Thèses sur le sujet « Host/non-Host »

In plant hosts, phytoplasmas induce physiological changes and in both hosts modulate plant-insect interactions. Previously, interactions have been examined with both hosts infected with phytoplasmas. Thus, it is unclear which organism the effect stems from or how phytoplasmas facilitate changes. To investigate phytoplasma manipulations of insect-plant interactions, the model Arabidopsis thaliana was used together with the fully sequenced Aster Yellows phytoplasma strain Witches’ Broom (AY-WB) and vector leafhopper Macrosteles quadrilineatus. I demonstrate possibility to differentiate effects of phytoplasma infection within plant and within insect hosts. To assess root cause of changes, AY-WB secreted effector proteins were examined, their roles within plants, and in manipulations of vector fecundity. One of the 56 secreted AY-WB proteins (SAPs) identified, SAP11, carries a nuclear localization signal and accumulates in plant cell nuclei (Bai et al. 2009). SAP11 is shown to reduce production of plant defense hormone jasmonic acid (Sugio et al. 2011). Stable expression of SAP11 and 3 other SAPs in Arabidopsis increase fecundity of M. quadrilineatus. In addition, phytoplasmas are known to affect non-host insect-plant interactions. Using the same approach, I demonstrate D. maidis survives and produces nymphs only on AY-WB-infected Arabidopsis. Furthermore, I show that whilst SAP11 has no effect on D. maidis survival, 3 other SAPs increase D. maidis survival and oviposition. These data suggest phytoplasmas utilize a suite of effector proteins to manipulate both host and non-host insect-plant interactions. Thus, AY-WB effector functions extend beyond direct interaction with plant hosts; they stimulate generation of insect vectors, and increase chance of uptake by novel insect hosts. This project highlights the value of using a model system in studying phytoplasma manipulation of their hosts and gives insight into development of evolutionary associations between phytoplasmas and vectors.

Significant differences were observed among 79 geographically diverse Arabidopsis accessions in response to the wheat powdery mildew pathogen, Blumeria graminis f.sp. tritici (Bgt) and the wheat leaf rust pathogen Puccinia triticina (Ptr). In response to Bgt genotypes classified into two major classes based on the degree of compatibility, Wc-1 an accession from Germany expressed significantly high frequency of penetration. Interestingly, in response to Ptr, a high frequency of guard cell death and sub-stomata vesicle formation (SVF) was observed on Wa-1, an accession from Poland. Attempted Ptr infection induced the production of reaction oxygen intermediates (ROI), nitric oxide, salicylic acid (SA) and camalexin. The expression of SA, jasmonic acid and ROI-dependent genes were also detected. Multiple small-to-medium effect quantitative trait loci (QTL) were identified that govern the expression of NMR in Arabidopsis against Ptr. In response to Bgt, a leaf collapse phenotype was observed in Ler when it was pre-treated with Cytochalasin E, an inhibitor of actin microfilament polymerization. Whereas, Col did not express a similar phenotype. This reaction showed a complicated genetic basis with the involvement of several genes. Our genetic analysis revealed two major QTLs on chromosomes one and three with the existence of episatsis effects. A role for ASYMMETRIC LEAVES1 (AS1) in plant immunity has recently been identified. My experiments showed a conserved regulatory function for NSPHAN, an orthologue of ASI gene in Nicotiana sylvestris when challenged with host and nonhost pathogens. This regulatory gene action remained consistent when the as1 mutant was coupled with key Arabidopsis defence related mutants.

Plant innate immunity is activated upon pathogen attack by recognizing their avirulent (avr) genes by Resistant (R) genes leading to R-gene resistance or host resistance. Another form of innate immunity is non-host resistance that is exhibited by a given plant species to most strains of a microbial species. R-gene resistance activates salicylic acid (SA) that is synthesized from methyl salicylic acid (MeSA) by Salicylic Acid Binding Protein 2 (SABP2). It was hypothesized that SABP2 plays the similar role in non-host resistance also. Growth experiments and non-host related gene analysis experiments were conducted on tobacco plants using P.s tabaci and P.s. phaseolicola that are host and non-host pathogens on tobacco respectively. Tobacco control plant C3 that expresses SABP2 and 1-2 that is RNAi silenced in SABP2 expression were used in this study. Results suggest that SABP2 may not have any significant role in tobacco non-host resistance.

Bluetongue virus (BTY) is an orbivirus of the Reoviridae family that infects sheep and other ruminants. BTY has three non-structural proteins, NS I, NS2 and NS3/3A. NS I forms tubular structures and its function is currently unknown. To investigate the role of NS I in BTY infection, the interactions of NS I with mammalian and insect cellular proteins, and BTY viral proteins, were examined. BTY NS I was identi tied as interacting with aldolase A, NUBP 1, Pyruvate kinase M2, cathespin B, SUM 0-1 and peptide TY7 using the yeast two-hybrid system, ELISA and immunofluorescence analysis. TY7 and NS I caused extensive cell death within 24h of co-expression; this cell death was not apoptosis and reduced BTY yield by 37%. The interaction of NS I with SUMO-I and its importance in BTY infection was confinned using siRNA to knockdown SUMO-I during BTY-IO infection. Knockdown of SUMO-I elicited a dramatic reduction in virus yield by 73%. NS I interactions with proteins of the insect vector Culicoides were also examined. A putative interaction between NS 1 and the ubiquitin activating enzyme El (UBA EI) ofCulicoides was identified during screening of a phage library, this has not been confirmed by other means. NS 1 interactions with other BTY proteins were analysed using immunoprecipitation and a strong interaction between NS 1 and YP7 was identified; this was confim1ed using the yeast two-hybrid system and immunoflourescence. Two main roles have been hypothesised for NS I from this data; firstly it is likely that NS I interaction with SUMO-I and UBA E I allows the targeting of specific proteins for sumoylation and ubiquitination allowing NS 1 to modify the host response to BTY infection. Secondly it is possible that NS I serves as an anchor for YP7 and virus cores allowing the build up of cores at the cytoskeleton in close proximity to YP2 for subsequent assembly and release. RNAi against NS J eliminated tubule formation but did not affect virus yield or YP7 and SUMO-J distribution and expression. It is therefore likely that the function of NS I does not rely on tubule fom1ation and that tubules are a form of storage for the active monomer of NSI.

Yellow rust, caused by Puccinia striiformis West., is an important foliar disease of wheat and barley throughout the world, and the development of resistant cultivars is the most economical and environmentally friendly method of control. Breeding for resistance to yellow rust has, for decades, been based on the use of race-specific resistance genes, which have shown to be short-lived. Non-host resistance has been studied as a possible source of durable resistance. Two major genes, as well as an undetermined number of minor genes, for non-host resistance to the barley attacking form of yellow rust, P. striiformis f. sp. hordei, have been previously detected in the wheat cultivar ‘Lemhi’. The present study aimed at quantifying and mapping those genes using QTL (quantitative trait loci) mapping procedures. For that purpose, an F2 population of 114 individuals resulting from the cross of resistant ‘Lemhi’ with ‘Chinese 166’, a wheat cultivar susceptible to barley yellow rust, was used as the mapping population. QTL effects and significance were estimated by means of interval mapping and MQM mapping procedures. A map for the F2 population was constructed which included 116 DNA markers (14 SSRs and 102 AFLPs). Two major QTLs have been mapped to chromosome arms 1DS (Psh1) and 2BL (Psh2), with significant LOD values. These two QTLs account for 76.7% of the phenotypic variance for resistance to barley yellow rust. Two other QTLs, with a minor effect, were mapped to chromosome arms 5AL (Psh3) and 6AL (Psh4), explaining 5.1% and 10.9% of the phenotypic variation, respectively. The QTL on 5A was derived from the susceptible variety, ‘Chinese 166’. In all cases the resistance towards P. striiformis f.sp. hordei was associated with a visual chlorosis/necrosis response typical of race-specific, host resistance.
A ferrugem amarela, cujo agente causal é Puccinia striiformis Westend, é uma doença particularmente importante nas produções de trigo e cevada em todo o mundo, principalmente em regiões de clima fresco e húmido (EVERSMEYER & KRAMER, 2000). Infecções severas deste patogénio podem causar drásticas reduções na altura da planta, no número de grãos por espiga, e no peso e qualidade dos grãos (MA & SINGH, 1996b). A espécie P. striiformis encontra-se dividida em formae speciales, em função do género vegetal que ataca. Por exemplo, o trigo é considerado hospedeiro para P. striiformis f. sp. tritici, a ferrugem amarela do trigo, mas não para a f. sp. hordei, a forma da ferrugem amarela que ataca a cevada. No entanto, a divisão de P. striiformis em formae speciales, e em particular a separação em f. sp. tritici e f. sp. hordei, tem sido fortemente questionada, uma vez que existem vários exemplos de formae speciales com capacidade de atacar genótipos de espécies que estão supostamente fora do seu leque de hospedeiros (hospedeiros ‘inapropriados’) (JOHNSON & LOVELL, 1994; CHEN et al., 1995). O desenvolvimento de cultivares resistentes à ferrugem amarela é actualmente considerado o melhor método de controlo da doença, tanto a nível económico como ambiental. No entanto, o melhoramento para a resistência a esta doença tem assentado, ao longo das últimas décadas, no uso de genes de resistência específica de planta hospedeira, que, na maioria dos casos, têm demonstrado baixa durabilidade (WELLINGS & MCINTOSH, 1990; BAYLES et al., 2000; SING & HUERTA-ESPINO, 2001). O uso generalizado de cultivares portadoras deste tipo de resistência resulta geralmente numa elevada pressão de selecção sobre o patogénio e na sua consequente evolução para novas formas de virulência (BROWN, 1995). Formas de resistência alternativas à resistência específica têm sido estudadas como possíveis fontes de resistência durável. A resistência de planta não hospedeira é considerada por vários autores, a forma mais eficaz de obter durabilidade (HEATH, 1991; CRUTE & PINK, 1996). Na sua generalidade, este tipo de resistência envolve um controlo genético complexo e uma multiplicidade de factores de defesa que impedem o microrganismo de formar uma interacção básica (compatível) com a planta (HEATH, 1991). No entanto, interacções não-hospedeiro entre espécies vegetais filogeneticamente próximas (como é o caso do trigo e da cevada) e formae speciales do mesmo patogénio (P. striiformis f. sp. hordei e P. striiformis f. sp. tritici) parecem envolver mecanismos de resistência semelhantes aos envolvidos na resistência específica de planta hospedeira, que geralmente estão associados ao retardamento do desenvolvimento do patogénio na fase pós-haustorial e à morte das células invadidas (reacção de hipersensibilidade) (NIKS, 1988; GARROOD, 2001). As estratégias de exploração da resistência de planta não hospedeira, assim como a sua durabilidade efectiva, irão, neste sentido, depender de a resistência ser controlada por mecanismos de defesa específicos ou não-específicos (HEATH, 2001). Torna-se, portanto, indispensável a existência de informação detalhada sobre os genes que controlam os mecanismos de resistência de planta não hospedeira, por forma a determinar a viabilidade do uso deste tipo de resistência como fonte de resistência durável. O progresso nos sistemas de marcadores moleculares de DNA e nos programas informáticos de análise genética tornou possível o mapeamento de genes e a identificação de QTLs (Quantitative Trait loci, loci para características quantitativas) com relativa precisão, o que permitiu uma revisão dos métodos de análise genética e das estratégias de melhoramento. A análise de QTLs, i.e., a dissecção genética de características quantitativas, atenta na determinação do número de loci envolvidos na resistência, assim como na localização no genoma da planta e contribuição para o fenótipo de cada um desses loci, através da associação entre a variação de marcadores genéticos numa população segregante e a variação fenotípica para a resistência apresentada por essa mesma população (MOHAN et al., 1997). A tecnologia de microsatélites ou SSRs (Simple Sequence Repeats, repetições de sequências simples), que consistem em repetições em tandem de motivos básicos de 2 a 6 bases (TAUTZ, 1989), emergiu na última década como o sistema de escolha no mapeamento molecular em plantas, e em particular no trigo. Tal ocorre devido ao elevado número de SSRs existente nos genomas das plantas, e porque nesta tecnologia se reúnem as principais vantagens dos diferentes sistemas de marcadores moleculares: são específicos do cromossoma, altamente informativos, co-dominantes, com uma boa cobertura do genoma e com elevado potencial de automatização (MORGANTE & OLIVIERI, 1993; RÖDER et al., 1995; POWELL et al., 1996a; KORZUN et al., 1997). Têm como principal inconveniente o elevado custo de identificação e produção (POWELL et al., 1996a). Vários mapas de ligação foram já desenvolvidos para o trigo baseados neste tipo de marcadores moleculares (DEVOS et al., 1995; PLASCHKE et al., 1995; RÖDER et al., 1995, 1998a, b; BRYAN et al., 1997; STEPHENSON et al., 1998; PESTSOVA et al., 2000; VARSHNEY et al., 2000; SOURDILLE et al., 2001; GUPTA et al., 2002), e têm sido amplamente usados na localização de genes e QTLs responsáveis por resistências a doenças, incluindo a resistência à ferrugem amarela (e.g. CHAGUÉ et al., 1999; PENG et al., 1999, 2000a, b; BOUKHATEM et al., 2002; SUN et al., 2002). Com base num cruzamento entre as cultivares de trigo ‘Lemhi’ (resistente à ferrugem amarela da cevada) e ‘Chinese 166’ (susceptível à doença), JOHNSON & LOVELL (1994) identificaram dois genes major, independentes e dominantes, responsáveis pela resistência de planta não hospedeira à ferrugem amarela da cevada na cv. ‘Lemhi’. Foi igualmente detectada a existência de um número indeterminado de genes minor, alguns dos quais com possível origem na cv. ‘Chinese 166’. Pretendeu-se com o presente trabalho: 1) desenvolver um mapa genético para uma população F2, constituída por 114 indivíduos, derivada do cruzamento ‘Lemhi’ x ‘Chinese 166’ usando marcadores do tipo SSR; 2) adicionar estes marcadores a um mapa de AFLPs previamente construído para a mesma população; e 3) localizar os genes responsáveis pela resistência do trigo à ferrugem amarela da cevada em segregação na população F2 ‘Lemhi’ x ‘Chinese 166’. Cento e dezoito indivíduos da população F2 ‘Lemhi’ x ‘Chinese 166’, assim como as plantas progenitoras desta população, foram previamente testados para resistência/susceptibilidade ao referido patogénio. ‘Lemhi’ apresentou um fenótipo totalmente resistente, enquanto ‘Chinese 166’ se apresentou moderadamente susceptível, o que confirmou a presença de gene(s) minor nesta cultivar. Os 118 indivíduos da F2 analisados fenotipicamente segregaram 115 resistentes : 3 susceptíveis, sugerindo que a resistência de ‘Lemhi’ à ferrugem amarela é efectivamente controlada por dois genes major. Foram testados 88 pares de primers de SSRs para a presença de polimorfismos entre ‘Lemhi’ e ‘Chinese 166’. Desta análise resultou um total de 41 SSRs polimórficos, que foram analisados em 114 indivíduos da população F2. Com base nestes SSRs e em 172 AFLPs (Amplified Fragment Length Polymorphisms, polimorfismos do comprimento dos fragmentos amplificados) anteriormente desenvolvidos para a mesma população, e recorrendo ao programa informático de análise genética JoinMap® versão 3.0 (VAN OOIJEN & VOORRIPS, 2001), foi construído um mapa molecular com 18 mapas de ligação, integrando 116 marcadores de DNA (14 SSRs e 102 AFLPs), e abrangendo 680 cM, com uma densidade média de 1 marcador por cada 6 cM. Os restantes 97 marcadores moleculares não foram integrados no mapa, provavelmente por, dada a extensão do genoma do trigo, não haver marcadores suficientes para criar ligação entre eles. Oito dos 18 grupos de ligação foram ancorados a seis cromossomas (1D, 2B, 3A, 5A, 6A e 6B) pela presença de SSRs. Uma vez que os restantes grupos de ligação não foram associados a nenhum QTL (ver parágrafo seguinte), não foram desenvolvidos esforços no sentido de identificar SSRs específicos para esses grupos de ligação. A identificação de QTLs foi efectuada usando o programa informático de análise de QTLs MapQTL™ versão 4.0 (VAN OOIJEN et al., 2002). Os efeitos dos QTLs e a sua significância para a variação fenotípica total da resistência à ferrugem amarela da cevada foram estimados pelos métodos Interval Mapping e MQM Mapping. Através do método Interval Mapping foram identificados dois QTLs major, localizados nos cromossomas 1DS (Psh1) e 2BL (Psh2), com origem na cv. ‘Lemhi’. Por forma a detectar possíveis QTLs minor mascarados por estes QTLs major, foi aplicado o método MQM Mapping. Neste método, recorre-se ao uso dos marcadores que flanqueiam os QTLs detectados por Interval Mapping como co-factores para eliminar o efeito daqueles e detectar QTLs minor. Após análise por MQM Mapping, foram localizados dois QTLs minor nos cromossomas 5AL (Psh3) e 6AL (Psh4), sendo que o QTL presente no cromossoma 5A deriva da variedade susceptível ‘Chinese 166’. Os quatro QTLs detectados explicam, no seu conjunto, 92,7% da variação fenotípica total da resistência à doença, o que indica que, provavelmente, todos os loci que contribuem para a resistência de planta não hospedeira foram identificados. Neste estudo, verificou-se que a resistência à ferrugem amarela da cevada estava associada a uma resposta fenotípica de clorose/necrose, típica de resistência específica de planta hospedeira. Para além disso, os genes Psh1 e Psh2, genes de resistência de planta não hospedeira à ferrugem amarela da cevada, foram identificados em regiões do genoma do trigo onde se pensa (no caso do Psh1) e onde se sabe (no caso de Psh2) existirem genes de resistência de planta hospedeira (genes Yr) à ferrugem amarela do trigo. Tendo em atenção estes factos, pode considerar-se a possibilidade de uma ligação entre genes Psh e genes Yr, que, a confirmar-se, pode levar a supor que se trata de genes que evoluíram de um mesmo gene de resistência ancestral, possuindo portanto estrutura e modo de acção semelhantes. Se tal se vier a verificar, então a durabilidade de ambos seria, também ela, semelhante. Patologistas e melhoradores teriam que repensar seriamente a validade da busca de genes de resistência de planta não hospedeira como fonte de resistência durável. A clonagem destes genes é, neste sentido, essencial para que estudos bioquímicos e de funcionamento dos genes possam ser posteriormente desenvolvidos, e para que seja determinada a viabilidade do uso dos genes Psh como genes de resistência com efeito duradouro.

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A brusone, causada por Pyricularia oryzae, é considerada uma doença economicamente importante para trigo na América do Sul. Uma das estratégias de manejo para minimizar as perdas causadas por essa doença é o uso de cultivares resistentes. Alternativamente, pode-se utilizar indutores de resistência para aumentar o nível basal de resistência do trigo à brusone. O presente estudo teve como objetivos: i) determinar as alterações fisiológicas e bioquímicas em plantas de trigo pulverizadas com uma concentração não fitotóxica do ácido α-picolinico (AP), o qual é uma toxina não seletiva produzida por P. oryzae e ii) verificar a manipulação metabólica exercido por P. oryzae quando infectando cultivares de trigo com diferentes níveis de resistência basal à brusone. Nas folhas de trigo pulverizadas com uma concentração não fitotóxica de AP (0.1 mg mL -1 ), os sintomas da brusone desenvolveram menos em associação com um melhor desempenho fotossintético, melhoria do metabolismo antioxidante e redução nas concentrações de H 2 O 2 , O 2 ●- e MDA. As cultivares BR-18 e EMBRAPA- 16 foram mais resistentes à brusone do que a cultivar BRS-Guamirim. O desempenho fotossintético das plantas infectadas foi alterado devido a limitações de natureza difusiva e bioquímica para uma eficiente fixação do CO 2 . Durante a fase assintomática da infecção por P. oryzae, mudanças drásticas no metabolismo de carboidratos e nos níveis de aminoácidos, compostos intermediários do ciclo de Krebs e poliaminas ocorreram nas plantas das três cultivares sugerindo, assim, uma manipulação metabólica exercida por P. oryzae. No entanto, um metabolismo antioxidativo mais eficiente foi importante para neutralizar os efeitos deletérios da infecção por P. oryzae em associação com maiores atividades da fenilalanina amônia liase e polifenoloxidase e maiores concentrações de compostos fenólicos e lignina. Com base nesses resultados e possível concluir que a concentração não fitotóxica de AP foi capaz de potencializar a defesa das plantas de trigo e reduzir a severidade da brusone. A infecção do trigo por P. oryzae ocasionou distúrbios no metabolismo primário das plantas e alguns deles foram semelhantes entre as cultivares independentemente do nível basal de resistência delas.
Blast, caused by Pyricularia oryzae, has become an economically important disease in wheat in South America. One of the management strategies for minimizing the losses caused by blast includes the use of resistant cultivars. Alternatively, the use of inducers of resistance showed the potentiation to increase wheat resistance to blast. This study aimed: i) to determine the physiological and biochemical alterations in wheat plants sprayed with a non-phytotoxic concentration of α-picolinic acid (PA), which is a non-host selective toxin produced by P. oryzae and ii) to establish the degree of metabolic manipulation exerted during the infection by P. oryzae on plants from cultivars with different levels of basal resistance to blast. The spray of leaves of plants with a non-phytotoxic concentration of PA (0.1 mg mL -1 ) resulted in less blast symptoms in association with a better photosynthetic performance, an improvement on the antioxidant metabolism and reduced concentrations of H 2 O 2 , O 2 ●- and malondialdehyde. The cultivars BR-18 and EMBRAPA-16 were more resistant to blast in comparison to cultivar BRS-Guamirim. The photosynthetic performance of the infected plants was altered due to diffusional and biochemical limitations for CO 2 fixation. During the asymptomatic phase of P. oryzae infection, drastic changes in the carbohydrates metabolism and on the levels of amino acids, intermediates compounds of Krebs cycle and polyamines occurred on plants from the three cultivars suggesting a metabolic manipulation exerted by the pathogen. However, amore efficient antioxidant metabolism was able to help the wheat plants to counteract against the deleterious effects of P. oryzae infection in association with great phenylalanine ammonia lyases and polyphenoloxidases activities and high concentrations of phenolics and lignin. Based on this information, it is possible to conclude that a non- phytotoxic concentration of PA elicited the activation of host defense mechanisms that reduced blast severity. Likewise, the infection of leaves by P. oryzae induced remarkable disturbances in the primary metabolism and some of them were conserved among the cultivars regardless of their basal level of resistance to blast.

Non-rutaceous plant species, potentially hosts to ‘Candidatus Liberibacter africanus’ (Laf) sensu lato, were sampled throughout the Cape Floristic Region from the Fynbos and Succulent Karoo biomes in South Africa, and tested for the presence of the insect-transmitted bacterial pathogen associated with ‘Citrus Greening disease’ (CG) in Citrus species (Rutaceae). Laf is considered a persistent problem to the production of citrus in South Africa as fruits produced from CG infected citrus are smaller in size, lopsided or misshaped and have a characteristic bitter taste. The information on the potential host range in indigenous and other plant species in South Africa is limited. In the current study three surveys were carried out during September 2017 (spring), January 2018 (summer), and August 2018 (winter) in the natural vegetation in Robertson, Worcester, Slanghoek, Vredendal, Lutzville and Klawer. Potential psyllid vectors were collected with vacuum sampling from approximately 20 randomly selected plant samples per plant species at each site. Branches and flowers, when available, of the same plants were collected for morphological identification. Leaf and petiole samples of 989 plant specimens, representing 19 plant families and 42 species, were collected. No typical galls induced on leaves by psyllid nymphs were observed on the plants. Psyllids were only collected from two Roepera foetida (Zygophyllaceae) plants. Of the 989 plant specimens of alternate host species tested for the presence of Liberibacters by real-time polymerase chain reaction (real-time PCR) assays 142 yielded a Ct value below the selected positive/negative threshold of 31 following the Liberibacter ‘Universal’ real-time PCR assay. Conventional PCR tests, including the amplification of the 16S rRNA, omp and rplJ genes of Laf, were conducted on these 142 plant specimens. Seven of these yielded very faint bands after gel electrophoresis analysis of the 16S rRNA conventional PCR test. Sanger sequencing of these suggested they were non-target amplicons. Therefore, none of the 42 plant species tested positive for ‘Ca. Liberibacter africanus’, the causal agent for CG in South African citrus. As a number of Atriplex semibaccata plants had yielded real-time PCR values <31 and yielded some amplicons with the 16S rRNA PCR, a sample with a DNA concentration above 250 ng/μl was selected for next-generation sequence (NGS) analysis using an Illumina HiSeq 2000 platform at Life Sequencing (Spain) to attempt to identify the presence of a potentially divergent Liberibacter. NGS data indicated that the bacterial entity amplified by the Liberibacter ‘Universal’ real-time PCR was not Liberibacter spp., but an, as yet, unidentified member of the Gammaproteobacteria. This bacterium may also be present in a number of other Atriplex samples tested during this study, including A. lindleyi and A. nummularia, which had also yielded amplicons in the real-time PCR assays. It may also be present in some of the other plant species testing positive in the real-time PCR test. The data generated from this study, and from the studies done in conjunction with this one, will be used for biological and epidemiological studies and the development of management strategies.
Dissertation (MSc)--University of Pretoria, 2019.
Microbiology and Plant Pathology
MSc
Unrestricted

Mycobacterium tuberculosis (M.tb), the causative agent of tuberculosis, is one of the leading causes of death due to a single infectious agent and results in more than 1 million human deaths every year. M.tb infection of the host initiates a local inflammatory response, resulting in the migration of a number of host plasma protein and non-protein factors to the site of infection. In addition, some of these factors are also produced locally at the site of infection. It is envisaged that these host factors are likely to come in direct contact with M.tb and immune cells and may modulate the outcome of the infection. In this study, a number of host factors including transferrin, lactoferrin, fibrinogen, C-reactive protein, alpha-2-macroglobulin (α2M), vitronectin, plasminogen, low-density lipoprotein (LDL), high-density lipoprotein (HDL), serotonin, L-alpha dipalmitoyl phosphatidylcholine (DPPC) and platelet activating factor C-16 (PAF C-16) were screened in vitro for their direct effect on the growth of mycobacteria using M.smegmatis as a model. As a result of this screening, PAF C-16, a phospholipid compound was identified that directly inhibited the growth of M.smegmatis and M.bovis BCG in a dose and time-dependent manner. Use of a range of PAF C-16 structural analogues, including Lyso-PAF, PAF C-18, Hexanolamino PAF, 2-O-methyl PAF & Pyrrolidino PAF, revealed that small modifications in structure did not alter the direct growth inhibition property of PAF C-16 and similar levels of M.smegmatis and M.bovis BCG growth inhibition were observed as compared to PAF C-16. Structural dissection of PAF C-16 suggested that the attachment of carbon tail to the glycerol backbone via ether bond at sn-1 position was important for its direct growth inhibition activity against mycobacteria. Microscopy and flow cytometry with PAF C-16 treated M.smegmatis and M.bovis BCG showed damage to the bacterial cell membrane. The addition of membrane-stabilizing agents, α-tocopherol, tween-80 and tween-20, partially mitigated the growth inhibitory effect of PAF C-16. These results suggested that the growth inhibition activity of PAF C-16 against mycobacteria is most likely due to its detergent-like effect, resulting in damage to the bacterial cell membrane. PAF C-16 and its structural analogues were also investigated for their effect on the growth of intracellular M.smegmatis inside THP1 cells. In vitro, PAF C-16, PAF C-18 and Hexanolamino PAF inhibited the growth of intracellular M.smegmatis, whereas, analogues such as Lyso-PAF and 2-O-methyl PAF failed to show any growth inhibitory effect, suggesting that the presence of acetyl group at sn-2 position was important for growth inhibition of intracellular M.smegmatis. Use of PAF receptor antagonists partially mitigated the inhibitory effect of PAF C-16 on the growth of intracellular M.smegmatis, suggesting this inhibition was through receptor-mediated signalling pathways. Blocking of PAF C-16 signalling pathway components such as phospholipase C and phospholipase A2, resulted in the increased survival of intracellular M.smegmatis. Arachidonic acid, a product of PAF C-16 signalling pathway directly inhibited the growth of M.smegmatis. Furthermore, inhibition of iNOS enzyme and antibody-mediated neutralization of TNF-α partially mitigated the inhibitory effect of PAF C-16 on intracellular M.smegmatis growth, suggesting that the production of NO and TNF-α were also involved in PAF C-16 induced intracellular growth inhibition. Overall, this study has identified PAF C-16, its structural analogues such as Lyso-PAF, PAF C-18, Hexanolamino PAF and other compounds including 1-O-hexadecyl-sn-glycerol, miltefosine and hexadecyl lactate with novel anti-mycobacterial activity. Further investigations are needed to demonstrate their effectiveness against M.tb both in vitro and in animal models to assess their therapeutic potential as anti-TB drugs.

Les virus infectent des représentants de tous les trois domaines, Eukarya, Bacteria et Archaea, mais nos connaissances sur les virus des Archées demeurent à leurs prémices. Nous avons choisi comme un hôte cible une archée aérobie Aeropyrum pernix, pH7. 0, 90-95 °C, afin d'élargir notre connaissance sur les virus des archées hyperthermophiles. Aucun virus du genre Aeropyrum n’avait encore été décrit avant le début de mon travail de thèse. A l’occasion de ce projet de recherche, 4 nouveaux virus d’A. Pernix ont été isolés et caractérisés. D'abord, la présence de 2 provirus putatifs a été prédite par analyse in silico dans le génome d’A. Pernix K1 et les 2 virus A. Pernix spindle-shaped virus 1 (APSV1) et A. Pernix ovoid virus 1 (APOV1) furent induits et isolés et leur génomes et morphotypes caractérisés. L’approche suivante était la recherche des virus infectant A. Pernix à partir d’échantillons environnementaux de sources chaudes situées proches de l’origine d’A. Pernix. Cela a conduit à la découverte, l’isolation et la caractérisation d’A. Pernix bacilliform virus 1 (APBV1) et d’Aeropyrum spring-shaped virus (ASPV). Le virus ASPV s’est avéré être le premier virus d’archée hyperthermophile avec un génome à ADN simple brin. Les séquences des quatre virus isolés ne montrent aucune similarité significative avec les génomes des autres virus connus. Ce résultat, ajouté aux propriétés morphologiques uniques des virus APBV1 et ASPV, a servi de base pour la proposition de 2 nouvelles familles, les Clavaviridiae (APBV1) et Spiraviridae (ASPV). L’ensemble des résultats obtenus contribuent à une meilleure compréhension de la diversité virale de notre planète
Viruses are known to infect members of all three domains, Eukarya, Bacteria and Archaea, but our knowledge about viruses infecting Archaea is still in its infancy. We have chosen an aerobic archaeon Aeropyrum pernix, growing optimally at pH7. 0, 90-95°C, as a target host to broaden our knowledge of hyperthermophilic archaeal viruses. No virus of the genus Aeropyrum, nor the order Desulfurococcales, had been described prior to the PhD project. In the course of the research, four novel viruses of A. Pernix have been isolated and studied. Firstly, the presence of two putative proviruses in the genome of A. Pernix K1 was predicted by in silico analysis, and the two viruses Aeropyrum pernix spindle-shaped virus 1 (APSV1), and Aeropyrum pernix ovoid virus 1 (APOV1) were induced and isolated, and their genomes and morphotypes were characterized. The next approach was based on the screening for viruses infecting A. Pernix in the environmental samples collected at hydrothermal areas close to the origin of A. Pernix. This resulted in the discovery, isolation and characterization of Aeropyrum pernix bacilliform virus 1 (APBV1), and Aeropyrum spring-shaped virus (ASPV). The virus ASPV is the first hyperthermophilic archaeal virus with a ssDNA genome. The genome sequences of all four isolated viruses show no significant similarity with genomes of other known viruses. This result, along with unique morphological properties of the viruses APBV1 and ASPV, served as a basis for the proposal of two novel virus families, the Clavaviridae (APBV1) and Spiraviridae (ASPV). Together, the results of the research contributed to a better understanding of the viral diversity on our planet

A pathosystem using Arabidopsis and wheat powdery mildew, Blumeria graminis f. sp. tritici (Bgt), for which Arabidopsis is a non-host, was employed to initiate the genetic dissection of non-host resistance (NHR). The EMS mutagenised population from the Arabidopsis line containing the GST1:LUC transgene, which can facilitate a high throughput mutant screening strategy, have been screened using an ultra low light imaging camera system. Following a mutant screen of approximately 100,000 M2 plants, a number of candidates have been identified that compromise the induction of the LUC transgene in response to attempted Bgt infection. Through this screening, nhr1 was isolated as a putative factor for non-host pathogen recognition. This mutant showed severely compromised GST1 induction and less hypersensitive cell death in response to Bgt inoculation, while exhibiting little difference against other host bacterial and fungal pathogens including Pseudomonas syringae p.v. tomato and Hyaloperonospora parasitica. In addition, nhr1 was sugar dependent in germination. We identified ads3 (ACTIVATED DISEASE SUSCEPTIBILTY 3), an Arabidopsis mutant showing drought resistant as well as disease susceptible phenotype against host and non-host pathogens. ads3 is emf1-D by enhanced expression of Embryonic Flower 1 (EMF1), which had been known as repressor of floral transition in plant. The susceptibility of emf1-D was recapitulated in transgenic Arabidopsis plants ectopically expressing EMF1. Conversely, conditionally decreased EMF1 level in the transgenic plant conveyed disease resistance. emf1-D was drought resistant and hypersensitive to abscisic acid (ABA). We show that ectopic expression of EMF1 modulates a ABA signalling, resulting in susceptibility to pathogens. The other plant link, which is the translationally controlled tumor protein (TCTP) over-expressing line, showed reduced NHR against Bgt. A leucine rich repeat receptor-like kinase (LRK) was isolated as a putative TCTP-interacting protein, and the KO line of the LRK showed less or delayed resistant response to non-host as well as host fungal pathogens. furthermore, the TCTP over-expressing line exhibited hypersensitivity towards ABA. These results suggest that ABA signalling could play a critical role in non-host resistance in plants.

Thermodynamic parameters for the complexation process involving substituted sodium benzoates with a, 13 and 7 cyclodextrins have been determined in water and in N,N-dimethylformamide at 298.15 K. The increasing negative enthalpies of complexation in N,N-dimethylformamide moving from a to 7-cyclodextrin reflects the effects of substituting the cyclodextrin ligand as the cyclodextrin cavity size increases. Single-ion parameters of the new benzoate-cyclodextrin anions have been derived and the results show that these anions are solvated much better in water than in N,N-dimethylformamide. Thermodynamic parameters for the complexation of 1:1 monosaccharide-cyclodextrin complexes have been determined in water at 298.15 K. The data show the ability of the cyclodextrins to selectively distinguish between the aldopentoses and aldohexoses. Thermogravimetric analysis and differential scanning calorimetry of an isolated monosaccharide-cyclodextrin complex reveal that N,N-dimethylformamide is interacting with cyclodextrin. nuclear magnetic resonance (NMR) and spectrophotometric studies together with computer modelled calculations suggest the formation of exclusion type complexes. stability constants, free energies, enthalpies and entropies of complexation of the D-amino acids and D-amino acid trifluoromethanesulphonates in methanol at 298.15 K are discussed. No significant variations were found in the free energies of complexation of the amino acids as a result of an entropy-enthalpy compensation effect. The transfer enthalpy of the amino acid cations are negative implying that these cations are enthalpically more stable in methanol than water. However, no complexation in water was found between these guest species and 18-crown-6. This is attributed to an interaction between 18-crown-6 and water as reflected in the transfer enthalpy of this ligand from water to methanol. Electrochemical, spectrophotometric, nuclear magnetic resonance and calorimetric studies on the interaction of a resorcinol-based calix[4]arene with amines in non-aqueous media (chloroform and benzonitrile) are reported as a contribution to the area of calixarene chemistry involving the generation of new electrolytes, resulting from a combination of proton transfer and hydrogen bonding from the calixarene to the amine. A summary and suggestions for further work are also given.

Swede midge, Contarinia nasturtii Kieffer (Diptera: Cecidomyiidae), is an invasive pest causing marketable losses on Brassica crops in the Northeastern United States and throughout southern Canada. Heading brassicas, like cauliflower and broccoli, are particularly susceptible because larvae feed concealed inside meristematic tissues of the plant, where head formation occurs. Our work details the development of a sustainable, affordable pest management tactic for swede midge – plant derived repellents. First, it was necessary to establish both a damage and marketability threshold for swede midge, so we developed a technique to manipulate larval density of swede midge on cauliflower, We asked: (1) What is the swede midge damage threshold? (2) How many swede midge larvae can render cauliflower crowns unmarketable? and (3) Does the age of cauliflower at infestation influence the severity of damage? We found that even a single larva causes mild twisting and scarring rendering cauliflower unmarketable 52% of the time, with more larvae causing more severe damage and additional losses, regardless of cauliflower age at infestation. Repellency is an important management approach to consider for swede midge. Since the host range of specialist insects appears constrained by plant phylogeny, we hypothesized that odors from less phylogenetically related plants would be more repellent to swede midge. To test our hypothesis, we performed no-choice and choice biological assays, asking: (1) How do essential oils from different plant species influence midge densities on broccoli? (2) What is the relationship between phylogenetic distance of non-host odors and larval densities on broccoli? Biological assays identified multiple essential oils that reduced larval densities, and phylogenetic analyses showed that less related plants were more effective. In addition to the biological assays, we tested 15 essential oils for their ability to repel gravid females from broccoli tissue in y-tube olfactometer assays. While most of the essential oils reduced the frequency at which females chose host plant meristems, wintergreen, thyme, lemongrass, eucalyptus lemon, garlic, cinnamon, and star anise were most effective. Additionally, we used chemical fingerprints (physical/chemical properties) from PubChem to compare the essential oil volatile compounds and develop an index for their similarity. We found that physicochemical similarity was predictive of repellency. Finally, for repellency to be an effective, long-term strategy, it was important to consider how and whether the repellent response of midges changes over time or previous experience. In our final chapter, we performed electroantennography trials testing how previous experience with garlic or eucalyptus lemon odor for one or 10 s influences the neurophysiological response of swede midge to host (broccoli) or non-host (garlic or eucalyptus lemon) odors. We asked: (1) Does previous experience with garlic or eucalyptus lemon influence the physiological response of swede midge to host or non-host odors? (2) Does the time of previous exposure to non-host odors influence their physiological response to host or non-host odors? Our findings show that swede midge, after 10 s of exposure to either repellent, was more responsive to repellents than host compounds, suggesting that the effectiveness of repellents will not diminish over time.

Magister Scientiae (Biodiversity and Conservation Biology)
To further investigate the strength of the association and the relative advantages of the association to both organisms, several manipulation experiments were set up. A cage experiment set up in the shallow subtidal zone showed that the coralline survived equally well without the winkle and did therefore not require the winkle or its empty shell for survival. A second controlled laboratory aquarium experiment was designed under both fluorescent (rich in blue light) and incandescent light (rich in red light) to ascertain whether the coralline had a preference for O. sinensis over the similar O. tigrina. This experiment was inconclusive as no recruitment was obtained under either of the light regimes. A third laboratory experiment was designed to determine whether the extra coralline weight had any possible advantage to the winkle, particularly against predation from the rock lobster Jasus lalandii. Results suggested that there were no apparent advantages to the winkle bearing the extra coralline load as adult O. sinensis bearing the coralline alga (3.7 ± 2.2 winkles 24hr-1) were equally prone to predation than those lacking the coralline (2.3 ± 1.9 winkles 24hr-1) (p = 0.184). Observations suggested instead that the convoluted nature of the coralline may indeed have promoted predation. We ultimately deduced that the high occurrence of the coralline on the shells of O. sinensis was probably due to the substantial overlap in the niches of the two organisms. This conclusion was supported by the high densities of juvenile O. sinensis combined with the high percent cover abundance of the coralline in intertidal rockpools. Understanding sexual reproduction in coralline algae as well as the life cycle of the winkle, ultimately provided insight into the postulated life cycle of this coralline-winkle association.
South Africa

Infection with leishmania parasites causes severe chronic and potentially fatal illness in millions of people annually. Nevertheless, leishmania-host interactions remain understudied, and available treatments are sub-optimal. Pivotal to the establishment of infection, parasite replication and development of clinical disease is the subversion of microbicidal activities of host macrophages by leishmania. The overall aim of this thesis was to enhance our understanding of the modus operandi of macrophage subversion and explore the involvement of parasite- and host-derived small non-coding RNAs in this process. My first objective was to investigate whether leishmania exosomes act as shuttle vehicles to export and deliver leishmania RNAs to host macrophages, where they may contribute to pathogenesis. We used high-throughput sequencing to characterize the transcriptome of leishmania exosomes and found that leishmania exosomes are selectively and specifically enriched in small RNAs derived almost exclusively from non-coding RNAs such as rRNAs and tRNAs. In depth analysis revealed the presence of tRNA-derived small RNAs, a novel RNA type with suspected regulatory functions. Exosomes protected their RNA cargo from degradation and were competent to deliver RNAs to macrophages. Furthermore, our results demonstrated a remarkably high degree of congruence in exosomal small non-coding RNA content between two distinct leishmania species, which argues for a conserved mechanism for exosomal RNA packaging in leishmania. My second objective was to investigate whether macrophage miRNA expression is modulated during leishmania infection. Here, I was interested to know whether targeting of the host RNAi machinery is a potential novel mechanism of pathogenesis used by leishmania to control macrophage phenotype and promote chronic infection. I profiled miRNA expression in human macrophages at later stages of infection using two independent technologies. The data showed that leishmania infection induced an overall down-regulation of miRNA expression in macrophages. This down-regulation was not caused through effects on synthesis or stability of Drosha and Dicer, two essential enzymes involved in miRNA maturation. Taken together, my findings suggest that both leishmania- and host-derived small non-coding RNAs may contribute to pathogenesis. They open up new avenues of research on small RNA pathways in leishmania infection biology, which may identify novel therapeutic approaches.
Medicine, Faculty of
Medicine, Department of
Experimental Medicine, Division of
Graduate

Dans le cadre d'une agriculture durable, la compréhension des mécanismes moléculaires qui déterminent la spécificité des pucerons pour les plantes est une étape essentielle dans le développement de stratégies de lutte contre les ravageurs. Cependant, les mécanismes conduisant à la compatibilité ou l'incompatibilité entre la plante et le puceron restent inconnus. Cette thèse visait à identifier les bases génétiques et les déterminants moléculaires impliqués dans la résistance du pois au puceron Acyrthosiphon pisum. La variabilité génétique naturelle de la résistance du pois aux biotypes adaptés et non-adaptés d'A. pisum a été identifiée en criblant une collection de 240 génotypes de pois. Une étude de génétique d'association a identifié le locus ApRVII contrôlant la résistance du pois aux biotypes adaptés et non adaptés d’A. pisumCette étude, couplée à des études transcriptomiques de génotypes de pois sélectionnés, a identifié des gènes candidats sous-jacents à ApRVII qui sont potentiellement impliqués dans la résistance du pois à A. pisum. Ces gènes indiquent l'implication des voies de biosynthèse de métabolites secondaires dans la résistance aux pucerons médiée par ApRVII. De plus, les études transcriptomiques ont identifié des voies moléculaires du pois spécifiquement réprimées pendant l'infestation par le biotype adapté, suggérant une possible manipulation du transcriptome du pois par l'infestation de pucerons. Les connaissances fournies au cours de cette thèse contribuent à une meilleure compréhension des mécanismes impliqués dans la compatibilité et l'incompatibilité entre les plantes et les pucerons
In the context of sustainable agriculture, understanding the molecular mechanisms that determine the specificity of aphids to plants is an essential step in developing pest management strategies. However, the mechanisms leading to compatibility or incompatibility between the plant and the aphid remain unknown. This thesis aimed to identify the genetic basis and molecular determinants involved in pea resistance to the aphid Acyrthosiphon pisum. The natural genetic variability of pea resistance to pea-adapted and non-adapted biotypes of the A. pisum was identified by screening a collection of 240 pea genotypes. An association genetics study identified the ApRVII locus controlling pea resistance to both adapted and non-adapted A. pisum biotypes.This study, coupled with transcriptomic studies of selected pea genotypes, identified candidate genes underlying ApRVII that are potentially involved in pea resistance to A. pisum. These genes indicated the involvement of biosynthetic pathways of secondary metabolites in ApRVII mediated resistance to the aphids. In addition, the transcriptomic studies identified pea molecular pathways specifically repressed during the infestation by the adapted biotype, suggesting a possible manipulation of pea transcriptome by the aphid infestation. The knowledge provided during this thesis contributes to a better understanding of the mechanisms involved in the compatibility and incompatibility between plants and aphids

Streptococcus uberis is an important cause of intramammary infection in dairy cattle. Strains of S. uberis appear to differ in their ability to cause disease based on previous epidemiological studies. We explored the pathogenicity of 2 strains of S. uberis, where one strain represented a putatively host-adapted type based on its ability to cause persistent infection and to spread from cow to cow in a lactating herd. This type was part of a clonal complex that is commonly associated with bovine mastitis. The other strain, which was isolated from a transient infection in a single animal in the same herd and did not belong to any known clonal complex, was selected as putatively non-adapted type. Cows (6 per strain) were experimentally challenged in a single hind quarter and the adjacent hind quarter was used as mock challenged control quarter. Both strains showed an equal ability grow in milk of challenge animals in vitro. All cows that were challenged with the putatively host-adapted strain developed clinical signs of mastitis, including fever and milk yield depression as well as elevated somatic cell count due to influx of polymorphonuclear leucocytes and lymphocytes. The cytokine response followed a specific order, with an increase in IL-1β, IL-6 and IL-8 levels at the time of first SCC elevation, followed by an increase in IL-10, IL-12p40 and TNF-α levels approximately 6 h later. In 4 of 6 animals, IL-17A was detected in milk between 57 and 168 h post challenge. The increase in IL-17A levels coincided with inversion of the pre-challenge CD4+:CD8+ T lymphocyte ratio, and was observed from 96 h post challenge. This was followed by normalisation of the CD4+:CD8+ ratio due to continued increase of the CD8+ concentration up to 312 h post challenge. Spontaneous resolution of infection was observed in 5 animals and coincided with a measurable IL-17A response in 4 animals, suggesting that IL-17 may be involved in the resolution of intramammary infection. To explore the mechanism of action of IL-17A we stimulated bovine PMN and bovine blood derived macrophages with recombinant IL-17A in vitro. IL-17A enhanced the killing ability of phagocyte toward the challenge strain. With the exception of minor elevation of IL-8 levels, no clinical, cytological or immunological response was detected in quarters challenged with the non-adapted strain. The observed strain specific pathogenicity was consistent across animals, implying that it is determined by pathogen factors rather than host factors. We further studied in vitro possible mechanisms involved in the differences observed between the two strains such as ability to adhere to the mammary epithelial cells, ability to resist to killing by phagocytes and ability to form biofilm. The adapted strain FSL Z1-048 showed an increased ability to adhere to the epithelial cells and an increase ability to resist to killing of monocyte derived macrophages. These mechanisms thus could potentially explain the in vivo observations.

Although social psychologists have made important strides towards understanding the effects of stigma on both individuals’ behaviours and their relationships with non-stigmatized groups, language patterns within this domain have largely been ignored. This thesis aims to address this gap by investigating the role that language patterns play in shaping the relationship between native and non-native speakers against the backdrop of an increasingly relevant context in which communicators with diverse language backgrounds interact: Immigration. Drawing on both communication accommodation theory (CAT) and intergroup contact theory, I investigate the processes by which language styles influence perceptions of both individuals and the groups they represent, as well as attempt to determine how language-based categorizations affect those whose language style deviates from majority group norms. Across six studies, I take the perspective of native speakers and demonstrate that perceptions of communicators based on their language are not uniform but are determined by factors including the style of language used and the speaker’s background. I then take the perspective of non-native speakers and, across two studies, show that negative perceptions of non-native accents can result in poorer interactions with the native speaking out-group as well as a reduced ability to comprehend and communicate in the host country’s language. In sum, the eight studies presented in this thesis demonstrate that perceptions related to one’s style of language can be detrimental to the relationship between native and non-native speakers and by extension host country natives and immigrants. Theoretical and practical implications are discussed.

Stem rust, caused by the macrocyclic fungal pathogen P. graminis (Pg), is one of the most devastating diseases of wheat and other small grains globally; and the emergence of new stem rust races virulent on deployed resistance genes brings urgency to the discovery of more durable sources of genetic resistance. Given its intrinsic durability and effectiveness across a broad range of pathogens, non-host resistance (NHR) presents a compelling strategy for achieving long-term rust control in wheat. However, NHR to Pg (Pg-NHR) remains largely unexplored as a protection strategy in wheat, in part due to the challenge of developing a genetically tractable system in which Pg-NHR segregates. In this dissertation, an investigation of Pg-NHR is undertaken via the pathogen's alternate (sexual) host, barberry ( Berberis spp.). Within the highly diverse Berberis genus, numerous species function as alternate hosts to Pg but others are non-hosts. European barberry (B. vulgaris L.), for example, is susceptible to Pg infection but Japanese barberry (B. thunbergii DC.) is a non-host. In this study, the nothospecies B. ×ottawensis C.K. Scheid, an inter-specific hybrid between Pg-susceptible B. vulgaris and Pg-resistant B. thunbergii, is explored as a possible means of mapping the gene(s) underlying the apparent Pg-NHR exhibited by B. thunbergii. The overall goal of this research is to contribute to the global search for novel sources of potentially durable stem rust resistance genes.

The first chapter describes a field study conducted in western Massachusetts, in which a natural population of B. ×ottawensis was characterized to determine if the hybrid can be used to genetically dissect the Pg-NHR exhibited by B. thunbergii. A population of 63 B. ×ottawensis individuals were clonally propagated, phenotyped for disease response to Pg via controlled inoculation using overwintered telia of Pg found on naturally infected E. repens, and genotyped using the de novo genotyping-by-sequencing (GBS) pipeline GBS-SNP-CROP. Controlled inoculation of a subset of 53 B. ×ottawensis accessions, verified via GBS to be true, first-generation hybrids, revealed 51% susceptible, 33% resistant, and 16% intermediate phenotypes. Although such variation in disease response within a natural population of F₁ hybrids could be explained by non-nuclear (cytoplasmic) inheritance of resistance, a similar pattern of segregation was observed in a population of B. ×ottawensis full-sibs, developed via controlled crosses. The results of this first chapter demonstrate not only that the Pg-NHR observed in B. thunbergii segregates among F₁ interspecific hybrids with Pg-susceptible B. vulgaris but that the resistance is likely nuclearly inherited. Therefore, at least in principle, the gene(s) underlying Pg-NHR in B. thunbergii should be mappable in an F₁ population derived from the controlled hybridization of the two parental species.

Building on the results of first chapter, the second chapter of this dissertation details the generation and use of a bi-parental B. ×ottawensis mapping population to develop genetic linkage maps for both parental species and begin mapping the gene(s) underlying Pg-NHR in B. thunbergii. Using 162 full-sib F₁ hybrids and a total of 15,411 sequence variants (SNPs and indels) identified between the parents via GBS, genetic linkage maps with 1,757 and 706 markers were constructed for B. thunbergii accession 'BtUCONN1' and B. vulgaris accession 'Wagon Hill', respectively. In each map, the markers segregated into 14 linkage groups, in agreement with the 14 chromosomes present in these Berberis spp. The total lengths of the linkage maps were 1474 cM (B. thunbergii) and 1714 cM (B. vulgaris), with average distances between markers of 2.6 cM and 5.5 cM. QTL analysis for Pg resistance led to the identification of a single QTL, dubbed QPgr-3S, on the short arm of chromosome 3 of B. thunbergii. The peak LOD score of QPgr-3S is 28.2, and the QTL spans 13 cM, bounded by the distal SNP marker M411 and proximal SNP marker M969. To gain further insight into the QPgr-3S region, a chromosome-level 1.2 Gb draft genome for B. thunbergii was assembled using long PacBio reads and Hi-C data. By anchoring the B. thunbergii linkage map to the draft genome, the 13 cM Q Pgr-3S region was found to correspond to ~3.4 Mbp, represented by 10 contigs. Using a 189.3 Mb transcriptome assembled from a multiple tissue library of RNA-seq data, the QPgr-3S region was found to contain 99 genes. To help narrow this list to candidate genes of highest priority for subsequent investigation, a combination of approaches was taken. Specifically, annotation of the QTL region and differential gene expression analysis led to the identification of 12 candidate genes within the region. (Abstract shortened by ProQuest.)

Dans cette thèse, j'ai étudié le rôle des sRNAs dérivés de l'hôte et du virus lors de l'infection du colza (Brassica napus, Canola) par la souche UK1 du virus de la mosaïque du navet (TuMV-UK1). En utilisant un dérivé de TuMV fusionné avec un gène codant pour la protéine fluorescente verte (TuMV-GFP), deux cultivars de colza (‘Drakkar’ et ‘Tanto’) qui diffèrent par leur susceptibilité à ce virus ont été identifiés. Le profil transcriptionnel des foyers d'infection locale, dans les feuilles de Drakkar et de Tanto, par séquençage nouvelle génération (NGS) a révélé de nombreux gènes exprimés de manière différentielle. Les mêmes échantillons d'ARN provenant de feuilles de Drakkar et de Tanto, traitées par des virus ou utilisées en contrôle, ont également servi à établir le profil NGS des sRNAs (sRNAseq) et de leurs cibles potentielles d'ARN (PAREseq). Les analyses bioinformatiques et leur validation in vivo, ont permis d’identifier les événements de clivage de transcrits impliquant des micro ARN (miRNA) connus et encore inconnus. Fait important, les résultats indiquent que TuMV détourne la voie du RNA silencing de l’hôte avec des siRNAs issus de son propre génome (vsiRNA) pour cibler les gènes de l’hôtes. Le virus déclenche également le ciblage à grande échelle des ARN messagers (ARNm) de l’hôte par l’activation de la production de siRNAs secondaires en phase, à partir de locus PHAS. À leur tour, les vsiRNAs et les siRNAs dérivés de l'hôte (hsRNAs) ciblent et clivent l'ARN viral par le complexe RISC. Ces observations éclairent le rôle des siRNAs dérivés de l'hôte et du virus dans la coordination de l'infection virale. Un autre chapitre de cette thèse est consacré à l'analyse des maladies induites par des virus en utilisant comme modèle de plante Arabidopsis, infectée par un tobamovirus, le virus de la mosaïque du colza (ORMV). De plus, ces observations ont permis de proposer un modèle dans lequel cette guérison dépend d’un adressage important de vsiRNAs secondaires antiviraux depuis leur source de production jusqu’à leurs tissus de destination, et l'établissement d'un apport en vsiRNAs capable de bloquer l'activité VSR impliquée dans la formation des feuilles symptomatiques
In this thesis, I investigated the role of host- and virus-derived sRNAs during infection of Rapeseed (Brassica napus, Canola) by the UK1 strain of Turnip mosaic virus (TuMV-UK1). By using a TuMV derivative tagged with a gene encoding green fluorescent protein (TuMV-GFP), two rapeseed cultivars (‘Drakkar’ and ‘Tanto’) that differ in susceptibility to this virus were identified. Transcriptional profiling of local infection foci in Drakkar and Tanto leaves by next generation sequencing (NGS) revealed numerous differentially expressed genes. The same RNA samples from mock- and virus- treated Drakkar and Tanto leaves were also used for the global NGS profiling of sRNAs (sRNAseq) and their potential RNA targets (PAREseq). The bioinformatic analysis and their in vivo validation led to the identification of transcript cleavage events involving known and yet unknown miRNAs. Importantly, the results indicate that TuMV hijacks the host RNA silencing pathway with siRNAs derived from its own genome (vsiRNAs) to target host genes. The virus also triggers the widespread targeting of host messenger RNAs (mRNAs) through activation of phased, secondary siRNA production from PHAS loci. In turn, both vsiRNAs and host-derived siRNAs (hsRNAs) target and cleave the viral RNA by the RISC-mediated pathway. These observations illuminate the role of host and virus-derived sRNAs in the coordination of virus infection. Another chapter of this thesis is dedicated to the analysis of virus-induced diseases by using Arabidopsis plants infected with the Oilseed rape mosaic tobamovirus (ORMV) as a model. Initially, the infected plants develop leaves with strong disease symptoms. However, at a later stage, disease-free, “recovered” leaves start to appear. Analysis of symptoms recovery led to the identification of a mechanism in which the VSR and virus derived-siRNAs play a central role. I used Arabidopsis mutants impaired in transcriptional and post-transcriptional silencing pathways (TGS and PTGS respectively) and a plant line carrying a promoter-driven GFP transgene silenced by PTGS (Arabidopsis line 8z2). Using various techniques able to monitor virus infection, small and long viral RNA molecules, VSR activity, as well as phloem-mediated transport with in these lines, this study led to the identification of genes required for disease symptoms and disease symptom recovery. Moreover, the observations allowed to propose a model in which symptoms recovery occurs upon robust delivery of antiviral secondary vsiRNAs from source to sink tissues, and establishment of a vsiRNA dosage able to block the VSR activity involved in the formation of disease symptoms

The fascinating field of molecular capsules has recently begun to see the creation of structures that, medicated by the encapsulation of guest molecules within their central cavity, are able to change the properties or reactivity of the substrate. The current capsule designs are however, prone to exchange of either part or whole ligands. This exchange or the capsule's subsequent disassembly can lead to loss of the cavity or modification of their external properties, and is a barrier to their more widespread application, a problem this work seeks to address by creating more a robust capsule structure. This thesis presents the design, synthesis and properties displayed by three novel capsules. All the species presented share a similar supramolecular tetrahedral structure, but their properties deviate significantly, showing either switchable behaviour, spin-crossover or a novel synthetic route to a kinetically inert structure. Improvements in the design have led to a final capsule that is water-soluble, robust, non-toxic and has been shown to encapsulate a range of guests. Chapter 1 includes an overview of the types of capsule constructed in literature and their possible application. The fundamental properties of these capsules are identified, with emphasis given to a discussion of mechanisms underlying their encapsulation phenomena. Chapter 2 describes efforts to construct a tetrahedral capsule based on iron(II) and an oxime ligand. While the use of an oxime motif achieved the aim of preventing exchange of the external groups, the capsule also displayed the surprising property of possessing a solvent responsive assembly-disassembly process. This potentially provides a basis for 'on-demand' encapsulation by being able to choose when to have hydrophobic cavity available for guests. Chapter 3 details the synthesis of a tetrahedral capsule containing iron (II) coordinated by a pyridyl-triazole bonding motif. the spin-crossover properties of the complex were initially demonstrated in the solid state, however, when in solution the capsule displayed the unusual ability of spin-crossover mediated structural rearrangement. Chapter 4 demonstrates the synthesis of a robust capsule. The synthetic route shown alleviates the problems surrounding the construction of inert species in a self-assembly process. Based around a cobalt (III) cation, the stability of the capsule to carious conditions is examined and its host-guest chemistry is explored, revealing some insights into the encapsulation behaviour of this structure.

Symmetrical cage-annulated podands were synthesized via highly efficient synthetic strategies. Mechanisms to account for the key reaction steps in the syntheses are proposed; the proposed mechanisms receive support from the intermediates that have been isolated and characterized. An unusual complexation-promoted elimination reaction was studied, and a mechanism is proposed to account for the course of this reaction. This unusual elimination may generalized to other rigid systems and thus may extend our understanding of the role played by the host molecules in "cation-capture, anion-activation" via complexation with guest molecules. Thus, host-guest interaction serves not only to activate the anion but also may activate the leaving groups that participate in the complexation. Complexation-promoted elimination provides a convenient method to desymmetrize the cage while avoiding protection/deprotection steps. In addition, it offers a convenient method to prepare a chiral cage spacer by introducing 10 chiral centers into the host system in a single synthetic step. Cage-annulated monocyclic hosts that contain a cage-butylenoxy spacer were synthesized. Comparison of their metal ion complexation behavior as revealed by the results of electrospray ionization mass spectrometry (ESI-MS), alkali metal picrate extraction, and pseudohydroxide extraction with those displayed by the corresponding hosts that contain cage-ethylenoxy or cage-propylenoxy spacers reveals the effect of the length of the cage spacer upon the host-guest behavior. A series of cage-annulated cryptands, cavitands and the corresponding non-cage-annulated model compounds have been synthesized. These host molecules display unusual behavior when examined by using ESI-MS techniques, i.e., they bind selectively to smaller alkali metal ions (i.e., Li+ and Na+), a result that deviates significantly from expectations based solely upon consideration of the size-fit principle. It seems likely that this behavior results from the effect of the host topology on host-guest behavior. A series of non-cage-annulated cryptands also have been synthesized. These compounds can serve as starting materials for cavitand construction.