The broad, long-term objective of this proposal is to characterize the roles of plant nitric oxide synthase (NOS) and its product NO in plant disease resistance. Having recently obtained a highly purified tobacco NOS-like activity, cloned the corresponding gene from Arabidopsis (canP), and demonstrated that its encoded protein (a variant P subunit of glycine decarboxylase) has NO synthesizing activity, the role of NOS and NO in disease resistance can be addressed rigorously. This will be accomplished by assessing resistance and defense gene expression in mutant or transgenic Arabidopsis and tobacco that overexpress or underexpress/lack the canP or NOS/canP gene. The regulation of variant P subunit expression will be assessed at the transcriptional and post-translational levels; whether its activity is regulated by interaction with other proteins also will be determined. Through genetic analyses with Arabidopsis mutants or transgenic plants with altered defense responses, canP (and NO production) will be positioned within the defense signaling pathway(s) relative to other signals or components. By rigorously establishing NO's importance in plant disease resistance, characterizing the enzyme and corresponding Arabidopsis and tobacco P subunit genes responsible for pathogen-induced NO production, and deciphering NO's position in the defense response pathway(s), important insights should be gained into plant signal transduction and disease resistance. These findings also may facilitate the development of new strategies for plant disease control, which, by reducing the use of pesticides, should benefit both human and environmental health. Moreover, elucidating the molecular mechanism through which the variant P subunit gene is activated in plants resisting infection will likely have a significant impact on our understanding of innate immunity in animals, including humans. This assumption is based on a growing body of evidence indicating that parts of the innate immune system are highly conserved among vertebrates, invertebrates and plants (see Blc). Furthermore, characterization and comparison of plant NOS's with their animal counterparts should provide valuable insights into how these enzymes work and how their activity is regulated.