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Versatile gene-specific sequence tags for Arabidopsis functional genomics: transcript profiling and reverse genetics applications.

ABSTRACT: Microarray transcript profiling and RNA interference are two new technologies crucial for large-scale gene function studies in multicellular eukaryotes. Both rely on sequence-specific hybridization between complementary nucleic acid strands, inciting us to create a collection of gene-specific sequence tags (GSTs) representing at least 21,500 Arabidopsis genes and which are compatible with both approaches. The GSTs were carefully selected to ensure that each of them shared no significant similarity with any other region in the Arabidopsis genome. They were synthesized by PCR amplification from genomic DNA. Spotted microarrays fabricated from the GSTs show good dynamic range, specificity, and sensitivity in transcript profiling experiments. The GSTs have also been transferred to bacterial plasmid vectors via recombinational cloning protocols. These cloned GSTs constitute the ideal starting point for a variety of functional approaches, including reverse genetics. We have subcloned GSTs on a large scale into vectors designed for gene silencing in plant cells. We show that in planta expression of GST hairpin RNA results in the expected phenotypes in silenced Arabidopsis lines. These versatile GST resources provide novel and powerful tools for functional genomics.

SUBMITTER: Hilson P

PROVIDER: S-EPMC528935 | biostudies-literature | 2004 Oct

REPOSITORIES: biostudies-literature

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Versatile gene-specific sequence tags for Arabidopsis functional genomics: transcript profiling and reverse genetics applications.

Hilson Pierre P Allemeersch Joke J Altmann Thomas T Aubourg Sébastien S Avon Alexandra A Beynon Jim J Bhalerao Rishikesh P RP Bitton Frédérique F Caboche Michel M Cannoot Bernard B Chardakov Vasil V Cognet-Holliger Cécile C Colot Vincent V Crowe Mark M Darimont Caroline C Durinck Steffen S Eickhoff Holger H de Longevialle Andéol Falcon AF Farmer Edward E EE Grant Murray M Kuiper Martin T R MT Lehrach Hans H Léon Céline C Leyva Antonio A Lundeberg Joakim J Lurin Claire C Moreau Yves Y Nietfeld Wilfried W Paz-Ares Javier J Reymond Philippe P Rouzé Pierre P Sandberg Goran G Segura Maria Dolores MD Serizet Carine C Tabrett Alexandra A Taconnat Ludivine L Thareau Vincent V Van Hummelen Paul P Vercruysse Steven S Vuylsteke Marnik M Weingartner Magdalena M Weisbeek Peter J PJ Wirta Valtteri V Wittink Floyd R A FR Zabeau Marc M Small Ian I

Genome research 20041001 10B

Microarray transcript profiling and RNA interference are two new technologies crucial for large-scale gene function studies in multicellular eukaryotes. Both rely on sequence-specific hybridization between complementary nucleic acid strands, inciting us to create a collection of gene-specific sequence tags (GSTs) representing at least 21,500 Arabidopsis genes and which are compatible with both approaches. The GSTs were carefully selected to ensure that each of them shared no significant similari ...[more]

PMID: 15489341

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Project description:Cyst nematodes are devastating plant parasites that become sedentary within plant roots and induce the transformation of normal plant cells into elaborate feeding cells with the help of secreted effectors, the parasitism proteins. These proteins are the translation products of parasitism genes and are secreted molecular tools that allow cyst nematodes to infect plants.We present here the expression patterns of all previously described parasitism genes of the soybean cyst nematode, Heterodera glycines, in all major life stages except the adult male. These insights were gained by analyzing our gene expression dataset from experiments using the Affymetrix Soybean Genome Array GeneChip, which contains probeset sequences for 6,860 genes derived from preparasitic and parasitic H. glycines life stages. Targeting the identification of additional H. glycines parasitism-associated genes, we isolated 633 genes encoding secretory proteins using algorithms to predict secretory signal peptides. Furthermore, because some of the known H. glycines parasitism proteins have strongest similarity to proteins of plants and microbes, we searched for predicted protein sequences that showed their highest similarities to plant or microbial proteins and identified 156 H. glycines genes, some of which also contained a signal peptide. Analyses of the expression profiles of these genes allowed the formulation of hypotheses about potential roles in parasitism. This is the first study combining sequence analyses of a substantial EST dataset with microarray expression data of all major life stages (except adult males) for the identification and characterization of putative parasitism-associated proteins in any parasitic nematode.We have established an expression atlas for all known H. glycines parasitism genes. Furthermore, in an effort to identify additional H. glycines genes with putative functions in parasitism, we have reduced the currently known 6,860 H. glycines genes to a pool of 788 most promising candidate genes (including known parasitism genes) and documented their expression profiles. Using our approach to pre-select genes likely involved in parasitism now allows detailed functional analyses in a manner not feasible for larger numbers of genes. The generation of the candidate pool described here is an important enabling advance because it will significantly facilitate the unraveling of fascinating plant-animal interactions and deliver knowledge that can be transferred to other pathogen-host systems. Ultimately, the exploration of true parasitism genes verified from the gene pool delineated here will identify weaknesses in the nematode life cycle that can be exploited by novel anti-nematode efforts.

Dataset Information

Versatile gene-specific sequence tags for Arabidopsis functional genomics: transcript profiling and reverse genetics applications.

Publications

Versatile gene-specific sequence tags for Arabidopsis functional genomics: transcript profiling and reverse genetics applications.

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