This thesis presents a more in-depth understanding of the interaction between the pathogenic fungus Sporisorium scitamineum and sugarcane, a disease known as \"cane smut\". The development of a long structure like a \"whip\" from the meristem of infected plants is the main characteristic of the disease, allowing the effective dispersion of teliospores in the field. Infected plants have a reduced sucrose content and juice quality, leading to considerable economic losses. In the first chapter, the gene expression profile of the pathogen during its development in planta - in the first moments of infection and after the emission of the whip - and in vitro was evaluated using the RNAseq technique. Were analyzed genes preferentially expressed in each condition, differentially expressed in comparison to its growth in vitro, and expressed only during interaction. The results allowed the identification of some potential pathogenicity mechanisms, active effectors and gene clusters expressed only during interaction. In the second chapter, the transient expression technique was used to determine the target cell compartment of some of the candidate effectors and to establish a viable protocol for the study of S. scitamineum proteins. The four putatively secreted genes most expressed during the initial moments of the interaction were fused to the gene encoding the fluorescent green protein (Citrine) and expressed in Nicotiana benthamiana. The results of confocal microscopy and westernblots indicated an accumulation of each candidate protein in the membrane, cytosol and/or nucleus, in addition to the occurrence of post-translational modifications. These data offer new study opportunities for the identification of plant proteins that interact with such effectors. In the third chapter, the transcriptional responses of sugarcane in the first moments of a compatible interaction and after the development of the whip were analyzed using again the data obtained from the dual RNAseq cane-smut. Among the main responses, was identified an increase in MADS-type transcription factors expression, indicating that the whip development may use a route similar to flowering, whose signaling seems to start as early as the colonization. In addition, whip development is accompanied by increased transcription of genes involved in energetic pathways, and hormones synthesis and signaling pathways. Genes encoding RGAs were differentially expressed and may be related to pathogen effector\'s recognition. In the fourth chapter, the metabolic profile of sugarcane was evaluated during disease progression, confirming that in the meristem of infected plants carbon allocation is channeled to energetic pathways, besides the regulation of several amino acids and changes in plant cell composition in response to whip development. Metabolomics approach also allowed the identification of a probable mycotoxin derived from S. scitamineum. The results obtained in this study contributed to increase the understanding of the interaction between S. scitamineum and sugarcane that is characterized by high complexity and specialization to the host, and can be used in a way to help the characterization of resistant varieties and contribute to the improvement of sugarcane with resistance to smut. / Esta tese apresenta uma compreensão mais aprofundada da interação entre o fungo patogênico Sporisorium scitamineum e a cana-de-açúcar, doença conhecida como \"carvão da cana\". O desenvolvimento de uma longa estrutura similar a um \"chicote\" a partir do meristema de plantas infectadas é a principal característica da doença, permitindo a efetiva dispersão dos teliósporos no campo. As plantas doentes apresentam um teor reduzido de sacarose e qualidade do sumo, levando a perdas econômicas consideráveis. No primeiro capítulo, o perfil de expressão gênica do patógeno durante o seu desenvolvimento in planta - nos primeiros momentos da infecção e após a emissão do chicote - e in vitro foi avaliado utilizando a técnica RNA-Seq. Foram analisados os genes preferencialmente expressos em cada condição, diferencialmente expressos em relação ao crescimento em meio de cultura, ou expressos apenas durante a interação. Os resultados permitiram a elaboração de hipóteses sobre os mecanismos de patogenicidade, sobre os genes candidatos a efetores ativos e a identificação de agrupamentos de genes expressos apenas durante a interação. No segundo capítulo, para determinar o compartimento celular alvo de alguns dos efetores candidatos e estabelecer um protocolo viável para o estudo de proteínas de S. scitamineum foi utilizada a técnica de expressão transiente. Os quatro genes mais expressos durante os momentos iniciais da interação que fazem parte do secretoma do fungo foram fusionados ao gene que codifica a proteína verde fluorescente (Citrina) e expressos em Nicotiana benthamiana. Os resultados de microscopia confocal e westernblots indicaram um acúmulo de cada uma das proteínas candidatas na membrana, citosol e/ou núcleo, além da ocorrência de modificações pós-traducionais. Esses dados oferecem novas oportunidades de estudo para a identificação de proteínas vegetais que interagem com tais efetores. No terceiro capítulo, as respostas transcricionais da cana-de-açúcar nos primeiros momentos de uma interação compatível e após o desenvolvimento do chicote foram analisadas utilizando novamente os dados obtidos a partir do dual RNAseq cana-carvão. Entre as principais respostas da cana destacou-se um aumento da expressão de genes que codificam fatores de transcrição do tipo MADS, indicando que o desenvolvimento do chicote pode usar uma rota semelhante à do florescimento, cuja sinalização parece iniciar logo nos primeiros momentos de colonização. Além disso, o desenvolvimento do chicote é acompanhado pelo aumento da transcrição de genes envolvidos em vias energéticas, e vias de síntese e sinalização hormonal. Genes que codificam para RGAs foram diferencialmente expressos e podem estar relacionados ao reconhecimento de efetores. No quarto capítulo, foi avaliado o perfil metabólico da cana-de-açúcar durante a progressão da doença, confirmando que no meristema de plantas infectadas ocorre um aumento da alocação de carbono em vias energéticas, além da regulação de vários aminoácidos e mudanças em relação à composição da parede celular em resposta ao desenvolvimento do chicote. A abordagem metabólica também permitiu a identificação de uma provável micotoxina derivada de S. scitamineum. Os resultados obtidos neste estudo contribuíram para aumentar a compreensão da interação entre S. scitamineum e a cana-de-açúcar que se caracteriza pela alta complexidade e especialização ao hospedeiro, e poderão ser utilizados de forma a auxiliar a caracterização de variedades resistentes e contribuir para o melhoramento da cana-de-açúcar com resistência ao carvão.

Les effecteurs TAL (« Transcription Activator-Like ») sont des protéines présentes majoritairement chez les bactéries phytopathogènes du genre Xanthomonas. Ces protéines bactériennes sont dirigées vers le noyau des cellules de la plante hôte où elles induisent l’expression de gènes. L’induction de gènes de « susceptibilité » de la plante est responsable de la maladie. Les effecteurs TAL sont capables de se lier à l’ADN grâce à un motif particulier consistant en une série de répétitions quasi-identiques s’enroulant autour de l’ADN et formant une super-hélice. Au sein des répétitions deux acides aminés localisés à l’intérieur de chaque boucle de la super-hélice interagissent directement et spécifiquement avec les nucléotides. Des combinaisons différentes de ces deux acides aminés se lient spécifiquement à certains nucléotides, selon un code unique.Une conséquence de cette interaction étroite est que les plantes et les bactéries co-évoluent selon une course aux armements où le génome de la plante se diversifie pour éviter d’être la cible des effecteurs TAL, tandis que les gènes tal se diversifient pour s’adapter à de nouvelles cibles. Les aspects évolutifs des effecteurs TAL sont encore largement inconnus, notamment comment la spécificité évolue vers de nouvelles cibles végétales. Cette thèse présente les premiers travaux sur la compréhension des mécanismes évolutifs des gènes tal, principalement abordés par la bioinformatique. Nous avons développé la suite de programmes « QueTAL » qui permet d’une part la construction d’arbres phylogénétiques basés soit sur la séquence des répétitions, soit sur la séquence des sites cibles, d’autre part la recherche de motifs de répétitions pouvant constituer les unités évolutives des effecteurs TAL. Cette suite bioinformatique est publique, en ligne, et activement utilisée par la communauté des scientifiques travaillant sur les effecteurs TAL des Xanthomonas.Ces programmes ont été appliqués (ainsi que d’autres approches) à plus de 900 séquences d’effecteurs TAL de 22 groupes bactériens. Nous avons mis en évidence i) une perte de diversité dans les répétitions chez les Xanthomonas, qui aurait des conséquence sur l’évolution de la structure des effecteurs TAL; ii) l’existence de groupes fonctionnels de gènes tal spécifiques à certains pathovars ; iii) un probable mécanisme évolutif reposant sur la recombinaison (principalement par conversion génique), révélé par le gain ou la perte de répétitions en blocs entiers. Notre hypothèse est que le moteur de la spécialisation des effecteurs TAL est la recombinaison de ces blocs entre gènes conduisant à une diversification fonctionnelle rapide vers de nouvelles cibles végétales.Nous avons ensuite analysé plus en détail la diversité des séquences TAL de souches africaines de Xanthomonas oryzae pv. oryzae (Xoo), agent de la bactériose vasculaire, maladie bactérienne la plus importante du riz. Nous avons montré qu’un gène tal résultant d’une conversion génique pouvait être fonctionnel, indiquant que ce mécanisme peut être un moteur évolutif chez les effecteurs TAL. Les données de transcriptomique et de gain de fonction ont permis de mettre en évidence un effecteurs TAL dont la virulence s’exerce par l’activation de deux gènes de susceptibilité, dont l’un n’avait jamais été décrit chez Xoo. Enfin nous présentons des résultats préliminaires sur les effets d’une déconstruction de TALome sur le transcriptome de riz ainsi que des travaux fonctionnels et évolutifs issus de collaborations sur d’autres Xanthomonas.Cette thèse offre un nouveau cadre conceptuel ainsi que de nouveaux outils pour l’analyse fonctionnelle et évolutive des effecteurs TAL qui devraient améliorer la mise au point de stratégies pour la résistance des plantes aux Xanthomonas. / Transcription activator-like (TAL) effectors are proteins found mainly in the genus of Xanthomonas phytopathogenic bacteria. These proteins enter the nucleus of cells in the host plant and can induce the expression of genes. The induction of “susceptibility” S genes in the plant will result in disease. TAL effectors are able to bind DNA thanks to a unique motif consisting of a series of nearly-identical repeats that wrap around the DNA forming a super-helix, in each repeat two amino-acids found in a loop on the inner side of the helix directly interact with nucleotides. Different combination of amino-acids in this loop bind specific nucleotides following a unique code.A consequence of this tight interaction is that plants and bacteria co-evolve following an arms race where the plant genome diversifies to avoid being targeted by the TAL effectors, while tal effector genes diversify to adapt to new targets.Various aspects of TAL effector evolution are still unknown, specially how does specificity arise towards certain targets in the host plant? As first steps towards answering this question, in these thesis we show the results of using primarily bioinformatic strategies to find evolutionary patterns in TAL effector sequences. We designed the suite “QueTAL” containing software for 1) the construction of phylogenetic trees based on repeat sequences, 2) comparison of predicted binding sites for TAL effectors, 3) identification of repeat motifs in TAL effector pairs. This suite was made publicly available and it is being actively used by the Xanthomonas research community.We used these programs along with other strategies to analyze variation in over 900 TAL effector sequences from 22 taxonomic groups finding 1) a loss of diversity of repeats through the Xanthomonas genus, which may impact the evolution of TAL effector architecture, 2) groups of TAL effector orthologs specific to certain taxonomic groups of pathovars that may share common functions, 3) evidence of repeat motifs shared and lost between TAL effectors hinting at extensive recombination (particularly gene conversion) events. We propose that the swapping of repeat blocks between TAL effectors is a motor for TAL effector specialization that allows for fast functional diversification through the acquisition of new targets in the host plants.We then analyzed in detail the diversity of TAL effector sequences in African strains of Xanthomonas oryzae pv. oryzae (Xoo), causing agent of bacterial leaf blight of rice, the most destructive bacterial disease in rice. We found indications of virulence activity of a TAL effector being the product of a gene conversion event, supporting our hypothesis of gene conversion as a motor of TAL effector evolution. We also used transcriptomic data and systematic gain-of-function assays to uncover a TAL effector that exerts a virulence role through the induction of two susceptibility genes, one of which represents a novel class of susceptibility gene in bacterial blight. Finally, we present partial results of transcriptomic analyses aimed at de-constructing the effects of each TAL effector from one strain on the rice transcriptome, as well as results from collaborative functional and evolutionary analyses in other groups of Xanthomonas.Altogether, this thesis offers a new conceptual framework and new tools for the analysis of TAL effector function and evolution, and we hope this will help in the design of strategies aimed at improving resistance to bacteria in agronomically important plants.

Nosema ceranae, a microsporidian, has been given much attention in recent years as it has been linked with Colony Collapse Disorder (CCD), which leads to the sudden deaths of honey bee colonies. It has been described that many pathogenic organisms secrete virulence factors in order to hijack its host`s cellular functions, but in most cases the underlying mechanisms of this process still remains to be deciphered. Cornman et al. (2009) have identified in N. ceranae a list of putative effector proteins (called secretome) destined to be secreted into the host, and I have taken this list for further investigation using a bioinformatical and experimental approaches. The principal aim of this project was to generate a N. ceranae ORFeome for genes predicted to be secreted, elucidate the function of effector candidates important for N. ceranae biology and/or pathogenicity, as well as to investigate any interactions between N. ceranae proteins and its host utilising two eukaryotic model organisms, budding yeast, S. cerevisiae, and fruit fly, D. melanogaster. A library of S. cerevisiae strains expressing N. ceranae proteins was generated utilising the Gateway® technology, and phenotypic and localisation screens were undertaken to investigate the N. ceranae secretome. Two N. ceranae ORFs, NcORF-15 (NcORF-02039) and NcORF-16 (NcORF-01159) encoding a putative thioredoxin and a hexokinase, respectively, were subjected to yeast complementation assays in order to assess their catalytic activity. NcORF-15, the putative thioredoxin, was able to rescue the sensitive phenotype of S. cerevisiae Δtrx2 under oxidative stress, whereas NcORF-16, the putative hexokinase, did not complement YSH7.4-3C, a triple knockout lacking hexokinase activity. A third N. ceranae effector candidate NcORF-4 (NcORF-00654), a putative proteasome subunit, was investigated for its nuclear localisation and protein interactions in both S. cerevisiae and D. melanogaster.

<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.

In this thesis, I set out to investigate the nature of nonhost resistance responses of Nicotianae sylvestris against Phytophthora capsici and P. infestans. Schulze-Lefert and Panstruga (2011) proposed that the inability of a pathogen to establish infection in nonhost plants could be a feature of the phylogenetic distance between host and nonhost plants. In distantly related plants PAMP triggered immunity is thought to be the major contribution to resistance as effectors are inappropriately attuned to perturb their orthologous plant targets. In contrast, effector triggered immunity (ETI) could be the major contributor to resistance in nonhost plants that are more closely related to the host plants. P. capsici and P. infestans can both infect Solanaceae plants including Solanum lycopersicum and N. benthamiana but both fail to cause disease or complete their life-cycle in N. sylvestris. Based on the hypothesis of Schulze-Lefert and Panstruga (2011), ETI should be contributing towards effective nonhost resistance responses in N. sylvestris against both pathogens. In addition, it is tempting to speculate that N. sylvestris, with a limited availability of functional resistance genes including Nucleotid binding-Leucine rich repeats (NB-LRRs), could be setup to recognise and responds to sequence-related effectors from P. infestans and P. capsici, rather than to have resistance genes that are specifically attuned to either pathogen. I conducted three research strands to test this theory. In Chapter 3 I used MCL clustering to classify 563 P. infestans and 515 P. capsici RXLR effector genes and defined families on the basis of sequence similarity. I found that the P. infestans and P. capsici RXLR complements are mostly species-specific. To investigate the role of ETI in nonhost resistance, 48 P. capsici and 82 P. infestans RXLR were screened for recognition by the nonhost plant N. sylvestris. Using this approach I identified 4 P. infestans and 8 P. capsici effectors that are consistently recognised in N. sylvestris (Chapter 4). Surprisingly, most of the recognised effectors are part of species-specific clusters. In Chapter 5 I established and implemented PathSeq, an enrichment and sequencing tool that facilitates the massively parallel study of naturally occurring diversity of pathogen effectors, including those that are recognised in N. sylvestris. In the same chapter I also used PathSeq and de novo prediction to expand the P. infestans RXLR complement from 563 to 1220 putative effectors. In this thesis I have shown that P. infestans and P. capsici effector set are diversifying at the sequence level. My data also suggests that ETI might play a part in nonhost resistance of N. sylvestris to P. capsici and P. infestans. Finally I have presented PathSeq, a tool that allows the study of the effectors set in multiple isolates at the time, and this, for a fraction of the cost of a full genome sequencing experiment.

Shiga toxin-producing <i>E. coli</i> (STEC) is an important food- and water-borne pathogen of humans, causing Hemorrhagic Colitis and Haemolytic Uremic Syndrome. Colonization of both cattle and human hosts is mediated through the action of effector molecules secreted via a type III secretion system (T3SS), which forms attaching and effacing lesions (A/E). The necessary effectors which form A/E by manipulation of host signalling and actin nucleation are present on a pathogenicity island called the Locus of Enterocyte Effacement (LEE).<p> It has been reported that vaccination of cattle with Type III-secreted proteins (T3SPs) from STEC O157 resulted in decreased shedding. In order to extend this to non-O157 STEC serotypes, we examined the serological cross-reactivity of T3SPs of serotypes O26:H11, O103:H2, O111:NM and O157:H7. Groups of cattle were vaccinated with T3SPs produced from each of the serotypes and the magnitude and specificity of the responses were measured resulting in limited cross reactivity. Overall, results suggest that vaccination of cattle with T3SPs as a means of reducing the risk of STEC transmission to humans will induce protection that is serotype specific.<p> To pursue the possibility of a cross-protective vaccine, we investigated the protective properties of a chimeric Tir protein against STEC serotypes. Several studies have reported that Tir is highly immunogenic and capable of producing high antibody titers. Potter and colleagues also demonstrated that the vaccination of cattle with ∆tir STEC O157 strain did not protect as well as the wildtype strain. We constructed thirty-mer peptides to the entire STEC O157 Tir protein, as well as to the intimin binding domain of the Tir protein from STEC serotype O26, O103 and O111. Using sera raised against STEC O157 and non-O157 T3SPs, we identified a number of immunogenic peptides containing epitopes unique to a particular serotype. Two different chimeric Tir proteins were constructed containing the STEC O157 Tir protein fused with six STEC non-O157 peptides with or without the Leukotoxin produced by <i>Mannheimia haemolytica</i>. However, the vaccination of mice with the chimeric protein did not protect against challenge with STEC O157 or STEC O111. These results suggest that to achieve cross protection against STEC serotypes using a recombinant protein vaccine, other immunogenic and protective antigens must also be included.<p> In order to identify other immunogenic and cross-protective antigens we cloned and expressed the genes coding for 66 effectors and purified each as histidine-tagged proteins. These included 37 LEE-encoded proteins and 29 non-LEE effectors. The serological response against each protein was measured by Western blot analysis and an enzyme-linked immunosorbent assay (ELISA) using sera from rabbits immunized with T3SPs from four STEC serotypes, experimentally infected cattle and human sera from 6 HUS patients. A total of 20 proteins were recognized by at least one of the STEC T3SP- vaccinated rabbits using Western blots. Sera from experimentally infected cattle and HUS patients were tested using an ELISA against each of the proteins. Tir, EspB, EspD, EspA and NleA were recognized by the majority of the samples tested. Overall, proteins such as Tir, EspB, EspD, NleA and EspA were highly immunogenic for both vaccinated and naturally infected subjects.<p> Based on the above results, two different mixtures of secreted proteins (5 proteins and 9 proteins) were used to vaccinate mice and test the level of shedding following challenge with STEC O157. Overall, the cocktail vaccine containing 9 immunogenic effectors including Tir, EspB, EspD, NleA and EspA was capable of reducing shedding as effectively as the current STEC T3SPs vaccine, Econiche®.

Shiga toxin-producing <i>E. coli</i> (STEC) is an important food- and water-borne pathogen of humans, causing Hemorrhagic Colitis and Haemolytic Uremic Syndrome. Colonization of both cattle and human hosts is mediated through the action of effector molecules secreted via a type III secretion system (T3SS), which forms attaching and effacing lesions (A/E). The necessary effectors which form A/E by manipulation of host signalling and actin nucleation are present on a pathogenicity island called the Locus of Enterocyte Effacement (LEE).<p> It has been reported that vaccination of cattle with Type III-secreted proteins (T3SPs) from STEC O157 resulted in decreased shedding. In order to extend this to non-O157 STEC serotypes, we examined the serological cross-reactivity of T3SPs of serotypes O26:H11, O103:H2, O111:NM and O157:H7. Groups of cattle were vaccinated with T3SPs produced from each of the serotypes and the magnitude and specificity of the responses were measured resulting in limited cross reactivity. Overall, results suggest that vaccination of cattle with T3SPs as a means of reducing the risk of STEC transmission to humans will induce protection that is serotype specific.<p> To pursue the possibility of a cross-protective vaccine, we investigated the protective properties of a chimeric Tir protein against STEC serotypes. Several studies have reported that Tir is highly immunogenic and capable of producing high antibody titers. Potter and colleagues also demonstrated that the vaccination of cattle with ∆tir STEC O157 strain did not protect as well as the wildtype strain. We constructed thirty-mer peptides to the entire STEC O157 Tir protein, as well as to the intimin binding domain of the Tir protein from STEC serotype O26, O103 and O111. Using sera raised against STEC O157 and non-O157 T3SPs, we identified a number of immunogenic peptides containing epitopes unique to a particular serotype. Two different chimeric Tir proteins were constructed containing the STEC O157 Tir protein fused with six STEC non-O157 peptides with or without the Leukotoxin produced by <i>Mannheimia haemolytica</i>. However, the vaccination of mice with the chimeric protein did not protect against challenge with STEC O157 or STEC O111. These results suggest that to achieve cross protection against STEC serotypes using a recombinant protein vaccine, other immunogenic and protective antigens must also be included.<p> In order to identify other immunogenic and cross-protective antigens we cloned and expressed the genes coding for 66 effectors and purified each as histidine-tagged proteins. These included 37 LEE-encoded proteins and 29 non-LEE effectors. The serological response against each protein was measured by Western blot analysis and an enzyme-linked immunosorbent assay (ELISA) using sera from rabbits immunized with T3SPs from four STEC serotypes, experimentally infected cattle and human sera from 6 HUS patients. A total of 20 proteins were recognized by at least one of the STEC T3SP- vaccinated rabbits using Western blots. Sera from experimentally infected cattle and HUS patients were tested using an ELISA against each of the proteins. Tir, EspB, EspD, EspA and NleA were recognized by the majority of the samples tested. Overall, proteins such as Tir, EspB, EspD, NleA and EspA were highly immunogenic for both vaccinated and naturally infected subjects.<p> Based on the above results, two different mixtures of secreted proteins (5 proteins and 9 proteins) were used to vaccinate mice and test the level of shedding following challenge with STEC O157. Overall, the cocktail vaccine containing 9 immunogenic effectors including Tir, EspB, EspD, NleA and EspA was capable of reducing shedding as effectively as the current STEC T3SPs vaccine, Econiche®.

Biotrophic plant pathogens lead to huge crop losses and they have great economical drawbacks on wheat and barley production. These pathogens rely on formation of haustoria and transfer of effector proteins into the host cells for generating disease. The main role of effector proteins is to disable plant defense mechanisms. Effector proteins contain N-terminal signal peptides and they have little sequence similarity between each other. It is vital to detect as many effector proteins as possible to understand infection and disease formation processes of biotrophic plant pathogens. To this end, sequencing of pathogen genomes are being emerged, the data will be invaluable for identifying the candidate effectors in terms of biological and biochemical roles in infection and more. There are some bioinformatics based methods available that can be utilized to classify and distinguish effectors from other pathogenic genes. It is important to understand how candidate effectors can be searched from Expressed Sequence Tags or transcriptome sequences. Hereby, our attempt is to present a pipeline in establishing a methodology. As a consequence, here we propose new candidate effectors. In plant-pathogen interactions also miRNAs are too proving to be an important factor which cannot be neglected. During disease infection, expression levels of miRNAs are varying which in turn may be a proof of miRNA regulation of pathogen genes. Therefore, cross-kingdom RNA interference may take place between plant and pathogen. We have tested plant pathogens for possible plant miRNA availability and found their most likely targets with in the pathogen genes.

Colletotrichum graminicola is a hemibiotrophic pathogen of maize that causes anthracnose leaf and stalk rot diseases. The pathogen penetrates the host and initially establishes an intracellular biotrophic infection, in which the hyphae are separated from the living host cell by a membrane that is elaborated by the host, apparently in response to pathogen signals. A nonpathogenic mutant (MT) of C. graminicola was generated that germinates and penetrates the host normally, but is incapable of establishing a normal biotrophic infection. The mutated gene is Cpr1, conserved in eukaryotes and predicted to encode a component of the signal peptidase complex. How can we explain why the MT is normal in culture and during early stages of pathogenicity, but is deficient specifically in the ability to establish biotrophy? To address this, first I characterized the insertion in the 3’ UTR of the MT strain in detail, something that had not been done before. The wild-type (WT) transcript did not differ from predictions, but the MT produced several aberrant transcript species, including truncated and non-spliced transcripts, and the normal one. Aberrant splicing of MT cpr1 was observed both in RNAseq transcriptome data and reverse-transcription polymerase chain reaction (RT-PCR), under different growth conditions and in planta. I also conducted a bioinformatic analysis of other conserved components of the secretory pathway in the MT and WT in planta. One explanation for nonpathogenicity of the MT is that it cannot cope with an increase in secretory activity during infection, and fails to produce necessary pathogenicity factors. With the transcriptome data, I was able to identify effector proteins that were expressed in the WT but not in the MT. Another possible explanation for the MT phenotype is that the MT can’t adapt to stress imposed by the plant. I developed a growth assay to characterize the effect of chemical stressors in vitro. The MT was more sensitive to most stressors, when compared to the WT. The transcriptome data indicates that the genes involved in different stress pathways are expressed in planta in both WT and MT, although very few genes are differentially expressed across the different growth stages.

Engineered DNA-binding proteins enable targeted manipulation of the genome. Zinc fingers are the most well characterized DNA-binding domain and for many years research has focused on understanding and manipulating the sequence-specificities of these proteins. Recently, major advances in the ability to engineer zinc finger proteins, as well as the discovery of a new class of DNA-binding domains - transcription activator-like effectors (TALEs), have made it possible to rapidly and reliably engineer proteins targeted to any sequence of interest. With this capability, focus has shifted to exploring the applications of this powerful technology. In this dissertation I explore three important applications of engineered DNA-binding proteins.