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The poplar rust disease is caused by fungi belonging to the Melampsora genus (Pucciniales, Basiodiomycota) that cause important damages in poplar plantations in France. These fungi achieve their complex life cycles on two different host plants. The secretion of molecules called effectors that alter cell processes and impair immunity are required to set a successful infection. A central theme of molecular phytopathology is to understand how these molecules function in the host cell. In this PhD thesis, the transcriptome analysis of M. larici-populina (Mlp) during its sexual cycle while infecting its two host plants, poplar and larch, revealed a majority of genes commonly expressed on both hosts and a fraction specifically expressed on each host, including genes encoding candidate effectors. Effectoromic screens developed on a panel of Mlp candidate effectors revealed two candidates of interest. The candidate effector MLP124017 interacts with proteins of the TOPLESS-RELATED PROTEINS family and presents a structure similar to NUCLEAR TRANSPORT FACTOR 2 LIKE proteins. The MLPCTP1 effector is translocated inside chloroplasts of the heterologous plant tobacco and poplar. The functions of these two effectors remain to be determined but the functional characterization initiated in this thesis opens new perspectives in term of diversity and roles of effectors in Pucciniales. The preliminary analysis of the M. allii-populina genome shows similar repertoires of genes and candidate effector genes compared with Mlp as well as an increased genome size due to transposable elements invasion, La maladie de la rouille foliaire du peuplier causée par des champignons du genre Melampsora (Pucciniales, Basidiomycètes) affecte largement les peupleraies en France. Ces champignons possèdent des cycles de vie complexes et infectent deux hôtes différents. La sécrétion de molécules appelées effecteurs est nécessaire lors du processus d'infection par le champignon afin de manipuler les processus de l’hôte et de contourner son immunité. La compréhension de leur rôle est centrale en phytopathologie moléculaire. Au cours de cette thèse, l’analyse du transcriptome de Melampsora larici-populina (Mlp) au cours de son cycle sexué lors de l'infection des deux hôtes, le peuplier et le mélèze, révèle la présence d'une majorité de gènes exprimés communément chez les deux hôtes et d'une fraction exprimée spécifiquement chez chaque hôte, notamment des gènes codant des effecteurs candidats. Des cribles fonctionnels réalisés sur un répertoire d’effecteurs candidats de Mlp ont révélé deux candidats d’intérêt. L’effecteur MLP124017 interagit avec des protéines de la famille TOPLESS-RELATED PROTEINS et présente une structure similaire à des protéines NUCLEAR TRANSPORT FACTOR 2 LIKE. L'effecteur MLPCTP1 est localisé dans les chloroplastes en système hétérologue tabac et chez le peuplier. Les fonctions de ces effecteurs restent à élucider mais ces travaux ouvrent de nouvelles perspectives quant à la diversité et au rôle des effecteurs chez les Pucciniales. L'analyse préliminaire du génome de M. allii-populina montre des répertoires comparables en gènes et en effecteurs candidats par rapport à Mlp ainsi qu'une expansion de la taille du génome due à l’invasion par des éléments transposables

Pinon, J., Frey, P., and Husson, C. 2006. Wettability of poplar leaves influences dew formation and infection by Melampsora larici-populina. Plant Dis. 90:177-184. The wettability of leaves of 60 poplar (Populus spp.) clones from sections Aigeiros (black) and Tacamahaca (balsam) and their hybrids was assessed by measuring the contact angle of calibrated water droplets on their abaxial leaf surface. The frequency and duration of dew on these clones were recorded in two nurseries. Black poplar leaves had the highest wettability, with relatively flat-shaped water droplets and a short drying time, in both the laboratory and nursery. Conversely, round-shaped water droplets on balsam poplars dried more slowly in the laboratory. In the nursery, dew was more frequent and persisted longer. First-generation intersectional hybrids behaved like balsam poplars, but successive backcrosses with black poplar (P. deltoides) resulted in clones with leaves that were more wettable and possessed less frequent and persistent dew. Infection by Melampsora larici-populina on two hybrid clones (‘Robusta’ and ‘Beaupre’), assessed by the number of uredinia in the laboratory, was dependent on the duration of leaf moisture. Maximum infection occurred when water was present on the leaves for 12 h. Leaf wettability should be considered as an additional trait when breeding poplar for durable resistance to M. larici-populina, as this characteristic is likely to be a useful defense against all pathotypes of the pathogen.

International audience; • In order to elucidate the genetic control of resistance to Melampsora larici-populina leaf rust in hybrid poplars, a Populus deltoides × P. trichocarpa F1 progeny was analysed for qualitative and quantitative rust resistances. • This progeny was evaluated for three components of quantitative resistance (latent period, uredinia number and uredinia size) to seven M. larici-populina strains in controlled conditions, and for one component of field susceptibility (rust colonization on the most infected leaf). • One qualitative resistance locus inherited from P. deltoides, R1, was localized on the genetic map. It segregates 1 : 1 in the F1 progeny and is effective against four of the studied strains. QTL analysis was performed separately on R1 and r1 genotype subsets. An additional detection was conducted on the entire F1 progeny for the three strains able to overcome R1 and for MAX2. A total of nine QTLs were detected. Two had large, broad-spectrum effects. One (RUS) is inherited from the P. trichocarpa parent; the other is inherited from P. deltoides and colocalized with R1. Seven QTLs had only limited and specific effects. Significant interaction effects were detected mainly between the two major QTLs. • Implications of these results for durable resistance breeding strategies, and possible benefits from the Populus genome sequence, are discussed.

1for correspondence) Summary The effects of poplar varietal mixture on infection levels caused by Melampsora larici-populina and on plant growth were studied during two consecutive growing seasons. The varietal mixture consisted of an intimate mixture of three poplar clones, Populus x interamericana ‘Boelare’, ‘Rap’ and Populus candicans ‘NNT’, expressing different race-specific resistances. Infection levels of M. larici-populina were assessed in monoclonal and polyclonal plots. Plant heights were determined at the end of each growing season. Infection levels in the growing season of the first year were not significantly different between monoclonal and polyclonal plots at the end of the epidemic. During the second growing season, a naturally occurring complex pathotype, able to infect clones ‘Boelare’ and ‘Rap’, invaded some plots prematurely and changed the composition of pathogen population. During this season, four infection assessments were conducted. Infection reduction in polyclonal plots compared with monoclonal plots was observed on ‘Boelare’ and P. candicans plants. Nevertheless, no significant difference was observed for growth between monoclonal and polyclonal plots. Biological factors which may be involved in the reduction of infection levels in polyclonal plots are dilution of susceptible individuals, physical barriers caused by resistant plants and cross-protection. A high auto-infection ratio may have partly hidden the positive effects of the clonal mixture.

Because of its mixed mating system and high potential gene flow, Melampsora larici-populina belongs to the group of pathogens with the highest evolutionary risk. Indeed, all qualitative resistances that have been selected by breeders during the last decades have been defeated. Although quantitative resistance (QR) is often considered more durable, a multidisciplinary approach is being conducted at INRA not only to identify the genes controlling QR but also to predict potential adaptation of the pathogen. Starting with a single Populus deltoides × P. trichocarpa F 1 pedigree, this work led to the identification of several QTLs with contrasted levels of effect and strain-specificity. Much attention is being paid to two major loci, R 1 and R US , that both map on LG XIX. Dominant allele R 1 is inherited from P. deltoides. It controls a qualitative resistance to M. larici-populina, and it was found to be statistically associated with QR levels in several P. deltoides × P. trichocarpa F 1 progenies. Dominant allele R US is inherited from P. trichocarpa. It has major effect of uredinia size and other QR components in both intraspecific and interspecific F 1 progenies. Fine mapping of R US is being conducted using a 1300 genotype F 1 host progeny, and a BAC library was constructed to generate a physical map of 2000 kb around R US . A promising BAC clone has been identified and sequenced. In parallel, an expressional approach based on the analysis of R US and r US infected material generated transcriptional and translational candidates. On the pathogen side, one strain was found to be able to circumvent R US , thus offering the opportunity to study the frequency and the mechanisms of adaptation to QR. Several populations of M. larici-populina that underwent contrasted selection pressures by R US are being characterized for their phenotypes and for their genotypes at several microsatellite loci. The first results demonstrate the existence of preadaptation in “wild” pathogen Populations and a clear selective pressure by the host.

International audience; The effect of RUS, a major quantitative resistance (QR) factor inherited from Populus trichocarpa, was assessed in two Populus deltoides · P. trichocarpa F1 progenies against eight isolates of Melampsora larici-populina, the causal agent of Eurasian poplar rust. Six isolates were identified on which RUS had no significant effect. The first RUS-defeating isolate identified suggested a pre-existing potential to overcome RUS in the pathogen’s populations. The P. deltoides genetic background made no difference either to the ability of a given isolate to overcome RUS or to the relative and absolute aggressiveness of the isolates. This study illustrates how extreme the isolate-specificity ofQR can be. It also yields insights into the relationship between size of uredinia and sporulation rate, discussing the epidemiological significance of spore production permm2 uredinium.

P.22 pour le second congrès Événement(s) lié(s) : - 2009 IUFRO Tree Biotechnology Conference; Whistler, BC (CAN) - (2009-06-28 - 2009-07-02); National audience; Leaf rust caused by Melampsora larici populina (Mlp) is a major disease for poplar trees in Europe. Using a segregating population populus deltoides x P. trichocarpa, Rus controlling size of the uredinia has been characterized. Rus, inherited from P. trichocarpo, explains between 42.4% and 82.8% of the phenotypic variation to different Mlp strains. We describe the construction of a fine genetic map using a segregating population of 1410 individuals with available SSR and markers derived from an AFLP-BSA approach. The alignment with the public available genome sequence (http://genome.jgi-psf.org/Poptr1 1/Poptr1 1.home.html) allowed locating Rus on a cluster of resistance genes in the linkage group XlX. To narrow down genomic region corresponding to the Rus locus, additional genetic markers including SSR, STS corresponding to positional candidate genes were developed. Seven hundred and thirteen more individuals were used to perform a high resolution mapping of the region. At this term, Rus is localised within a 0.5 cM interval between the closely genetic markers SRR_84 and L29_1. To physically clone the gene, a 14X BAC library of the male P. trichocorpa has been constructed. Chromosome walking allowed the construction of two local physical maps corresponding to the two haplotypes Rus VS rus. Two BAC clones carrying Rus were selected and sequenced. Rus was defined into a 45kb interval.

International audience; Forty-one microsatellite markers were developed for the poplar rust fungus Melampsora larici-populina. The genome sequence of M. larici-populina strain 98AG31 was screened in silico for microsatellite loci using Magellan software. Thirty-nine loci, evaluated on a sexual population of 35 M. larici-populina isolates sampled on larch trees in the French Alps, were identified to be highly polymorphic. These markers revealed two to 34 alleles, with an average of 7.23 alleles per locus. The estimated heterozygosity ranged from 0.029 to 0.958 across the 41 microsatellite loci. These markers will be highly useful for population genetics studies of M. larici-populina.

International audience; The course of the infection and development of the biotrophic fungus Melampsora larici-populina on leaf tissue from the hybrid poplar Populus deltoides × P. nigra ‘Ogy’ was monitored at the histological level. Leaf disks were inoculated with one of two rust physiological races (E1 and E2), resulting in interactions that were either incompatible (race E1) or compatible (race E2). In the compatible interaction, the fungus rapidly colonized the leaf without inducing any apparent host response. Symptoms appeared on the leaf several days after inoculation just prior to spore dissemination. The incompatible interaction was characterized by the early collapse and disorganization of cytoplasm of infected cells 17 h after inoculation and within 2 h after the appearance of the first haustoria. Resistance to M. larici-populina was mediated through a hypersensitive response, since it was extremely localized and involved only the few cells that were in the immediate vicinity of each infected cell.

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International audience; Qualitative resistance to Melampsora larici-populina leaf rust inherited from North American species Populus deltoides did not allow for durable control of this pathogen in interspecific hybrid cultivars. Despite significant levels of strain-specificity, quantitative resistance would exert lower selection pressures on the pathogen populations, and hence could be more durable. Previous studies restricted to a large P. × interamericana (i.e., P. deltoides × Populus trichocarpa) F1 family revealed that the presence of R1, a segregating defeated qualitative resistance gene inherited from P. deltoides, had major beneficial effects on quantitative resistance. The present study was based on 14 F1 families from a 4 × 5 P. deltoides × P. trichocarpa factorial mating design where at least four defeated qualitative resistances segregate 1:1. Even though quantitative resistance assessments were conducted in the laboratory with a M. larici-populina strain able to overcome these qualitative resistances, their presence had a significant effect on the mean level and on the genetic variability for quantitative resistance. One unprecedented result is the identification of a defeated qualitative resistance which presence is associated with lower levels of quantitative resistance. Possible inferences on the nature of the genetic relationship between both resistance types are discussed.

International audience; The foliar rust caused by the fungus Melampsora larici-populina Kleb. is the most devastating disease affecting the European poplar plantations. We have identified a factor of quantitative resistance, Rus, inherited from the genotype Populus trichocarpa 101-74, which can reduce by 60% the level of infection in laboratory and which has a significant effect in field. This factor is localized on the genetic map of P. trichocarpa 101-74 based on the analysis of one F1 interspecific family P. deltoïdes x P. trichocarpa. Map based cloning of Rus is under development, fine mapping and construction of BAC contig are underway. The alignments with the sequence of P. trichocarpa Nisqually-1 makes it possible to target the area of interest. We defined an exploratory zone of approximately 400 kb on which we already detected 28 genes. Ten of them are re-annotated as Resistance Gene Analogs (RGAs). Predicted genes have been investigated as candidate genes for fine mapping and association study. Association between Rus and those candidate genes has been first investigated in a collection of 94 individuals of P. trichocarpa. The collection was phenotyped for rust resistance under control conditions and in the field. The individuals have been genotyped by EcoTILLING.

National audience; La rouille foliaire causée par Melampsora larici-populina Kleb. (Mlp) est une des principales maladies affectant les peupleraies européennes. Nous avons identifié un facteur de résistance quantitative à Mlp hérité du génotype Populus trichocarpa 101-74, Rus, qui peut réduire de 60% le niveau de sporulation après inoculation contrôlée et qui a un effet significatif en conditions naturelles d'infection. Ce facteur est localisé sur une carte génétique basée sur l'analyse d'une famille Fi Populus deltoïdes x P.trichocarpa. Un travail de clonage positionnel du facteur Rus est en cours de réalisation. Pour identifier des marqueurs plus proches de Rus, nous avons utilisé la technique de Bulk Segregant Analysis (BSA). Une puissance de tri importante (15 bulks de génotypes contrastés Rus et rus, et 114 couples d'amorces AFLP testés) a permis d'amplifier 7 marqueurs discriminants les génotypes Rus et rus. Ces 7 marqueurs et 2 marqueurs AFLP déjà cartographiés ont été clonés, séquencés pour permettre le développement de marqueurs STS et le génotypage à moindre coût de la famille étendue à 1300 génotypes Fi. Le marqueur le plus proche est situé à 2.9 cM. La cartographie de ces bandes et de 5 marqueurs microsate1lites additionnels ainsi que les résultats des alignements avec la séquence de P. trichocarpa Nisqually-1 permet de cibler la région d'intérêt. Nous avons défini une zone exploratoire d'environ 400 kb sur laquelle nous avons déjà détecté des analogues de gènes de résistance de type NBS-LRR. Une banque BAC du clone d'intérêt 101-74 comptant environ 52 000 clones a été construite et organisée en pool ligne puis en pool plaque pour optimiser la recherche de BAC portant Rus. Un ensemble de 70 locus micro satellite répartis sur l'ensemble du génome, choisis d'après les données de cartographie génétique ont été utilisés pour confirmer la couverture de la banque préalablement estimée à 14X. Les marqueurs génétiques, les plus proches du gène sont utilisés pour cribler la banque par PCR sur pool plaque puis la recherche de la coordonnée dans la plaque est menée. La carte physique locale sera initiée après la réalisation des empreintes de chaque clone BAC sélectionné. Parallèlement, une annotation expertisée de la région de 500 000 bases du chromosome XIX de Nisqually-1 entre les bases 800 000 et 1 300 000 est en cours pour identifier les gènes dans cette zone. L'objectif ici étant de développer de nouveaux marqueurs dans les gènes pour faire le lien entre locus d'intérêt et gène.

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National audience; Les rouilles à Melampsora larici-populina sont des ennemis majeurs du Peuplier. Mieux comprendre les mécanismes génétiques de la relation hôte – pathogène et mettre sur le marché des nouveaux clones minimisant l’impact du champignon est un enjeu essentiel pour la populiculture. La sélection de variétés à résistance complète s’est avérée vaine du fait du fort potentiel d’adaptation de l’agent pathogène. Dans l’objectif d’explorer des formes de résistance potentiellement plus durables, des tests en laboratoire d’évaluation de la résistance partielle ont été mis au point par les améliorateurs forestiers de l’Inra d’Orléans. Des protocoles d’inoculation sur disques foliaires excisés permettent d’accéder à trois composantes épidémiologiques clés : la latence infectieuse, le nombre d’urédosores et leur taille. Ce travail, couplé à des expérimentations en pépinière, des travaux de cartographie génétique et de détection de QTL, permettra d’identifier l’emplacement puis la nature des gènes qui contribuent aux résistances partielles présentes chez le Peuplier.

Depuis une dizaine d'années, les peupleraies cultivées en Europe ont un problème récurrent dû à Melampsora larici-populina (Mlp), agent d’une rouille foliaire. Les méthodes de culture "intensives" des peupleraies, les effets de mode des cultivars couplés à une sélection basée sur des résistances qualitatives ont provoqué l'apparition de races de Mlp qui contournent toutes ces résistances. Les sélectionneurs se tournent à présent sur la recherche de gènes ayant une pression de sélection moindre sur l'agent pathogène ; les gènes de résistance quantitative sont une bonne alternative. La recherche des déterminants génétiques de la résistance quantitative a mis en évidence un facteur génétique majeur hérité de Populus trichocarpa (RUS) qui contrôle la taille des urédosores de Mlp. Pour permettre le clonage positionnel de ce facteur génétique il faut déterminer la localisation précise de ce facteur contrôlant la résistance quantitative à Mlp chez le peuplier.[br/] Pour ce faire, nous avons densifié la zone du génome contenant RUS par un criblage d'amorces AFLP1 en utilisant la technique BSA2. Les marqueurs les plus intéressants ont été clonés et séquencés. Les séquences nucléotidiques obtenues ont ensuite été ancrées sur la séquence du génome de P. trichocarpa Nisqually-1. En parallèle, nous avons construit des amorces spécifiques STS3 pour cartographier finement la région de RUS.[br/] Huit bandes s'avèrent discriminantes sur 62 couples d'amorces testées. L'ancrage de 9 marqueurs sur la séquence de Nisqually-1 confirme que le facteur RUS se situe sur le groupe de liaison XIX. Nous avons réussi à développer 3 marqueurs STS valides pour la cartographie fine du locus de RUS. Nous avons cartographié à l’aide de 1429 individus les marqueurs STS E1M4-1S, E6M5-1S et E21M18-1S respectivement à 8.1, 30.3 et 9.3 cM de RUS. Cependant il reste encore à améliorer les amorces STS des autres marqueurs qui ne ségrègeaient pas sur les descendants. Cela permettra d’améliorer la précision de la carte génétique à cet endroit précis et d’obtenir des marqueurs suffisamment proches pour faciliter le criblage d’une banque BAC développée par ailleurs., Since ten years, poplars cultivated in Europe have a recurring problem due to Melampsora larici-populina (Mlp), agent of a leaf rust. The "intensive" methods of poplars cultivation, the fashion effects of cultivars coupled with a selection based on qualitative resistances provoked the appearance of races of Mlp which by-pass all resistances. Breeders turn toward the research for genes having a lesser selection pressure on the pathogenic agent: the quantitative resistance genes are a good alternative. The research for genetic factors of quantitative resistance put in evidence a major genetic factor inherited from Populus trichocarpa (RUS) which controls the size of Mlp uredinia. To allow positionnal cloning of RUS it is necessary to determine the precise localization of this factor controlling the quantitative resistance to Mlp to the poplar.[br/] To do it, we have densified the zone of the genome containing RUS by a screening of AFLP primers using the BSA technique. The most interesting markers were cloned and sequenced. Nucleotidic sequences obtained were then anchored on the sequence of the genome of P. trichocarpa Nisqually-1. In parallel, we built specific STS primers in order to map finely the region of RUS.[br/] Eight bands were discriminating on 62 primers combinations. The anchoring of 9 markers on the sequence of Nisqually-1 confirms that the factor RUS is located on the linkage group XIX. We managed to develop 3 valid STS markers for fine mapping of the locus RUS. We mapped, with 1429 individuals, our STS markers, E1M4-1S, E6M5-1S and E21M18- 1S respectively at 8.1, 30.3 and 9.3 cM of RUS. However, it still necessary to improve the other STS markers which not segregate in the progenies. It will allow to improve the precision of the genetic map in this place and to obtain markers enough close to facilitate the screening of a BAC library developed elsewhere

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A plant's capability to develop ectomycorrhizal symbiosis is under the control of both genetic and environmental factors. In order to determine the roles played by these different factors, we have performed a quantitative genetic analysis of the ability of poplar trees to form ectomycorrhizas. Quantitative genetics were applied to an interspecific family of poplar for which the two parental genetic maps had already been described, and for which data analyses concerning fungal aggressors were obtained. Quantitative trait loci (QTL) related to ectomycorrhiza formation were identified and located in the genetic maps of the two parents. One QTL was located at a linkage group of the genetic map of Populus trichocarpa showing a high concentration of several QTL involved in the pathogenic interaction with the fungus Melampsora larici-populina, the causal agent of leaf rust.

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Not available, Les protéines de réserve végétatives (VSPS) majeures de populus x euramericana ont été mises en évidence dans l'écorce, le bois, les bourgeons et les racines. Si dans la tige, les VSPS de p. euramericana montrent peu de spécificité tissulaire, la vacuole constitue leur seul site de stockage. Les protéines de réserve végétatives de l'écorce sont des protéines solubles, majeures durant la phase hivernale, et composées d'un seul type de sous-unités de 32, 34, 36 et 38 kda. Elles constituent probablement les glycoformes d'un polypeptide de 29 kda portant des chaines oligosaccharidiques de nature, de nombre, ou de longueur variable. D'autre part, nous avons montré qu'il existe aussi des homologies structurales et immunologiques entre les VSPS du bois et de l'écorce de p. euramericana. La biosynthèse des VSPS de p. euramericana apparait strictement régulée. Elle intervient lors du changement de photopériode en jours courts, et durant l'hydrolyse massive des protéines foliaires. Toutefois, l'accumulation des VSPS se déroule aussi lorsque les relations source-puits sont affectées par une attaque parasitaire des feuilles par melampsora larici-populina

Les populations raciales de L larici-populina ont ete etudiees pendant 4 ans (pres de 7000 identifications), dans plusieurs pepinieres de l'est de la France. La race E1 est ubiquiste en France (et tres probablement en Europe occidentale), la race E2 existe au moins dans la moitie nord de la France, en Belgique, aux Pays-Bas, et la race E3 semble presente dans l'ouest et le sud-ouest de la France et en Italie, dans la vallee du Po. Les races E2 et E3 ont une frequence irreguliere d'une annee a l'autre sur le meleze, l'hote alternant (tableau I). Il existe un certain lien entre les populations raciales du meleze et celles des peupliers (tableau I et II) (...)

Not available, Les protéines de réserve végétatives (VSPS) majeures de populus x euramericana ont été mises en évidence dans l'écorce, le bois, les bourgeons et les racines. Si dans la tige, les VSPS de p. euramericana montrent peu de spécificité tissulaire, la vacuole constitue leur seul site de stockage. Les protéines de réserve végétatives de l'écorce sont des protéines solubles, majeures durant la phase hivernale, et composées d'un seul type de sous-unités de 32, 34, 36 et 38 kda. Elles constituent probablement les glycoformes d'un polypeptide de 29 kda portant des chaines oligosaccharidiques de nature, de nombre, ou de longueur variable. D'autre part, nous avons montré qu'il existe aussi des homologies structurales et immunologiques entre les VSPS du bois et de l'écorce de p. euramericana. La biosynthèse des VSPS de p. euramericana apparait strictement régulée. Elle intervient lors du changement de photopériode en jours courts, et durant l'hydrolyse massive des protéines foliaires. Toutefois, l'accumulation des VSPS se déroule aussi lorsque les relations source-puits sont affectées par une attaque parasitaire des feuilles par melampsora larici-populina

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