Discoidin domain receptors(DDRs)are single-pass transmembrane proteins belonging to receptor tyrosine kinases(RTKs)family,which are activated by collagen ligands with unusual slow,sustained kinetics,distinguishing the...Discoidin domain receptors(DDRs)are single-pass transmembrane proteins belonging to receptor tyrosine kinases(RTKs)family,which are activated by collagen ligands with unusual slow,sustained kinetics,distinguishing them from canonical RTKs.While DDRs play critical roles in cell adhesion,differentiation,and cancer progression,their activation mechanisms remain partly understood.Here,we investigated the transmembrane domains(TMDs)of DDR1 and DDR2 to elucidate their interaction dynamics in membrane.Using bacterial adenylate cyclase two-hybrid(BACTH)assays,we demonstrated robust homotypic interactions and even stronger heterotypic associations between DDRTMDs.NMR spectroscopy of DDR1TMD and DDR2TMD reconstituted in lipid bilayer-mimetic bicelles showed obvious chemical shift alterations,further validating the stability of their heterocomplex formation.Systematic mutagenesis identified leucine zipper motifs rather than GXXXA motifs mediated both homo-and hetero-associations of DDR1TMD and DDR2TMD.These findings demonstrated the TMD as a critical mediator of DDRs oligomerization and revealed their interaction patterns within membrane.Our study advances the understanding of DDR signaling regulation and highlights transmembrane domain interactions as potential targets for modulating DDR-related pathologies.展开更多
The basic leucine zipper （bZIP） transcription factors form a large gene family that is important in pathogen defense, light and stress signaling, etc. The Completed whole genome sequences of model plants Arabidopsis...The basic leucine zipper （bZIP） transcription factors form a large gene family that is important in pathogen defense, light and stress signaling, etc. The Completed whole genome sequences of model plants Arabidopsis （Arabidopsis thaliana）, rice （Oryza sativa） and poplar （Populus trichocarpa） constitute a valuable resource for genome-wide analysis and genomic comparative analysis, as they are representatives of the two major evolutionary lineages within the angiosperms： the monocotyledons and the dicotyledons. In this study, bioinformatics analysis identified 74, 89 and 88 bZIP genes respectively in Arabidopsis, rice and poplar. Moreover, a comprehensive overview of this gene family is presented, including the gene structure, phylogeny, chromosome distribution, conserved motifs. As a result, the plant bZIPs were organized into 10 subfamilies on basis of phylogenetic relationship. Gene duplication events during the family evolution history were also investigated. And it was further concluded that chromosomal/segmental duplication might have played a key role in gene expansion of bZIP gene family.展开更多
Plant trichomes originate from epidermal cells.In this work,we demonstrated that a homeodomain-leucine zipper(HD-Zip)gene,Gh_A06G1283(Gh HD-1A),was related to the leaf trichome trait in allotetraploid cotton and could...Plant trichomes originate from epidermal cells.In this work,we demonstrated that a homeodomain-leucine zipper(HD-Zip)gene,Gh_A06G1283(Gh HD-1A),was related to the leaf trichome trait in allotetraploid cotton and could be a candidate gene for the T_1 locus.The ortholog of GhHD-1A in the hairless accession Gossypium barbadense cv.Hai7124 was interrupted by a long terminal repeat(LTR)retrotransposon,while GhHD-1A worked well in the hairy accession Gossypium hirsutum acc.T586.Sequence and phylogenetic analysis showed that GhHD-1A belonged to the HD-Zip IV gene family,which mainly regulated epidermis hair development in plants.Silencing of GhHD-1A and its homoeologs GhHD-1D in allotetraploid T586and Hai7124 could significantly reduce the density of leaf hairs and affect the expression levels of other genes related to leaf trichome formation.Further analysis found that GhHD-1A mainly regulated trichome initiation on the upper epidermal hairs of leaves in cotton,while the up-regulated expression of GhHD-1A in different organs/tissues also altered epidermal trichome development.This study not only helps to unravel the important roles of GhHD-1A in regulating trichome initiation in cotton,but also provides a reference for exploring the different forms of trichome development in plants.展开更多
Transcription factors play key roles in plant development and stress responses through their interaction with cis-elements and/or other transcription factors. Homeodomain associated leucine zipper proteins (HD-Zip) co...Transcription factors play key roles in plant development and stress responses through their interaction with cis-elements and/or other transcription factors. Homeodomain associated leucine zipper proteins (HD-Zip) constitute a family of transcription factors that are characterized by the presence of a DNA-binding domain closely linked with leucine zipper motif functioning in dimer formation. This type of association is unique to plants and considered as an excellent candidate to activate developmental responses to altering environmental conditions. Cotton is the most important fiber plant with a lot of local and commercial uses in the world. HD-Zip proteins not only have key roles in different stages of vascular and inter-fascicular fiber differentiation of cotton but also are suggested to have an important role against abiotic stress that is one of the key factors limiting cotton productivity. Plants have developed various strategies to manage stress conditions through a combination of metabolic, physiological and morphological adaptations. These adaptive changes rely largely on alterations in gene expression. Therefore, transcriptional regulators play a crucial role in stress tolerance. Being a transcription factor HD-Zip might be a useful target for genetic engineering to generate multiple stress tolerance in susceptible plants. In the following chapter, we discussed how the HD-Zip proteins would play a useful role for cotton development both in fiber production and stress adaptation.展开更多
One day,a Swiss inventor named George de Mestral was walking in the woods.Later,he saw that many burrs had stuck to his wool pants.He wondered why they were hard to pull off. He looked at the burrs under a microscope,...One day,a Swiss inventor named George de Mestral was walking in the woods.Later,he saw that many burrs had stuck to his wool pants.He wondered why they were hard to pull off. He looked at the burrs under a microscope,and saw that each burr was made up of tiny hooks.展开更多
The Arabidopsis （Arabidopsis thaliana L.） genome encodes for four distinct classes of homeodomain leucinezipper （HD-ZIP） transcription factors （HD-ZIPI to HD-ZIPIV）, which are all organized in multi-gene familie...The Arabidopsis （Arabidopsis thaliana L.） genome encodes for four distinct classes of homeodomain leucinezipper （HD-ZIP） transcription factors （HD-ZIPI to HD-ZIPIV）, which are all organized in multi-gene families. HD-ZIP transcription factors act as sequence-specific DNA-binding proteins that are able to control the expression level of target genes. While HD-ZIPI and HD-ZIPII proteins are mainly associated with environmental responses, HD-ZIPIII and HD- ZIPIV are primarily known to act as patterning factors. Recent studies have challenged this view. It appears that several of the different HD-ZlP families interact genetically to align both morphogenesis and environmental responses, most likely by modulating phytohormone-signaling networks.展开更多
A homeodomain leucine-zipper （HD-Zip） gene BnHB6 （GenBank accession No. AY336103） was isolated from oilseed rape （Brassica napus L.） following drought treatment through rapid amplification of cDNA ends （RACE）....A homeodomain leucine-zipper （HD-Zip） gene BnHB6 （GenBank accession No. AY336103） was isolated from oilseed rape （Brassica napus L.） following drought treatment through rapid amplification of cDNA ends （RACE）. The full-length cDNA of BnHB6 was 1 611 bp and contained a 936-bp open reading frame encoding 311 amino acids. Sequence analysis indicated that BnHB6 belonged to the HD-Zip I subfamily. High-stringency Southern boltting analysis showed that BnHB6 appeared in rape as a single copy but had homologous genes. Semi-quantitative reverse transcription-polymerase chain reaction （RT-PCR） analysis revealed that BnHB6 was expressed in several tissues tested under control conditions, but that expression was significantly upregulated in shoots by mannitol, NaCI, cold treatment, anaerobic culture, wounding, H2O2, abscisic acid （ABA）, and salicylic acid （SA） treatments, but not by ultraviolet treatment. Further RT- PCR analysis revealed that BnHB6 was a late-responsive gene, the expression of which was not activated by NaCI, cold treatment, H2O2, ABA, and SA at an early time point （20 min） of treatment in the shoot. However, after a certain period of treatment, the induced expression culminated and then declined until the next peak occurred. Tissue-specific analysis revealed that BnHB6 was expressed at certain levels in the roots, shoots, and flowers, and the roots were found to respond to the osmotic stimuli more rapidly than shoots to increase the expression of BnHB6. The present study implies that BnHB6 plays a positive role as a regulator of biotic and abiotic stresses on growth during seedling establishment.展开更多
Salt acclimation, which is induced by previous salt exposure, increases the resistance of plants to future exposure to salt stress. However, little is known about the underlying mechanism, particularly how plants stor...Salt acclimation, which is induced by previous salt exposure, increases the resistance of plants to future exposure to salt stress. However, little is known about the underlying mechanism, particularly how plants store the"memory" of salt exposure. In this study, we established a system to study salt acclimation in Arabidopsis thaliana. Following treatment with a low concentration of salt, seedlings were allowed to recover to allow transitory salt responses to subside while maintaining the sustainable effects of salt acclimation. We performed transcriptome profiling analysis of these seedlings to identify genes related to salt acclimation memory. Notably, the expres-sion of Basic-leucine zipper 17 (bZIP17) and Hmg-CoA reductase degradation 3A (HRD3A), which are important in the unfolded protein response (UPR) and endoplasmic reticulum-associated degradation (ERAD), respectively, increased following treatment with a low concentration of salt and remained at stably high levels after the stimulus was removed, a treatment which improved plant tolerance to future high-salinity challenge. Our findings suggest that the upregulated expression of important genes involved in the UPR and ERAD represents a "memory" of the history of salt exposure and enables more potent responses to future exposure to salt stress, providing new insights into the mechanisms underlying salt acclimation in plants.展开更多
The stability of GCN4 leucine zipper and its four mutants in guanidine hydrochloride was detected to verify the contributions of different a position amino acid residues in polypeptide sequences to the forming and sta...The stability of GCN4 leucine zipper and its four mutants in guanidine hydrochloride was detected to verify the contributions of different a position amino acid residues in polypeptide sequences to the forming and stability of parallel coiled coils. The changes of the circular dichroism spectra show that the displace- ment of the a position polar asparagine and the increase of asparagine in the GCN4 leucine zipper can reduce the α-helix content of the coiled coil structure. The mutants are less stable than the natural peptide in guanidine hydrochloride. The results show that the interaction between the polar asparagine contributes to the conformational stability of the coiled coil. Both the conformation and the number of polar residues in the coiled coil also affect the α-helix content and its resistance to the denaturant. The conclusions provide evidence describing the folding process of proteins including coiled coils in vivo.展开更多
Glucocorticoid-induced tumor-necrosis factor receptor(GITR)and its ligand,GITRL,play significant roles in regulating immune responses.It is clear that human soluble GITRL(hsGITRL)transduces signal activity through mul...Glucocorticoid-induced tumor-necrosis factor receptor(GITR)and its ligand,GITRL,play significant roles in regulating immune responses.It is clear that human soluble GITRL(hsGITRL)transduces signal activity through multiple oligomerization states.To develop human soluble trimeric GITRL protein as a potential therapeutic target,we explored the link of the isoleucine-zipper(ILZ)motif to the N-terminus of the human soluble GITRL with two leucine sequences.hsGITRL,with the ILZ motif(ILZ-hsGITRL),was firstly expressed in Escherichia coli,which exhibited a predominant trimer when identified by Sephadex G-100 filtration and non-reducing SDS–polyacrylamide gel electrophoresis(SDS-PAGE).The significantly higher biological activity of the ILZ-hsGITRL compared with hsGITRL was confirmed by CD41 T proliferation,interferon-c(IFN-c)secretion and binding activity assay.To reveal and compare the underlying mechanisms,the level of extracellular signal-regulated kinase-1/2(ERK1/2)phosphorylation was examined,indicating that ILZ-hsGITRL induced more persistent and stronger ERK1/2 activation than hsGITRL.In conclusion,the incorporation of an ILZ motif could markedly improve the costimulation of hsGITRL.展开更多
WRKY proteins are transcriptional regulators involved in plant responses to biotic and abiotic stresses, metabolisms, and developmental processes. In the present study, we isolated a WRKY cDNA, OsWRKY89 from a rice cD...WRKY proteins are transcriptional regulators involved in plant responses to biotic and abiotic stresses, metabolisms, and developmental processes. In the present study, we isolated a WRKY cDNA, OsWRKY89 from a rice cDNA library. The deduced polypeptide contains 263 amino acid residues with a potential leucine zipper structure in its N-terminus, sharing low identity with other known WRKY members. OsWRKY89 and three deletion derivatives from its N-terminal were expressed in high levels in Escherichia coli as a C-terminally six-histidine-tagged fusion protein, and purified by employing one-step affinity chromatography on a Ni-NTA column. The recombinant OsWRKY89 protein was found to bind specially to sequences harboring W box cis elements by using electrophoretic mobility shift assays. This binding activity was decreased significantly by deletion of the leucine zipper-like structure in the N-terminal of Os- WRKY89. Using a yeast two-hybrid assay system, we found that the leucine zipper motif of OsWRKY89 was involved in the protein-protein interaction. Further deletion to remove partial WRKY domain abolished completely the interaction between the expressed protein and the W boxes, indicating that the WRKY domain is essential to the DNA-binding. These data strongly suggest that the leucine zipper-like motif of OsWRKY89 plays a significant role in the protein-protein and DNA-protein interactions.展开更多
基金supported by the National Natural Science Foundation of China(32471354 to T.C.and 82260400 to J.L)Natural Science Foundation of Hainan Province(No.822RC703 to J.L)。
文摘Discoidin domain receptors(DDRs)are single-pass transmembrane proteins belonging to receptor tyrosine kinases(RTKs)family,which are activated by collagen ligands with unusual slow,sustained kinetics,distinguishing them from canonical RTKs.While DDRs play critical roles in cell adhesion,differentiation,and cancer progression,their activation mechanisms remain partly understood.Here,we investigated the transmembrane domains(TMDs)of DDR1 and DDR2 to elucidate their interaction dynamics in membrane.Using bacterial adenylate cyclase two-hybrid(BACTH)assays,we demonstrated robust homotypic interactions and even stronger heterotypic associations between DDRTMDs.NMR spectroscopy of DDR1TMD and DDR2TMD reconstituted in lipid bilayer-mimetic bicelles showed obvious chemical shift alterations,further validating the stability of their heterocomplex formation.Systematic mutagenesis identified leucine zipper motifs rather than GXXXA motifs mediated both homo-and hetero-associations of DDR1TMD and DDR2TMD.These findings demonstrated the TMD as a critical mediator of DDRs oligomerization and revealed their interaction patterns within membrane.Our study advances the understanding of DDR signaling regulation and highlights transmembrane domain interactions as potential targets for modulating DDR-related pathologies.
文摘The basic leucine zipper （bZIP） transcription factors form a large gene family that is important in pathogen defense, light and stress signaling, etc. The Completed whole genome sequences of model plants Arabidopsis （Arabidopsis thaliana）, rice （Oryza sativa） and poplar （Populus trichocarpa） constitute a valuable resource for genome-wide analysis and genomic comparative analysis, as they are representatives of the two major evolutionary lineages within the angiosperms： the monocotyledons and the dicotyledons. In this study, bioinformatics analysis identified 74, 89 and 88 bZIP genes respectively in Arabidopsis, rice and poplar. Moreover, a comprehensive overview of this gene family is presented, including the gene structure, phylogeny, chromosome distribution, conserved motifs. As a result, the plant bZIPs were organized into 10 subfamilies on basis of phylogenetic relationship. Gene duplication events during the family evolution history were also investigated. And it was further concluded that chromosomal/segmental duplication might have played a key role in gene expansion of bZIP gene family.
基金supported by the National Natural Science Foundation of China (31471539)the Jiangsu Collaborative Innovation Center for Modern Crop Production Project, China (No.10)
文摘Plant trichomes originate from epidermal cells.In this work,we demonstrated that a homeodomain-leucine zipper(HD-Zip)gene,Gh_A06G1283(Gh HD-1A),was related to the leaf trichome trait in allotetraploid cotton and could be a candidate gene for the T_1 locus.The ortholog of GhHD-1A in the hairless accession Gossypium barbadense cv.Hai7124 was interrupted by a long terminal repeat(LTR)retrotransposon,while GhHD-1A worked well in the hairy accession Gossypium hirsutum acc.T586.Sequence and phylogenetic analysis showed that GhHD-1A belonged to the HD-Zip IV gene family,which mainly regulated epidermis hair development in plants.Silencing of GhHD-1A and its homoeologs GhHD-1D in allotetraploid T586and Hai7124 could significantly reduce the density of leaf hairs and affect the expression levels of other genes related to leaf trichome formation.Further analysis found that GhHD-1A mainly regulated trichome initiation on the upper epidermal hairs of leaves in cotton,while the up-regulated expression of GhHD-1A in different organs/tissues also altered epidermal trichome development.This study not only helps to unravel the important roles of GhHD-1A in regulating trichome initiation in cotton,but also provides a reference for exploring the different forms of trichome development in plants.
文摘Transcription factors play key roles in plant development and stress responses through their interaction with cis-elements and/or other transcription factors. Homeodomain associated leucine zipper proteins (HD-Zip) constitute a family of transcription factors that are characterized by the presence of a DNA-binding domain closely linked with leucine zipper motif functioning in dimer formation. This type of association is unique to plants and considered as an excellent candidate to activate developmental responses to altering environmental conditions. Cotton is the most important fiber plant with a lot of local and commercial uses in the world. HD-Zip proteins not only have key roles in different stages of vascular and inter-fascicular fiber differentiation of cotton but also are suggested to have an important role against abiotic stress that is one of the key factors limiting cotton productivity. Plants have developed various strategies to manage stress conditions through a combination of metabolic, physiological and morphological adaptations. These adaptive changes rely largely on alterations in gene expression. Therefore, transcriptional regulators play a crucial role in stress tolerance. Being a transcription factor HD-Zip might be a useful target for genetic engineering to generate multiple stress tolerance in susceptible plants. In the following chapter, we discussed how the HD-Zip proteins would play a useful role for cotton development both in fiber production and stress adaptation.
文摘One day,a Swiss inventor named George de Mestral was walking in the woods.Later,he saw that many burrs had stuck to his wool pants.He wondered why they were hard to pull off. He looked at the burrs under a microscope,and saw that each burr was made up of tiny hooks.
基金funded by the Deutsche Forschungsgemeinschaft, an International Reintegration Grant of the European Union, the European Research Council and the German Ministry for Agriculture
文摘The Arabidopsis （Arabidopsis thaliana L.） genome encodes for four distinct classes of homeodomain leucinezipper （HD-ZIP） transcription factors （HD-ZIPI to HD-ZIPIV）, which are all organized in multi-gene families. HD-ZIP transcription factors act as sequence-specific DNA-binding proteins that are able to control the expression level of target genes. While HD-ZIPI and HD-ZIPII proteins are mainly associated with environmental responses, HD-ZIPIII and HD- ZIPIV are primarily known to act as patterning factors. Recent studies have challenged this view. It appears that several of the different HD-ZlP families interact genetically to align both morphogenesis and environmental responses, most likely by modulating phytohormone-signaling networks.
基金Sino-UK Science and Technology Collaboration Fund, Shanghai Agriculture Committee，国家自然科学基金
文摘A homeodomain leucine-zipper （HD-Zip） gene BnHB6 （GenBank accession No. AY336103） was isolated from oilseed rape （Brassica napus L.） following drought treatment through rapid amplification of cDNA ends （RACE）. The full-length cDNA of BnHB6 was 1 611 bp and contained a 936-bp open reading frame encoding 311 amino acids. Sequence analysis indicated that BnHB6 belonged to the HD-Zip I subfamily. High-stringency Southern boltting analysis showed that BnHB6 appeared in rape as a single copy but had homologous genes. Semi-quantitative reverse transcription-polymerase chain reaction （RT-PCR） analysis revealed that BnHB6 was expressed in several tissues tested under control conditions, but that expression was significantly upregulated in shoots by mannitol, NaCI, cold treatment, anaerobic culture, wounding, H2O2, abscisic acid （ABA）, and salicylic acid （SA） treatments, but not by ultraviolet treatment. Further RT- PCR analysis revealed that BnHB6 was a late-responsive gene, the expression of which was not activated by NaCI, cold treatment, H2O2, ABA, and SA at an early time point （20 min） of treatment in the shoot. However, after a certain period of treatment, the induced expression culminated and then declined until the next peak occurred. Tissue-specific analysis revealed that BnHB6 was expressed at certain levels in the roots, shoots, and flowers, and the roots were found to respond to the osmotic stimuli more rapidly than shoots to increase the expression of BnHB6. The present study implies that BnHB6 plays a positive role as a regulator of biotic and abiotic stresses on growth during seedling establishment.
基金supported by the National Natural Science Foundation of China (31771489 and 31571384)
文摘Salt acclimation, which is induced by previous salt exposure, increases the resistance of plants to future exposure to salt stress. However, little is known about the underlying mechanism, particularly how plants store the"memory" of salt exposure. In this study, we established a system to study salt acclimation in Arabidopsis thaliana. Following treatment with a low concentration of salt, seedlings were allowed to recover to allow transitory salt responses to subside while maintaining the sustainable effects of salt acclimation. We performed transcriptome profiling analysis of these seedlings to identify genes related to salt acclimation memory. Notably, the expres-sion of Basic-leucine zipper 17 (bZIP17) and Hmg-CoA reductase degradation 3A (HRD3A), which are important in the unfolded protein response (UPR) and endoplasmic reticulum-associated degradation (ERAD), respectively, increased following treatment with a low concentration of salt and remained at stably high levels after the stimulus was removed, a treatment which improved plant tolerance to future high-salinity challenge. Our findings suggest that the upregulated expression of important genes involved in the UPR and ERAD represents a "memory" of the history of salt exposure and enables more potent responses to future exposure to salt stress, providing new insights into the mechanisms underlying salt acclimation in plants.
基金Supported by the National Natural Science Foundation of China (No. 30170199) and the Basic Research Foundation of Tsinghua University (No. JC2003050)
文摘The stability of GCN4 leucine zipper and its four mutants in guanidine hydrochloride was detected to verify the contributions of different a position amino acid residues in polypeptide sequences to the forming and stability of parallel coiled coils. The changes of the circular dichroism spectra show that the displace- ment of the a position polar asparagine and the increase of asparagine in the GCN4 leucine zipper can reduce the α-helix content of the coiled coil structure. The mutants are less stable than the natural peptide in guanidine hydrochloride. The results show that the interaction between the polar asparagine contributes to the conformational stability of the coiled coil. Both the conformation and the number of polar residues in the coiled coil also affect the α-helix content and its resistance to the denaturant. The conclusions provide evidence describing the folding process of proteins including coiled coils in vivo.
基金supported by the National Natural Science Foundation of China(Grant No.30871193 and No.30972748)Natural Science Foundation of Jiangsu Province(Grant No.BK2004405)+2 种基金Natural Science Foundation of Jiangsu Province Educational Commission(Grant No.08KJB320002)Research Foundation of Jiangsu Province Health Department(Grant No.H200952)Top Talent Program of Jiangsu University and SCI-Tech Innovation Team of Jiangsu University。
文摘Glucocorticoid-induced tumor-necrosis factor receptor(GITR)and its ligand,GITRL,play significant roles in regulating immune responses.It is clear that human soluble GITRL(hsGITRL)transduces signal activity through multiple oligomerization states.To develop human soluble trimeric GITRL protein as a potential therapeutic target,we explored the link of the isoleucine-zipper(ILZ)motif to the N-terminus of the human soluble GITRL with two leucine sequences.hsGITRL,with the ILZ motif(ILZ-hsGITRL),was firstly expressed in Escherichia coli,which exhibited a predominant trimer when identified by Sephadex G-100 filtration and non-reducing SDS–polyacrylamide gel electrophoresis(SDS-PAGE).The significantly higher biological activity of the ILZ-hsGITRL compared with hsGITRL was confirmed by CD41 T proliferation,interferon-c(IFN-c)secretion and binding activity assay.To reveal and compare the underlying mechanisms,the level of extracellular signal-regulated kinase-1/2(ERK1/2)phosphorylation was examined,indicating that ILZ-hsGITRL induced more persistent and stronger ERK1/2 activation than hsGITRL.In conclusion,the incorporation of an ILZ motif could markedly improve the costimulation of hsGITRL.
基金This work was supported by the State Basic Research and Development Plan(G200001 6203)the National Natural Science Foundation of China(Grant Nos.30370139&30471122).
文摘WRKY proteins are transcriptional regulators involved in plant responses to biotic and abiotic stresses, metabolisms, and developmental processes. In the present study, we isolated a WRKY cDNA, OsWRKY89 from a rice cDNA library. The deduced polypeptide contains 263 amino acid residues with a potential leucine zipper structure in its N-terminus, sharing low identity with other known WRKY members. OsWRKY89 and three deletion derivatives from its N-terminal were expressed in high levels in Escherichia coli as a C-terminally six-histidine-tagged fusion protein, and purified by employing one-step affinity chromatography on a Ni-NTA column. The recombinant OsWRKY89 protein was found to bind specially to sequences harboring W box cis elements by using electrophoretic mobility shift assays. This binding activity was decreased significantly by deletion of the leucine zipper-like structure in the N-terminal of Os- WRKY89. Using a yeast two-hybrid assay system, we found that the leucine zipper motif of OsWRKY89 was involved in the protein-protein interaction. Further deletion to remove partial WRKY domain abolished completely the interaction between the expressed protein and the W boxes, indicating that the WRKY domain is essential to the DNA-binding. These data strongly suggest that the leucine zipper-like motif of OsWRKY89 plays a significant role in the protein-protein and DNA-protein interactions.
