Abstract
BackgroundNitrogen dioxide (NO2) triggers hypersensitive response (HR)-like cell death in Arabidopsis thaliana. A high-throughput mutant screen was established to identify genes involved in this type of programmed cell death.ResultsAltogether 14,282 lines of SALK T-DNA insertion mutants were screened. Growing 1000 pooled mutant lines per tray and simultaneous NO2 fumigation of 4 trays in parallel facilitated high-throughput screening. Candidate mutants were selected based on visible symptoms. Sensitive mutants showed lesions already after fumigation for 1 h with 10 ppm (ppm) NO2 whereas tolerant mutants were hardly damaged even after treatment with 30 ppm NO2. Identification of T-DNA insertion sites by adapter ligation-mediated PCR turned out to be successful but rather time consuming. Therefore, next generation sequencing after T-DNA-specific target enrichment was tested as an alternative screening method. The targeted genome sequencing was highly efficient due to (1.) combination of the pooled DNA from 124 candidate mutants in only two libraries, (2.) successful target enrichment using T-DNA border-specific 70mer probes, and (3.) stringent filtering of the sequencing reads. Seventy mutated genes were identified by at least 3 sequencing reads. Ten corresponding mutants were re-screened of which 8 mutants exhibited NO2-sensitivity or -tolerance confirming that the screen yielded reliable results. Identified candidate genes had published functions in HR, pathogen resistance, and stomata regulation.ConclusionsThe presented NO2 dead-or-alive screen combined with next-generation sequencing after T-DNA-specific target enrichment was highly efficient. Two researchers finished the screen within 3 months. Moreover, the target enrichment approach was cost-saving because of the limited number of DNA libraries and sequencing runs required. The experimental design can be easily adapted to other screening approaches e.g. involving high-throughput treatments with abiotic stressors or phytohormones.
Highlights
Nitrogen dioxide (NO2) triggers hypersensitive response (HR)-like cell death in Arabidopsis thaliana
The exposure chamber allowed NO2 treatment of 4 trays in parallel, i.e. fumigation of all 14 trays with one Identification of mutated genes by adapter ligationmediated PCR Adapter ligation-mediated PCR is the standard method employed by the SALK institute to define T-DNA insertion sites [6]
It is based on digestion of the DNA by the restriction enzyme Ase1, Ase adapter ligation, and selective amplification of T-DNA-containing fragments using the T-DNA left border-specific primer LBb1 and the adapter-specific primer AP2 before sequencing
Summary
Nitrogen dioxide (NO2) triggers hypersensitive response (HR)-like cell death in Arabidopsis thaliana. Several PCR-based methods such as the adapter ligation-mediated PCR rely on insert-specific primers to selectively amplify the T-DNA/genomic DNA junction before identification of the mutated gene by sequencing [6]. Insert specific biotinylated 70mer probes can be employed for the isolation of T-DNAcontaining DNA fragments by streptavidin beads [7]. Combining this target enrichment with generation sequencing (NGS) allowed the simultaneous identification of multiple insertion sites in a complex pool of DNA from as much as 64 different mutants [7]. Adapter ligation-mediated PCR and target enrichment followed by NGS (i.e. targeted genome sequencing) are suitable for high-throughput mappings of mutation sites [6, 7]
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