Fluorescence-microscopy Screening and Next-generation Sequencing: Useful Tools for the Identification of Genes Involved in Organelle Integrity

Abstract

This protocol describes a fluorescence microscope-based screening of Arabidopsis seedlings and describes how to map recessive mutations that alter the subcellular distribution of a specific tagged fluorescent marker in the secretory pathway. Arabidopsis is a powerful biological model for genetic studies because of its genome size, generation time, and conservation of molecular mechanisms among kingdoms. The array genotyping as an approach to map the mutation in alternative to the traditional method based on molecular markers is advantageous because it is relatively faster and may allow the mapping of several mutants in a really short time frame. This method allows the identification of proteins that can influence the integrity of any organelle in plants. Here, as an example, we propose a screen to map genes important for the integrity of the endoplasmic reticulum (ER). Our approach, however, can be easily extended to other plant cell organelles (for example see 1,2 ), and thus represents an important step toward understanding the molecular basis governing other subcellular structures. Video LinkThe video component of this article can be found at http://www.jove.com/video/3809/ Protocol EMS TreatmentArabidopsis thaliana seeds are mutagenized using as mutagen agent ethyl methane sulfonate (EMS) 3,4 , which induces into the genome C-to-T changes resulting in C/G to T/A mutations 5-7 . Confocal Screening of M2 and M3 PopulationsIn this section we describe the observation of seedlings with a confocal or fluorescence microscope as described earlier 8 1. Weigh 0.8 g Arabidopsis seeds (~40,000 seeds) carrying the organelle fluorescent marker (specifically, in this study ssGFPHDEL (signal sequence-GFP-HDEL tetrapeptide) has been used as an ER marker). 2. Transfer the seeds in a 50-ml falcon tube, then add 25 ml distilled water. 3. Add 0.2% (v/v) ethyl methane sulfonate. 4. Incubate for 16 hours on nutating mixer at low speed. 5. Aspirate the liquid and discard it into a flask containing 1.0 M NaOH to inactivate the EMS. 6. Add 25 ml water to the Falcon tube containing the seeds, close, and invert 5 times to wash the seeds; wait until all the seeds have settled, then aspirate the water and discard into the 1.0 M NaOH flask. 7. Repeat the washing step up to 10 times. 8. After the final wash, re-suspend the seeds in a minimal amount of water. 9. Proceed to seed sterilization adding 25 mL of 10 % bleach, shake vigorously for 30 s. Let seeds settle to bottom, then pour off bleach and rinse with 25 mL of sterile water. Pour off the sterile water and add 25 mL 70 % ethanol. Shake tubes for 30 s. Let seeds settle to bottom. Pour off ethanol and rinse with 25 mL of sterile water. Repeat twice the wash with sterile water, then pour seeds on a plastic Petri dish containing 3 MM filter paper and let dry under the hood. Store at 4°C for 2 days. 10. Plate the seeds on ½ MS phytagel (half concentration of Murashige and Skoog medium), 150-mm Petri dish (~250-300 seeds for plate). 11. Grow M1 seeds on plate for 2 weeks, then tr...

Golgi Traffic and Integrity Depend on N-Myristoyl Transferase-1 in Arabidopsis

“…To identify the mutation responsible for the abnormal phenotype, we crossed STINGY with Landsberg erecta (Ler) to generate a mapping population that was used for rough mapping with a GeneChip Arabidopsis ATH1 genome array strategy (Borevitz, 2006;Stefano et al, 2012aStefano et al, , 2012b) (see Methods). The mutation was mapped on chromosome 5 between 22.9 and 24.6 Mb.…”

“…The mutation was mapped on chromosome 5 between 22.9 and 24.6 Mb. Subsequently, we sequenced the genomic DNA of STINGY by next-generation sequencing, as described earlier (Marti et al, 2010;Stefano et al, 2012aStefano et al, , 2012b. Within the rough-mapped region, we identified only one typical G/C to A/T EMS transition (Maple and Møller, 2007).…”

“…The leaf discs were grouped in clusters of five and processed for genomic DNA extraction. The genomic DNA was then quantified (Borevitz, 2006) and prepared for GeneChip Arabidopsis ATH1 hybridization for rough mapping, as described earlier (Stefano et al, 2012a). Identification of the single nucleotide polymorphisms in the rough-mapped region was achieved by Next Generation genome sequencing, as described previously (Marti et al, 2010), using an Illumina Genome Analyzer II (Bentley et al, 2008).…”

Nanoparticles in Dentistry: Evidence and Future

“…Nanoparticles can be used for the delivery of antibiotics and anti-inflammatory agents directly within the subgingival environment. However, only minimal research has been conducted to test its effectiveness in eliminating bacteria and toxicity in the periodontium, none of which have researched the penetration through the epithelial cell barrier [18][19][20].…”