This PhD examined the epidemiology of two potentially devastating plant pathogens, Phytophthora ramorum and P. kernoviae, at Benmore Botanic Garden in Argyll & Bute and Brodick Castle Garden in Arran. Spore traps, river baiting, bait plants and soil sampling were used to both confirm the presence of, and measure the amount of inoculum in the environment in order to quantify the relationship between inoculum levels and disease development. P. ramorum was detected in spore traps at high levels under a sporulating host throughout the year at Benmore. Also, findings at sites where infected hosts had been removed before the study led to the conclusion that the low level spore traps detect inoculum from soil splash. Rhododendron and Vaccinium bait plants were also infected with P. ramorum via soil splash at sites within Benmore where there was no sporulating host present. P. kernoviae was detected in spore traps at Brodick throughout the year but only where there was a sporulating host overhead. P. kernoviae infected bait plants only where an infected host is overhead. Water baiting confirmed the presence of P. ramorum in two streams at Benmore but P. kernoviae was not detected using this method despite the large-scale P. kernoviae infection at Brodick. Inoculum continued to be detected in soil in areas of both gardens where infected hosts had been removed two years previously, confirming that both of these pathogens can survive in soil for a considerable period. A number of statistical models were produced to describe conditions required for P. ramorum sporulation and infection. Sporulation occurred during warmer and wetter conditions and infection of bait plants occurred in wet conditions and where an infected host is in close proximity. A statistical model was also used to produce a P. ramorum risk map, uniquely at the garden scale, to enable garden managers at Benmore to visualise the areas most at risk within their garden. The findings of this study have clear management implications for the control of disease establishment and spread within the garden setting.

As the global population rises, the demand for food increases which underscores a need for improvement in food security. Disease pressures are a major concern surrounding sustainable agriculture. Static crop populations, containing little to no genetic diversity, are vulnerable to diverse pathogen populations. Wild relatives of crop plants are a reservoir for new disease resistance traits that can be introgressed into cultivated crops. The identification of novel disease resistance is of paramount importance because pathogen co-evolution is not only defeating current resistance genes (R genes) but chemical controls as well. Phytophthora sojae (P. sojae), the causal agent of Phytophthora root and stem rot disease, reduces soybean harvests worldwide. We developed an approach to screen for new R genes that recognize core effectors from P. sojae. We expect R genes identified by these screens to be durable because P. sojae requires core effectors for virulence. We utilized effector-based screening to probe Glycine soja germplasm with core RXLR effectors from P. sojae to search for novel R genes. We developed segregating populations from crosses of P. sojae resistant G. soja germplasm with susceptible G. max cultivar Williams to determine inheritance of potential R genes in germplasm that responded to core effectors. We are using marker assisted breeding to map disease resistance traits in recombinant inbred (RI) lines. To better understand pathosystems, we examined host resistance and susceptibility using bioinformatics. We analyzed the interaction between Arabidopsis thaliana ecotype Col-0 and Hyaloperonospora arabidopsidis isolate Emwa1 using a publicly available RNA time-course experiment. We describe a new algorithm to sort genes into time-point specific clusters using activation and repression parameters. Gene ontology annotations were used to identify defense genes with unique expression profiles, and A. thaliana null mutants for these genes were significantly more susceptible to Emwa1 than wild-type. We plan to use these tools to rapidly identify and guide introgression of durable disease resistance into crop species. / Ph. D.

Effector proteins are exported to the interior of host cells by numerous plant pathogens. Effector proteins have been well characterized in bacteria. However, the mechanisms through which these effectors promote virulence are largely unknown. Bioinformatic analysis of genome sequences from oomycete pathogens Phytophthora sojae, P. ramorum, P. infestans and Hyaloperonospora arabidopsidis (Hpa) have led to the identification of a large number of candidate effector genes. These effector genes have characteristic motifs (signal peptide, RxLR and dEER) that target the effectors into plant cells. Although these effector genes are very diverse, certain genes are conserved between P. sojae and H. arabidopsidis, suggesting that they play important roles in pathogenicity. The goal of my first project was to characterize a pair of conserved effector candidates from Hpa and P. sojae. We hypothesized that these effectors have important conserved roles with regard to infection. We found that the Hpa effector was expressed early during the course of infection of Arabidopsis and triggered an ecotype-specific defense response in Arabidopsis, suggesting that it was recognized by host surveillance proteins. Both the effectors from Hpa and P. sojae respectively could suppress immunity triggered by pathogen associated molecular patterns (PTI) and by effectors (ETI) in planta. They also enhanced bacterial virulence in Arabidopsis when delivered by the Type III secretion system. Similar results were seen with experiments with transgenic Arabidopsis expressing the effectors. My second project showed that a different Hpa effector protein, HaRxL10, targets the Jasmonate-Zim Domain (JAZ) proteins that repressed responses to the phytohormone jasmonic acid (JA). This manipulation activates a regulatory cascade that reduces accumulation of a second phytohormone, salicylic acid (SA) and thereby attenuates immunity. This virulence mechanism is functionally equivalent to but mechanistically distinct from activation of JA-SA crosstalk by the bacterial JA mimic coronatine. These results reveal a new mechanism underpinning oomycete virulence and demonstrate that the JA-SA crosstalk is an Achilles\' heel that is manipulated by unrelated pathogens through distinct mechanisms. / Ph. D.

<i>Phytophthora infestans</i>, the oomycete pathogen responsible for late blight of potato and tomato, is regarded as the biggest threat to global potato production and is thought to cost the industry around £6 billion annually. Traditionally, fungicides have been used to control the disease, but this is both economically and environmentally costly, as multiple chemical applications may be required during a single growing season. <i>P. infestans</i> has rapidly overcome genetic resistances introduced into cultivated potato from wild species. This provides the rationale for developing artificial resistance genes to create durable resistance to late blight disease.<i>Phytophthora</i> species secrete essential effectors into plant cells that target critical host cellular mechanisms to promote disease. One such <i>P. infestans</i> effector is AVR3a^KI which is recognised by the potato R3a protein, a member of the CC-NB-LRR type resistance gene family. However, the closely related virulent form, AVR3a^EM, which is homozygous in more than 70% of wild <i>P. infestans</i> isolates, evades this recognition. Domain swapping experiments have revealed that the LRR domain of R3a is involved in recognition of AVR3a^KI, as the CC-NB domain of an R3a-paralog which does not mediate recognition of AVR3a^KI, is able to induce a HR when combined with the LRR of wild-type R3a. However, a chimeric protein consisting of the CC-NB domain of a more distantly-related homolog of R3a and the LRR of domain of R3a, is unable to recognise AVR3a^KI, suggesting that function is achieved only when the different domains of an R protein are attuned to recognition and signalling. Gain-of-function variants of <i>R3a</i> (<i>R3a*</i>), engineered by an iterative process of error-prone PCR, DNA fragmentation, re-assembly of the leucine rich repeat (LRR)-encoding region of <i>R3a</i>, are able to recognise both forms of AVR3a. This gain-of-recognition is accompanied by a gain-of-mechanism, as shown by a cellular re-localisation from the cytoplasm to prevacuolar compartments upon perception of recognised effector forms. However, R3a* variants do not confer resistance to AVR3a^EM-carrying isolates of <i>P. infestans</i>.Future efforts will target the NB-ARC domain of R3a, in a bid to fine-tune the intra-cellular signalling of gain-of-recognition R3a* variants. It is hoped that a shuffled <i>R3a*</i> gene, capable of conferring resistance to <i>P. infestans</i> isolates harbouring AVR3a^EM, will provide durable late blight resistance when deployed in the field in combination with other mechanistically different R proteins.

Several oomycete species of the genus Saprolegnia are recognized as devastating fish pathogens and are responsible for the loss of millions of fish annually for the aquaculture industry. Until recently, these pathogens were kept in check using malachite green; however, due to its toxicity, this chemical has now been banned from use. Saprolegnia parasitica is recognized as the major pathogen of aquaculture fish species. The industry is struggling to predict and control S. parasitica outbreaks in fish hatcheries and there is a need for new knowledge regarding the population genetic structure of this pathogen. Random amplified microsatellites were used to compare isolates of S. parasitica collected from a variety of hatchery locations during the period of November 2009 - August 2011, in order to determine the level of genetic variability and determine changes in genetic diversity over time. Allele frequencies of scored characters were graphically compared. Population genetic diversity was measured using Nei’s genetic distance, Shannon’s Information Index, number of polymorphic loci and phylogenetic trees. Due to the presence of Saprolegnia parasitica in the facilities tested, it appears to be ubiquitous in aquaculture facilities and treatment and prevention will be an ongoing concern in aquaculture management. Overall, genetic diversity of S. parasitica isolates was determined to be low with at least some sexual recombination occurring over time. There was a diversity of genotypes collected from the same hatchery on a single day, indicating there was not a single genotype present at a given time point. Genetic profiling, such as used here, could provide facility managers with a new approach to develop a series of best practices to control sporadic outbreaks of disease. Use of these genetic markers and close monitoring of S. parasitica genotypes will permit early detection and sanitation protocols. / Graduate / 2015-04-24 / 0476 / 0792 / 0369 / cren06@uvic.ca

Submitted by Rosana Amâncio (rosana.amancio@ufcg.edu.br) on 2018-07-11T20:50:26Z No. of bitstreams: 1 Dissertação Thamara - Capa Dura.pdf: 2260125 bytes, checksum: 530ae87f1e4a9200aafe1cb3102cff39 (MD5) / Made available in DSpace on 2018-07-11T20:50:26Z (GMT). No. of bitstreams: 1 Dissertação Thamara - Capa Dura.pdf: 2260125 bytes, checksum: 530ae87f1e4a9200aafe1cb3102cff39 (MD5) Previous issue date: 2016-08-19 / CNPq / A gomose, provocada principalmente pelo oomiceto Phytophthora parasitica, é uma das mais graves doenças que acometem culturas de citros no âmbito mundial. Durante a interação, plantas induzem cascatas de sinalização a fim de induzir respostas de defesa. Contudo, P. parasitica secreta proteínas efetoras capazes de modular estas respostas por parte do hospedeiro, a fim de promover a infecção. No gênero Citrus, espécies comercialmente importantes são suscetíveis a infecção por este patógeno e a resistência a gomose é encontrada na espécie de citros Poncirus trifoliata. Considerando a escassez de informações acerca do patossistema citros-P. parasitica, o presente trabalho objetivou analisar, por meio de RT-qPCR, a expressão quantitativa de genes efetores apoplásticos e citoplasmáticos de P. parasitica e da cascata de defesa em citros, durante interações com espécies suscetíveis e resistentes, Citrus sunki e P. trifoliata, respectivamente. Dos 17 genes efetores estudados, 10 apresentaram expressão quantitativa relativa diferencial ao nível de significância induzida em P. parasitica após inoculação em raízes de P. trifoliata, sendo 06 apoplásticos e 04 citosólicos. Os perfis de expressão dos 17 genes efetores de P. parasitica apresentaram dois picos máximos de expressão, indicativos da síntese de novo desses genes ao longo dos pontos temporais de interação, sendo o acúmulo dos transcritos mais precoce sobre P. trifoliata (as 6 h.a.i.) e mais tardio sobre C. sunki (as 96 h.a.i.). Os elevados níveis de expressão de genes efetores em P. parasitica induzidos por C. sunki as 96 h.a.i. devem corresponder a fase necrotrófica de vida do oomiceto, consequentemente devido ao sucesso na penetração das células vegetais suscetíveis e acúmulo de biomassa do patógeno. A presença de hifas intracelulares no córtex de raízes de C. sunki foi abundantemente visualizada em micrografias as 96 h.a.i., a qual deve ocorrer como consequência da suscetibilidade da planta ao patógeno. Seis grupos hierárquicos de genes co-regulados foram formados a partir dos perfis de expressão dos 17 genes efetores em P. parasitica, os quais são reagrupados de modo diferente de acordo com a interação com C. sunki ou com P. trifoliata, indicando que o patógeno foi capaz de reconhecer entre hospedeiros suscetível ou resistente e sintetizar seletivamente quais efetores e em que intensidade devem ser segregados. As raízes de C. sunki expressaram 10 componentes de cascatas de resistência mediada pelo SA em resposta não bem-sucedida a infecção por P. parasitica. A supressão por P. parasitica da expressão de 05 genes de cascatas de resistência mediada pelo SA foi observada em raízes de P. trifoliata e deve indicar tentativas do patógeno de burlar com a imunidade da planta. Entretanto, a resistência de P. trifoliata a P. parasitica não deve utilizar genes envolvidos na cascata de resistência mediada pelo SA, mas sim genes PR-5 e calose sintase, envolvendo barreiras bioquímicas e estruturais. Portanto, o presente trabalho fornece uma nova visão para o entendimento acerca do processo de modulação de efetores de P. parasitica em interações suscetíveis e resistentes e, a maneira como estes hospedeiros respondem mediante interação / The gummosis, mainly caused by the oomycete Phytophthora parasitica, is one of the most serious diseases affecting citrus crops worldwide. During the interaction, plants induce signaling cascades in order to induce defense responses. However, P. parasitica secrets effector proteins capable of modulating these host responses in order to promote the infection. In Citrus genus, commercially important species are susceptible to infection by this pathogen and the gummosis resistance is achieved in Poncirus trifoliata citrus species. Considering the lack of information on citrus-P. parasitica pathosystem, this study aimed to analyze, through RT-qPCR, the quantitative expression of P. parasitica effector and citrus defense genes during citrus-P. parasitica susceptible and resistant interactions, with Citrus sunki and P. trifoliata, respectively. As results, P. parasitica was able to recognize among susceptible or resistant host and selectively synthesize which effectors and in that intensity should be expressed. Of the 17 studied effector genes, 10 showed quantitative relative differential expression at significance level induced in P. parasitica after inoculation in trifoliate orange roots, being 06 apoplastics and 04 cytosolics. The expression profiles for the 17 effector genes in P. parasitica had two maximum peaks of expression, that are indicative of de novo synthesis of these genes along the time points of interaction, showing transcript accumulation earlier on P. trifoliata (at 6 h.a.i.) and later on C. sunki (at 96 h.a.i.). High levels of the effector gene expression in P. parasitica induced by C. sunki at 96 h.a.i. must match the necrotrophic phase of life of this oomycete, consequently due to their successful penetration into the susceptible plant cells and pathogen biomass accumulation. The presence of intracellular hyphae in cortex of C. sunki roots was abundantly visualized in the micrographs at 96 h.a.i., which may occur as a result of the plant susceptibility to the pathogen. Six hierarchical groups of co-regulated genes were formed from the expression profiles of the 17 effector genes in P. parasitica, which are grouped differently according to interact with C. sunki or P. trifoliata, indicating that the pathogen was able to recognize between susceptible or resistant host and selectively synthesize which effectors and in that intensity should be segregated. The roots of C. sunki expressed 10 components of the cascade resistance mediated by SA in response not successful to P. parasitica infection. The suppression by P. parasitica of the expression of 05 genes of the cascade resistance mediated by SA was found in P. trifoliata roots, and must indicate pathogen attempts to circumvent with the immunity of the plant. However, P. trifoliata resistance to P. parasitica should not use genes involved in the resistance cascade mediated by SA, but instead PR-5 and callose synthase genes, involving biochemical and estructural barriers. In conclusion, this study provides a new insight into the understanding of the effectors of modulation process of P. parasitica in susceptible and resistant interactions and how these hosts respond through interaction.

Les oomycètes représentent un groupe de microorganismes eucaryotes filamenteux distincts phylogénétiquement des champignons incluant de nombreuses espèces phytopathogènes. CBEL est une glycoprotéine pariétale de Phytophthora parasitica constituée d'une répétition de deux régions séparées par un linker. Chaque région protéique est constituée d'un domaine protéique de liaison à la cellulose (CBM1) et un motif PAN /Apple impliqué dans des interactions protéines-protéines ou protéines-polysaccharides. Cette étude doctorale porte sur la caractérisation de la protéine CBEL et plus particulièrement de ses CBM1s ainsi que sur l'évaluation et optimisation du potentiel de cette protéine à : (i) stimuler les défenses naturelles des plantes (ii) augmenter l'activité de glycosides hydrolases. Dans la première partie de ce travail doctoral différents tests visant à reproduire un traitement éliciteur externe sur plante entière ont pour cela été développés. Ces tests ont permis de mettre en évidence que formulée en présence de surfactants CBEL est capable d'induire diverses réponses de défense chez A. thaliana. Une production en masse de cette protéine a été réalisée dans la levure Pichia pastoris et la bactérie Escherichia coli dans l'optique d'une future application agronomique. Les protéines recombinantes CBELcol et CBELpic produite dans ces différents systèmes d'expression présentent des profils de glycosylation différents de celui de la protéine native CBELnat. Alors que ces protéines semblent se lier de manière identique à la cellulose les différents tests d'élicitation développés au cours de ce travail mettent en évidence des variations dans leur activité élicitrice suggérant que la nature des résidus glucidiques présents sur cette glycoprotéine peut avoir un impact sur sa capacité induire des réponses de défenses en application externe. Lors de la deuxième partie de ce travail de thèse la capacité de CBEL à interagir avec différents substrats cellulosiques a été caractérisée. Les résultats obtenus ont permis de montrer que CBEL se lie avec une haute affinité à la cellulose cristalline avicel et que la présence de CBM1 fonctionnels est nécessaire à cette interaction. De manière intéressante, le CBM1-1 et CBM1-2 ne semblent pas contribuer de manière égale à cette interaction. Par ailleurs la laison de CBEL à la cellulose induit des perturbations structurales sur le substrat et permet d'améliorer l'activité de la xylanase XynB de Talaromyces versatilis sur paille de blé. En outre une xylanase chimère possédant dans sa séquence le CBM1-1 de CBEL possède également une activité augmentée sur paille blé. L'ensemble de ces résultats met en évidence le potentiel de CBEL et de son CBM1-1 pour l'amélioration de l'activité de glycoside hydrolases utilisables par exemple en bioraffinerie. En dernier lieu un travail de caractérisation structurale de la protéine CBEL a également été entamé au cours de cette étude. L'enveloppe de la protéine CBEL en solution à notamment été déterminée par SAXS (Small Angle X-ray Scattering) et un modèle 3D de cette protéine a été obtenu. / Oomycetes are fungal like microorganisms evolutionary distinct from true fungi that include pathogens of plants. CBEL is a cell wall glycoprotein isolated from the oomycete Phytophthora parasitica that is composed of two distinct regions linked by a threonine/proline rich linker. Each region owns a cellulose binding module (CBM1) and a PAN-Apple domain involved in protein-protein or proteins-polysaccharides interactions. Since CBEL is able to induce defense responses in numerous plant species, its use for the development of products able to protect crops has been envisaged. For this purpose we analysed the effect of an external CBEL treatment on plants. We found that in the presence of surfactants CBEL is able to induce cytosolic calcium changes, defense gene expression, and cell death on A. thaliana. CBEL application for crop protection requires the development of economically reliable production processes. In the case of proteinaceous elicitors, an attractive strategy to obtain large amount of elicitors is to express them in heterologous hosts such as bacteria or yeasts. CBELcol and CBELpic were produced respectively in E. coli and in P. pastoris. CBELcol is unglycosylated whereas CBELpic displays a glycosylation profile distinct from the native protein (CBELnat). We found that all these proteins are able to bind crystalline cellulose. On the other side we found that the elicitor activity of CBELpic is distinct from CBELnat and CBELcol suggesting that the glycosylation on CBEL can have an impact on its ability to induce plant defense responses after external treatment on A. thaliana. In the second part of this work the two CBMs (1-1 and 1-2) that form part of CBEL have been submitted to detailed characterization, first to better quantify their interaction with cellulose and second to determine whether these CBMs can be useful for biotechnological applications, such as biomass hydrolysis. A variety of biophysical techniques were used to study the interaction of the CBMs with various substrates and the data obtained clearly indicate that CBEL's CBM1-1 exhibits much greater cellulose binding ability than CBM1-2. Engineering of the family 11 xylanase from Talaromyces versatilis (TvXynB), an enzyme that naturally bears a family 1 CBM, has produced two variants. The first one lacks a CBM, whereas the second contains the CBEL CBM1-1 in the place of the natural CBM1. The study of these enzymes has revealed that wild type TvXynB binds to cellulose, probably via its CBM1, and that the substitution of its CBM by oomycetal CBM1-1 does not affect its activity on this substrate. Moreover, the presence of CBEL during the hydrolysis of wheat straw actually potentiates the action of TvXynB, a result that is consistent with the hypothesis that CBM1-1 can alter cellulose surface fibres rather like some other members of CBM family 1.

Dans l’objectif de redynamiser la culture des protéagineux, il est primordial d’améliorer la gestion du risque lié à Aphanomyces euteiches, agent responsable de la pourriture racinaire du pois. Le manque de connaissances sur les mécanismes d’infection d’A. euteiches constitue un frein à l’élaboration de stratégies de contrôle durable. Dansl’optique d’étudier les premières étapes du processus infectieux d’A. euteiches, laconstruction d’une souche A. euteiche-GFP a été entreprise. Transfecter A. euteiches aura permis d’assurer (i) la production de protoplastes par digestion enzymatique du mycélium puis (ii) d’insérer le gène gfp par la méthode de transfection chimique PEG-CaCl2 et (iii) de constater l’insertion stable du gène gfp dont l’expression s'est avérée transitoire. En parallèle, dans l’objectif d’apporter des éléments de réponses quant aux rôles des exsudats racinaires et du Root Extracellular Trap (RET) dans les réactions de défense du pois, une étude comparée des interactions entre Pisum sativum (plante sensible) et Vicia Faba (plante tolérante) au pathogène A.euteiches a été réalisée durant les premières phases de l’infection. Alors que de nombreuses variations au niveau de la composition polysaccharidique du RET et des exsudats ont été observées chez le pois, la féverole a présenté des modifications marginales. Chez le pois, l’infection est intense et rapide alors qu’elle semble réduite chez la féverole. La féverole repousse les zoospores tandis que le pois infecté les attire davantage. La féverole semble pouvoir protéger le pois au travers de mécanismes de communication qu’il convient de caractériser. L’ensemble de ces résultats semblent prometteurs dans le développement de méthode de lutte contre la pourriture racinaire du pois. Enfin, l’étude des propriétés bio-physico-chimiques des sols susceptibles de conditionner l’apparition de la maladie ont permis de confirmer la corrélation positive entre la densité d’inoculum du pathogène et le potentiel infectieux (PI) dans des sols naturellement infestés : les sols possédant des teneurs élevées en sable ou en calcium s'avèrent défavorables au développement de la maladie. Cette étude a aussi permis de mettre en évidence une influence possible des communautés microbiennes des sols, susceptibles d’influencer le processus infectieux d’A.euteiches / Aphanomyces euteiches is a pathogenic oomycete considered to be the most damaging root disease of pea crops in the world and there is currently no registered pesticide for its control. Crop management is the most efficient tool to control root rot, and avoidance of infested soil seems to be the optimal solution. Mechanisms related to A. euteiches root colonization remain poorly understood. In order to better understand A.euteiches infectious cycle, a polyethylene glycol (PEG) – calcium chloride (CaCl2) transformation protocol has been perfected in order to stably express the reporter gene GFP. The data show for the first a transient expression of green fluorescent protein (GFP) which can be observed in A.euteiches mycelium, a Saprolegnia oomycete. Vector pGFPN, containing the ham34 promoter and terminator of the Peronospora oomycete Bremia Lactucae, was introduced in A.euteiches protoplasts. Transient expression of GFP could be observed in A. euteiches mycelium by confocal microscopy. qPCR analyses confirmed the actual gfp gene insertion in its genome. Meanwhile, the influence of both pea and faba bean root extracellular trap (RET) and root exudates has been explored for A. euteiches zoospores by chemotaxis assays, microscopic observations and oomycete DNA quantification. Reciprocally pea and faba bean roots responses to A. euteiches infection have been studied at early stage of infection by biochemical analysis of cell wall polymer content in the RET and root exudates. Whereas infected pea root exudates stimulated A.euteiches zoospores attraction, faba bean exudates had a repellent effect on zoospores. In response to infection, arabinogalactan protein content of root pea exudates was altered. Interestingly, A.euteiches colonization was less intense on faba bean root surface and protect pea root at early stage of infection. Finally, the correlation between inoculum quantity in infested and the inoculum potential (IP) in field has been confirmed by qPCR. Analyses of the influence of abiotic soil parameters on the disease showed that a high calcium concentration or sand content negatively impact the IP. Furthermore, microbial communities proved to play a role in the expression of the disease in some soils. Metagenomics could be applied in order to provide new directions in managing this disease.

No description available.

No description available.