<p> As the root microbiome’s role in plant defenses against herbivory becomes clearer, scientific focus has lingered on a single side of plant defenses: resistance. Its counterpart, tolerance, is comparatively overlooked despite its power as an evolutionarily sustainable mitigator of herbivore damage. This thesis seeks to supplement our limited understanding of the extent to which tolerance to herbivory may be influenced by rhizosphere microbial communities. First, in an agricultural field setting, I (1) quantified domesticated tomato cultivar and wild ancestor tolerance to herbivory form the specialist tobacco hornworm (<em>Manduca sexta</em>) and (2) characterized the bacterial and fungal rhizosphere communities associating with high and low tolerance plants. In a subsequent greenhouse experiment, I grew these same tomato lines in either sterilized or unsterilized soil and re-challenged plants with tobacco hornworms to tease apart the contributions from host plant and rhizosphere microbiome in expressing tolerance to herbivory. In the field, wild tomato lines excelled at tolerating hornworm herbivory, while their domesticated counterparts suffered 26% yield losses under herbivory. Rhizosphere community characteristics were most reliably shaped by timepoint of rhizosphere sampling, and more subtly by tomato line and herbivory treatments. Fungal and bacterial community traits that associated with high tolerance lines include (1) high diversity, (2) resistance to community shifts under herbivory, and (3) the abundance of ASVs belonging to <em>Strenotrophomonas</em>, <em>Sphingobacterium</em>, and <em>Sphingomonas</em>. When re-challenging these lines with hornworm herbivory in the greenhouse, expressed tolerance to tobacco hornworm damage was inverted from field trends. Though wild lines suffered yield losses when grown in +microbiome treatments, we found no consistent interactions between herbivory and microbiome treatments that might indicate that +microbiome treatments either helped or hampered plant expression of tolerance to herbivory under greenhouse conditions. These experiments shed light on what role, if any, the rhizosphere microbiome plays in plant tolerance to herbivory. Ultimately, understanding the qualities of tolerance-conferring microbiomes can (1) open avenues through which plant defenses may be amended in pest management, either through microbial inoculants or plant breeding efforts aimed at enhancing crop recruitment of beneficial microbiomes; and (2) ameliorate our understanding of the tripartite interactions between host plants, their rhizospheres, and their specialist herbivores. </p>

Thesis (MSc (Viticulture and Oenology. Wine Biotechnology))--University of Stellenbosch, 2002. / Pathogen devastation of food products has been the topic of extensive research efforts worldwide. Fungal infections are foremost amongst these pests, contributing not only to losses in product yield, but also significantly affecting the quality thereof. It is not surprising then that producers of these foodstuffs and their derived products continually strive towards the highest possible product quality. Therefore, it remains imperative that satisfactory methods are implemented to control these fungal pathogens. The current strategies are all hampered by drawbacks, and severe crop losses are still experienced. New technologies are being explored; one such technology is the genetic transformation of plant species. This method has enabled scientists to introduce foreign genes, with known functions and predictable outcomes, into plants. Genes identified to be involved in disease resistance have become the focus of numerous research efforts concerned with the improvement of the plant's innate defence response. This study aimed to enhance disease resistance to fungal pathogens by means of the genetic transformation of two genes previously shown to be involved in disease resistance. These genes encode polygalacturonase-inhibiting proteins (PGIPs) from Phaseolus vulgaris and resveratrol synthase from Vitis vinifera. PGIPs specifically inhibit the action of fungal polygalacturonases (PGs), which are enzymes responsible for the hydrolytic breakdown of plant cell walls. These enzymes were also found to be the first enzymes that are secreted by fungal pathogens during infection of the host plant. Additionally, PGIP-PG interaction results in the existence of molecules involved in the activation of plant defence responses. Resveratrol, the product of resveratrol synthase, exerts its antifungal action by destruction of the microbial cellular membranes. These mentioned genes were transformed alone, and in combination, into Nicotiana tabacum and the resultant transgenic lines were evaluated for enhanced disease resistance and for possible synergistic effects between the transgenes. Several independent transgenic lines were regenerated with genes integrated into the tobacco genome. Almost all the plants harbouring only pgip or vst1 genes also expressed these genes at a high frequency. Some non-expressing lines were identified from the transgenic plants that had integrated both genes, but several lines were obtained expressing both transgenes. Good correlations were observed between transgene product activity and enhanced resistance to the fungus Botrytis cinerea in an antifungal in planta assay. Lines showing the highest PGIP activity and resveratrollevels were more resistant to the pathogen, leading to disease resistance of up to 80% seven days after inoculation in comparison to an untransformed control. These lines maintained their strong inhibition, even three weeks post-inoculation, showing a complete halt in disease development and fungal growth. These results provide good indications of the efficacy of these transgenes in the upregulation of plant defence. However, the study will have to be expanded to include even more transgenic lines to elucidate the possible synergistic effects of these genes. In an additional pilot study, genes encoding for precursors and for the formation of resveratrol were introduced into the yeast Saccharomyces cerevisiae. The resultant recombinant yeast strains were evaluated for their ability to produce the phenolic substance, resveratrol. This compound has been implicated in beneficial aspects relating to human health, including positive effects on atherosclerosis and platelet aggregation as a direct result of its antioxidant and anti-inflammatory activities. Recombinant yeast strains were constructed that expressed genes coding for coenzyme A ligase and resveratrol synthase. These strains were shown to be able to produce the phenolic compound resveratrol from the precursors present in the yeast as well as from the products introduced with the transformation. The resveratrol was complexed with an added glucose moiety. These results are extremely positive, considering the possibility of manipulating wine yeasts to produce resveratrol during the wine fermentation, thereby adding to the health aspects of both red and white wine. This is the first report of the production of this compound by the introduction of genes necessary for its biosynthesis in a foreign host. This study has confirmed the importance of PGIPs and resveratrol in the effort to enhance disease resistance in plants through genetic transformation technology. It has also shown that the health benefits of resveratrol could be exploited more optimally in the wine industry, by producing wine yeasts with the ability to synthesise this important antioxidant.

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

M.Sc. (Biochemistry) / Lipopolysaccharide (LPS) is a complex lipoglycan that is found in the outer membrane of Gram-negative bacteria and is composed of three regions namely the fatty acid Lipid A, a core region of short oligosaccharide chains and an O-antigen region of polysaccharides. When LPS is recognized as a microbe-/pathogen-associated molecular pattern (M/PAMP), it not only induces an innate immune response in plants but also stimulates the development of defence responses such as the immediate release of reactive oxygen species/intermediates (ROS/I), pathogen-related (PR) gene expression and activation of the hypersensitive response (HR), resulting in stronger subsequent pathogen interactions. The identification and characterisation of the elusive LPS receptor/receptor complex in plants is thus of importance, since understanding the mechanism of perception and specific signal transduction pathways will clarify, and lead to the advancement of, basal resistance in plants in order to decrease crop plant losses due to pathogen attack. In mammals, LPS binds to a LPS binding protein (LBP) to form a LPS-LBP complex which is translocated to myeloid differentiation 2 (MD2) with the presence/absence of its co-receptor, a glycosylphosphatidylinositol (GPI)-linked protein, CD14. The interaction occurs on the host membrane and triggers an inflammatory defence response through the signalling cascade activated by the interaction with Toll-like receptor 4 (TLR4). A similar LPS-receptor interaction is, however, unknown in plants. To address the LPS perception mechanism in plants, biological binding studies with regard to concentration, incubation time and temperature, affinity, specificity and saturation were conducted in Arabidopsis thaliana protoplasts using LPS labeled with Alexa 488 hydrazide. Quantum dots (Qdots), which allow non-covalent hydrophobic labeling of LPS, were further also employed in similar Arabidopsis protoplast binding studies. These studies were conducted by fluorescence determination through the use of a BD FACS Aria flow cytometer. Although Alexa-labeling does not affect the biological activity in mammalian studies, the same cannot necessarily be said for plant systems, and hence Qdots were included to address this question. The conjugation of Qdots to LPS was confirmed by transmission electron microscopy (TEM) and results illustrated higher fluorescence values as compared to Alexa-LPS fluorescence analysis. Furthermore, inhibition of the perception process is also reported using Wortmannin and Brefeldin A as suitable endo- and exocytosis inhibitors. Affinity, specificity and saturability as well as the role of endo- and exocytosis inhibition in LPS binding to protoplasts was ultimately demonstrated by both fluorophores, with the use of Qdots as a label proving to be a more sensitive strategy in comparison to the conventional Alexa 488 hydrazide label.

Les études entreprises depuis une douzaine d'années indiquent que le monoxyde d'azote (NO) est un médiateur physiologique impliqué dans de nombreux processus chez les plantes, incluant la germination, le développement des racines, la fermeture des stomates ou encore la réponse adaptative aux stress biotiques et abiotiques. Malgré cet important panel de fonctions, les mécanismes sous-jacents aux effets du NO ont été peu appréhendés et restent pour l'essentiel énigmatiques. Le travail présenté dans ce manuscrit s'inscrit dans cette problématique et a consisté en l’identification et la caractérisation de protéines cibles du NO chez le tabac dans le contexte de stress biotiques et abiotiques. Nous avons démontré que la cryptogéine, un éliciteur des réactions de défense, induit la S-nitrosylation rapide et transitoire de plusieurs protéines dans des suspensions cellulaires de tabac. Après purification, une douzaine de ces protéines ont été identifiées via une analyse par spectrométrie de masse. Celles-ci incluent notamment une protéine chaperonne de la famille des AAA-ATPase nommée CDC48 (Cell Division Cycle 48). Cette dernière a fait l'objet d'une étude structure/fonction approfondie afin d'appréhender l'impact de sa S-nitrosylation. Après avoir vérifié que la protéine recombinante était S-nitrosylable in vitro, nous avons démontré que ce processus n'affecte pas la structure secondaire de la protéine mais induit des modifications locales de sa structure tertiaire et une inhibition de son activité ATPasique. Le résidu cystéine 526, localisé dans le second domaine ATPasique de la protéine, a été identifié comme site probable de S-nitrosylation. Cette localisation stratégique pourrait expliquer l'effet inhibiteur du NO sur l'activité enzymatique de CDC48. La dernière partie de ce travail a été centrée sur l'analyse des mécanismes par lesquels le NO active la protéine kinase NtOSAK (Nicotiana tabacum stress activated protein kinase) chez le tabac. Nous avons démontré que NtOSAK forme un complexe constitutif avec la glycéraldéhyde 3 phosphate deshydrogénase (GAPDH). En réponse à un stress salin, le NO promeut l'activation de NtOSAK via la phosphorylation de deux résidus serine localisés dans la boucle d'activation de l'enzyme. De plus, il induit une S-nitrosylation rapide de la GAPDH, ce processus n'affectant pas la formation du complexe. Notre hypothèse est que ce complexe constituerait une plateforme de signalisation régulée par le NO et pouvant recruter les protéines cibles de NtOSAK lors de la réponse au stress salin. / Studies conducted over the past ten years indicate that nitric oxide (NO) is a physiological mediator involved in many physiological processes in plants, including germination, root development, stomatal closure or responses against biotic or abiotic stresses. Despite this important range of functions, the mechanisms underlying the effects of NO in plants remain largely unknown. The present work aims at identifying and functionally characterizing NO target proteins in tobacco in the context of biotic and abiotic stresses. We demonstrated that cryptogein, an elicitor of defense responses, induces a rapid and transient S-nitrosylation of several proteins in tobacco cell suspensions. After purification, a dozen of these proteins have been identified through mass spectrometry analysis. These proteins include CDC48 (Cell Division Cycle 48), a chaperone-like protein belonging to the AAA-ATPase family. The regulation of CDC48 by NO was deeply investigated using a combination of structural and biochemical analyses. Once the in vitro S-nitrosylation of CDC48 was confirmed, we next demonstrated that this process does not affect the secondary structure of the protein but induces local changes in its tertiary structure together with an inhibition of its ATPase activity. The cysteine residue 526, located in the second ATPase domain of the protein, was identified as a probable S-nitrosylation site. This crucial localization may explain the inhibitory effect of NO on CDC48 enzymatic activity. The last part of this work was focused on the analysis of the mechanisms underlying the NO-dependent activation of the protein kinase NtOSAK (Nicotiana tabacum stress activated protein kinase) in tobacco. We demonstrated that NtOSAK forms a constitutive complex with glyceraldehyde 3-phosphate dehydrogenase (GAPDH). In response to salt stress, NO promotes the activation of NtOSAK via the phosphorylation of two serine residues located in the activation loop of the enzyme. Moreover, it induces a rapid S-nitrosylation of GAPDH. Interestingly, this latter process does not affect the complex formation. Our hypothesis is that once S-nitrosylated, GAPDH might act as a phosphorelay recruiting protein substrates for NtOSAK.

M.Sc. / Since plants are stationary, their immune systems have adapted to their environments to enable them to overcome or respond appropriately to various environmental, physical and physiological stresses that they may encounter by developing complicated defense mechanisms. The plant defense response activates complex biochemical and structural changes in plant cells. Heat stress per se, appears to be a priority stress response in plants, and increased disease susceptibility may be a result of this response. In this study, altered gene expression levels mediated by a heat treatment in Arabidopsis thaliana seedlings were analyzed. Seedlings were exposed to a heat stress of 42C for 30 minutes, followed by a 2.5 hour recovery period at 25ºC. RNA that was isolated from the heat stress treated plants as well as control plants (untreated) was used to perform suppression subtractive hybridization (SSH) in order to obtain a forward and a reverse DNA library. The forward SSH library represented the genes that were up-regulated due to the heat shock and the reverse SSH library represented the down-regulated genes. Sequenced clones from these libraries were BLAST against the A. thaliana genome using the Genbank database and the Accession numbers retrieved were then used for Bioinformatics analysis to obtain functionality of the genes found. The bioinformatics tools used were TAIR tools, DAG graphs and FatiGO and genes were categorized into the biological processes, molecular functions and cellular components. The TAIR tools and FatiGO were then used to analyze microarray data obtained for a similar study, in order to compare the genes identified with SSH. The genes coding for photosystem IID, serine-type peptidase, phospholipase D α, a S-locus protein kinase, regulator of chromosome condensation (RCC1) and Glucose-6-phosphate translocator are prominently up-regulated whilst other genes encoding photosystem I, plastocyanin-like mavicyanin, carbohydrate trans-membrane transporter MSS1, zinc finger C3HC4 ring family protein, ubiquitin conjugating enzyme 35 (UBC35) and integral membrane family proteins are significantly down-regulated. The FatiGO results helped to assign functionality to the genes that were found. For the SSH forward library, the cellular protein metabolic pathway was the most highly expressed term (19.21%), whereas in the microarray data, the term „positive regulation of response to stimulus‟ and membrane disassembly had a 100% expression. The reverse SSH data (down-regulation) found phosphate metabolic process as the most highly expressed term with an expression of 44.36% ix and the microarray data (negative fold-change) found the term photorespiration to be the most highly expressed with 93.54% expression. These high levels of negative expression indicate the down-regulation of these processes in the cell during heat shock. From these results it can be assumed that at the onset of a heat stress, the plant‟s immediate response is to activate pathways of regulation as a response to the stimulus as a self-protection mechanism, and repress other pathways such as photorespiration in order to preserve its energy such as ATP. These findings suggest that the plant is well equipped to overcome stress in its environment by activation/repression of specific organelles and pathways in the system, in order to maintain its equilibrium. Studies such as these can prove to be helpful to solve the interesting question of how a plant overcomes various environmental stresses in order to prevent disease susceptibility.

The earwigs (Dermaptera) contains mostly nocturnal insects, which are considered subsocial due to formation of aggregated family groups and maternal care. Species of Dermaptera have been shown to play an important role as generalist predators in different crop systems worldwide. Despite that, the knowledge about their behavior and insect-plant interactions remains poorly understood. The earwig Doru luteipes (Dermaptera: Forficulidae) is one of the most important generalist predators in different crops, especially in maize (Zea mays ssp. mays) by voraciously consuming eggs and larvae of Spodoptera frugiperda (Lepidoptera: Noctuidae) and Diatraea saccharalis (Lepidoptera: Crambidae), key pests in this crop. Maize is one of most produced cereals in the word. However, great losses are registered annually due to pest attacks, despite the use of pesticides. Studies have demonstrated that domesticated plants such as maize have reduced defenses against herbivores in detriment to intensely selecting for rapid growth and high yield, when compared with their wild ancestors\' species known as teosintes (Zea spp.). This thesis focuses on the study of the trophic interactions which occur in the system \'Zea plants - herbivores caterpillars - predator earwig\' and is divided in four chapters. In the Chapter 1 is present a general introduction and the thesis outline. In the Chapter 2, we studied through scent collection and multiple-choice bioassays, the communication between D. luteipes individuals which influences their predatory behavior. Our results indicate that earwigs can produce and use chemical cues by conspecifics to orientate them towards foraging resources. In the Chapter 3, we investigated the predation behavior of earwigs during photo- and scotophase and their preference for maize volatiles induced by S. frugiperda or D. saccharalis at different time intervals (early and old damage). Behavior, olfactometry bioassays and plant volatile collections were conducted. As was hypothesized, earwigs showed a predation activity only during night. Besides that, they were attracted by maize odors produced by both caterpillars and showed significant preference to early-damage odors over old-damage. Therefore, it is considered that early-damage volatiles (Green Leaf Volatiles-GLVs) could be the key compounds of D. luteipes attraction. In the last part (Chapter 4) was explored how domestication of Zea plants influences insect-plants interactions through plant defense. Experiments about food utilization by caterpillars, olfactometry bioassays and plant volatile collections were conducted. Interestingly, as in the previous chapter, we suggest that GLVs have an important role in earwig attraction. The results show that life-history of Zea plants influences plant defense and consequently the plant-insect interactions. In conclusion, our findings bring a new contribution to the knowledge about plant defenses in a tritrophic context and predatory strategies in D. luteipes, which would promote alternatives to optimize the conservation and biological control of pest by predators in the field. / As tesourinhas (Dermaptera) são em sua maioria insetos noturnos considerados sub-sociais devido ao cuidado maternal e à formação de grupos familiares agregados. Espécies de Dermaptera possuem um papel importante como predadores generalistas em diferentes culturas em todo mundo. Apesar disso, o conhecimento sobre seu comportamento e as interações inseto-planta permanecem pouco estudadas. A tesourinha Doru luteipes Scudder (Dermaptera: Forficulidae) é um dos predadores mais frequentes em diferentes culturas, especialmente no milho (Zea mays ssp. mays), devido ao voraz consumo de ovos e lagartas de Spodoptera frugiperda e Diatraea saccharalis, pragas chave nesta cultura. O milho é um dos cereais mais produzidos no mundo, embora grandes perdas sejam registradas anualmente devido ao ataque de pragas, apesar do uso de agroquímicos. Estudos recentes demostraram que plantas domesticadas tais como o milho têm reduzido suas defesas contra os herbívoros em detrimento à intensa seleção de cultivares de rápido crescimento e alta produtividade, quando comparados com seus ancestrais selvagens conhecidos como teosintos (Zea spp.). Esta tese foca no estudo das interações tróficas que ocorrem no sistema \'plantas Zea - lagartas herbívoras - tesourinhas predadoras\' e foi dividida em quatro capítulos. No Capítulo 1, apresenta-se uma introdução geral e o esboço da tese. No Capítulo 2, a partir da coleta de voláteis de adultos de D. luteipes e ensaios de múltipla escolha, foi estudada a comunicação química entre estes indivíduos que influencia o comportamento de predação. No Capítulo 3 foi investigado o comportamento de predação das tesourinhas durante a foto e escotofase, e a preferência delas pelos voláteis do milho induzidos por S. frugiperda ou D. saccharalis, em diferentes intervalos de tempo (dano recente ou dano tardio). As tesourinhas mostraram atividade predatória apenas durante a noite, e foram atraídas pelos odores do milho atacados pelas duas lagartas. O dano recente (voláteis de folhas verdes-VFVs) contêm os compostos chaves para a atração de D. luteipes. No Capítulo 4 foi explorado como a domesticação das plantas Zea influenciam as interações com insetos por meio das defesas de plantas. Assim como no capitulo anterior, os VFVs apresentaram um importante papel na atração das tesourinhas. Os resultados indicaram que a história de vida do gênero Zea influenciou as defesas das plantas e consequentemente, as interações inseto-planta. Em conclusão, o presente trabalho traz novas contribuições ao conhecimento sobre defesas de plantas num contexto tritrófico, bem como às estratégias de predação em D. luteipes, o qual pode gerar ferramentas para otimizar a conservação e controle biológico de pragas por predadores em campo.

Abstract not available

The broad aim of the work described in this thesis was to use the arbuscular mycorrhizal (AM) defective rmc tomato to explore the development and function of different types of fungus-plant interfaces (phenotypes) and to characterize the cellular modifications preceding colonization of rmc by a range of different AM fungi. Three main patterns of colonization with rmc have been described: 1) Pen- phenotype in which the AM fungus is restricted to the root surface with several attempts to penetrate the epidermal cells without success; 2) Coiphenotype where AM fungi penetrate the epidermis but cannot develop cortical colonization; and 3) Myc+ phenotype (with G. intraradices WFVAM23), where the AM fungus penetrates the cortex and forms a “normal” colonization after a delayed penetration of the epidermal cells (Review of literature). Little is known about cellular interactions, nutrient transfer or the ability of the fungi to complete their life cycles in the different phenotypes. These aspects were the main foci of this work. In addition further fungal isolates were screened to asses their ability to colonize rmc. The first experiments involved compartmented pots to follow the fungal life cycle, production of external mycelium and spores in the different rmc phenotypes (Chapter 3). The results showed that in the Pen- and Coiphenotypes, AM fungi are unable to form spores to complete the life cycle. However, in the Coi-phenotype, the fungus remained alive up to week 18, suggesting that some C transfer occurred. The fungus forming the Myc+ phenotype, G. intraradices WFVAM23, was able to produce spores, although they were significantly smaller than those produced with the wild-type tomato. The results suggested that arbuscules are essential for completion of the fungal life cycle. Labeled 32P was used to determine whether arbuscules are also essential for P transfer (Chapter 4). A compartmented pot system was used in which only fungal hyphae but not roots could obtain 32P. 32P was found in the shoots of rmc inoculated with S. calospora (Coi- phenotype), indicating that interfaces other than arbuscules can be involved in transfer of P. A nurse pot system was used to obtain synchronized colonization to determine how long AM fungi stay alive during the interactions with rmc and to elucidate the cellular modifications preceding colonization of rmc by a range of different AM fungi (Chapter 5). The results showed that rmc did attract the AM fungi, that the plant nucleus moved to the middle of the plant cell only after fungal penetration of plant roots and that callose deposition in rmc was not involved in blocking the AM fungi. Fourteen AM fungi with different taxonomic affiliations and fourteen different G. intraradices isolates were screened to try to relate phylogeny of AM fungi with phenotypes in rmc (Chapter 6). There were a large number of interactions, depending on the inoculated AM fungi, and although there were some similarities in the rmc phenotypes within phylogenetic groups, there was no clear relationship between phylogeny and development of interactions with rmc. This study showed the following. 1) Arbuscules/arbusculate coils are necessary for the completion of the AM fungal cycle. However, intraradical hyphae also participate in transfer of both P and C as demonstrated with the Coi- phenotype. 2) rmc clearly attracted AM fungi and the fungi stay alive and induce plant cellular responses such as nuclear movement only after penetrating rmc roots. 3) Plant defense responses such as callose deposition are not involved in blocking AM fungi in rmc; and 4) there was no relationship between the phenotypes described in rmc and phylogeny of the Glomeromycota. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1292816 / Thesis(Ph.D.)-- School of Earth and Environmental Sciences, 2007.

Orientadores: Marcos Antonio Machado, Juliana de Freitas Astúa / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-19T08:50:41Z (GMT). No. of bitstreams: 1 Kubo_KarenSumire_D.pdf: 11235043 bytes, checksum: fb3c7d7e7af4e5fc35ef4a27c3364fda (MD5) Previous issue date: 2011 / Resumo: O Citrus leprosis virus C (CiLV-C) é o agente causal da leprose dos citros, uma doença incomum transmitida pelo ácaro Brevipalpus phoenicis. Uma vez que o CiLV-C permanece confinado em lesões localizadas nas folhas, ramos e frutos sem causar infecção sistêmica, o vetor precisa se alimentar nestas lesões para adquirir o vírus. O objetivo deste trabalho foi analisar os perfis de expressão diferencial entre um genótipo resistente (tangor 'Murcott') e um suscetível (laranja 'Pera') em resposta à leprose dos citros e identificar os possíveis mecanismos de resistência envolvidos na resistência à doença. Por esse motivo, antes da instalação dos experimentos biológicos, nós aperfeiçoamos a detecção do CiLV-C em seu vetor, para certificação da aquisição viral. O experimento biológico incluiu quatro grupos: genótipo resistente ou suscetível infestados com ácaros virulíferos ou avirulíferos para CiLV-C. Com o intuito de se identificar genes diferencialmente expressos, nós utilizamos lâminas de microarranjo com sondas baseadas na base de dados do Citrus EST (CitEST). As análises estatísticas foram realizadas por two-way ANOVA considerando os fatores genótipo e a infecção pelo CiLV-C, de maneira a se encontrar respostas envolvidas na resistência ao CiLV-C. Os resultados foram interpretados por Gene Set Enrichment Analysis (GSEA). Os resultados sugerem que existem receptor-like proteins (RLP) e receptor-like kinase (RLK) que podem reconhecer o vírus ou o ácaro, ativando uma resposta de defesa baseada na assinatura de 'Ca POT.2+' e ativação da via do ácido salicílico. Estudos adicionais ainda são necessários para verificar se a resposta de defesa pode estar relacionada à resistência sistêmica adquirida (SAR) / Abstract: Citrus leprosis virus C (CiLV-C) is the causal agent of citrus leprosis, an unusual disease transmitted by the mite Brevipalpus phoenicis. Since CiLV-C remains confined in localized lesions in leaves, stems and fruits without causing systemic infection, the vector needs to feed in these lesions to acquire the virus. The aim of this work was to analyze the differential gene expression profiles between resistant ('Murcott' tangor) and susceptible ('Pera' sweet orange) citrus genotypes in response to CiLV-C, and to identify possible mechanisms involved in disease resistance. For this reason, before the biological experiments were set, we improved the detection of CiLV-C in the mite vector to ensure virus acquisition. The biological experiment consisted in four groups: susceptible or resistant genotype infested with CiLV-C viruliferous or nonviruliferous mites. In order to identify differentially expressed genes, we used microarray chips designed using the Citrus EST database (CitEST). The statistical analysis was performed by two-way ANOVA considering the genotype and the infection by CiLV-C, aiming to find defense responses against CiLV-C. The results were interpreted by Gene Set Enrichment Analysis (GSEA) and led to the hypothesis that receptor-like proteins (RLP) and receptor-like kinase (RLK) may recognize the virus or the mite triggering a defense response based on 'Ca POT.2+' signature and activation of Salicylic acid pathway (SA). Further studies are necessary to evaluate if the defense response could be related to the development of systemic acquired resistance (SAR) / Doutorado / Bioquimica / Doutor em Biologia Funcional e Molecular