DGK5ß-derived phosphatidic acid regulates ROS production in plant immunity by stabilizing NADPH oxidase.

In plant immunity, phosphatidic acid (PA) regulates reactive oxygen species (ROS) by binding to respiratory burst oxidase homolog D (RBOHD), an NADPH oxidase responsible for ROS production. Here, we analyze the influence of PA binding on RBOHD activity and the mechanism of RBOHD-bound PA generation. PA binding enhances RBOHD protein stability by inhibiting vacuolar degradation, thereby increasing chitin-induced ROS production. Mutations in diacylglycerol kinase 5 (DGK5), which phosphorylates diacylglycerol to produce PA, impair chitin-induced PA and ROS production. The DGK5 transcript DGK5ß (but not DGK5α) complements reduced PA and ROS production in dgk5-1 mutants, as well as resistance to Botrytis cinerea. Phosphorylation of S506 residue in the C-terminal calmodulin-binding domain of DGK5ß contributes to the activation of DGK5ß to produce PA. These findings suggest that DGK5ß-derived PA regulates ROS production by inhibiting RBOHD protein degradation, elucidating the role of PA-ROS interplay in immune response regulation.

Antepenultimate residue at the C-terminus of NADPH oxidase RBOHD is critical for its function in the production of reactive oxygen species in Arabidopsis.

Production of reactive oxygen species (ROS) is a conserved immune response primarily mediated by NADPH oxidases (NOXs), also known in plants as respiratory burst oxidase homologs (RBOHs). Most microbe-associated molecular patterns (MAMPs) trigger a very fast and transient ROS burst in plants. However, recently, we found that lipopolysaccharides (LPS), a typical bacterial MAMP, triggered a biphasic ROS burst. In this study, we isolated mutants defective in LPS-triggered biphasic ROS burst (delt) in Arabidopsis, and cloned the DELT1 gene that was shown to encode RBOHD. In the delt1-2 allele, the antepenultimate residue, glutamic acid (E919), at the C-terminus of RBOHD was mutated to lysine (K). E919 is a highly conserved residue in NADPH oxidases, and a mutation of the corresponding residue E568 in human NOX2 has been reported to be one of the causes of chronic granulomatous disease. Consistently, we found that residue E919 was indispensable for RBOHD function in the MAMP-induced ROS burst and stomatal closure. It has been suggested that the mutation of this residue in other NADPH oxidases impairs the protein's stability and complex assembly. However, we found that the E919K mutation did not affect RBOHD protein abundance or the ability of protein association, suggesting that the residue E919 in RBOHD might have a regulatory mechanism different from that of other NOXs. Taken together, our results confirm that the antepenultimate residue E is critical for NADPH oxidases and provide a new insight into the regulatory mechanisms of RBOHD.