PsCRN108 interferes with plant <i>HSP</i> gene expression.

<p>(A) Relative transcript levels of <i>AtHSP90</i>.<i>1</i> in transgenic <i>Arabidopsis</i> leaves. The transcript levels of <i>AtHSP90</i>.<i>1</i> (<i>AT5G52640</i>) in stable transgenic <i>Arabidopsis</i> leaves expressing <i>GFP</i> or <i>PsCRN108</i> were measured by qRT-PCR using RNA samples extracted from <i>P</i>. <i>capsici</i> inoculated leaves (10 hpi) and mock-treated leaves. The relative transcript levels during infection were normalized to those of mock-treated leaves expressing <i>GFP</i> using <i>Actin2</i> as an internal reference. Values are means ± SEM of three independent biological replicates (<i>**</i>, P<0.01; <i>t</i>-test). (B) Relative transcript levels of <i>NbHsp90-1</i>. The transcript levels of <i>NbHsp90-1</i> in <i>N</i>. <i>benthamiana</i> leaves expressing <i>GFP</i>, <i>PsCRN108</i> or the indicated <i>PsCRN108</i> variants were measured by qRT-PCR using RNA samples extracted from <i>P</i>. <i>capsici</i> inoculated leaves (10 hpi) and mock-treated leaves. The relative transcript levels during infection were normalized to those of mock-treated leaves expressing GFP using <i>NbEF1α</i> gene as an internal reference. Values are means ± SEM of three independent biological replicates (<i>**</i>, P<0.01 compared with GFP; Dunnett's test). (C) Relative transcript levels of <i>GmHsp90A1</i>. Hypocotyls of etiolated soybean cultivar (Williams) were challenged with zoospores of the indicated <i>P</i>. <i>sojae</i> lines, and RNA was extracted at 10 hpi. The transcript levels of <i>GmHsp90A1</i> in soybean inoculated with transgenic <i>P</i>. <i>sojae</i> lines were normalized to levels of those of mock-treated soybean using <i>ACT20</i> gene as an internal reference. Values are the means ± SEM of three independent biological replicates (**, P<0.01; *, P<0.05 compared with the WT; Dunnett's test). (D) Schematic of the constructs used in the GUS activity assay. A -2000 bp promoter region of <i>AtHSP90</i>.<i>1</i> were fused to <i>GUS</i>. “ATG” is the translational start site for the <i>GUS</i> gene. The 35S min promoter fused to <i>GUS</i> (p35S min:GUS) was used for normalization of the relative GUS activity. (E) Relative GUS enzyme activity in <i>N</i>. <i>benthamiana</i>. The constructs of pAtHSP90.1:GUS and p35S min:GUS were transiently introduced into <i>N</i>. <i>benthamiana</i> leaves together with constructs encoding GFP, PsCRN108 or the indicated PsCRN108 variants. The leaves were immersed in a solution containing <i>P</i>. <i>capsici</i> zoospores 48 h post infiltration, and the protein was extracted at 3 hpi. GUS activity levels were measured using a fluorometric assay. Each column represents the ratio from the pAtHSP90.1:GUS construct relative to that from the p35S min:GUS construct. Values are the mean ± SEM of three independent biological replicates with three technological replicates each (<i>**</i>, P<0.01 compared with GFP; Dunnett's test).