Abstract
Plasma membrane NADPH oxidase, RBOH (Respiratory Burst Oxidase Homolog), plays a pivotal role in reactive oxygen species (ROS) production in response to pathogen attacks. We indicated that a potato calcium-dependent protein kinase (StCDPK5) activates StRBOHB by direct phosphorylation of the N-terminal region (Kobayashi et al. 2007 Plant Cell). However, how StCDPK5 recognizes StRBOHB as a substrate remains to be defined. Tomato SlCDPK2 was identified as a CDPK that phosphorylates ACC synthase (SlACS2). Agrobacterium-mediated transient expression of SlCDPK2 did not induce StRBOHB-mediated oxidative burst in Nicotiana benthamiana, whereas bimolecular fluorescence complementation (BiFC) analyses showed that both StCDPK5 and SlCDPK2 interact with StRBOHB on plasma membrane. To determine the domains required for StRBOHB substrate specificity, a series of chimeric genes was constructed by reciprocal exchange of domains between StCDPK5 and SlCDPK2. The expression of chimeric genes including both variable and kinase domains of StCDPK5 induced StRBOHB-mediated oxidative burst. These results indicate that variable and kinase domains of StCDPK5 are responsible for activation of StRBOHB.
