OsbZIP53 Negatively Regulates Immunity Response by Involving in Reactive Oxygen Species and Salicylic Acid Metabolism in Rice

doi:10.1016/j.rsci.2023.12.002

Abstract

Abstract:

The basic region/leucine zipper (bZIP) transcription factors play important roles in plant development and responses to abiotic and biotic stresses. OsbZIP53 regulates resistance to Magnaporthe oryzae in rice by analyzing APIP5-RNAi transgenic plants. To further investigate the biological functions of OsbZIP53, we generated osbzip53 mutants using CRISPR/Cas9 editing and also constructed OsbZIP53 over-expression transgenic plants. Comprehensive analysis of phenotypical, physiological, and transcriptional data showed that knocking-out OsbZIP53 not only improved disease resistance by inducing a hypersensitivity response in plants, but also regulated the immune response through the salicylic acid pathway. Specifically, disrupting OsbZIP53 increased H₂O₂ accumulation by promoting reactive oxygen species generation through up-regulation of several respiratory burst oxidase homologs (Osrboh genes) and weakened H₂O₂ degradation by directly targeting OsMYBS1. In addition, the growth of osbzip53 mutants was seriously impaired, while OsbZIP53 over-expression lines displayed a similar phenotype to the wild type, suggesting that OsbZIP53 has a balancing effect on rice immune response and growth.

Key words: OsbZIP53, hypersensitive response, reactive oxygen species metabolism, rice immunity, salicylic acid, transcription factor

Wu Lijuan, Han Cong, Wang Huimei, He Yuchang, Lin Hai, Wang Lei, Chen Chen, E Zhiguo. OsbZIP53 Negatively Regulates Immunity Response by Involving in Reactive Oxygen Species and Salicylic Acid Metabolism in Rice[J]. Rice Science, 2024, 31(2): 190-202.

Figures/Tables 7

Fig. 1. Phylogenetic tree of OsbZIP53 and its homologs in plants. Homologs are indicated by NCBI accession numbers. Monocot homologs are marked with dots with distinctive color.

Fig. 2. Expression pattern and subcellular localization of OsbZIP53. A, Expression of OsbZIP53 in different rice tissues detected by qRT-PCR. Root and shoot were at 14 d after germination, stem and leaf were at 60 d after germination, panicle was 5‒8 cm in length, and seed was at 12 d after fertilization. The ACTIN gene was used as an internal standard, and relative expression levels were quantified using the 2-ΔΔCT threshold cycle method. Error bars indicated standard deviation. Three biological replicates were conducted for each sample. B, Expression of OsbZIP53 induced by different hormone and stress treatments. Seven-day-old rice seedlings were given the treatments (stress or hormones) for 3 h. CK, Control; ABA, 50 μmol/L abscisic acid; SA, 2 mmol/L salicylic acid; H2O2, 100 mmol/L H2O2; PEG, 20% polyethylene glycol 6000; NaCl, 100 mmol/L NaCl. Data were means and standard deviations of triplicates. The stars above the bars indicated the significant differences under treatments with respect to control in the t-test (*, P < 0.05; **, P ≤ 0.01). C, Subcellular localization of OsbZIP53 transiently expressed in rice protoplasts. Ghd7-CFP was used as a nuclear localization marker. GFP, Green fluorescent protein; CFP, Cyan fluorescent protein. Scale bars, 5 μm.

Fig. 3. Phenotypes of CRISPR/Cas9-induced OsbZIP53 mutants and over-expression transgenic plants. A, Schematic organization of the coding region of OsbZIP53, the sgRNA target site was underlined, the protospacer adjacent motif (PAM) was shown in orange, and the insertion and deletion mutations were indicated in red. The bottom was the sequencing results correspond to the mutation type. B, Relative expression of OsbZIP53 in wild type (WT) and over-expression transgenic lines (OsbZIP53Ox-1 and OsbZIP53Ox-3). The leaves from 8-week-old WT and OsbZIP53 mutant lines were used for qRT-PCR. The ACTIN gene was used as an internal standard, and relative expression levels were quantified using the 2-ΔΔCT threshold cycle method. Three biological replicates were employed for each sample.C, Morphology of different age leaves in WT and transgenic lines (osbzip53cr-1 and OsbZIP53Ox-3). L1, L2 and L3 indicated the upmost leaf, the second upmost leaf and the third upmost leaf, respectively. Scale bar, 2 cm.D, Whole plant morphology in WT and transgenic lines. Scale bars, 10 cm.E, Number of functional leaves on each tiller of osbzip53cr-1 and WT. F, Number of tillers per plant in osbzip53cr-1 and WT. G, 3,3ʹ-Diaminobenzidine (DAB) staining to show H2O2 accumulation in WT and transgenic plants. Scale bar, 1 cm.H, H2O2 content in the leaves of osbzip53cr-1 and WT. Data in B, E, F, and H are Mean ± SD (n = 3). ** (P ≤ 0.01) are detected by the t-test between the transgenic plants and WT.

Fig. 4. Analysis of reactive oxygen species (ROS)-associated gene expression and enzyme activity. A, Relative expression levels of respiratory burst oxidase homolog revealed by qRT-PCR osbzip53cr mutant and wild type (WT). B, Relative expression levels of ROS-associated genes in osbzip53cr mutant and WT. C, Determination of superoxide dismutase (SOD) and peroxidase (POD) activities in osbzip53cr mutant and WT. The leaves from 6-week-old WT and osbzip53cr mutant lines were used for qRT-PCR. The ACTIN gene was used as an internal standard, and relative expression levels were quantified using the 2-ΔΔCT threshold cycle method. Data are Mean ± SD (n = 3). * (P < 0.05) and ** (P ≤ 0.01) indicate significant differences between osbzip53cr and WT in the t-test.

Fig. 5. OsMYBS1 may act as putative direct downstream target gene of OsbZIP53. A, Genome browser tracks showing OsbZIP53 binding signal around the OsMYBS1 locus by chromatin immunoprecipitation (ChIP)-seq. Blue boxes represent exons, yellow box represents binding sequence, and arrows represent transcription directions. B, Binding of maltose-binding protein (MBP)-OsbZIP53 to the probe around the OsMBYS1 locus in an electrophoretic mobility shift assay. MBP alone served as a control. The experiment was repeated three times.C, Relative expression levels of OsMBYS1 in osbzip53cr and wild type (WT). The leaves from 6-week-old WT and OsbZIP53 mutant lines were used for qRT-PCR. The ACTIN gene was used as an internal standard, and relative expression levels were quantified using the 2-ΔΔCT threshold cycle method.D, Diagram of constructs used in transcriptional activity assay. REN, Renilla. OsMYBS1exo2 indicates the sequence located on rice chromosome 1 at 18 756 774‒18 757 035 bp, which overlaps with the end of the second exon of OsMYBS1. E, Relative LUC (luciferase) activity represents the transcriptional activity. The activities of firefly LUC (LUC) and Renilla LUC (REN, as a control) were assayed, and the ratio of LUC/REN was calculated to represent the relative LUC activity. ‘1 + 3’ and ‘2 +3’ represent the combinations of effector 1 and reporter 3 and effector 2 and reporter 3 in D, respectively. Data in C and E are Mean ± SD (n = 3). ** indicates highly significant differences (P ≤ 0.01) detected by the t-test.

Fig. 6. Repression of OsbZIP53 increases resistance to Magnaporthe oryzae. A, Lesions in the leaves of osbzip53cr (cr-1 and cr-2), OsbZIP53Ox (Ox-1 and Ox-3), and wild type (WT) at 7 d after post-inoculation. Scale bar, 0.5 cm. B, Relative fungal biomass of osbzip53cr, OsbZIP53Ox and WT in A. The relative fungal biomass was estimated by DNA-based quantitative PCR using the CT value of M. oryzae transposable element MoPot2 relative to that of the rice Ubiquitin gene. C, Lesions in the leaves of OsbZIP53Ox and WT under natural disease state in the field. Scale bar, 1 cm. D, Average number of lesions formed in OsbZIP53Ox and WT under natural disease state in the field. Data are Mean ± SD (n = 3). ** indicates highly significant differences at P ≤ 0.01 detected by the t-test.

Fig. 7. Defense responses are activated in ObZIP53 mutant plants. A, Salicylic acid content in leaves of osbzip53cr-1 and wild type (WT). B, Schematic map of genes involved in processes related to defense response. The color scale of genes indicates the relative transcriptional level |log2 (fold change)| of the genes in osbzip53cr-1 in comparison to the WT plants based on RNA-seq results. MAPK, Mitogen-activated protein kinase.C and D, Relative expression levels of defense response-related genes (C) and OsWRKY45 pathway-related genes (D) revealed by qRT-PCR. Leaves from 6-week-old WT and OsbZIP53 mutant lines were used. The ACTIN gene was used as an internal standard, and relative expression levels were quantified using the 2-ΔΔCT threshold cycle method. Data are Mean ± SD (n = 3). ** indicates highly significant differences at P ≤ 0.01 detected by the t-test.

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