Search

R2R3-MYB protein is the most abundant class of MYB transcription factor family in plants. The transcript profiles of two E4 races of Melampsora larici-populina-tolerant poplars and an intolerant poplar were investigated to characterise the role of the R2R3-MYB family genes in the poplar–E4 interaction. In this study, 217 R2R3-MYBs were identified, and 83 R2R3-MYB genes were assigned to 22 different coexpression modules by weighted gene coexpression network analysis. Most R2R3-MYB genes were unchanged in the early period of E4 infection in both tolerant and intolerant poplars. However, there were obvious increases in differentially expressed R2R3-MYB genes in tolerant poplars at 2 and 4 dpi when defence responses occurred, suggesting that differently expressed R2R3-MYB genes at these time points may play an important role in poplar resistance to E4 infection. In total, 34 R2R3-MYB genes showed differential expression at 2 and 4 dpi between tolerant and intolerant poplars. Among them, 16 differentially expressed R2R3-MYB genes were related to 43 defence-related genes that had significant differences between tolerant and intolerant poplars. There might be coregulatory relationships between R2R3-MYBs and other TFs during poplar–E4 interaction. Some differentially expressed R2R3-MYB genes were related to genes involved in flavonoid biosynthesis and IAA or free SA signal transduction and might help activate defence response during poplar–E4 interaction. MYB194 could be an important node in the convergence of IAA and SA signalling.

Melampsora larici-populina (Mlp) is a devastating pathogen of poplar trees, causing the defoliating poplar leaf rust disease. Genomic studies have revealed that Mlp possesses a repertoire of 1184 small secreted proteins (SSPs), some of them being characterized as candidate effectors. However, how they promote virulence is still unclear. This study investigates the candidate effector Mlp37347’s role during infection. We developed a stable Arabidopsis transgenic line expressing Mlp37347 tagged with the green fluorescent protein (GFP). We found that the effector accumulated exclusively at plasmodesmata (PD). Moreover, the presence of the effector at plasmodesmata favors enhanced plasmodesmatal flux and reduced callose deposition. Transcriptome profiling and a gene ontology (GO) analysis of transgenic Arabidopsis plants expressing the effector revealed that the genes involved in glucan catabolic processes are up-regulated. This effector has previously been shown to interact with glutamate decarboxylase 1 (GAD1), and in silico docking analysis supported the strong binding between Mlp37347 and GAD1 in this study. In infection assays, the effector promoted Hyalonoperospora arabidopsidis growth but not bacterial growth. Our investigation suggests that the effector Mlp37347 targets PD in host cells and promotes parasitic growth.

Rust fungi cause epidemics that threaten the production of important plant species, such as wheat and soy. Melampsora larici-populina (Mlp) causes the poplar rust and encodes at least 1184 candidate effectors (CEs) whose functions are poorly known. In this study, we sequenced the transcriptome and used mass spectrometry to analyze the metabolome of Arabidopsis plants constitutively expressing 14 Mlp CEs and of a control line to discover alterations leading to plant susceptibility. We found 2299 deregulated genes across the experiment. Genes involved in pattern-triggered immunity, such as FRK1, PR1, RBOHD, and WRKY33, as well as AUX/IAA genes were down-regulated. We further observed that 680 metabolites were deregulated in at least one CE-expressing transgenic line, with “highly unsaturated and phenolic compounds” and “peptides” enriched among down- and up-regulated metabolites. Interestingly, transgenic lines expressing unrelated CEs had correlated patterns of gene and metabolite deregulation, while expression of CEs belonging to the same family deregulated different genes and metabolites. Thus, our results uncouple effector sequence similarity and function. This supports that effector functional investigation in the context of their virulence activity and effect on plant susceptibility requires the investigation of the individual effector and precludes generalization based on sequence similarity.

Transcription factors (TFs) have been shown to play important roles in determining poplar susceptibility. In this study, the transcript profiles of five resistance-related TF groups at different time points were investigated to study the roles of TFs in the compatible interaction between &lsquo, Robusta&rsquo, (Populus nigra &times, P. deltoides) and the virulent E4 race of Melampsora larici-populina. The susceptibility test indicated that the parasitic process of E4 could be divided into two representative time periods: the infection phase and the production phase. Bioinformatics analysis showed that in these two phases, E4 infection induced a network of TFs in &lsquo, Although some TFs responded rapidly and positively, most TFs did not respond to E4, especially during the infection phase. The ethylene, jasmonic acid, and auxin pathways were downregulated, while a calcium-binding protein was upregulated. No other significantly changed phytohormone-related genes were found, which was consistent with the pathological process in the absence of an immune response, suggesting that the lack of response of most TFs during the infection phase of E4 is related to the susceptibility of &lsquo

Melampsora larici-populina causes serious poplar foliar diseases called rust worldwide. Salicylic acid (SA) and jasmonic acid (JA) are important phytohormones that are related to plant defence responses. To investigate the transcriptome profiles of SA- and JA-related genes involved in poplar rust interaction, two tolerant poplars and one intolerant poplar were selected for this study. Weighted gene coexpression network analysis (WGCNA) was applied to characterize the changes in the transcriptome profiles and contents of SA and JA after infection with the virulent E4 race of M. larici-populina. In response to infection with the E4 race of M. larici-populina, tolerant symptoms were correlated with the expression of genes related to SA and JA biosynthesis, the levels of SA and JA, and the expression of defence-related genes downstream of SA and JA. Tolerant poplars could promptly regulate the occurrence of defence responses by activating or inhibiting SA or JA pathways in a timely manner, including regulating the expression of genes related to programmed cell death, such as Kunitz-type trypsin inhibitor (KTI), to limit the growth of E4 and protect themselves. WGCNA suggested that KTI might be regulated by a Cytochrome P450 family (CYP) gene. Some CYPs should play an important role in both JA- and SA-related pathways. In contrast, in intolerant poplar, the inhibition of SA-related defence signalling through increasing JA levels in the early stage led to continued inhibition of a large number of plant–pathogen interaction-related and signalling-related genes, including NBS-LRRs, EDS1, NDR1, WRKYs, and PRs. Therefore, timely activation or inhibition of the SA or JA pathways is the key difference between tolerant and intolerant poplars.

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

The TIFY domain contains approximately 36 conserved amino acids that form the core motif TIF[F/Y]XG, and they were reported to play important roles in plant growth, tissue development and defence regulation. Moreover, more and more evidence has shown that some members of the TIFY gene family perform their functions by modulating plant hormone signalling pathways. Poplar trees are found worldwide, and they comprise approximately 30 species. Benefit from the importance of poplar and its advanced platform, this tree is considered to be the model perennial plant. Here, we conducted a genome-wide identification of TIFY genes in poplar, and 24 TIFY genes were found. These 24 TIFY genes were assigned to different subfamilies according to the presence or absence of domains and motifs that they harboured. Careful analyses of their locations, structures, evolution and duplication patterns revealed an overview of this gene family in poplar. The expression profiles of these 24 TIFY genes were then analysed in different tissues using publicly available expression data; their expression profiles following different JA/SA treatments and infection with leaf rust pathogen were also carefully examined by qRT-PCR assays. Based on their expression profiles, the functions of a number of TIFY genes could be predicted. By performing this study, we have provided valuable information for further functional characterisation of TIFY genes in poplar and candidate genes for the improvement of poplar disease resistance.

International audience; The rust fungi Melampsora larici-populina (Mlp) and Melampsora allii-populina (Map) are the main phytosanitary constraints for commercial poplar cultivation in Europe. Although Mlp is more aggressive and prevalent than Map, the two species may co-infect the same poplar tree or even the same poplar leaf, making the epidemiological surveys of each species difficult to achieve. In this study, a new duplex real-time PCR assay targeting each species was developed, based on single-copy genes. This test proved to be specific, inclusive and was successfully used to detect and quantify each species, starting from urediniospore samples or directly from infected poplar leaves, with or without visible symptoms. This new molecular tool was also assessed for comparative studies of time-course infection experiments on artificially inoculated poplar leaf discs. These studies showed that the growth dynamics of Map were significantly slower when the two species were co-inoculated on the same leaf disc, confirming that Map is less aggressive than Mlp.

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.

Most rust fungi have a complex life cycle involving up to five different spore-producing stages. The telial stage that produces melanized overwintering teliospores is one of these and plays a fundamental role for generating genetic diversity as karyogamy and meiosis occur at that stage. Despite the importance of telia for the rust life cycle, almost nothing is known about the fungal genetic programs that are activated in this overwintering structure. In the present study, the transcriptome of telia produced by the poplar rust fungus Melampsora larici-populina has been investigated using whole genome exon oligoarrays and RT-qPCR. Comparative expression profiling at the telial and uredinial stages identifies genes specifically expressed or up-regulated in telia including osmotins/thaumatin-like proteins (TLPs) and aquaporins that may reflect specific adaptation to overwintering as well numerous lytic enzymes acting on plant cell wall, reflecting extensive cell wall remodeling at that stage. The temporal dynamics of karyogamy was followed using combined RT-qPCR and DAPI-staining approaches. This reveals that fusion of nuclei and induction of karyogamy-related genes occur simultaneously between the 25 and 39 days post inoculation time frame. Transcript profiling of conserved meiosis genes indicates a preferential induction right after karyogamy and corroborates that meiosis begins prior to overwintering and is interrupted in Meiosis I (prophase I, diplonema stage) until teliospore germination in early spring.

Fungal colonization can significantly affect the secondary metabolism of the host plants. We tested the impact of a common below-ground symbiosis, i.e., ectomycorrhiza formation, on poplar leaf chemical components that are involved in the defence against a common disease, i.e., rust fungi, in N-deficient soil. A rust-susceptible poplar clone (Populus trichocarpa x deltoides 'Beaupre') was (a) non-associated with ectomycorrhizal fungus (EM) Hebeloma mesophaeum (Pers.) Quelet MUN and non-infected with rust fungus Melampsora larici-populina Kleb. (isolate 98AG31), (b) associated with EM, (c) inoculated with rust fungus and (d) associated with EM and inoculated with rust fungus. Poplar leaves were analysed by photometric and mass spectrometric techniques (liquid chromatography-tandem mass spectrometry (LC-MS/MS), pyrolysis-field ionization mass spectrometry (Py-FIMS)). Both rust infection and mycorrhiza formation led to increased proportions of condensed tannins in relation to total phenolics (13% in the control, 18-19% in the fungal treatments). In contrast, salicylic acid concentration (6.8 mu g g(-1) in the control) was higher only in the rust treatments (17.9 and 25.4 mu g g(-1) with rust infection). The Py-FIMS analysis revealed that the rust-infected treatments were significantly separated from the non-rust-infected treatments on the basis of six flavonoids and one lipid. The relative abundance of these components, which have known functions in plant defence, was decreased after rust infection of non-mycorrhizal plants, but not in mycorrhizal plants. The results indicate that the ectomycorrhizal formation compensated the rust infection by a decrease in the flavonoid syntheses. The study provides new evidence for an interactive response of mycorrhizal colonization and infection with rust fungi in the metabolism of poplar.

International audience; Fungal plant pathogens, especially rust fungi (Pucciniales), are well known for their complex life cycles, which include phases of sexual and asexual reproduction. The effect of asexual multiplication on population genetic diversity has been investigated in the poplar rust fungus Melampsora larici-populina using a nested hierarchical sampling scheme. Four hierarchical levels were considered: leaf, twig, tree and site. Both cultivated and wild poplar stands were sampled at two time points at the start and end of rust epidemics. A total of 641 fungal isolates was analysed using nine microsatellite markers. This study revealed that the genetic signature of asexual multiplication in the wild poplar stand was seen only at lower hierarchical levels (leaf and twig). Moreover, we observed an erosion of clonal structure through time, with an increase in both gene and genotypic diversity. New genotypes contributed to host infection over time, which demonstrates the importance of allo-infection in the epidemic process in this host-pathogen system. Compared with the wild stands, the nearly lack of detection of clonal structure in the cultivated stands reflects the higher infection level on cultivated poplars. More generally, this genetic analysis illustrates the utility of population genetics approach for elucidating the proportion of asexual reproduction in the multiplication of isolates during an epidemic, and for proper quantification of asexual dispersal in plant pathogens.

International audience; The advent of molecular epidemiology has greatly improved our ability to identify the population sources and track the pathogen movement. Yet the wide spatial and temporal scales usually considered are useful only to infer historical migration pathways. In this study, Bayesian genetic assignments and a landscape epidemiology approach were combined to unravel genetic origin and annual spread during a single epidemic of a plant pathogen: the poplar rust fungus [i]Melampsora larici-populina[/i]. The study focused on a particular area—the Durance River valley—which enabled inoculum sources to be identified and channelled spread of the epidemic along a one-dimensional corridor. Spatio-temporal monitoring of disease showed that the epidemic began in the upstream part of the valley and spread out downstream. Using genetic assignment tests, individuals collected at the end of the epidemic were sorted into two genetic groups; very few hybrids were detected, although individuals from both groups coexisted locally downstream in the valley. The epidemic originated from two genetically distinct inoculum sources. Individuals of each group then dispersed southwards along the Durance River and became mixed in poplar riparian stands. These two genetic groups were found previously at a wider spatial scale and proved to result from distinct evolutionary histories on either wild or cultivated poplars. This study showed that the two groups can mix during an epidemic but do not hybridize because they then reproduce asexually. In general, the methods employed here could be useful for elucidating the genetic origin and retracing the colonization history and migration pathways of recent epidemics.

Many factors can impact the genetic structures and population dynamics of fungal plant pathogens. Here we illustrated some of them at different spatial scales for the poplar rust fungus Melampsora larici-populina. At the scale of France, two main genetic groups were found. The first one infects only wild hosts and results from natural evolution of rust populations. The second one was formed after the R7 resistance breakdown, which is carried by the cultivar ?Beaupré?. This group exhibited a high proportion of virulent 7 individuals and presented signs of selection and demographic expansion; these signs indicate the recent invasion of individuals from this group on both wild and cultivated poplars in the northern half of France. At a regional scale, we focused on a contact zone between the ?cultivated? and the ?wild? genetic groups, located in the Alps. Upstream the Durance River valley, the influence of landscape has been highlighted by the effect of the Ecrins range which protects the ?wild? group located on the east side from the invasion of individuals from the ?cultivated? group, which arise from the northwest. Downstream the valley the annual epidemic wave was shown to be composed of admixed individuals from ?wild? and ?cultivated? groups, originating from both sides of the Ecrins range. Lastly we assessed the genetic consequences of the colonization wave. We evidenced a gradual increase of genetic differentiation with the epidemic source and a loss of genetic diversity. This work highlights the need of combining population genetics and epidemiology to characterize demographic processes and their genetic consequences, La structure génétique et la dynamique des populations des champignons phytopathogènes peuvent être influencées par de nombreux facteurs, ce que nous avons illustré à différentes échelles chez Melampsora larici-populina, l'agent de la rouille du peuplier. A l'échelle de la France, deux principaux groupes génétiques se différencient. Le premier, inféodé aux hôtes sauvages, est le produit de l'évolution des populations en conditions naturelles. Le second, formé suite au contournement de la résistance R7 portée par le cultivar "Beaupré", se caractérise par une forte proportion d"individus virulents 7 et présente des signatures de sélection et d'expansion démographique, témoins de l'invasion de ce groupe sur tous les peupliers (y compris sauvages) de la moitié nord de la France. A une échelle plus restreinte, nous avons examiné une zone de contact située dans les Alpes, où la délimitation entre les groupes « cultivé » et « sauvage » était la plus marquée. En testant l'effet du paysage, nous avons montré que le massif des Ecrins protégeait le groupe « sauvage » situé à l'est, en amont de la vallée de la Durance, de l'invasion des individus au profil « cultivé » venant du nord-ouest. Il n'en est pas de même dans la partie aval de cette vallée, colonisée annuellement par une vague épidémique, issue de ces deux groupes génétiques présents en amont de part et d'autre du Massif des Ecrins. En dernier lieu nous avons examiné les conséquences génétiques des évènements de colonisation, qui se traduisent par une augmentation de la différenciation par rapport à la source de l'épidémie ainsi qu'une érosion de la diversité génétique. Ce travail original a permis de souligner l'importance de combiner les approches d'épidémiologie et de génétique des populations pour caractériser au mieux les processus démographiques et leurs conséquences génétiques

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.

Association studies aim at linking phenotypic variation with allelic variation in candidate genes and benefit from several generations of recombination in natural populations to identify causative polymorphisms. Such association studies are under development on Populus nigra L., with emphasis on nucleotide polymorphism associated to quantitative resistance to Melampsora larici-populina Kleb (Mlp), the causal agent of leaf rust. Populus nigra, the natural host of Mlp, is widely represented in European riparian ecosystems, where the occurrence and impact of the fungus are highly variable. Geographical patterns of nucleotide diversity in genes involved in the host-pathogen interaction is expected from local co-evolution. As a first step, Single Nucleotide Polymorphisms (SNPs) have been identified in a discovery panel of twenty-one P. nigra genotypes covering French natural range. Sequencing was done on both functional and positional candidate resistance genes (closeness to mapped QTLs and resistance genes). An automated procedure for primer design allowed us to obtain successful amplification in P. nigra for 90% of gene fragments. Average nucleotide variation in P. nigra is high (1 SNP/97 pb). A collection of more than 1100 P. nigra genotypes collected in different natural populations mainly in France but also in Germany, Netherlands, Italy and Spain was evaluated for rust resistance under natural inoculation in a clonal test in France and under artificial inoculations with different strains in leaf-disk bioassays. Even if most of the genetic variation for rust resistant could be observed at the individual (genotype) level, significant differentiation between natural populations was detected. The high level of genetic diversity observed at both gene and phenotypic levels, the rapid linkage disequilibrium decay between SNP is offering a favourable situation to identify interesting resistance alleles and to avoid false positive associations.

International audience; High levels of variability for partial resistance to Melampsora larici-populina leaf rust in black poplar (Populus nigra L.) across Europe offers unique opportunity to identify favourable alleles through association studies. Nucleotide polymorphism (SNP and indels) within candidate genes has been explored in a discovery panel of 21 French P.nigra genotypes. Phenotyping for field and laboratory resistance components revealed significant diversity at both the intra and inter-metapopulation levels, with a suspected North vs. South geographical contrast.

International audience; Broad-spectrum resistance to Melampsora larici-populina (Mlp) leaf rust is a major challenge for poplar breeding in Northern Europe. When no rust epidemic was evident in the natural Populus nigra populations, cultivation of inter-specific clones carrying qualitative resistances have strongly modified both qualitative and quantitative pathogenicity components of Mlp populations in cultivated areas. In monoclonal poplar plantations, durability of rust resistance will depend strongly on (1) resistance mechanisms pyramided in a given genotype, (2) genetic diversity present in Mlp populations submitted to these high selection pressures and (3) possibility of recombination rate associated with sexual reproduction on larch. Recent results on genetic and molecular bases of Mlp-poplar interaction and multidisciplinary research projects in development will be presented. Although quantitative resistance (QR) is often considered more durable, a multidisciplinary approach is being conducted at INRA not only to identify optimal genetic constructions for broad spectrum resistance but also to predict potential adaptation of the pathogen. Starting with a single Populus deltoides x P. trichocarpa F1 pedigree, much attention is being paid to two major loci, R1 and RUS, that both map on LG XIX. Dominant allele R1 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 x P. trichocarpa F1 progenies. Dominant allele RUS is inherited from P. trichocarpa. It has major effect of uredinia size and other QR components in both intraspecific and interspecific F1 progenies. Fine mapping of RUS is being conducted using a 1410 genotype F1 host progeny, and a BAC library was constructed to generate a physical map of 2000 kb around RUS. A promising BAC clone has been identified and sequenced. In parallel, an expressional approach based on the analysis of RUS and rUS infected material generated transcriptional and translational candidates. Opportunities offered by genetic variability available in Populus nigra, the natural host of Mlp, are currently explored in inter-specific crosses and in a new F1 mapping pedigree. Moreover, a collection of more than 1100 P.nigra genotypes collected in different natural European populations has been recently evaluated for rust resistance to initiate an association genetic study. On the pathogen side, several populations of M. larici-populina that underwent contrasted selection pressures by RUS 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. All these results will be discussed in the framework of poplar breeding strategies and poplar cultivar deployment.

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.

International audience; Dispersal has a great impact on the genetic structure of populations, but remains difficult to estimate by direct measures. In particular, gradual and stochastic dispersal are often difficult to assess and to distinguish, although they have different evolutionary consequences. Plant pathogens, especially rust fungi, are suspected to display both dispersal modes, though on different spatial scales. In this study, we inferred dispersal capacities of the poplar rust fungus Melampsora larici-populina by examining the genetic diversity and structure of 13 populations from eight European and two overseas countries in the Northern hemisphere. M. larici-populina was sampled from both cultivated hybrid poplars and on the wild host, Populus nigra. The populations were analyzed with 11 microsatellite and 8 virulence markers. Although isolates displayed different virulence profiles according to the host plant, neutral markers revealed little population differentiation with respect to the type of host. This suggests an absence of reproductive isolation between populations sampled from cultivated and wild poplars. Conversely, studying the relationship between geographic and genetic structure allowed us to distinguish between isolation by distance (IBD) patterns and long distance dispersal (LDD) events. The European populations exhibited a significant IBD pattern, suggesting a regular and gradual dispersal of the pathogen over this spatial scale. Nonetheless, the genetic differentiation between these populations was low, suggesting an important gene flow on a continental scale. The two overseas populations from Iceland and Canada were shown to result from rare LDD events, and exhibited signatures of strong founder effects. Furthermore, the high genetic differentiation between both populations suggested that these two recent introductions were independent. This study illustrated how the proper use of population genetics methods can enable contrasted dispersal modes to be revealed.