Fig. 5: AsHSFA2c negatively regulates AsAGO1 through transcriptional inhibition. | Communications Biology

Fig. 5: AsHSFA2c negatively regulates AsAGO1 through transcriptional inhibition.

From: Comprehensive co-expression network reveals the fine-tuning of AsHSFA2c in balancing drought tolerance and growth in oat

Fig. 5

a The credibility evaluation of DAP-seq peaks showed that peaks below the IDR threshold were displayed in red (non-reproducible peaks), and peaks above were displayed in black (biologically reproducible peaks). b Three different secondary position weight matrices (PWM) represented the obtained motifs in the experiment, with a Z score indicated. c A significant binding signal of AsHSFA2c was identified in the AsAGO1 promoter by DAP-seq. d EMSA assay showed that AsHSFA2c protein bound the promoter of AsAGO1. GST-AsHSFA2c was incubated with 5′-biotin-labeled DNA probes with AsAGO1 promoter sequence (Biotin-AsAGO1) or mutated AsAGO1 promoter sequence (Biotin-mAsAGO1). e Luciferase assay showed that AsHSFA2c inhibited the AsAGO1 promoter. N.benthamiana plants were co-inoculated with p35s::AsHSFA2c-bd/p35s::AsHSFA2c and pAsAGO1::LUC. The p35s::AsHSFA2c-bd plasmid contains only the transcriptional binding domain of AsHSFA2c and lacks its transcriptional activation domain. The sample was collected at 48 hpi. A cooled CCD imaging apparatus (Roper Scientific) was used to capture luciferase images. Scale bar, 0.5 cm. qRT-PCR detection of LUC expression level. N.benthamiana plants were co-inoculated with p35s::AsHSFA2c-bd and pAsAGO1::LUC as a negative control. Error bars represent the SD of three biological replicates. f Relative expression of genes selected from DAP-seq in TRV1TRV2 and AsHSFA2c knockdown lines. Error bars represent the SD of three biological replicates. g, h qRT-PCR detection of AsAGO1 expression level under drought (g) and IAA treatment (h). The X-axis represents processing time. Error bars represent the SD of three biological replicates. i, j The malfunction of AsAGO1 inhibited plant growth and positively regulated plant drought tolerance. “Watered” represented well-watered conditions. “After Drought” represented a 2-day period of recovery with full irrigation post-drought treatment (i). Scale bars, 5 cm. Statistics on plant height (error bars represent the SD of 15 biological replicates) and survival rate (error bars represent the SD of three biological replicates) of AsAGO1 knock-down strains (j). k Chlorophyll contents detection statistics of AsAGO1 knockdown lines. Error bars represent the SD of 15 biological replicates. l Water loss rate statistics of AsAGO1 knock-down lines. Error bars represent the SD of three biological replicates. Student’s t-test was performed to determine statistical significance. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; ns, p ≥ 0.05.

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