ABSTRACT
TGA family of transcription factors play important roles in the systemic acquired resistance (SAR) in plants. In SAR, TGA7 binds to the activation sequence-1 (as-1) in the promoter region of SAR related genes and regulates their expressions in an NPR1 dependent manner. Despite its important roles in plant immunity, the molecular mechanism for DNA binding of TGA7 remains unclear. In the present work, we resolved the crystal structure of TGA7 dimers at a resolution of 2.06 Å, in which each monomer binds one molecule of palmitate. Further biochemical studies revealed that TGA7 specifically binds to the TGACG boxes of as-1 DNA in the form of homodimers, and it has specific requirements for the relative spacing and orientation of the two TGACG boxes. Moreover, we built a TGA7-DNA complex model and confirmed by site-directed mutagenesis that amino acid residue R109 in the DNA binding domain (DBD) of TGA7 is a key residue responsible for DNA recognition. Our work offers a good example for structural and functional studies of TGA proteins, and provides key clues to understand the DNA binding mechanism of TGA proteins in the SAR.
Subject(s)
Arabidopsis , Arabidopsis/genetics , Transcription Factors/genetics , Gene Expression Regulation , Plant Immunity , Protein DomainsABSTRACT
Sclerotinia stem rot (SSR), caused by Sclerotinia sclerotiorum, is among the most devastating diseases in Brassica napus worldwide. Conventional breeding for SSR resistance in Brassica species is challenging due to the limited availability of resistant germplasm. Therefore, genetic engineering is an attractive approach for developing SSR-resistant Brassica crops. Compared with the constitutive promoter, an S. sclerotiorum-inducible promoter would avoid ectopic expression of defense genes that may cause plant growth deficits. In this study, we generated a S. sclerotiorum-inducible promoter. pBnGH17D7, from the promoter of B. napus glycosyl hydrolase 17 gene (pBnGH17). Specifically, 5'-deletion and promoter activity analyses in transgenic Arabidopsis thaliana plants defined a 189 bp region of pBnGH17 which was indispensable for S. sclerotiorum-induced response. Compared with pBnGH17, pBnGH17D7 showed a similar response upon S. sclerotiorum infection, but lower activity in plant tissues in the absence of S. sclerotiorum infection. Moreover, we revealed that the transcription factor BnTGA7 directly binds to the TGACG motif in pBnGH17D7 to activate BnGH17. Ultimately, pBnGH17D7 was exploited for engineering Sclerotinia-resistant B. napus via host-induced gene silencing. It induces high expression of siRNAs against the S. sclerotiorum pathogenic factor gene specifically during infection, leading to increased resistance.
Subject(s)
Arabidopsis , Ascomycota , Brassica napus , Brassica , Brassica napus/genetics , Brassica napus/metabolism , Plant Diseases/genetics , Plant Proteins/genetics , Plant Proteins/metabolism , Plant Breeding , Ascomycota/physiology , Brassica/metabolism , Arabidopsis/genetics , Arabidopsis/metabolism , Gene SilencingABSTRACT
BACKGROUND: The TGACG-binding (TGA) family has 10 members that play vital roles in Arabidopsis thaliana defense responses and development. However, their involvement in controlling flowering time remains largely unknown and requires further investigation. RESULTS: To study the role of TGA7 during floral transition, we first investigated the tga7 mutant, which displayed a delayed-flowering phenotype under both long-day and short-day conditions. We then performed a flowering genetic pathway analysis and found that both autonomous and thermosensory pathways may affect TGA7 expression. Furthermore, to reveal the differential gene expression profiles between wild-type (WT) and tga7, cDNA libraries were generated for WT and tga7 mutant seedlings at 9 days after germination. For each library, deep-sequencing produced approximately 6.67 Gb of high-quality sequences, with the majority (84.55 %) of mRNAs being between 500 and 3,000 nt. In total, 325 differentially expressed genes were identified between WT and tga7 mutant seedlings. Among them, four genes were associated with flowering time control. The differential expression of these four flowering-related genes was further validated by qRT-PCR. CONCLUSIONS: Among these four differentially expressed genes associated with flowering time control, FLC and MAF5 may be mainly responsible for the delayed-flowering phenotype in tga7, as TGA7 expression was regulated by autonomous pathway genes. These results provide a framework for further studying the role of TGA7 in promoting flowering.