Unfolded Protein Response Enhances Plant Resistance to Disease by Regulating Tryptophan Metabolism

doi:10.13560/j.cnki.biotech.bull.1985.2025-0275

Abstract

Abstract:

Objective To investigate the relationship between endoplasmic reticulum stress (ERS) induced unfolded protein response (UPR), tryptophan metabolism, and plant disease resistance, and to clarify the regulatory mechanisms of the transcription factors bZIP28 and bZIP60 in tryptophan metabolism and their impact on plant disease resistance. Method RNA-seq and qRT-PCR were used to analyze the expressions of genes involved in the tryptophan metabolism pathway under ER stress conditions. The synthesis of tryptophan-derived metabolites, camalexin and IAA, was measured under ER stress. The expressions of bZIP28 and bZIP60 after pathogenic infection and the disease resistance of plants overexpressing these genes were assessed. Yeast one-hybrid assays and dual-luciferase reporter assays were conducted to examine the regulation of WRKY33 by bZIP28/60. Result ER stress triggered the UPR, leading to the up-regulation of tryptophan metabolic genes and increased levels of camalexin and IAA. Pathogenic infection up-regulated the expressions of bZIP28 and bZIP60. Plants overexpressing bZIP28/60 demonstrated enhanced resistance to disease. bZIP28/60 indirectly regulated the expression of WRKY33, with the core regulatory sequence located within the -364 to -566 bp region of the WRKY33 promoter. Conclusion ER stress modulates the expression of WRKY33 through the transcription factors bZIP28 and bZIP60, resulting in elevated levels of camalexin and IAA, thereby enhancing resistance to pathogen infection and alleviating ER stress.

Key words: ER stress, unfolded protein response, bZIP28, bZIP60, WRKY33, tryptophan metabolism, camalexin, IAA

CAO Yuan-yuan, ZHOU Shu-hao, ZHANG Hai-rong, CUI Xiao-na. Unfolded Protein Response Enhances Plant Resistance to Disease by Regulating Tryptophan Metabolism[J]. Biotechnology Bulletin, 2025, 41(8): 155-164.

Figures/Tables 6

Fig. 1 Expressions of tryptophan metabolism genes under TM treatment or in the lew1 mutantA: RNA-seq heatmap illustrates the gene expressions. The color gradient indicates the log2fold change. B: Tryptophan metabolism pathway. C: Seedlings were treated with 5 μg/mL TM, and the relative expressions of tryptophan metabolism genes were detected by qPCR. The data indicate the mean fold difference ± SD of three biological replicates. Statistically significant differences (P<0.05) are indicated by distinct letters using Student's t-test, the same below

Fig. 2 Disease resistance treated with TM and auxin response after TM treatment and in the lew1 mutantA: Camalexin levels: 14-day-old seedlings were treated with 5 μg/mL TM for 24 h, with DMSO treatment as control, then the Camalexin contents were quantified using HPLC. B: Phenotypes after B.cinerea infection: Leaves were treated with 5 μg/mL TM, using DMSO treatment as control, followed by adding B. cinerea spore suspension onto the leaves, then the phenotypes were observed after 3 d; wrky33 mutant and WRKY33 over-expression plants were used as controls, bar = 5 mm. C: The lesion sizes on the leaves in figure B were measured using Image J. D: Elongation phenotype of hypocotyls of wild type and lew1 mutant after 5 d of dark treatment. E: The hypocotyl lengths in figure D were measured using Image J software.. F: GUS staining of 4-day-old seedlings was conducted to detect the response of DR5::GUS in WT and lew1 mutant. WT: Wild type; TM: tunicamycin; DMSO: dimethyl sulfoxide. * indicates significant differences (P<0.05); ** indicates significant differences (P<0.01), the same below

Fig. 3 Expression of bZIP28 and bZIP60 after Alternaria brassicicola and Botrytis cinerea infectionA: Gene expressions after Botrytis cinerea infection for different times. B: Gene expressions after Alternaria brassicicola infection for different times. 6-day-old WT seedlings were transferred into MS liquid medium and subsequently cultured under continuous light conditions for 6 d. The seedlings were then immersed in suspension containing 8×104 spores/mL of either A. brassicicola or B. cinerea for 0, 4, 8, 12, and 24 h. RT-qPCR analysis was performed to determine the relative expressions of bZIP28, bZIP60, and WRKY33. The data represent the mean fold difference ±SD of three biological replicates, Statistically significant differences (P<0.05) are indicated by distinct letters. Student's t-test was used to determine the P-values. hpi: Hours post injection

Fig. 4 Effects of bZIP28 and bZIP60 overexpression on plant resistance to diseaseA:Phenotypes of ZIP28 and bZIP60 over-expressed plants infected with B. cinerea, bar = 2 mm, OE#4/#6 and OE#3/#8 indicate different transgenic lines. B: Expression of bZIP28 and bZIP60 in over-expressed plants, ** indicates significant difference. C: Statistical analysis of lesion size in bZIP28 and bZIP60 overexpressed plants after Botrytis cinerea infection

Fig. 5 Transcription regulatory roles of bZIP28 and bZIP60 on WRKY33 detected using dual-luciferase reporter assayA: Seedlings were treated with 5 μg/mL TM for 24 h, and the relative expressions of genes were detected by RT-qPCR. B: Constructed vector used in the dual-luciferase reporter assay: Full indicates the full-length WRKY33 promoter, the promoter is truncated into segments “a” (-1 to -566 bp) and “b” (-566 to -1724bp); segment “a” in the promotor is further truncated into “a1” (-364 to -566 bp) and “a2” (-1 to -364 bp). C: bZIP28 or bZIP60 expression vector and constructs fused with full-length WRKY33 promoter reporter vector were co-transformed into Arabidopsis protoplasts to measure the ratio of luciferase enzyme activity, with EV (empty vector) as a control. D: Regulation of WRKY33 promoter segments “a” and “b” by bZIP28 and bZIP60. E: Regulation of WRKY33 promoter segments “a1” and “a2” by bZIP28 and bZIP60

Fig. 6 Transcription regulatory roles of bZIP28 and bZIP60 on WRKY33 detected using yeast one-hybrid assayDifferent segments of the WRKY33 promoter were separately fused to pHIS2 and co-transformed into Y187 with either pGADT7-bZIP28D or pGADT7-bZIP60S. The empty vector AD served as a negative control, while co-transformation of pHIS2-53 and AD-53 served as a positive control

References 43

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