Rice mitogen-activated protein kinase interactome analysis using the yeast two-hybrid system.
Mitogen-activated protein kinase (MAPK) cascades support the flow of extracellular signals to intracellular target molecules and ultimately drive a diverse array of physiological functions in cells, tissues, and organisms by interacting with other proteins. Yet, our knowledge of the global physical MAPK interactome in plants remains largely fragmented. Here, we utilized the yeast two-hybrid system and coimmunoprecipitation, pull-down, bimolecular fluorescence complementation, subcellular localization, and kinase assay experiments in the model crop rice (Oryza sativa) to systematically map what is to our knowledge the first plant MAPK-interacting proteins. We identified 80 nonredundant interacting protein pairs (74 nonredundant interactors) for rice MAPKs and elucidated the novel proteome-wide network of MAPK interactors. The established interactome contains four membrane-associated proteins, seven MAP2Ks (for MAPK kinase), four MAPKs, and 59 putative substrates, including 18 transcription factors. Several interactors were also validated by experimental approaches (in vivo and in vitro) and literature survey. Our results highlight the importance of OsMPK1, an ortholog of tobacco (Nicotiana benthamiana) salicyclic acid-induced protein kinase and Arabidopsis (Arabidopsis thaliana) AtMPK6, among the rice MAPKs, as it alone interacts with 41 unique proteins (51.2% of the mapped MAPK interaction network). Additionally, Gene Ontology classification of interacting proteins into 34 functional categories suggested MAPK participation in diverse physiological functions. Together, the results obtained essentially enhance our knowledge of the MAPK-interacting protein network and provide a valuable research resource for developing a nearly complete map of the rice MAPK interactome.
Department of Molecular Biology, College of Life Sciences, Sejong University, Gunja-dong, Gwangjin-gu, Seoul 143-747, Republic of Korea.
Raksha
R
Singh
Mi-Ok
MO
Lee
Jae-Eun
JE
Lee
Jihyun
J
Choi
Ji Hun
JH
Park
Eun Hye
EH
Kim
Ran Hee
RH
Yoo
Jung-Il
JI
Cho
Jong-Seong
JS
Jeon
Randeep
R
Rakwal
Ganesh Kumar
GK
Agrawal
Jae Sun
JS
Moon
Nam-Soo
NS
Jwa
D016960:Agrobacterium tumefaciens / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins / Q000378:metabolism; D004789:Enzyme Activation; D057075:Enzyme Assays / Q000379:methods; D015723:Gene Library; D057166:High-Throughput Screening Assays; D047468:Immunoprecipitation; D008164:Luminescent Proteins / Q000378:metabolism; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D020928:Mitogen-Activated Protein Kinases / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D019697:Onions / Q000378:metabolism; D012275:Oryza / Q000201:enzymology* / Q000235:genetics; D010766:Phosphorylation; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism; D025941:Protein Interaction Mapping / Q000379:methods; D060066:Protein Interaction Maps; D020543:Proteome / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014026:Nicotiana / Q000235:genetics / Q000378:metabolism; D014157:Transcription Factors; D020798:Two-Hybrid System Techniques
D001426:Bacterial Proteins; D008164:Luminescent Proteins; D008565:Membrane Proteins; D010940:Plant Proteins; D020543:Proteome; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; C054562:yellow fluorescent protein, Bacteria; D020928:Mitogen-Activated Protein Kinases