Figure 5.

eQTL analysis of PtoWRKY68 expression under well-watered and drought-stress conditions. A) Manhattan plots of eQTL analysis with expression of PtoWRKY68 under well-watered and drought-stress conditions. Horizontal dashed line, significance threshold (P < 3.3 × 10^–7). Arrowhead, candidate gene. B) Venn diagram showing the overlap of well-watered and drought-stress genes identified by eQTL analysis. C) FPKM values of PtoSVP.3 under well-watered and drought-stress conditions. In box plots, center line represents the median, box limits denote the upper and lower quartiles, whickers indicate the interquartile range, and dots are outliers. Error bars are ± SD. Statistical analysis was performed using the t-test, *P < 0.05. D) Scatter diagram of the correlation between the expression of PtoSVP.3 and PtoWRKY68 under drought stress. Dashed line represents linear regression. E) Schematic of the locations of CArG motifs in the promoter of PtoWRKY68. Solid squares, CArG motifs; short black lines, CArG Probe 1 (CP1); CP2 and CP3 indicate the probes used in F). F) EMSA. Relative binding affinity of SVP.3 to CArG motifs. Reciprocal competitive EMSA of the binding of recombinant SVP.3 protein to CArG motifs using the indicated biotin- labeled probes and unlabeled competitors. For each probe, 50× and 100× excess competitor was added. G) DLRA of the interaction between PtoSVP.3 and the PtoWRKY68 promoter. H) Relative luciferase activity by DLRA in N. benthamiana leaves. Quantification was performed by normalizing firefly luciferase (LUC) activity to that of Renilla luciferase (REN), 35S:REN was used as the internal control. Relative luciferase activities using PtoSVP.3-pGreenII62-SK as the effector compared to the control effector (pGreenII62-SK empty vector). Error bars are ± SD. Statistical analysis was performed using the t-test (n = 8); statistically significant differences are indicated by **P < 0.01. Three independent transfection experiments were performed.