Intragenic suppressor of Osiaa23 revealed a conserved tryptophan residue crucial for protein-protein interactions.
The Auxin/Indole-3-Acetic Acid (Aux/IAA) and Auxin Response Factor (ARF) are two important families that play key roles in auxin signal transduction. Both of the families contain a similar carboxyl-terminal domain (Domain III/IV) that facilitates interactions between these two families. In spite of the importance of protein-protein interactions among these transcription factors, the mechanisms involved in these interactions are largely unknown. In this study, we isolated six intragenic suppressors of an auxin insensitive mutant, Osiaa23. Among these suppressors, Osiaa23-R5 successfully rescued all the defects of the mutant. Sequence analysis revealed that an amino acid substitution occurred in the Tryptophan (W) residue in Domain IV of Osiaa23. Yeast two-hybrid experiments showed that the mutation in Domain IV prevents the protein-protein interactions between Osiaa23 and OsARFs. Phylogenetic analysis revealed that the W residue is conserved in both OsIAAs and OsARFs. Next, we performed site-specific amino acid substitutions within Domain IV of OsARFs, and the conserved W in Domain IV was exchanged by Serine (S). The mutated OsARF(WS)s can be released from the inhibition of Osiaa23 and maintain the transcriptional activities. Expression of OsARF(WS)s in Osiaa23 mutant rescued different defects of the mutant. Our results suggest a previously unknown importance of Domain IV in both families and provide an indirect way to investigate functions of OsARFs.
College of Life and Environmental Sciences, Hangzhou Normal University, Hangzhou, China ; State Key Laboratory of Plant Physiology and Biochemistry, College of Life Science, Zhejiang University, Hangzhou, China.
Jun
J
Ni
State Key Laboratory of Plant Physiology and Biochemistry, College of Life Science, Zhejiang University, Hangzhou, China ; Agricultural Crops Molecular Improving Lab, Liaoning Academy of Agricultural Sciences, Shenyang, China.
Zhenxing
Z
Zhu
Agricultural Crops Molecular Improving Lab, Liaoning Academy of Agricultural Sciences, Shenyang, China.
Gaohang
G
Wang
College of Life and Environmental Sciences, Hangzhou Normal University, Hangzhou, China.
Yanxia
Y
Shen
College of Life and Environmental Sciences, Hangzhou Normal University, Hangzhou, China.
Yanyan
Y
Zhang
State Key Laboratory of Plant Physiology and Biochemistry, College of Life Science, Zhejiang University, Hangzhou, China.
Ping
P
Wu
D016297:Mutagenesis, Site-Directed; D009154:Mutation; D012275:Oryza / Q000235:genetics*; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism*; D030821:Plants, Genetically Modified; D016133:Polymerase Chain Reaction; D011485:Protein Binding; D014364:Tryptophan / Q000378:metabolism*; D020798:Two-Hybrid System Techniques
D010940:Plant Proteins; D014364:Tryptophan