Pathogen-induced binding of the soybean zinc finger homeodomain proteins GmZF-HD1 and GmZF-HD2 to two repeats of ATTA homeodomain binding site in the calmodulin isoform 4 (GmCaM4) promoter.
Calmodulin (CaM) is involved in defense responses in plants. In soybean (Glycine max), transcription of calmodulin isoform 4 (GmCaM4) is rapidly induced within 30 min after pathogen stimulation, but regulation of the GmCaM4 gene in response to pathogen is poorly understood. Here, we used the yeast one-hybrid system to isolate two cDNA clones encoding proteins that bind to a 30-nt A/T-rich sequence in the GmCaM4 promoter, a region that contains two repeats of a conserved homeodomain binding site, ATTA. The two proteins, GmZF-HD1 and GmZF-HD2, belong to the zinc finger homeodomain (ZF-HD) transcription factor family. Domain deletion analysis showed that a homeodomain motif can bind to the 30-nt GmCaM4 promoter sequence, whereas the two zinc finger domains cannot. Critically, the formation of super-shifted complexes by an anti-GmZF-HD1 antibody incubated with nuclear extracts from pathogen-treated cells suggests that the interaction between GmZF-HD1 and two homeodomain binding site repeats is regulated by pathogen stimulation. Finally, a transient expression assay with Arabidopsis protoplasts confirmed that GmZF-HD1 can activate the expression of GmCaM4 by specifically interacting with the two repeats. These results suggest that the GmZF-HD1 and -2 proteins function as ZF-HD transcription factors to activate GmCaM4 gene expression in response to pathogen.
Park
Hyeong Cheol
HC
Division of Applied Life Science (BK21 program), Plant Molecular Biology and Biotechnology Research Center, Gyeongsang National University, Jinju, 660-701, Korea.
Kim
Man Lyang
ML
Lee
Sang Min
SM
Bahk
Jeong Dong
JD
Yun
Dae-Jin
DJ
Lim
Chae Oh
CO
Hong
Jong Chan
JC
Lee
Sang Yeol
SY
Cho
Moo Je
MJ
Chung
Woo Sik
WS
D000595:Amino Acid Sequence; D001665:Binding Sites; D002147:Calmodulin / Q000235:genetics*; D018076:DNA, Complementary / Q000737:chemistry / Q000302:isolation & purification; D018506:Gene Expression Regulation, Plant; D018398:Homeodomain Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000378:metabolism; D010802:Phylogeny; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D020033:Protein Isoforms / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D013025:Glycine max / Q000235:genetics* / Q000378:metabolism / Q000382:microbiology; D020080:Tandem Repeat Sequences; D015533:Transcriptional Activation; D016335:Zinc Fingers
D002147:Calmodulin; D018076:DNA, Complementary; D018398:Homeodomain Proteins; D009687:Nuclear Proteins; D010940:Plant Proteins; D020033:Protein Isoforms