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The basidiomycete cereal pathogens Ustilago hordei and U. maydis are closely related and possess genomes with a high degree of homology and synteny. I report on the disparity of the RNAi phenomenon between U. hordei and U. maydis. Using an RNAi expression vector I targeted both a GUS transgene and an endogenous mating-type gene and confirmed the presence of double-stranded (ds)RNA in transgenic cells of both species. However, down-regulation of the GUS gene and production of siRNAs were seen only in U. hordei. The biological effect was a reduction in GUS protein and activity, and reduced mating only in U. hordei. In support of this experimental evidence, homologs to Dicer and Argonaute were found in the U. hordei genome but not in the published U. maydis genome. Interestingly, preliminary U. hordei sequences reveal conservation and synteny in U. maydis in the regions spanning these loci, with the only noticeable difference being the lack of Dicer and Argonaute genes in U. maydis. U. maydis also appears to differ from U. hordei with respect to genes presumed to be involved in transcriptional gene silencing and also has far fewer transposons in its genome. Efforts to clone the avirulent gene UhAvr1 led to a locus containing a large number of small proteins predicted to be secreted. This locus appears to be heterochromatic and is orthologous to the largest cluster of secreted proteins in U. maydis. Other laboratories have reported that deletion of this cluster in U. maydis results in a dramatic reduction in virulence. Genetic evidence for an avirulence gene at this locus in U. hordei suggests that the locus may also be important for U. hordei. Differences between these two smut fungi at this locus and at others identified in this study point to key differences in gene regulation and genome evolution.

The basidiomycete cereal pathogens Ustilago hordei and U. maydis are closely related and possess genomes with a high degree of homology and synteny. I report on the disparity of the RNAi phenomenon between U. hordei and U. maydis. Using an RNAi expression vector I targeted both a GUS transgene and an endogenous mating-type gene and confirmed the presence of double-stranded (ds)RNA in transgenic cells of both species. However, down-regulation of the GUS gene and production of siRNAs were seen only in U. hordei. The biological effect was a reduction in GUS protein and activity, and reduced mating only in U. hordei. In support of this experimental evidence, homologs to Dicer and Argonaute were found in the U. hordei genome but not in the published U. maydis genome. Interestingly, preliminary U. hordei sequences reveal conservation and synteny in U. maydis in the regions spanning these loci, with the only noticeable difference being the lack of Dicer and Argonaute genes in U. maydis. U. maydis also appears to differ from U. hordei with respect to genes presumed to be involved in transcriptional gene silencing and also has far fewer transposons in its genome. Efforts to clone the avirulent gene UhAvr1 led to a locus containing a large number of small proteins predicted to be secreted. This locus appears to be heterochromatic and is orthologous to the largest cluster of secreted proteins in U. maydis. Other laboratories have reported that deletion of this cluster in U. maydis results in a dramatic reduction in virulence. Genetic evidence for an avirulence gene at this locus in U. hordei suggests that the locus may also be important for U. hordei. Differences between these two smut fungi at this locus and at others identified in this study point to key differences in gene regulation and genome evolution.

The basidiomycete cereal pathogens Ustilago hordei and U. maydis are closely related and possess genomes with a high degree of homology and synteny. I report on the disparity of the RNAi phenomenon between U. hordei and U. maydis. Using an RNAi expression vector I targeted both a GUS transgene and an endogenous mating-type gene and confirmed the presence of double-stranded (ds)RNA in transgenic cells of both species. However, down-regulation of the GUS gene and production of siRNAs were seen only in U. hordei. The biological effect was a reduction in GUS protein and activity, and reduced mating only in U. hordei. In support of this experimental evidence, homologs to Dicer and Argonaute were found in the U. hordei genome but not in the published U. maydis genome. Interestingly, preliminary U. hordei sequences reveal conservation and synteny in U. maydis in the regions spanning these loci, with the only noticeable difference being the lack of Dicer and Argonaute genes in U. maydis. U. maydis also appears to differ from U. hordei with respect to genes presumed to be involved in transcriptional gene silencing and also has far fewer transposons in its genome. Efforts to clone the avirulent gene UhAvr1 led to a locus containing a large number of small proteins predicted to be secreted. This locus appears to be heterochromatic and is orthologous to the largest cluster of secreted proteins in U. maydis. Other laboratories have reported that deletion of this cluster in U. maydis results in a dramatic reduction in virulence. Genetic evidence for an avirulence gene at this locus in U. hordei suggests that the locus may also be important for U. hordei. Differences between these two smut fungi at this locus and at others identified in this study point to key differences in gene regulation and genome evolution. / Science, Faculty of / Botany, Department of / Graduate

Leptosphaeria maculans est l'agent de la nécrose du collet des crucifères, principale maladie fongique du colza (Brassica napus). Lorsque des variétés de colza possédant la résistance Rlm7 ont été commercialisées au début des années 2000, la totalité des souches européennes étaient avirulentes vis-à-vis de ce gène de résistance. A l'inverse, AvrLm3 était considéré comme absent de ces populations. Un précédent projet de thèse réalisé dans l'équipe a montré la rapide capacité d'adaptation de L. maculans à la pression de sélection exercée par Rlm7 (Daverdin et al. 2012) et le phénotypage des populations isolées lors de cette étude avait montré que 98% des souches virulentes vis-à-vis de Rlm7 étaient avirulentes vis-à-vis de Rlm3. L'hypothèse d'un masquage du phénotype avirulent dû à la présence d'AvrLm4-7 a alors été validée par la complémentation d'une souche avirulente vis-à-vis de Rlm3 par AvrLm4-7. Sur ces bases, l'objectif de mon projet de thèse était d'identifier AvrLm3 afin d'analyser l'antagonisme entre les phénotypes AvrLm3 et AvrLm4-7, ainsi que les mécanismes menant au contournement de Rlm3 et Rlm7.Le gène AvrLm3 avait été identifié comme génétiquement lié à AvrLm4-7. Toutefois il n'avait pas été possible d'identifier un gène candidat. Par la combinaison d'approches de génétique et de génomique (clonage positionnel, RNA-seq, séquençage de novo d'une souche avirulente et de clones BAC), j'ai pu identifier AvrLm3, dont la séquence était absente du génome de référence de L. maculans. AvrLm3 est très fortement exprimé lors des phases précoces de l'infection et code pour une petite protéine sécrétée et ne présentant pas d'homologies avec d'autres protéines fongiques. Ce gène a donc les caractéristiques classiques des gènes codant pour des effecteurs, mais présente la particularité d'être le premier gène d'avirulence de L. maculans localisé en région télomérique. Bien que le mécanisme par lequel la présence du gène AvrLm4-7 supprime la reconnaissance d'AvrLm3 dans l'interaction entre B. napus et L. maculans n'ait pas été élucidé au terme de ma thèse, plusieurs hypothèses ont pu être invalidées, notamment l'absence de régulation de l'expression d'AvrLm3 par AvrLm4-7. De plus, une approche double hybride a mis en évidence une absence d'interaction directe entre les deux protéines d'avirulence. L'isolement de souches de L. maculans issues de parcelles expérimentales en 2012 et 2013 a mis en évidence le contournement actuel de la résistance Rlm7, avec environ 10 % de souches virulentes. Le phénotypage de ces collections a confirmé la présence majoritaire d'un allèle avirulent d'AvrLm3 chez celles-ci, moins de 0,5 % des souches isolées étant virulentes à la fois vis-à-vis de Rlm3 et de Rlm7. L'analyse de 592 souches d'origine mondiale a montré qu'AvrLm3 est toujours présent chez L. maculans, sous diverses formes alléliques, suggérant un rôle majeur d'AvrLm3 dans la fitness fongique. La résurgence du phénotype avirulent vis-à-vis de Rlm3 suite au contournement de Rlm7 fait envisager l'opportunité d'exploiter l'interaction entre les deux gènes d'avirulence pour une gestion plus durable des gènes de résistance de B. napus, par l'alternance de cultivars possédant Rlm3 et Rlm7 ou le pyramidage de ces deux gènes dans des variétés de colza. De plus, le rôle démontré d'AvrLm3 et d'AvrLm4-7 dans l'agressivité lors de l'infection suppose un fort coût de fitness lié à la virulence. Toutefois, des mécanismes originaux de compensation permettant au champignon d'échapper à la reconnaissance par Rlm3 et Rlm7 tout en conservant a priori fonctionnelle la fonction effectrice d'AvrLm3 et AvrLm4-7 ont été identifiés. Les résultats obtenus lors de ce travail de thèse ont permis la meilleure caractérisation d'une interaction gène-pour-gène inhabituelle. La diversité des mécanismes moléculaires permettant à L. maculans de contourner la résistance Rlm3 illustre la complexité de la course aux armements entre les plantes et les agents pathogènes. / Leptosphaeria maculans is a Dothideomycete responsible for stem canker on oilseed rape (Brassica napus). Genetic control, encompassing mostly the use of major resistance genes, is the most effective method to control this pathogen. When the first cultivars harboring Rlm7 have been deployed in the early 2000's, all of the European isolates were avirulent towards this resistance gene and AvrLm3 was considered to be absent from the populations. In 2012, Daverdin et al. showed that L. maculans has the ability to rapidly overcome the Rlm7 resistance whenever a strong selection pressure is applied. The phenotyping of isolates sampled by Daverdin et al. also showed that more than 98 % of the isolates virulent towards Rlm7 had become avirulent towards Rlm3. This result led to the hypothesis that the presence of AvrLm4-7 can suppress AvrLm3 recognition by Rlm3 and this was validated by the complementation of an isolate avirulent towards Rlm3 with a functional allele of AvrLm4-7. AvrLm3 is genetically linked to AvrLm4-7, at a distance of 20 cM. However, no candidate gene was identified before the beginning of my PhD. The objective of my PhD project was to identify AvrLm3, in order to better understand the antagonistic relationship between AvrLm3 and AvrLm4-7 phenotypes. The combination of genetic and genomic approaches (genetic mapping, RNA-seq, de novo sequencing of an isolate avirulent towards Rlm3 and BAC clone sequencing) allowed me to identify AvrLm3, whose sequence was absent from the reference genome assembly. AvrLm3 has common characteristics with others genes encoding fungal effector: it codes for a small, cysteine-rich protein, is highly expressed at early infection stages and shows no homology with others fungal genes. Although the mechanism allowing the suppression of AvrLm3 recognition due to the presence of AvrLm4-7 has not been elucidated, several hypotheses were invalidated: the presence of AvrLm4-7 has no impact on AvrLm3 expression and the two avirulence proteins do not interact physically.The sampling of field isolates in 2012 and 2013 showed the currently ongoing breakdown of Rlm7, with ca. 10% of virulent isolates, and the resurgence of the AvrLm3 phenotype in these populations, only 0.5 % of isolates being virulent towards both Rlm3 and Rlm7. A large collection of isolates from worldwide origin was genotyped for AvrLm3. It revealed that all isolates possess AvrLm3, with a high level of allelic diversity. The resurgence of the avirulent phenotype towards Rlm3 following the breakdown the Rlm7, along with the important role of AvrLm3 and AvrLm4-7 in fungal fitness, suggest that the antagonistic relationship between these two avirulence phenotypes could be an opportunity to propose original strategies to increase the durability of Rlm3 and Rlm7, by alternating both resistance genes at the landscape level or using pyramiding strategies. However, we identified novel isoforms of AvrLm4-7 allowing the fungus to escape Rlm7 recognition, while maintaining the suppression of Rlm3 recognition effective. This work allowed to better characterize an unusual gene-for-gene relationship. Indeed, only one other example of antagonism between avirulence phenotypes has been identified to date, in the phytopathogenic fungus Fusarium oxysporum (Houterman et al., 2008). The diversity of mechanisms allowing L. maculans to overcome the Rlm3 resistance illustrates perfectly the complexity of the arms race between plants and pathogens.

Leptosphaeria maculans, agent de la nécrose du collet des crucifères, est un agent pathogène majeur du colza (Brassica napus). La lutte génétique est aujourd’hui le procédé le plus utilisé afin de protéger les cultures des attaques de ce champignon. Cette méthode se base principalement sur l’utilisation de cultivars possédant des gènes de résistance spécifique (Rlm) qui permettent le déclenchement des réactions de défense de la plante parla reconnaissance directe ou indirecte des produits des gènes d’avirulence correspondants (AvrLm) présents dans la population pathogène. Plusieurs de ces résistances ont déjà été massivement déployées en France et dans le monde, connaissant dans un premier temps un fort succès commercial grâce à la protection fournie, suivie d’une perte d’efficacité très rapide. Avant cette thèse, le nombre d’études au champ des processus impliqués dans le contournement d’un gène de résistance était très limité, en particulier chez les champignons. L’objectif de cette thèse était d'étudier l’évolution moléculaire du gène d’avirulence AvrLm4-7sous pression de sélection, en profitant de son clonage et de la commercialisation récente de cultivars Rlm7, afin d’obtenir une étude précoce et détaillée des mécanismes moléculaires à l’origine du contournement d’une résistance spécifique. Le gène AvrLm4-7 présente l’originalité de coder pour une protéine responsable d’une double spécificité d’interaction vis-à-vis des gènes Rlm4 et Rlm7. Dans un premier temps, j’ai pu valider par mutagenèse dirigée le rôle primordial de l’acide aminé 120 dont la mutation affecte la reconnaissance d’AvrLm4 par Rlm4 sans toutefois altérer la reconnaissance d’AvrLm7 par Rlm7.Le contournement de la résistance Rlm7 a été ensuite analysé à l’aide d’une importante collection de souches prélevée sur deux sites expérimentaux indépendants (Grignon ; Versailles) sur une période de trois ans. Sur le premier site était cultivée une variété Rlm7 en monoculture avec un travail du sol simplifié tandis que sur le second site, le mode de culture incluait rotation culturale et enfouissement par labour des résidus de cultures. Il a ensuite été montré que, au contraire de la reconnaissance AvrLm4/Rlm4, un grand nombre d’évènements de mutation peuvent être à l’origine de la virulence d’une souche vis-à-vis de Rlm7. L’analyse moléculaire des souches virulentes et avirulentes de cette collection a ainsi permis de répertorier sept catégories d’évènements de mutation. La grande majorité des cas concerne la délétion d’AvrLm4-7 mais des mutations dues au RIP et plusieurs autres évènements de mutation provoquant l’introduction prématurée de codons stop dans la séquence codante du gène sont aussi observés. La majorité de ces évènements de mutation sont liés à la reproduction sexuée du champignon et ont lieu au sein même de la parcelle d’étude. Le phénotypage de cette collection a par ailleurs révélé un fort contraste entre les deux sites expérimentaux, démontrant ainsi l’importance des pratiques culturales dans le maintien de l’efficacité de la résistance Rlm7 dans le temps. En effet, après trois années de culture de cultivars Rlm7, la fréquence des souches virulentes a7 dans les populations du site de Versailles reste inférieure à 1 % contre environ 30 % sur le site de Grignon. Finalement, le phénotypage de la collection de souches a également montré que le contournement de Rlm7 s’accompagnait dans plus de 98% des souches de la résurgence de l’avirulence AvrLm3. Par l’étude de cette collection et par croisements génétiques, j’ai pu montrer que AvrLm3 n’était pas un nouvel allèle d’avrLm4-7 mais un second gène situé en région télomérique à 19.3 cM d’AvrLm4-7. J’ai également démontré une interaction fonctionnelle antagoniste entre AvrLm4-7 et AvrLm3 qui empêche la reconnaissance Rlm3 /AvrLm3 en présence d’AvrLm4-7 et explique la restauration de l’avirulence AvrLm3 lors de la perte de l’avirulence AvrLm7.Par une association originale de biologie moléculaire, de génétique des populations et d’agronomie, j’ai ainsi pu apporter une nouvelle illustration à la course aux armements entre un agent pathogène et sa plante hôte, les gènes AvrLm3 et AvrLm4-7 utilisant deux stratégies distinctes afin d’échapper à la reconnaissance de leurs gènes de résistance spécifiques. / Leptosphaeria maculans is a filamentous ascomycete causing stem canker of oilseed rape (Brassica napus). This disease is often controlled by the use of B. napus cultivars harbouring major resistance genes (Rlm). Direct or indirect recognition of the corresponding avirulence protein (AvrLm) in the pathogen triggers plant defence reactions. Several resistances have been massively deployed in France and worldwide, they initially showed commercial success due to the protection provided and in a second time, a very fast decrease of efficiency (resistance breakdown).Prior to this thesis, field studies of resistance gene breakdown mechanisms were rare, especially for fungi. The purpose of this PhD thesis was to study the molecular evolution of the avirulence gene AvrLm4-7 under selection pressure, by exploiting our knowledge of the gene, and the recent release of Rlm7 cultivars, to obtain an early and detailed study of the molecular mechanisms involved in a resistance gene breakdown. AvrLm4-7 induces resistance responses in plant harbouring either Rlm4 or Rlm7 and I validated by targeted point mutagenesis the central role of the amino acid 120 in the avrLm4-7-Rlm4 interaction. Its mutation prevents AvrLm4-7 recognition by Rlm4 without affecting avrLm4-7-Rlm7 recognition.Loss of avirulence towards Rlm7 was then studied by the analysis of an important isolate collection originating from two independent French experimental fields (Grignon; Versailles) over three years. In the first field was cropped Rlm7 cultivars in monoculture with low tillage agronomical practices whereas crop rotation and ploughing were done in the second field. In contrast to AvrLm4-Rlm4 evolution, a great number of mutations were found to explain the “gain” of virulence towards Rlm7. Seven mutational event categories were found. The great majority of these categories involve AvrLm4-7 deletion but mutation due to RIP and several other mutational events causing premature apparition of stop codons in the coding sequence of the gene were observed too. The majority of these events are linked to the sexual reproduction of the fungus and occurs in the experimental field.In addition, our work showed the importance of the cultural practices in preserving Rlm7 efficacy. Indeed, after three years using Rlm7 cultivars, a7 frequency was below 1% whereas representing around 30% of the isolates observed in Grignon.Finally, phenotyping of the isolate collection also showed the resurgence of an A3 phenotype linked with the loss of AvrLm7 avirulence in more than 98% of the isolates. Genetic analysis and collection phenotyping showed that AvrLm3 is not a new AvrLm4-7 allele but a distinct gene located in a telomeric region at 19.3 cM of AvrLm4-7. I also demonstrated that an antagonistic interaction between AvrLm4-7 and AvrLm3 exists: the presence of Avrlm4-7 prevents Rlm3 to detect AvrLm3 and explains the surge of the AvrLm3 avirulence along with the loss of the AvrLm7 avirulence.By an original association of molecular biology, population genetic and agronomy, this work provided a new illustration of the plant-pathogen arms race, AvrLm3 and AvrLm4-7 using two different strategies to escape their respective resistance genes.

Pendant l’infection, les agents phytopathogènes sécrètent un arsenal de molécules, appelées effecteurs, éléments clés de la pathogénie qui modulent l’immunité innée de la plante et facilitent l’infection. Leptosphaeria maculans est le champignon responsable de la nécrose du collet du colza. Plus de 650 gènes codant des effecteurs potentiels ont été identifiés dans son génome, dont 7 ont un rôle reconnu dans l’avirulence du champignon. Les effecteurs fongiques correspondent principalement à de petites protéines potentiellement sécrétées (PPS), n’ayant pas d’homologues dans les bases de données et pas de motifs connus. Par conséquent, leur fonction biologique est difficile à prédire, et très peu de choses sont connues sur le mode d’action des effecteurs de L. maculans au cours de l’infection.L’objectif de ma thèse était de caractériser fonctionnellement des effecteurs de L. maculans afin de mieux comprendre leur rôle au cours du processus infectieux. Cette caractérisation fonctionnelle a consisté en : i) la détermination de la localisation subcellulaire de ces effecteurs dans Nicotiana benthamiana et Arabidopsis thaliana ; ii) la recherche de cibles végétales ciblées par ces effecteurs ; et iii) la détermination des processus cellulaires impactés par ces effecteurs par expression stable dans A. thaliana et tests de suppression de mort cellulaire dans N. benthamiana. Quatre effecteurs ont été choisis pour cette étude : AvrLm10-1, AvrLm10-2, AvrLm4-7 et AvrLm3.AvrLm10-1 et AvrLm10-2 sont tous les deux nécessaires pour induire une reconnaissance par le gène de résistance Rlm10. Des orthologues d’AvrLm10-1 et AvrLm10-2 ont été identifiés chez des Dothidéomycètes et des Sordariomycètes phytopathogènes ainsi que plusieurs paralogues exprimés spécifiquement pendant l’infection chez L. maculans. AvrLm10-1 et AvrLm10-2 présentent toutes les deux une localisation nucléo-cytoplasmique. Une interaction physique entre AvrLm10-1 et AvrLm10-2 a été mise en évidence, ainsi qu’une interaction potentielle de ces deux protéines avec une protéine PR1 (Pathogenesis-related 1) et une cystéine-protéase végétale.AvrLm4-7 est reconnu par deux gènes de résistance, Rlm4 et Rlm7, et sa présence empêche la reconnaissance d’AvrLm3 par Rlm3. AvrLm4-7 est capable de supprimer la mort cellulaire provoquée aussi bien par des inducteurs généraux de la mort cellulaire que par des inducteurs de la PAMP-Triggered Immunity (PTI) et de l’Effector-Triggered Immunity (ETI). AvrLm4-7 présente une localisation nucléo-cytoplasmique, qu’il soit exprimé avec ou sans son peptide signal, ce qui suggère un mode d’action intracellulaire. AvrLm4-7 interagit potentiellement avec une protéine ribosomale végétale, de la même manière qu’un effecteur de Blumeria graminis avec lequel il partage des analogies structurales. Cependant, des lignées d’A. thaliana exprimant AvrLm4-7 de façon constitutive ne présentent aucune différence morphologique ou de sensibilité aux maladies comparativement à l’écotype sauvage Col0.AvrLm3 est un gène d’avirulence très conservé dans les populations de L. maculans dont la reconnaissance par le gène de résistance Rlm3 est supprimée en présence d’AvrLm4-7. AvrLm3 est capable de supprimer la mort cellulaire associée à la PTI et à l’ETI. Cet effecteur est localisé dans l’apoplasme des cellules foliaires lorsqu’il est exprimé avec son peptide-signal, suggérant un mode d’action extracellulaire. AvrLm3 interagit potentiellement avec une myrosinase-associated proteine sécrétée impliquée dans le système myrosinase-glucosinolate, suggérant qu’AvrLm3 perturberait la synthèse des glucosinolates, ce qui est un mode d’action inédit pour un effecteur d’agent phytopathogène.Cette thèse a permis de mieux comprendre le mode d’action des effecteurs de L. maculans et de proposer de nouvelles stratégies de contrôle des maladies fongiques. / During infection, plant pathogens secrete an arsenal of molecules collectively known as effectors that circumvent plant innate immunity and trigger infection. The phytopathogenic fungus Leptosphaeria maculans is the causal agent of stem canker of oilseed rape. More than 650 putative effector-encoding genes have been identified in its genome, 7 of them corresponding to avirulence genes. Fungal effectors mainly correspond to small secreted proteins (SSP) with no known homologs and no predicted functions. Their biological function is therefore difficult to predict, and very little is known about the mode of action of L. maculans effectors during infection.The objective of my thesis was to elucidate the role of L. maculans effectors during infection through their functional characterization which included: i) the determination of their subcellular localization in Nicotiana benthamiana et Arabidopsis thaliana; ii) a search for their plant targets; and iii) the determination of the cellular processes targeted by those effectors through their stable expression in A. thaliana and by testing their ability to suppress cell-death in N. benthamiana. We investigated four effectors in that study: AvrLm10-1, AvrLm10-2, AvrLm4-7 and AvrLm3.AvrLm10-1 and AvrLm10-2 are both necessary to trigger recognition by the Rlm10 resistance gene. We have identified orthologs for AvrLm10-1 and AvrLm10-2 in Dothideomycetes and Sordariomycetes phytopathogens, and several paralogs in L. maculans which are expressed specifically during oilseed rape infection. AvrLm10-1 and AvrLm10-2 both have a nucleo-cytoplasmic localization. AvrLm10-1 and AvrLm10-2 physically interact, and may also interact with a PR1 (Pathogenesis-related 1) protein and a secreted cysteine-protease. AvrLm4-7 is recognized by two resistance genes, Rlm4 and Rlm7, and suppresses recognition of AvrLm3 by Rlm3. AvrLm4-7 suppresses cell-death triggered by general inducers, PAMP-Triggered Immunity (PTI) and Effector-Triggered Immunity (ETI). AvrLm4-7 has a nucleo-cytoplasmic localization, whether expressed with or without its signal peptide, suggesting an intracellular mode of action. One of the potential plant targets for AvrLm4-7 is a ribosomal protein, just like a Blumeria graminis effector with structural analogy to AvrLm4-7. Transgenic lines of A. thaliana constitutively expressing AvrLm4-7 do not show any morphological phenotypes or any difference in their susceptibility to diverse fungal pathogens. AvrLm3 is an avirulence gene strongly conserved in L. maculans populations. Recognition of AvrLm3 by Rlm3 is suppressed by the presence of AvrLm4-7. AvrLm3 suppresses cell-death triggered by several inducers of PTI and ETI. AvrLm3 is localized in plant apoplasm when expressed with its signal peptide, suggesting an extracellular localization. AvrLm3 potentially interacts with a secreted myrosinase-associated protein implicated in the myrosinase-glucosinolate system, suggesting that AvrLm3 could disturb glucosinolate production, which is a novel mode of action never described for a plant pathogen effector.My thesis allowed us to improve our knowledge on fungal effector function during infection and to propose new strategies for plant diseases management

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No. of bitstreams: 2 Dissertação - Lorena Ferreira Peixoto - 2014.pdf: 2832669 bytes, checksum: ed2f415262daf1618ecdf785ad9bfb22 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2014-08-15 / Fundação de Amparo à Pesquisa do Estado de Goiás - FAPEG / Rice is a worldwide cultivated and consumed grain, playing an important role on the diet of half of the world’s population. Several losses in production and grain quality have been reported due to biotic factors, such as rice blast, caused by Magnaporthe oryzae, which is the major disease in rice crops. One of the most effective ways to control this disease is the use of resistant cultivars. However, the high genetic variability of the pathogen results in a rapid resistance loss. The discovery of highly fertile, hermaphrodites individuals outside of the rice center of origin, suggests that sexual reproduction may contribute to this genetic variability, which influences the appropriate control strategies. M. oryzae reproduction studies begins with the determination of mating types, controlled by two idiomorphic genes (MAT1-1 e MAT1-2), along with the sexuality (hermaphrodite, female or male) and fertility (number of perithecia). Another important approach under investigation for this crop is the detection of avirulence genes from M. oryzae, to understand the pathogen variability. Our study focused on the investigation of MAT1-1 or MAT1-2 genes, and the presence of the avirulence gene AVR1-CO39in field isolates collected from all rice production regions from Brazil. Sexuality and fertility were also characterized. 208 selected isolates were cultivated in PDA medium and the fungus mycelia were used for DNA extraction and PCR detection of the above-mentioned genes. For the sexual characterization, 106 field isolates were paired in Petri dishes containing rice bran medium with two reference isolates: KA-3 (MAT1-1) and GUY11 (MAT1-2), known worldwide for their mating type and high fertility. The AVR1-CO39 gene was only detected in two field isolates. One of them was able to infect the rice cultivar CO39, which has the resistance gene Pi-CO39(t). A mutation on AVR1-CO39 gene could impair the recognition of its effector by Pi-CO39(t) protein. Only one mating type (MAT1-2) was observed on the 208 field isolates. It was also observed that, among the 106 analyzed isolates, one (0,94%) was identified as a female; three (2,8%) as hermaphrodite, 62 (57,9%) as male; and 41 (38,3%) were not determined, considered infertile. We also observed the formation of perithecia inside of rice leaves. Despite the predominance of one mating type among rice field isolates, there is a possibility that sexual reproduction may occur as the other idiomorphic gene (MAT1-1) is present on field isolates collected from other Poaceae. The identification of highly fertile hermaphrodites and fertile-female individuals in this study also highlight this possibility. / O arroz é cultivado e consumido em todos os continentes, desempenhando um importante papel na dieta de mais da metade da população mundial. O seu cultivo vem sofrendo perdas na produção e na qualidade de grãos, devido a fatores bióticos como a brusone, causada pelo fungo Magnaporthe oryzae, que é a principal doença da cultura do arroz, representando uma ameaça à segurança alimentar mundial. O uso de cultivares resistentes é considerado o método mais efetivo para o controle da doença, porém, a alta variabilidade do patógeno resulta em uma rápida suplantação da resistência. Com a descoberta de isolados de alta fertilidade, hermafroditas, fora do centro de origem do arroz, sugere-se que a reprodução sexuada possa estar contribuindo para esta variabilidade genética, o que consequentemente influencia as estratégias apropriadas de controle. O estudo da reprodução sexuada em M. oryzae inicia-se com a definição dos tipos compatíveis, característica controlada pelo gene mating type com dois idiomorfos (MAT1-1 e MAT1-2); além das características como sexualidade (hermafrodita, fêmea ou macho) e fertilidade (número de peritécios). Outra abordagem de grande importância para a cultura é a detecção de genes de avirulência de M. oryzae, visando estudos de sua variabilidade. Dessa forma, o objetivo desta pesquisa foi investigar a presença dos genes MAT1-1 ou MAT1-2, e do gene de avirulência AVR1-CO39, em isolados coletados em todas as regiões produtoras de arroz do Brasil, além de caracterizá-los quanto à sexualidade e fertilidade. Foram selecionados 208 isolados que forma cultivados em BDA e seus micélios utilizados para extração de DNA e detecção dos genes citados. Para a caracterização sexual, 106 isolados de campo foram pareados em placa de Petri, contendo meio de farelo de arroz, com dois isolados: KA-3 (MAT1-1) e GUY11 (MAT1-2), os quais apresentam mating types e alta fertilidade, conhecidos mundialmente. O gene AVR1-CO39 foi detectado em apenas dois isolados, e um deles é patogênico à cultivar CO39, portadora do gene de resistência Pi-CO39(t), levantando a possibilidade de que uma mutação possa ter ocorrido, como deleção, o que impossibilita o reconhecimento do efetor pela proteína do gene Pi-CO39(t). Apenas um tipo compatível (MAT1-2) foi observado nos 208 isolados de campo. Foi observado também que, entre os 106 isolados analisados, um (0,94%), foi identificado como fêmea; três (2,8%), como hermafroditas; 62 (57,9%), como machos; e 41 (38,3%), como não determinados, sendo considerados inférteis. Observou-se também a formação de peritécios no interior da folha de arroz. Apesar do predomínio de um mating type, entre os isolados do arroz, há a possibilidade de ocorrência da reprodução sexuada, devido à presença do outro idiomorfo (MAT1-1) em isolados coletados de outras gramíneas, juntamente com a presença de hermafroditas e da fêmea-fértil, com alta fertilidade, de isolados MAT1-2 identificados nesse trabalho.

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.