P0386 Identification of key genes involved in cell death and defense responses to pathogens in Arabidopsis using Next Generation Sequencing Strategy and the lesion mimic mutant vad1

Mehdi Khafif , CNRS-INRA, castanet-tolosan, France
The molecular mechanisms underlying programmed cell death in plants are still poorly understood, especially those related to the Hypersensitive Response, a form of programmed cell death associated with disease resistance. We previously identified an Arabidopsis lesion mimic mutant vad1 (for vascular associated death) which exhibits propagative HR-like lesions along the vascular system, and presents an increased resistance to different strains of Pseudomonas. VAD1 might constitute a new component of the signaling pathways leading to HR/resistance. Using a combination of molecular, genetic and biochemical approaches, we previously showed that VAD1 could act as an integrative node in hormonal signaling, with ET acting in concert with SA, and as a positive regulator of cell death propagation (Lorrain et al., 2004; Bouchez et al., 2007). This mutant has been used as the starting point for the identification of new actors of cell death pathways, by search for suppressors of vad1-associated phenotypes (svd). 27 mutants were isolated: 15 without any lesions, 12 with different lesions. Use of Next-Generation Sequencing (NGS) technologies allows to map mutations responsible for phenotypes of interest. Thus comparisons were made between svd118, svd142 and Ws-4 (wild type), after a 100 bp paired-end sequencing, More than 30 Gigabases were generated using an Illumina® HiSeq2000® sequencer. For each line and an average coverage between 30 and 80X was obtained. We generated WS-4 reference contigs a combination of SOAPdenovo and velvet assemblers. Comparison between svd mutant sequences and WS-4 led to the identification of a few candidate mutations using SHORE tools.