Fig 1.
Characterization of TaWRKY58 expression in wheat.
A. Domain architecture of TaWRKY58. (B–F) Relative expression levels of TaWRKY58 in response to: (B) UV radiation, (C) 10 mM SA, (D) 20% (w/v) PEG6000, (E) 200 mM NaCl, and (F) F. graminearum infection (48 hpi). Asterisks (*) above bars indicate significant differences (P ≤ 0.05) relative to controls (Student’s t-test). Three biological repeats were performed for each mutant.
Fig 2.
Nuclear localization of TaWRKY58.
A. Schematic prediction of TaWRKY58 subcellular localization. The red five-pointed star represents the predicted location of the TaWRKY58 protein. B. Confocal microscopy images of wheat protoplasts expressing UBI::TaWRKY58-GFP. “BF” refers to brightfield microscope, “GFP” represents the fluorescence signal of GFP, and “AF” stands for the fluorescence signal of chloroplast. Scale bar: 20 μm.
Fig 3.
Phenotypic effects of the TaWRKY58 mutation in wheat.
A. Gene structure of wild-type TaWRKY58 and the position of the premature stop codon (T4-2223, C1593T) in Δtawrky58. B. Sanger sequencing chromatogram confirming the Δtawrky58 mutation. C. Plant phenotypes at Zadoks stage 60. Scale bar: 10 cm. D. Plant height measurements at Zadoks stage 65 for wild-type (WT) and Δtawrky58 homozygous lines (n ≥ 10 plants per genotype). E. Relative TaWRKY58 expression in transgenic lines versus WT at Zadoks stage 13 (Z13), six biological replicates were performed for each plant. F. Greenhouse phenotypes of different mutations at Z60. Scale bar: 10 cm. G. Plant height measurements of different mutations at Z65 under greenhouse conditions. H. GA content in spikelets and rachises of different mutations at Zadoks stage 60. All data are presented as the mean ± SD unless otherwise stated (n ≥ 3 biological replicates).
Fig 4.
Effect of TaWRKY58 on biotic and abiotic stress in wheat.
A. Symptoms of FHB in spikes at 12 days after inoculation with F. graminearum. Red lines mark the extent of symptom spread. B. Number of infected spikelets per spike at 12 days post-inoculation with F. graminearum. Each mutant line was inoculated using over 30 spikes. C. Number of infected spikelets at 10 days after inoculation. Each transgenic line was inoculated with 20 spikes. D. SA accumulation in wheat. Samples were collected 48 hours post-inoculation at Zadoks growth stage 65; water-inoculated plants served as the control. FW, fresh weight. E. Phenotypic responses of different genotypes to 200 mM NaCl treatment for 30 days at Zadoks stage 65. Scale bar: 10 cm. F. ABA content in leaves of plants subjected to 200 mM NaCl for 7 days at Zadoks stage 13; water-treated plants were used as control. G. Soluble sugar accumulation in leaves after 7 days of 20% PEG6000 treatment at Zadoks stage 13. H. Phenotypes under drought stress applied for 30 days at Zadoks stage 65. Scale bar: 10 cm. I. Soluble sugar accumulation in leaves at Fig 4H. J. GA content in spikelets and rachis of plants from Fig 4H. All data are presented as the mean ± SD unless otherwise stated (n ≥ 3 biological replicates).
Fig 5.
Identification of TaWRKY58 target genes by DAP-seq.
A. Distribution of TaWRKY58 binding peaks across genomic features. B. Enrichment of TaWRKY58 peaks within ±1 kb of transcription start sites (TSS). C. De novo identified binding motif (Motif1) significantly enriched in TaWRKY58 peaks (E-value = 2.6e-4164). D. Positional distribution frequency of Motif1 relative to peak centers. E. Top 10 enriched molecular functions among Motif1-containing target genes (GO analysis).
Fig 6.
Molecular mechanism of TaWRKY58-mediated transcriptional repression.
A. Relative expression levels of TaLRR and TaBCS1 in the rachis of WT Δtawrky58 mutant at Zadoks stage 60, six biological replicates were performed for each plant. B. Schematic diagram of EMSA probes design. Probes were biotin labeled. Competition experiments were carried out by adding excessive unlabeled probes. C. The key interaction sites between TaWRKY58 and motif1 were predicted through AlphaFold. D. EMSA showing recombinant TaWRKY58 binding to TaLRR promoter. Competitor: unlabeled probe. E. EMSA of TaWRKY58 binding to TaBCS1 promoter and mutant probes (M1-M4). Underline indicates binding loss with M1. F. Dual-LUC assay in N. benthamiana: 35S::TaWRKY58 represses proTaLRR::LUC. Vectors: pGreen0800-proTaLRR::LUC + pGreen62sk (± TaWRKY58). G. Normalized LUC/REN ratios in rice protoplasts confirming repression of proTaLRR::LUC and proTaBCS1::LUC by TaWRKY58, six biological replicates were performed for each plant.
Fig 7.
Functional analysis of TaLRR and TaBCS1 in wheat.
A-B. Sanger sequencing chromatograms confirming the mutation sites in the Δtalrr (A) and Δtabcs1 (B) mutants. C. Phenotypic comparison of wild-type (J411), Δtalrr, and Δtabcs1 plants at Zadoks growth stage 19. D. Plant height measurements of the indicated genotypes at Zadoks growth stage 65. E. the expression levels of TaLRR and TaBCS1 in J411 leaves after 7 days of 20% PEG6000 treatment at Zadoks stage 13. F. Phenotypes under drought stress (30 days) followed by rehydration for 12 days. G. Soluble sugar content in leaves of each genotype after 7 days of 20% (w/v) PEG6000 treatment at Zadoks growth stage 13. H. GA content in rachis of plants at Fig 7F. All data are presented as the mean ± SD unless otherwise stated (n ≥ 3 biological replicates).
Fig 8.
Schematic model of TaWRKY58-mediated negative regulation of flowering and drought stress response in wheat.