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. 2016 Jun 17;28(7):1662–1681. doi: 10.1105/tpc.16.00105

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© 2016 American Society of Plant Biologists. All rights reserved.

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Figure 6. — Molecular Interaction between MED18 and HLS1 and Their Synergistic Action on Target Gene Expression.

(A) and (B) Interaction between MED18 and HLS1 in co-IP assay in N. benthamiana (A) and transgenic Arabidopsis (B) plants. In (A), HLS1-HA was transiently coexpressed with MED18-MYC by agroinfiltration in N. benthamiana leaves. The empty vector expressing MYC was used as a negative control. In (B), transgenic Arabidopsis plants stably expressing HLS1-HA and MED18-MYC were used in the co-IP assays. Anti-HA beads were used to precipitate HLS1-HA protein. Anti-HA (α-HA) and anti-MYC (α-MYC) antibodies were used to detect protein accumulation in input or immunoprecipitated (IP) samples.

(C) Synergistic action of HLS1 and MED18 in the regulation of ABI5 or WRKY33 expression. The schematic diagram shows plasmid constructs used in transcriptional activation assay. The CaMV 35S promoter driving the luciferase reporter gene (35S:LUC) and the WRKY33 or ABI5 promoter fused with GUS reporter gene (pWRKY33/pABI5:GUS) are used as an internal control and a reporter, respectively. 35S promoter driving expression of HLS1 tagged with HA (HLS1-HA) is used as an effector. The bar graphs show the mean relative GUS activity from expression of the various plasmids depicted in the schematic. The mean values from protoplasts transfected with empty vector, pWRKY33/pABI5:GUS, and 35S:LUC were set to 1 as an internal control. The GUS signal is normalized with the LUC signal. The data represent mean values ± se (n = 3) from two independent biological replicates, and statistically significant differences are indicated by different letters (least squares means post hoc test: P < 0.05).

(D) MED18-mediated WRKY33 expression is dependent on HLS1. Relative gene expression is normalized to ACT2. The relative expression in wild-type plants at 0 h is set to 1.

(E) MED18 recruitment to transcription start site and 3′-coding regions of WRKY33 is enhanced by inoculation with B. cinerea in an HLS1-dependent manner. The enrichment of the WRKY33 gene in the wild type at 0 h is set to 1 as a background control in the ChIP-qPCR assay.

(F) Ectopic expression of MED18 rescues disease phenotype of hls1 mutant. The disease lesion size was determined after drop inoculation with B. cinerea. The data represent mean values ± se (n = 20). Statistically significant differences are indicated by asterisks compared with wild-type plants (Student’s t test: *P < 0.05 and ***P < 0.001).

In (D) and (E), the data represent mean values ± se (n = 3), and the statistically significant differences are marked by different letters (least squares means post hoc test: P < 0.05). pABI5:GUS and pWRKY33:GUS, reporter GUS fused with ABI5 or WRKY33 promoter region, respectively; HLS1, HLS1 tagged with HA; MED18, MED18 tagged with MYC. MED18; WT, overexpressing MED18 in wild-type background. MED18; hls1, overexpressing MED18 in the hls1 mutant background.