Membrane-associated transcripts in Arabidopsis; their isolation and characterization by DNA microarray analysis and bioinformatics.

Membrane-associated, integral membrane and secreted proteins are of key importance in many cellular processes. For most of the 28,952 predicted proteins in Arabidopsis, the actual subcellular localization has not been demonstrated experimentally. So far, their potential membrane-association has been deduced from algorithms that predict transmembrane domains and signal peptides. However, the comprehensiveness and accuracy of these algorithms is still limited. The majority of membrane-associated and secreted proteins is synthesized on membrane-bound polysomes. Therefore, the isolation and characterization of mRNA associated with membrane-bound polysomes offers an experimental tool for the genome-wide identification of these proteins. Here we describe an efficient method to isolate mRNA from membrane-bound polysomes and report on the validation of the method to enrich for transcripts encoding membrane-associated and secreted proteins. The sensitivity and reproducibility of the isolation method was investigated by DNA microarray analysis. Pearson correlations between transcript levels obtained from three replicate isolations showed that the method is highly reproducible. A significant enrichment for mRNAs encoding proteins containing predicted transmembrane domains and signal peptides was observed in the membrane-bound polysomal fraction. In this fraction, 301 transcripts were classified by gene ontologies as 'cellular component unknown', and potentially encode previously unrecognized secreted or membrane-associated proteins.

Identification of arabidopsis loci required for susceptibility to the downy mildew pathogen Hyaloperonospora parasitica.

Plants are susceptible to a limited number of pathogens. Most infections fail due to active defense or absence of compatibility. Many components of the plant's surveillance system and defense arsenal have been identified in the last decades. However, knowledge is limited on compatibility; in particular, the role of plant factors in the infection process. To gain insight into these processes, we have initiated an Arabidopsis thaliana mutant screen for reduced susceptibility to the downy mildew pathogen Hyaloperonospora parasitica. Ethyl methane sulfonate (EMS) mutants were generated in the highly susceptible Arabidopsis line Ler eds1-2. Eight downy mildew-resistant (dmr) mutants were analyzed in detail, corresponding to six different loci. Microscopic analysis showed that, in all mutants, H. parasitica growth was severely reduced. Resistance of dmr3, dmr4, and dmr5 was associated with constitutive expression of PR-1. Furthermore, dmr3 and dmr4, but not dmr5, also were resistant to Pseudomonas syringae and Golovinomyces orontii, respectively. However, enhanced activation of plant defense was not observed in dmr1, dmr2, and dmr6. We postulate that, in these susceptibility mutants, cellular processes are disrupted which are required for H. parasitica infection. This interesting new set of mutants provides a basis to elucidate the molecular processes underlying susceptibility to downy mildew in Arabidopsis.

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

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.