Expressed Sequence Tags from the oomycete Plasmopara halstedii, an obligate parasite of the sunflower

<p>Abstract</p> <p>Background</p> <p>Sunflower downy mildew is a major disease caused by the obligatory biotrophic oomycete <it>Plasmopara halstedii</it>. Little is known about the molecular mechanisms underlying its pathogenicity. In this study we used a genomics approach to gain a first insight into the transcriptome of <it>P. halstedii</it>.</p> <p>Results</p> <p>To identify genes from the obligatory biotrophic oomycete <it>Plasmopara halstedii </it>that are expressed during infection in sunflower (<it>Helianthus annuus </it>L.) we employed the suppression subtraction hybridization (SSH) method from sunflower seedlings infected by <it>P. halstedii</it>. Using this method and random sequencing of clones, a total of 602 expressed sequence tags (ESTs) corresponding to 230 unique sequence sets were identified. To determine the origin of the unisequences, PCR primers were designed to amplify these gene fragments from genomic DNA isolated either from <it>P. halstedii </it>sporangia or from <it>Helianthus annuus</it>. Only 145 nonredundant ESTs which correspond to a total of 373 ESTs (67.7%) proved to be derived from <it>P. halstedii </it>genes and that are expressed during infection in sunflower. A set of 87 nonredundant sequences were identified as showing matches to sequences deposited in public databases. Nevertheless, about 7% of the ESTs seem to be unique to <it>P. halstedii </it>without any homolog in any public database.</p> <p>Conclusion</p> <p>A summary of the assignment of nonredundant ESTs to functional categories as well as their relative abundance is listed and discussed. Annotation of the ESTs revealed a number of genes that could function in virulence. We provide a first glimpse into the gene content of <it>P. halstedii</it>. These resources should accelerate research on this important pathogen.</p

A major invasion of transposable elements accounts for the large size of the Blumeria graminis f.sp. tritici genome

Powdery mildew of wheat (Triticum aestivum L.) is caused by the ascomycete fungus Blumeria graminis f.sp. tritici. Genomic approaches open new ways to study the biology of this obligate biotrophic pathogen. We started the analysis of the Bg tritici genome with the low-pass sequencing of its genome using the 454 technology and the construction of the first genomic bacterial artificial chromosome (BAC) library for this fungus. High-coverage contigs were assembled with the 454 reads. They allowed the characterization of 56 transposable elements and the establishment of the Blumeria repeat database. The BAC library contains 12,288 clones with an average insert size of 115kb, which represents a maximum of 7.5-fold genome coverage. Sequencing of the BAC ends generated 12.6Mb of random sequence representative of the genome. Analysis of BAC-end sequences revealed a massive invasion of transposable elements accounting for at least 85% of the genome. This explains the unusually large size of this genome which we estimate to be at least 174Mb, based on a large-scale physical map constructed through the fingerprinting of the BAC library. Our study represents a crucial step in the perspective of the determination and study of the whole Bg tritici genome sequenc

Cross-Kingdom RNAi of Pathogen Effectors Leads to Quantitative Adult Plant Resistance in Wheat

Cross-kingdom RNA interference (RNAi) is a biological process allowing plants to transfer small regulatory RNAs to invading pathogens to trigger the silencing of target virulence genes. Transient assays in cereal powdery mildews suggest that silencing of one or two effectors could lead to near loss of virulence, but evidence from stable RNAi lines is lacking. We established transient host-induced gene silencing (HIGS) in wheat, and demonstrate that targeting an essential housekeeping gene in the wheat powdery mildew pathogen (Blumeria graminis f. sp. tritici) results in significant reduction of virulence at an early stage of infection. We generated stable transgenic RNAi wheat lines encoding a HIGS construct simultaneously silencing three B.g. tritici effectors including SvrPm3(a1/f1), a virulence factor involved in the suppression of the Pm3 powdery mildew resistance gene. We show that all targeted effectors are effectively downregulated by HIGS, resulting in reduced fungal virulence on adult wheat plants. Our findings demonstrate that stable HIGS of effector genes can lead to quantitative gain of resistance without major pleiotropic effects in wheat

Hybridization of powdery mildew strains gives rise to pathogens on novel agricultural crop species

Throughout the history of agriculture, many new crop species (polyploids or artificial hybrids) have been introduced to diversify products or to increase yield. However, little is known about how these new crops influence the evolution of new pathogens and diseases. Triticale is an artificial hybrid of wheat and rye, and it was resistant to the fungal pathogen powdery mildew (Blumeria graminis) until 2001 (refs. 1,2,3). We sequenced and compared the genomes of 46 powdery mildew isolates covering several formae speciales. We found that B. graminis f. sp. triticale, which grows on triticale and wheat, is a hybrid between wheat powdery mildew (B. graminis f. sp. tritici) and mildew specialized on rye (B. graminis f. sp. secalis). Our data show that the hybrid of the two mildews specialized on two different hosts can infect the hybrid plant species originating from those two hosts. We conclude that hybridization between mildews specialized on different species is a mechanism of adaptation to new crops introduced by agriculture

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

Recognition of Phytophthora elicitins and of the Ralstonia solanacearum PopA protein in tobacco

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Phytophthora elicitins and the Ralstonia solanacearum PopA protein elicit the hypersensitive response in tobacco through different recognition mechanisms

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