FONZIE: An optimized pipeline for minisatellite marker discovery and primer design from large sequence data sets

<p>Abstract</p> <p>Background</p> <p>Micro-and minisatellites are among the most powerful genetic markers known to date. They have been used as tools for a large number of applications ranging from gene mapping to phylogenetic studies and isolate typing. However, identifying micro-and minisatellite markers on large sequence data sets is often a laborious process.</p> <p>Results</p> <p>FONZIE was designed to successively 1) perform a search for markers via the external software Tandem Repeat Finder, 2) exclude user-defined specific genomic regions, 3) screen for the size and the percent matches of each relevant marker found by Tandem Repeat Finder, 4) evaluate marker specificity (i.e., occurrence of the marker as a single copy in the genome) using BLAST2.0, 5) design minisatellite primer pairs via the external software Primer3, and 6) check the specificity of each final PCR product by BLAST. A final file returns to users all the results required to amplify markers. A biological validation of the approach was performed using the whole genome sequence of the phytopathogenic fungus <it>Leptosphaeria maculans</it>, showing that more than 90% of the minisatellite primer pairs generated by the pipeline amplified a PCR product, 44.8% of which showed agarose-gel resolvable polymorphism between isolates. Segregation analyses confirmed that the polymorphic minisatellites corresponded to single-locus markers.</p> <p>Conclusion</p> <p>FONZIE is a stand-alone and user-friendly application developed to minimize tedious manual operations, reduce errors, and speed up the search for efficient minisatellite and microsatellite markers departing from whole-genome sequence data. This pipeline facilitates the integration of data and provides a set of specific primer sequences for PCR amplification of single-locus markers. FONZIE is freely downloadable at: <url>http://www.versailles-grignon.inra.fr/bioger/equipes/leptosphaeria_maculans/outils_d_analyses/fonzie</url></p

Two independent approaches converge to the cloning of a new Leptosphaeria maculans avirulence effector gene, AvrLmS-Lep2.

Brassica napus (oilseed rape, canola) seedling resistance to Leptosphaeria maculans, the causal agent of blackleg (stem canker) disease, follows a gene-for-gene relationship. The avirulence genes AvrLmS and AvrLep2 were described to be perceived by the resistance genes RlmS and LepR2, respectively, present in B. napus 'Surpass 400'. Here we report cloning of AvrLmS and AvrLep2 using two independent methods. AvrLmS was cloned using combined in vitro crossing between avirulent and virulent isolates with sequencing of DNA bulks from avirulent or virulent progeny (bulked segregant sequencing). AvrLep2 was cloned using a biparental cross of avirulent and virulent L. maculans isolates and a classical map-based cloning approach. Taking these two approaches independently, we found that AvrLmS and AvrLep2 are the same gene. Complementation of virulent isolates with this gene confirmed its role in inducing resistance on Surpass 400, Topas-LepR2, and an RlmS-line. The gene, renamed AvrLmS-Lep2, encodes a small cysteine-rich protein of unknown function with an N-terminal secretory signal peptide, which is a common feature of the majority of effectors from extracellular fungal plant pathogens. The AvrLmS-Lep2/LepR2 interaction phenotype was found to vary from a typical hypersensitive response through intermediate resistance sometimes towards susceptibility, depending on the inoculation conditions. AvrLmS-Lep2 was nevertheless sufficient to significantly slow the systemic growth of the pathogen and reduce the stem lesion size on plant genotypes with LepR2, indicating the potential efficiency of this resistance to control the disease in the field

Epigenetic Control of Effector Gene Expression in the Plant Pathogenic Fungus Leptosphaeria maculans

Plant pathogens secrete an arsenal of small secreted proteins (SSPs) acting as effectors that modulate host immunity to facilitate infection. SSP-encoding genes are often located in particular genomic environments and show waves of concerted expression at diverse stages of plant infection. To date, little is known about the regulation of their expression. The genome of the Ascomycete Leptosphaeria maculans comprises alternating gene-rich GC-isochores and gene-poor AT-isochores. The AT-isochores harbor mosaics of transposable elements, encompassing one-third of the genome, and are enriched in putative effector genes that present similar expression patterns, namely no expression or low-level expression during axenic cultures compared to strong induction of expression during primary infection of oilseed rape (Brassica napus). Here, we investigated the involvement of one specific histone modification, histone H3 lysine 9 methylation (H3K9me3), in epigenetic regulation of concerted effector gene expression in L. maculans. For this purpose, we silenced the expression of two key players in heterochromatin assembly and maintenance, HP1 and DIM-5 by RNAi. By using HP1-GFP as a heterochromatin marker, we observed that almost no chromatin condensation is visible in strains in which LmDIM5 was silenced by RNAi. By whole genome oligoarrays we observed overexpression of 369 or 390 genes, respectively, in the silenced-LmHP1 and -LmDIM5 transformants during growth in axenic culture, clearly favouring expression of SSP-encoding genes within AT-isochores. The ectopic integration of four effector genes in GC-isochores led to their overexpression during growth in axenic culture. These data strongly suggest that epigenetic control, mediated by HP1 and DIM-5, represses the expression of at least part of the effector genes located in AT-isochores during growth in axenic culture. Our hypothesis is that changes of lifestyle and a switch toward pathogenesis lift chromatin-mediated repression, allowing a rapid response to new environmental conditions

External Lipid PI3P Mediates Entry of Eukaryotic Pathogen Effectors into Plant and Animal Host Cells

Coverage of RAD sequences. (PDF 224 kb

Pouvoir pathogene de Leptosphaeria maculans et reaction hypersensible de Brassica spp: : intervention d'une pathotoxine, la sirodesmine PL, et d'une phytoalexine, la brassilexine

SIGLECNRS T Bordereau / INIST-CNRS - Institut de l'Information Scientifique et TechniqueFRFranc

Analyse de l'environnement et clonage du gène AvrLm4-7 chez l'ascomycète Leptosphaeria maculans

Leptosphaeria maculans est un champignon ascomycète qui constitue le principal agent pathogène du colza (Brassica napus), avec lequel il développe des interactions de type gène-pour-gène. L objectif de ces travaux de thèse était le clonage du gène d avirulence AvrLm4-7 appartenant au cluster génétique regroupant les gènes AvrLm3-AvrLm4-AvrLm7-AvrLm9. Une approche de clonage positionnel a permis la définition d une zone de 238 kb abritant le gène AvrLm4-7, qui a été entièrement séquencée. Son analyse bioinformatique a mis en évidence une organisation en isochores riches en bases G+C contenant des gènes de "ménage", et d isochores riches en bases A+T correspondant à une mosaïque de rétrotransposons. Le gène AvrLm4-7 a été identifié au milieu de séquences répétées, et a été validé par complémentation fonctionnelle comme responsable des spécificités d interactions vis-à-vis des résistances Rlm7 et Rlm4. Il code pour une protéine de 143 acides aminés, riche en cystéine, potentiellement sécrétée hors des cellules fongiques, et produite de façon constitutive, avec une sur-expression au cours des stades précoces de l infection. L analyse d isolats naturels de L. maculans a mis en évidence que l acquisition de la double virulence avrLm4-avrLm7 pouvait s effectuer par délétion du gène, insertion d un élément transposable dans la séquence codante, ou inactivation suite à la dégénérescence de la séquence par RIP. Le séquençage du gène chez des isolats AvrLm4-AvrLm7 et avrLm4-AvrLm7 a révélé l existence d un polymorphisme nucléotidique ciblé sur trois bases. Plusieurs éléments indiquent l implication de l une de ces mutations ponctuelles dans la spécificité liée à la résistance Rlm4.Leptosphaeria maculans is the ascomycete fungus responsible for stem canker (blackleg disease) of oilseed rape (Brassica napus). Gene-for-gene interactions are the rule between L. maculans and oilseed rape, with 9 avirulence (AvrLm) genes genetically identified in L. maculans. The AvrLm7 gene belongs to the genetic cluster AvrLm3-AvrLm4-AvrLm7-AvrLm9, and the aim of this PhD thesis was to clone AvrLm7, and to characterize its genomic environment. Map based cloning allowed us to delineate a 238 kb region encompassing AvrLm7. The region was composed of mosaic of G+C-rich and A+T-rich isochors. G+C-rich isochors contained numerous housekeeping genes, whereas A+T-rich isochors corresponded to mosaic of retrotransposons. Following complementation of a virulent isolate, AvrLm7 was isolated within an A+T-rich isochore. The AvrLm7 sequence was shown to induce resistance responses in plants harbouring either Rlm7 or Rlm4, and the gene was therefore renamed AvrLm4-7. It encodes a 143 amino-acids cystein-rich protein, predicted to be secreted outside of the fungus cells. AvrLm4-7 is contitutively expressed, and strongly overexpressed during the early stages of infection. L. maculans natural isolates were analysed to evaluate the mechanisms responsible for the loss of AvrLm7 and AvrLm4. These mechanisms correspond either to deletion of the gene, or inactivation events, i.e., insertion of a transposable element within the coding sequence, or RIP-induced inactivation. Sequencing of alleles in AvrLm4-AvrLm7 and avrLm4-AvrLm7 isolates revealed three main nucleotide polymorphisms, one of these leading to the loss of the AvrLm4.ORSAY-PARIS 11-BU Sciences (914712101) / SudocSudocFranceF

Génomique comparative et évolutive au sein du complexe d'espèces Leptosphaeria maculans-Leptosphaeria biglobosa

Leptosphaeria maculans brassicae (Lmb) est un champignon filamenteux de la classe des Dothideomycètes faisant partie du complexe d espèces Leptosphaeria maculans-Leptosphaeria biglobosa composé d agents pathogènes des crucifères. Lmb est particulièrement adapté au colza (Brassica napus) et provoque la maladie qui lui est la plus dommageable : la nécrose du collet. Dans le but de mieux comprendre et contrôler cette maladie, l équipe d accueil a initié un projet de génomique visant à identifier de façon systématique les gènes impliqués dans le pouvoir pathogène. Les premières données génomiques montraient deux aspects très importants et potentiellement spécifiques de Lmb : (i) tous les gènes d'avirulence caractérisés expérimentalement étaient localisés dans de grandes régions riches en bases AT et composées d'éléments transposables (ET), (ii) ces régions riches en AT préfiguraient une structure génomique particulière, qui, si elle se généralisait à l'ensemble du génome, aurait été totalement inédite chez un micro-organisme eucaryote. La première partie de cette thèse présente la description du génome de Lmb en se focalisant sur sa structure en isochores, résultant d une invasion du génome par des ET qui ont ensuite été inactivés par un mécanisme de défense spécifique aux champignons ascomycètes, le RIP (Repeat-Induced Point mutation). Puis, l impact potentiel de cette structure sur la diversification et l évolution des protéines jouant un rôle clé lors de l interaction agent pathogène-plante a été évalué, mettant ainsi en avant l existence d un génome à deux vitesses . Afin de mieux comprendre le rôle potentiel joué par les ET au niveau des capacités d adaptation de Lmb au colza, une étude de génomique comparative et évolutive de cinq membres du complexe d espèces a été réalisée. Ce travail montre que Lmb est la seule espèce du complexe dont le génome a été envahi par les ET, et que ces derniers sont impliqués dans (i) des réarrangements intrachromosomiques potentiellement liés à la spéciation entre Lmb et l espèce la plus proche, (ii) la présence de gènes espèce-spécifiques et (iii) des déplacements dans des régions génomiques très dynamiques de gènes codant des effecteurs. Les travaux constituant cette thèse participent à la généralisation du concept selon lequel un lien fort existe chez les champignons filamenteux phytopathogènes entre ET et gènes impliqués dans la pathogenèse ou l adaptation à l hôte.Leptosphaeria maculans brassicae (Lmb) is a filamentous ascomycete from class Dothideomycetes. It belongs to the Leptosphaeria maculans-Leptosphaeria biglobosa species complex which comprises pathogens of crucifers. Lmb is specifically adapted to oilseed rape (Brassica napus) and is responsible for the most damaging disease of this crop: stem canker . In order to better understand and control the disease, the host team initiated a genomic project aiming at systematically identify genes involved in pathogenicity, analyse genome plasticity and evaluate their incidence on adaptability to host. Preliminary genome data firstly showed that all characterized avirulence genes were localized in large AT-rich regions, mainly composed of Transposable Elements (TEs). In addition, these AT-rich regions were the first hints that the Lmb genome may present a very unusual structure compared to other microorganisms. The first part of this thesis describes the Lmb genome with a special focus on its isochore structure, which is the result of a massive TE invasion of the genome followed by an inactivation of TEs by an ascomycete-specific defense mechanism called RIP (Repeat-Induced Point mutation). The potential impacts of this genome structure on diversification and evolution of proteins involved in the plant-pathogen interaction were assessed and highlighted the existence of a two speed genome. To better understand how TEs are involved in adaptation of Lmb towards oilseed rape, a comparative and evolutionary genomic analysis of five members of the species complex was conducted. This study shows that Lmb is the only species of the complex with genome invaded by TEs at such an extent, and that TEs are involved in (i) intrachromosomal rearrangements putatively related to the speciation event between Lmb and its closest relative species, (ii) the presence of species-specific genes, (iii) translocations of effector genes into highly dynamic genomic regions. Our data contribute to the generalization of the two speed genome concept in filamentous phytopathogens postulating that highly plastic regions of the genome are enriched in genes involved in niche adaptation and that a strong link exists between TEs and genes involved in pathogenesis or host adaptation.PARIS11-SCD-Bib. électronique (914719901) / SudocSudocFranceF

Caractérisation de mutants de Leptosphaeria maculans obtenus par agrotransformation et affectés dans leur pouvoir pathogène

Leptosphaeria maculans est l ascomycete responsable de la maladie la plus dommageable sur colza, la nécrose du collet. C est un champignon hémibiotrophe au cycle parasitaire complexe et exemplaire des stratégies d infection des Dothidéomycètes. Le déterminisme moléculaire du pouvoir pathogène de L. maculans est actuellement mal connu et seuls deux gènes impliqués dans le pouvoir pathogène étaient décrits et validés au début de cette thèse. De façon à accroître notre compréhension du déterminisme du pouvoir pathogène, une stratégie de mutagenèse insertionnelle aléatoire via Agrobacterium tumefaciens a été mise en place. L intérêt de cette approche pour l identification de mutants affectés dans leur pouvoir pathogène et étiquetés a tout d abord été validé, puis nous avons développé une analyse phénotypique et moléculaire plus détaillée de trois des mutants de la collection.Les trois mutants sélectionnés (m210, m20 et m186) sont non pathogènes et affectés à des étapes différentes du processus infectieux : la phase de germination à la surface de l hôte pour m210, la phase de colonisation biotrophe pour m20 et la transition biotrophie-nécrotrophie pour m186. Pour chacun des mutants, une intégration unique de l ADN-T a été mise en évidence. Dans les trois cas, celle-ci a eu lieu dans des régions promotrices, conduisant à une dérégulation de l expression des gènes adjacents au site d intégration (sur-expression pour m186, réduction de 40% pour m210, sur-expression in vitro et sous-expression in planta pour m20). L implication de chacun des gènes responsables du phénotype mutant, ainsi que leur fonction et leur rôle dans le pouvoir pathogène ont ensuite été analysés par différentes approches (bio-informatique, complémentation fonctionnelle, silencing, etc ).The Ascomycete Leptosphaeria maculans is responsible for the most damaging disease of oilseed rape (Brassica napus), stem canker. This hemibiotrophic fungus possess a complex parasitic cycle and is representative of Dothideomycete infection strategies. The molecular determinism of L. maculans pathogenicity is currently poorly understood with only two pathogenicity genes described and validated at the beginning of this thesis. Thus, the first objective of this PhD was to initiate a random insertional mutagenesis strategy using Agrobacterium tumefaciens-mediated transformation in order to increase our knowledge on L. maculans pathogenicity factors. We first validated the robustness of this approach to identify loss-of pathogenicity tagged mutants, then we performed a more detailed phenotypic and molecular characterisation of three mutants of the collection. The three non pathogenic mutants selected (m20, m210 and m186) are affected in three different steps of the infection process: the germination phase on the host surface for m210, the biotrophic colonisation phase for m20 and the switch between biotrophy and necrotrophy for m186. A single copy integration event of the T-DNA occurred in each mutant. In addition, all integrations took place in promoter regions, leading to a deregulation of the expression of the closest genes (over-expression for m186, 40% reduction of expression for m210, and over-expression in vitro and reduction in planta for m20). Identification of the gene responsible for the mutant phenotype, along with its intrinsic function and role in pathogenicity were then analysed using various approaches (bioinformatics, functional complementation, silencing, etc...).ORSAY-PARIS 11-BU Sciences (914712101) / SudocSudocFranceF

Invasion by Leptosphaeria maculans (phoma stem canker on brassicas) : from genome to worldwide crop

Leptosphaeria maculans and L. biglobosa, which cause phoma stem canker disease, are related pathogens of brassicas that were originally considered as one species but occupy slightly different ecological niches and are now reproductively isolated. Globally, the invasion by the more damaging L. maculans into areas where only L. biglobosa was present occurred in North America in the 1980s and Eastern Europe in the 1990s, whereas there are still areas of the world, such as China, where only the less damaging pathogen L. biglobosa is present. The threat to Chinese oilseed rape production from L. maculans was assessed by using models developed to describe the spread (in space and time) of L. maculans across Alberta, Canada. In addition, the worldwide population of L. biglobosa is much more variable than that of L. maculans. Further evidence is provided by the massive invasion, dated 5-20 million years ago, of the genome of L. maculans by only a few repeated element families. Short-term strategies to prevent occurrence of severe phoma stem canker epidemics in China include training of extension workers to recognise symptoms of the disease and use of PCR-based diagnostics to detect the pathogen on imported seed. Long-term strategies include the introduction of durable QTL-mediated resistance to L. maculans into Chinese oilseed rape cultivars and exploitation of new genomic information about L. maculans and L. biglobosa as a component of an integrated disease management programmeNon peer reviewe