OsTGA2 confers disease resistance to rice against leaf blight by regulating expression levels of disease related genes via interaction with NH1

Abstract

How plants defend themselves from microbial infection is one of the most critical issues for sustainable crop production. Some TGA transcription factors belonging to bZIP superfamily can regulate disease resistance through NPR1-mediated immunity mechanisms in Arabidopsis. Here, we examined biological roles of OsTGA2 (grouped into the same subclade as Arabidopsis TGAs) in bacterial leaf blight resistance. Transcriptional level of OsTGA2 was accumulated after treatment with salicylic acid, methyl jasmonate, and Xathomonas oryzae pv. Oryzae (Xoo), a bacterium causing serious blight of rice. OsTGA2 formed homo- and hetero-dimer with OsTGA3 and OsTGA5 and interacted with rice NPR1 homologs 1 (NH1) in rice. Results of quadruple 9-mer protein-binding microarray analysis indicated that OsTGA2 could bind to TGACGT DNA sequence. Overexpression of OsTGA2 increased resistance of rice to bacterial leaf blight, although overexpression of OsTGA3 resulted in disease symptoms similar to wild type plant upon Xoo infection. Overexpression of OsTGA2 enhanced the expression of defense related genes containing TGA binding cis-element in the promoter such as AP2/EREBP 129, ERD1, and HOP1. These results suggest that OsTGA2 can directly regulate the expression of defense related genes and increase the resistance of rice against bacterial leaf blight disease.

Fig 1. Expression of OsTGA transcription factors in rice.

(A and B) Expression profiles of OsTGA genes in rice seedlings in response to treatments of exogenous SA at 2 mM (A) and MeJA at 100 μM (B) (C and D) Expression profiles of OsTGAs upon Xoo challenge. Leaves inoculated with Xanthomonas oryzae pv. oryzae (Xoo) KACC10331 (C) or inoculated with sterile water (D) were collected. Total RNA extracted from these samples was used for cDNA synthesis followed by qRT-PCR analysis. Bars represent mean ± standard deviation. Data are obtained from three independent replicated experiments. Gray, white, and black boxes indicate OsTGA2, OsTGA3, and OsTGA5, respectively.

Fig 2. Identification of DNA binding sequences of OsTGA2.

(A and B) Identification of DNA-binding sequences of OsTGA2. (A) Summary of putative OsTGA2 DNA binding sequences. Q9 protein-binding microarray analysis was performed using OsTGA2-DsRed fusion protein. A total of 113 probe sequences that produced high signal intensities were selected for further analysis. (B) Signal intensities of TGACGTA sequence and single nucleotide substitution derivatives using Q9 protein-binding microarray. W, Wild-type sequence; 1 to 9, single nucleotide substitution variants (number corresponds to the position of the nucleotide substitution). One probe set that was irrelevant to GGGAAA sequences was used as negative control (N). (C) Sequence logo of TGA binding site based on the information weight matrix model. The height of each letter within a stack is proportional to its frequency at that position in the binding site. Letters are sorted with the most frequent one on the top. The sequence logo was created using WebLogo software (http://weblogo.berkeley.edu). (D) Rice TGA2 binds to TGACGT sequences in gel mobility shift assay. Nucleotide sequences of oligonucleotides used as probe and competitors are depicted. Unlabeled wild-type (Wt) and mutated (M1, M2, M3) oligonucleotides were included as competitors.

Fig 3. OsTGAs form dimers.

(A)Yeast two-hybrid experiments demonstrate dimerization of OsTGAs. OsTGA2, OsTGA3, and OsTGA5 proteins fused to GAL4 DNA-binding domain (BD) and GAL4 activation domain (AD) expressed in yeast stain YH190. Cells were grown on selective media for 3 days at 30°C before pictures were taken. (B) Visualization of protein-protein interactions in rice protoplast by BiFC assay.

Fig 4. OsTGAs interacts with OsNHs.

(A) Interaction of OsTGAs with OsNHs in yeast. OsTGA proteins fused to GAl4-binding domain (BD) were expressed in combination with OsNHs fused to GAL4 activation domain (AD) in yeast stain YH190. Cells were grown on selective media supplemented with 3-AT for 3 days at 30°C before pictures were taken. (B) Visualization of nYFP-OsNHs and cFYP-OsTGAs interactions in rice protoplast by BiFC (Bar is 20 μm).

Fig 5. Overexpression of OsTGA2 confers resistance to rice against bacterial leaf blight.

(A and B) OsTGA2 and OsTGA3 overexpressing transgenic plants were inoculated with Xoo strain KACC10331 when plants were 6-week old. Northern blotting were done to confirm the overexpression in each lines. Three independent experiments were performed and lesion lengths were measure at 3 weeks after inoculation (B). Pictures were taken 3 weeks after inoculation (A). (C-E) Three lines of T3 generation OsTGA2 overexpressing transgenic plants (4–34, 6–5, 8–3) were infected with Xoo. LogCFU/leaf was measured in each line on 10 and 18 day postinoculation (dpi) (E).

Fig 6. Validation of up-regulated genes using Real-time PCR analysis.

Total RNA was extracted from two-week old WT or OsTGA2 overexpressing transgenic plants treated with 2 mM SA and used for cDNA synthesis followed by Q-RT PCR to check expression levels of stress related genes: (A) AP2/EREBP129, (B) ERD1, (C) indole-3-acetate O-methyltransferase 1-like, jasmonate O-methyltransferase, (D) HSP70-HSP90 organizing protein 1, (E) DEAD-box ATP-dependent RNA helicase 12-like, (F) peptidyl-prolyl cis-trans isomerase FKBP65, (G) YSL13, (H) glycine-rich RNA-binding, (I) Conserved hypothetical protein (Os08g0351300), (J) Conserved hypothetical protein (Os08g0395700), (K) UDP-glucuronosyl/UDP-glucosyltransferase family protein, (L) auxin-induced protein 5NG4.