Protein interactome analysis of 12 mitogen-activated protein kinase kinase kinase in rice using a yeast two-hybrid system.
The mitogen-activated protein kinase (MAPK) cascade is composed at least of MAP3K (for MAPK kinase kinase), MAP2K, and MAPK family modules. These components together play a central role in mediating extracellular signals to the cell and vice versa by interacting with their partner proteins. However, the MAP3K-interacting proteins remain poorly investigated in plants. Here, we utilized a yeast two-hybrid system and bimolecular fluorescence complementation in the model crop rice (Oryza sativa) to map MAP3K-interacting proteins. We identified 12 novel nonredundant interacting protein pairs (IPPs) representing 11 nonredundant interactors using 12 rice MAP3Ks (available as full-length cDNA in the rice KOME (http://cdna01.dna.affrc.go.jp/cDNA/) at the time of experimental design and execution) as bait and a rice seedling cDNA library as prey. Of the 12 MAP3Ks, only six had interacting protein partners. The established MAP3K interactome consisted of two kinases, three proteases, two forkhead-associated domain-containing proteins, two expressed proteins, one E3 ligase, one regulatory protein, and one retrotransposon protein. Notably, no MAP3K showed physical interaction with either MAP2K or MAPK. Seven IPPs (58.3%) were confirmed in vivo by bimolecular fluorescence complementation. Subcellular localization of 14 interactors, together involved in nine IPPs (75%) further provide prerequisite for biological significance of the IPPs. Furthermore, GO of identified interactors predicted their involvement in diverse physiological responses, which were supported by a literature survey. These findings increase our knowledge of the MAP3K-interacting proteins, help in proposing a model of MAPK modules, provide a valuable resource for developing a complete map of the rice MAPK interactome, and allow discussion for translating the interactome knowledge to rice crop improvement against environmental factors.
Department of Molecular Biology, College of Life Sciences, Sejong University, Gunja-dong, Gwangjin-gu, Seoul, Republic of Korea.
Raksha
R
Singh
Jae-Eun
JE
Lee
Sarmina
S
Dangol
Jihyun
J
Choi
Ran Hee
RH
Yoo
Jae Sun
JS
Moon
Jae-Kyung
JK
Shim
Randeep
R
Rakwal
Ganesh Kumar
GK
Agrawal
Nam-Soo
NS
Jwa
D020930:MAP Kinase Kinase Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012275:Oryza / Q000235:genetics* / Q000378:metabolism; D010940:Plant Proteins / Q000032:analysis* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D025941:Protein Interaction Mapping / Q000379:methods*; D040901:Proteomics / Q000379:methods*; D015203:Reproducibility of Results; D020798:Two-Hybrid System Techniques
D010940:Plant Proteins; D020930:MAP Kinase Kinase Kinases; C090757:mitogen-activated protein kinase kinase kinase 12
Interactome; MAP3Ks; Plant proteomics; Protein-protein interactions; Rice; Translational proteomics; Yeast two hybrid