Characterization of mutants impaired in Piriformospora indica-induced cytoplasmic calcium elevation in Arabidopsis roots

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A 129 D compound from the root-colonizing beneficial endophyte Piriformospora indica (129Piri), which mimics growth promotion of the fungus, induces cytoplasmic Ca2+ ([Ca2+]cyt) elevation and phosphatidic acid (PA) biosynthesis in Arabidopsis roots. Using transgenic Arabidopsis expressing the Ca2+ sensor aequorin, we isolated EMS mutants impaired in [Ca2+]cyt elevation to 129Piri. The Ca2+ response to flg22 is not impaired. Two classes of mutants are investigated: cam1 (ca2+ mutant1) completely lacks [Ca2+]cyt elevation in response to 129Piri, but also to the protein toxin of Alternaria brassicae (toxAlt). cam1 plants do not respond to P. indica and are highly susceptible to A. brassicae infections. caf (ca2+ friendly mutants) show biphasic [Ca2+]cyt elevation patterns in response to 129Piri, but a wildtype (WT) response to toxAlt. The aims are: (a) Chemical synthesis of 129Piri and analysis in WT and mutants, (b) continuation of map-based cloning of cam1, (c) analysis of 129Piri- and toxAlt-induced responses in cam1 including a comparative microarray analysis, (d) molecular and genetic characterization of three caf mutants, (e) MAP-based cloning of a caf gene. (f) We will test whether PA synthesis can be stimulated in the Ca2+ mutants by 129Piri and toxAlt, (g) and whether shingolipids and plastids (CAS protein) control [Ca2+]cyt elevation.