Minisymposium 13: Membrane Transport

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Abs # M1304: Plant cell Ca²⁺ currents and downstream nitric oxide generation: unraveling a plant innate immunity signaling cascade

Presenter:	Berkowitz, Gerald       Contact Presenter
Authors	Ali, Rashid  (A)   Tsaltas, Dimitrios  (A)   Lemtiri-Chlieh, Fouad  (A)   Leng, Qiang  (A)   von Bodman, Susanne  (A)   Berkowitz, Gerald  (A)
Affiliations:	(A): University Of Connecticut

A symphony of cytosolic signaling molecules (including Ca²⁺, nitric oxide (NO), cyclic nucleotides (cNMP), and calmodulin (CaM)) have been suggested as early components of the hypersensitive response (HR) to pathogen infection. However, specific interactions amongst these cytosolic secondary messengers, and delineation of their roles in the signal cascade are at present unclear. Here, we provide insight into how plants translate perception of a pathogen into a signal cascade leading to an innate immune response, and identify new steps in this process. We show that the cyclic nucleotide gated channel (CNGC) AtCNGC2 conducts Ca²⁺ across the plant cell membrane, and provide a model linking this Ca²⁺ current to downstream NO production. NO is a critical signaling molecule invoking plant innate immune response to pathogens. The CNGC blocker Gd³⁺ reduced plant HR to avirulent pathogen. Patch clamp studies demonstrated the presence of a cNMP-dependent inward Ca²⁺ current in wild type (WT) arabidopsis that was not present in cells of a AtCNGC2 loss-of-function mutant. We found that addition of an NO donor overcame the absence of HR in the AtCNGC2 mutant, indicating lack of innate immune response to avirulent pathogens in this mutant is due to a block of NO generation downstream from the AtCNGC2-dependent Ca²⁺ signal. Patch clamp recordings of WT arabidopsis guard cells demonstrated that the bacterial elicitor lipopolysaccharide (LPS) activates a CNGC Ca²⁺ current. Monitoring of in vivo NO generation in WT leaf guard cells using a fluorescent dye indicated that this pathogen-induced cell membrane inward Ca²⁺ current is linked to NO generation due to build up of cytosolic Ca²⁺/CaM, leading to activation of AtNOS1, an enzyme involved in NO synthesis. The same rise in cytosolic Ca²⁺/CaM in response to LPS that activates AtNOS1 leads to a down-regulation of the CNGC channel, restricting inward Ca²⁺ current. This interplay of cytsolic secondary molecules, then, allows for the generation of a cellular Ca²⁺ spike involved in this signaling pathway. A model will be presented detailing these newly identified steps in the plant innate immunity signaling pathway. Supported by NSF awards 0344141 and 0211687.