PsCRN108–HSE interaction inhibits binding to HSE of plant AtHsfA1a.

<p>(A) Schematic of the constructs used in the GUS activity assay. The HSE and HSEm were fused to the 35S minimal promoter and <i>GUS</i>. “ATG” is the translational start site for the GUS gene. The 35S minimal promoter fused to GUS was used for normalization of the relative GUS activity. (B) HSE-driven GUS activity in <i>N</i>. <i>benthamiana</i> was suppressed by PsCRN108. Leaves transiently expressing PsCRN108 or PsCRN108 mutants together with the GUS constructs were challenged with <i>P</i>. <i>capsici</i> zoospores for 3 h. Then, GUS activities were measured by fluorometric assays. The data in each column are fold changes normalized to the control (p35S min:GUS). Bars indicate standard errors from three biological replicates (<i>*</i>, P<0.05 compared with GFP; Dunnett’s test). (C) Pull-down of PsCRN108 by HSE. Biotinylated HSE (100 fmol) was incubated with 150 μg indicated proteins in the presence of streptavidin agarose. Upper panel, Western blot analysis of the input proteins using an anti-MBP antibody. Middle panel, Western blot detection of un-bound proteins before elution. Lower panel, proteins pulled-down by HSE. MBP and PsCRN108-HhHm were used as negative controls and AtHsfA1a as the positive control. (D) Inhibition of binding between AtHsfA1a and HSE by PsCRN108 as determined by DNA pull-down assay. Biotinylated HSE (100 fmol) was first incubated with 150 μg GST:PsCRN108 proteins (first panel) in the presence of streptavidin agarose, and then 150 μg MBP (negative control) or 150 μg MBP:AtHsfA1a (second panel) proteins were added after washing of the unbound protein. PsCRN108 (third panel), but not AtHsfA1a (fourth panel), was found to bind to HSE by Western blotting. (E) Western blot analysis of GFP- and HA- fusion proteins transiently expressed in <i>N</i>. <i>benthamiana</i> 48 h after infiltration. The protein sizes are expressed in kDa. PS, Ponceau S staining. (F) Inhibition of binding between AtHsfA1a and HSE by PsCRN108 as determined by <i>in vivo</i> ChIP assay. <i>N</i>. <i>benthamiana</i> leaves expressing the constructs as indicated were infected with <i>P</i>. <i>capsici</i> zoospores for 10 h, and then used for ChIP assays with anti-GFP antibodies. qPCR analysis was performed on immunoprecipitated DNA using primers specific for the <i>NbHsp90</i>-1 promoter (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005348#ppat.1005348.s007" target="_blank">S7 Fig</a>). Values for the ChIP samples were first normalized to the input control and then divided by the no-antibody control to obtain the fold enrichment values. Values are the means ± SEM of three independent biological replicates (**, P<0.01 compared with the indicated control; Dunnett’s test).