Forty-one rice cDNAs encoding protein kinases were fused to the tandem affinity purification (TAP) tag and expressed in transgenic rice plants. The TAP-tagged kinases and interacting proteins were purified from the T1 progeny of the transgenic rice plants and identified by mass spectrometry. Ninety-five percent of the TAP-tagged kinases were recovered. Fifty-six percent of the TAP-tagged kinases were found to interact with other rice proteins. A number of these interactions were consistent with known protein complexes found in other species, validating the TAP-tag method in rice plants. Phosphorylation sites were identified on four of the kinases that interacted with either 14-3-3 proteins or cyclins.

Eighty-eight rice (Oryza sativa) cDNAs encoding rice leaf expressed protein kinases (PKs) were fused to a Tandem Affinity Purification tag (TAP-tag) and expressed in transgenic rice plants. The TAP-tagged PKs and interacting proteins were purified from the T1 progeny of the transgenic rice plants and identified by tandem mass spectrometry. Forty-five TAP-tagged PKs were recovered in this study and thirteen of these were found to interact with other rice proteins with a high probability score. In vivo phosphorylated sites were found for three of the PKs. A comparison of the TAP-tagged data from a combined analysis of 129 TAP-tagged rice protein kinases with a concurrent screen using yeast two hybrid methods identified an evolutionarily new rice protein that interacts with the well conserved cell division cycle 2 (CDC2) protein complex.

Plants uniquely contain large numbers of protein kinases, and for the vast majority of the 1,429 kinases predicted in the rice (Oryza sativa) genome, little is known of their functions. Genetic approaches often fail to produce observable phenotypes; thus, new strategies are needed to delineate kinase function. We previously developed a cost-effective high-throughput yeast two-hybrid system. Using this system, we have generated a protein interaction map of 116 representative rice kinases and 254 of their interacting proteins. Overall, the resulting interaction map supports a large number of known or predicted kinase-protein interactions from both plants and animals and reveals many new functional insights. Notably, we found a potential widespread role for E3 ubiquitin ligases in pathogen defense signaling mediated by receptor-like kinases, particularly by the kinases that may have evolved from recently expanded kinase subfamilies in rice. We anticipate that the data provided here will serve as a foundation for targeted functional studies in rice and other plants. The application of yeast two-hybrid and TAPtag analyses for large-scale plant protein interaction studies is also discussed.