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

Abstract

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.

Figure 1.

Identification of a pathogen-responsive protein-binding site in the GmCaM-4 promoter. (A) Schematic diagram of DNA fragments derived from the GmCaM-4 promoter used for gel mobility shift assay (EMSA) and their patterns of binding with nuclear extracts prepared from W82 soybean suspension culture cells treated with MgCl₂ (lanes 2, MgCl₂) or Pseudomonas syringae pv glycinea carrying avrA (Psg. avrA) (lanes 3, Psg. avrA). None (lanes 1) represents free DNA probe. The positions of fragments upstream of the GmCaM4 transcriptional start site are indicated. (B) Oligonucleotides (A2-1–A2-7) corresponding to the GmCaM-4 promoter A2 region that were used as competitors. (C) Competitive gel mobility shift assay. The DNA binding reaction was performed by pre-incubating unlabeled competitors (A2-1–A2-7) and then adding the ³²P-labeled A-2 fragment as a probe. A 100-fold molar excess of competitor DNA was added to each reaction mixture. Free (arrow) and protein-complexed (arrowhead) probes were separated on an 8% polyacrylamide gel and visualized by autoradiography.

Figure 2.

DNase I footprint analysis of the A2 region of the GmCaM-4 promoter bound by pathogen-responsive proteins. End-labeled fragments were digested with DNase I after incubation with 0, 5, 10, 20 or 25 μg nuclear proteins from Psg. avrA-treated W82 soybean cells. The resulting DNA fragments were purified and electrophoresed on a 6% non-denaturing polyacrylamide sequencing gel. GA, G + A sequencing ladder (non-coding strand); lane 1, no nuclear protein; lanes 2–5, 5, 10, 20 and 25 μg nuclear protein, respectively. The DNase I-protected region is indicated by a solid bar and the sequence is shown.

Figure 3.

Isolation of cDNA clones encoding proteins that bind to the A2 region of the GmCaM4 promoter. (A) Strategy for cDNA isolation by selection in yeast. Soybean cDNA libraries constructed in the yeast expression vector pAD-GAL4-2.1 were transformed into YM4271 cells carrying the dual reporter genes HIS3 and lacZ under the control of four tandem repeats of the A2 region of the GmCaM-4 promoter. The yeast expression cDNA libraries encode an N-terminal fusion of the GAL4 activation domain (AD) to soybean proteins. P_minHIS3 indicates the minimal promoter of the HIS3 gene, and P_minCYC1 indicates the CYC1 minimal promoter. P_ADH1 indicates the alcohol dehydrogenase 1 (ADH1) promoter, and T_ADH1 indicates the ADH1 terminator. (B) Specific interactions of GmZF-HD1 and −2 with the A2 region of the GmCaM-4 promoter. Yeast transformants harboring the indicated clones (upper left) were grown in the presence of 20 mM (upper right) or 60 mM (lower left) 3-AT. Nine of the 16 positive clones are shown in this figure. Numbers indicate representative clones as seen in Table 1. The β-galactosidase activity of yeast colonies grown in YPD medium was determined by a filter-lift assay (lower right).

Figure 4.

Amino acid sequence alignment and phylogenetic analysis of GmZF-HD1 and −2. (A) Alignment of the GmZF-HD1 and −2 amino acid sequences with the Flaveria trinervia (FtHB1) and Arabidopsis thaliana (AtHB24 and AtHB26) homeodomain proteins. The highly conserved domains Ia, Ib and II are boxed, identical residues are indicated with white letters in black boxes, dashes indicate the absence of residues, and the trihelix-DNA binding regions of the homeodomain are underlined. (B) Phylogenetic tree of plant homeodomain proteins. The GenBank accession numbers for the following proteins are given in parenthesis: GmHB1 (AW760234), ATHB24 (AC006439), ATHB28 (AL049862.1), ATHB25 (AB011479), OsHB1 (AC007858), FbHB4 (Y18581), FbHB2 (Y18579), FtHB1 (Y18577), FbHB3 (Y18580), ATHB26 (AB011483), ATHB27 (AB007647), BEL1 (U39944), ATH1 (X80126), ATHB2 (X68145), ATHB6 (X67034), PRHA (L21991), GL2 (L32873), ATML1 (U37589), STM (U32344), KN1 (X61308), KNAT3 (X92392).

Figure 5.

Expression pattern of the GmZF-HD1 mRNA in soybean. (A) Northern blot analysis of GmZF-HD1 gene expression in various soybean tissues. (B) Time-course of expression of the GmZF-HD1 transcript after pathogen (Psg. avrA) treatment in soybean suspension cells. Total RNA (20 μg) prepared from apical hypocotyls (Apical), elongating hypocotyls (Elongating), mature hypocotyls (Mature), plumule, seed, root and soybean suspension cells treated with pathogen (Psg. avrA) were separated on a 1.5% formaldehyde/agarose gel, transferred onto a nylon membrane and hybridized with a ³²P-labeled GmZF-HD1 cDNA probe. To verify equal loading of total RNA, rRNAs were visualized by staining with ethidium bromide.

Figure 6.

The DNA-binding abilities of GmZF-HD1 and −2. (A) Schematic representation of GmZF-HD1 and −2 GST fusion constructs. Numbers indicate the positions of boundaries in fusion constructs. Amino acid positions of domain boundaries are indicated. (B) Interaction of the 30-nt sequence containing the A2-2 region of the GmCaM-4 promoter with recombinant GmZF-HD1 and −2 proteins. The ³²P-labeled 30-nt oligonucleotide was incubated in the presence of recombinant GmZF-HD1 or −2 protein or GST alone. All lanes contain 10 μg of each bacterial extract. Free (arrow) and protein-complexed (arrowhead) probes were separated on an 8% polyacrylamide gel and visualized by autoradiography.

Figure 7.

Specific interaction of the GmZF-HD1 protein with 30-nt oligonucleotides derived from the GmCaM4 promoter. (A) Nucleotide sequences of a 30-nt fragment of the GmCaM-4 promoter (WT) and mutated fragments (M1 to M6) used as probes or competitors. (B) DNA-binding ability of recombinant GmZF-HD1 with 30-nt DNA (WT) and mutant fragments (M1 to M6). The radioactive probes were incubated in the presence of recombinant GmZF-HD1 or GST alone. (C) Competitive gel mobility shift assay using 30-nt DNA (WT) and mutated DNA fragments (M1 to M6) as competitors. The DNA binding reaction was performed with recombinant GST-fused GmZF-HD1 protein and ³²P-labeled 30-nt DNA fragment (WT) in the absence or presence of unlabeled competitors. Each competitor DNA (200-fold molar excess) was added to a reaction mixture. Free (arrow) and protein-complexed (arrowhead) probes were separated on an 8% polyacrylamide gel and visualized by autoradiography.

Figure 8.

Direct interaction with and activation of the GmCaM4 promoter by the GmZF-HD1 protein. (A) Gel mobility super-shift assay using an anti-GmZF-HD1 antibody. Various concentrations of polyclonal antibodies raised against the GST::GmZF-HD1 protein were incubated with the protein–DNA complex formed with the ³²P-labeled 30-nt fragment of GmCaM-4 promoter and nuclear extracts from pathogen-treated soybean suspension culture cells. Lane 1 contains preimmune serum, lane 2 contains 1 μg affinity-purified GmZF-HD1 antibody, and lane 3 contains nuclear extracts alone. Nuclear extracts from pathogen-treated cells were preincubated with 0.1 μg (lane 4), 0.3 μg (lane 5), 0.5 μg (lane 6), 0.7 ug (lane 7) or 1 μg (lane 8) affinity-purified GmZF-HD1 antibody before adding the ³²P-labeled 30-nt fragment of GmCaM-4 promoter as a probe. Free (arrow) and protein-complexed (arrowhead) probes were separated on an 8% polyacrylamide gel and visualized by autoradiography. (B) Schematic representation of the effector and reporter constructs used in transient expression assays in Arabidopsis protoplasts. In the effector construct, GmZF-HD1 cDNA was inserted between the CaMV 35S promoter and a Nos terminator (Nos-T). In the reporter construct, four tandem repeats of the A2 region containing the 30-nt fragment of the GmCaM-4 promoter were subcloned upstream of a minimal TATA promoter-GUS construct. (C) Effect of the GmZF-HD1 protein on GmCaM-4 promoter activity in Arabidopsis protoplasts. The empty vector without GmZF-HD1 and the A2 region of the GmCaM-4 promoter were used as controls. For normalization of transformation efficiency, the CaMV 35S promoter-luciferase (LUC) plasmid was co-transfected in each experiment. The data are presented as mean ± SE of results from three independent samples. The numbers show the relative increase in expression compared to the value obtained with the vector control.