak filtrujące orzęski z rodzaju Euplotes nie odżywiające się sinicami nitkowatymi, czy wrotki z rodzaju Philodina, dla których sinice nitkowate są tylko pokarmem alternatywnym, nie mają takiego wpływu. Na podstawie przeprowadzonych badań można wysunąć wniosek, że istnieje sygnał chemiczny informujący sinicę nitkowatą o obecności w środowisku zagrażających jej orzęsków i pozwalający jej na uruchomienie mechanizmów obronnych takich jak ograniczone rozprzestrzenianie czy produkcja pochewek, a będących przygotowaniem do szybkiej i skutecznej obrony w sytuacji gdy orzęski zaczynają na niej żerować. Sygnał ten jest specyficzny i wydzielany tylko przez orzęski wyspecjalizowane w odżywianiu się sinicami nitkowatymi.

Plants are continuously subjected to biotic stresses such as herbivory and pathogens. Consequently they have evolved many defense mechanisms. Inducible defenses that are activated only after insect infestation are one type of plant adaptation to herbivory. Many plant species possess arrays of inducible defenses, including the accumulation of toxic phytochemicals and antinutritive proteins that function to deter herbivory. Inducible defenses are generally activated at the transcriptional level and they can occur at the whole plant level, which presumably protects the plant from future herbivory. The genus Populus which includes both aspens and poplars, is an important tree for forestry but often undergoes severe defoliation by herbivores. Outbreaks of forest tent caterpillar (Malacosoma disstria, FTC) and the subsequent massive defoliation of its natural host, trembling aspen (Populus tremuloides Michx.), are known to periodically occur in North America. Within aspen populations, however, individual clones show variation in susceptibility to FTC, and this suggests the importance of innate defenses of aspen. Although it has been known that aspen leaves contain phenolic phytochemicals as defensive compounds, the involvement of defensive proteins was not known when this work began. Therefore, one aim of this study was to investigate protein-based induced defenses in trembling aspen, using a molecular approach. In order to initiate investigation of protein-based induced defenses in trembling aspen, genes for polyphenol oxidase (PPO) and trypsin inhibitor (Tl), known defense-related genes in other plant species, were isolated and characterized. Both PPO and TI were transcriptionally activated in aspen foliage by FTC herbivory, artificial tissue damage, and methyl jasmonate, a signal molecule for inducible defenses. In time course analyses, it was demonstrated that PPO and TI mRNAs accumulated within several hours in both wounded leaves and unwounded leaves of the same plant. This was consistent with the wound response previously reported from other plant species including hybrid poplar (Populus trichocarpa x P. deltoides) and is indicative of the presence of signaling mechanisms for systemic induction of defense proteins in trembling aspen. To further obtain insight into mechanisms for inducible defenses, signal molecules for induction of defenses were investigated using a model system, poplar suspension cultures, based on the observation that plant cell cultures often show rapid alkalinization of the medium in response to defense-related signal molecules. Using the alkalinization assay system, two different alkalinization factors were purified from poplar leaf extracts. First, three 5 kD peptides causing rapid alkalinization, the rapid alkalinization factors (RALFs), were isolated and further characterized at the molecular level. RALF appears to be a novel hormone-like peptide that was also recently characterized from tobacco. In contrast to other known alkalinization factors, RALF did not induce defenses such as the expression of phenylalanine ammonia lyase. Based on the expression profile of RALF genes, it was predicted that RALF may be involved in general cellular signaling such as growth and development rather than defense signaling. A second alkalinization peptide causing slower alkalinization, slow alkalinization factor (SALF), was also isolated and partially sequenced by Edman degradation. Database searches of the obtained peptide sequence revealed that SALF seems to be derived from the N-terminus of a known protein, photosystem 1 centre protein subunit D. Although it is not yet clear whether the SALF peptide is a defense-related signal in poplar, it is hypothesized that this breakdown product of a known protein may act as a biologically active signal in plants. Overall, this thesis presents: 1) the first demonstration of protein-based inducible defenses in trembling aspen at molecular level; 2) the discovery of novel peptide molecules with alkalinization activity in suspension cultures of poplar cells.
Graduate

Antipredator defenses are ubiquitous in aquatic ecosystems. In the widely studied Chaoborus-Daphnia predator-prey system, Daphnia elicit a variety of phenotypically plastic responses to Chaoborus including: morphological, life history, and behavioral responses. While these inducible defenses benefit the prey, metal contaminants have been shown to interfere with chemosensory functions, thereby inhibiting antipredator defenses and decreasing survivorship. However, in lakes with a history of metal contamination, such as Kelly Lake in Sudbury, Ontario, there is evidence to suggest that Daphnia may have adapted to high, ambient copper concentrations. Using seven distinct Daphnia clones that were hatched from resting eggs from Kelly Lake, we examined morphological and life history traits when clones were exposed to either a nominal concentration of copper, kairomone, or a combination of both. As expected, clones displayed a variety of inducible responses in both kairomone-control and kairomone-copper treatments, which was attributed to genetic variability. Expected trade-offs in life history traits were not always observed, suggesting that inducible traits may be coupled. Furthermore, in contradiction to life history theory, one clone exhibited both increased somatic growth and increased reproductive output, indicating that clones likely adopted adaptive strategies to stressors rather than elicitng trade-offs in traditional traits. Our results indicate that environmentally relevant copper concentrations do not inhibit the induction of antipredator defenses in Daphnia from Kelly Lake, and we conclude that Kelly Lake Daphnia have developed an adaptive tolerance to copper. Adaptation to copper contamination may have implications for resilience in natural Kelly Lake populations.
Thesis (Master, Biology) -- Queen's University, 2012-04-03 19:33:59.137

4 ABSTRACT Characteristic high helmets in Daphnia cucullata, which increase resistance of Daphnia against various invertebrate predators, can be induced by chemical cues released by these invertebrates but also by small-scale turbulence. I evaluated the response of D. cucullata to both factors in several clones originating from two riverine pools differing in their predator regime. In a large pool, the dominant invertebrate predator is the cladoceran Leptodora kindtii, and the Daphnia population there shows a typical cyclomorphosis. In the second (small) pool, Daphnia do not produce helmets, despite the presence of the phantom midge larvae Chaoborus flavicans. I compared phenotypic changes of Daphnia cucullata clones from these pools to Chaoborus kairomones and to a small-scale turbulence in laboratory experiments. Kairomones induced significantly longer helmets in clones from both pools; however, only clones form the large pool reacted also to turbulence. As all daphnids in the experiments responded to chemical cues from Chaoborus, I assume that either the kairomone dose under natural conditions in the small pool is too low to induce helmets or the phenotypic response of the local population is mediated by other factors. On the other hand, the variation of response to small-scale turbulence suggests that...