The mitogen-activated protein (MAP) kinase cascade plays an important role in the regulation of plant growth and development as well as biotic and abiotic stress responses. Rice MAP kinase 5 (OsMPK5), an orthologue of Arabidopsis AtMPK3, is an abscisic acid- and stress-inducible kinase that positively regulates rice tolerance to abiotic stresses but negatively modulates host resistance to pathogen infection. However, it is unknown how OsMPK5 regulates downstream signaling components and influence rice biotic and abiotic stress responses. To elucidate the mechanism of OsMPK5-mediated signal transduction in rice, therefore, it is critical to identify its potential protein targets or substrates. In this study, an in-situ solid phase phosphorylation screen was conducted to isolate potential OsMPK5 substrates. A rice protein expression library was screened using purified recombinant OsMPK5 and ³²P γ-ATP. The phosphorylation screen yielded over a dozen putative substrates with potential roles in apoptosis, ion transportation, transcription, phosphorylation and dephosphorylation, etc. Strikingly, many of these putative substrates or their orthologues from other plant species have been previously shown to be involved in biotic and abiotic stress responses. In this study, two of the putative substrates were further characterized by a combination of molecular, biochemical and genetic approaches.

The first putative substrate is a transcription factor called OsEIL1, which is an important signaling component of ethylene signal transduction. In vitro protein binding and kinase assays reveal that OsMPK5 interacts with and phosphorylates OsEIL1 in vitro. In addition, OsMPK5 protein kinase activity is negatively correlated with the OsEIL1 protein level and downstream OsERF1 and OsPR5 expression in transgenic rice. In addition, transgenic analysis showed that OsEIL1 positively regulated OsERF1 and OsPR5 expression and increased rice disease resistance against the rice blast infection. This suggests that OsMPK5 mediates biotic stress responses by negatively regulating OsEIL1 stability and ethylene signaling transduction.

The second interacting protein is a rice calcium-dependent protein kinase (OsCDPK5) which belongs to a large family of protein kinases with diverse roles in plant growth and development as well as biotic and abiotic stress responses. In vitro kinase assays demonstrated the phosphorylation of OsMPK5 by OsCDPK5 in a calcium-dependent manner. The autophosphorylation activity of OsCDPK5 as well as its ability to phosphorylate OsMPK5 were positively regulated by calcium concentration in vitro. Furthermore, OsCDPK5 was found to physically interact with OsMPK5 in vitro and in vivo. RT-PCR and RNA blot analyses showed that the expression of OsCDPK5 in rice was up-regulated by ACC and wounding but down-regulated by ABA. To determine the potential role of OsCDPK5 in plant stress tolerance, transgenic RNAi lines were generated via the Agrobacterium-mediated rice transformation. Similar to the silencing effect of OsMPK5 in rice, suppression of OsCDPK5 enhanced the expression of ethylene-responsive defense genes, suggesting that OsCDPK5 may potentially regulate rice biotic and abiotic stress tolerance.