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. 2017 Jul 31;8:1343. doi: 10.3389/fpls.2017.01343

FIGURE 2.

FIGURE 2

SlWRKY3 subcellular localization, trans-regulation properties and DNA-binding specificities. (A) Transient expression of YFP-SlWRKY3 fusion protein and control YFP in tomato leaf protoplasts. (a,a′) YFP-SlWRKY3 fluorescence and bright field/YFP fluorescence, respectively. (b,b′) Control YFP fluorescence and bright field/YFP fluorescence, respectively. Scale bar indicates 5 μM. (B) SlWRKY3 transactivation ability. SlWRKY3 coding region was fused to GAL4-DBD into the pGBKT7 vector carrying the nutritional marker TRP1 as reporter gene. The resulting plasmid, and the empty pGBKT7 vector used as negative control, were transformed into the yeast strain Y8930 harboring ADE2 and HIS3 reporter genes. Yeasts were separately grown on SD (synthetic dropout) medium lacking either tryptophan (a,b; pGBKT7 or GAL4-DBD-SlWRKY3, respectively), or adenine and histidine (a′,b′; pGBKT7 or GAL4-DBD-SlWRKY3, respectively). (C) Position weight matrix (PWM) representation of the top scoring 8-mer obtained in “seed-and-wobble” algorithm. (D) Box plot of the distribution of enrichment scores (E-scores) of all possible 8-mers containing the indicated elements. Boxes represent quartiles 25–75%, black line represents the median of the distribution (quartile 50%) and bars indicate quartiles 1 to 25% (above) and 75 to 100% (below). The higher and sharper distributions of E-scores corresponding to TTGACT and TTGACC motifs reflect higher binding affinity of SlWRKY3 to these elements. (E) Box plot of the distribution of E-scores of all possible 8-mers containing the indicated elements varying at their 5′-end nucleotide, showing highest affinity of SlWRKY3 for the DNA element GTTGACY. Representation of the boxes is as in (D). (F) E-scores of the 8-mers indicated in the Figure. Each point represents the average of two different E-scores, corresponding to NGTTGACC and NGTTGACT. Highest affinity was observed for CGTTGACY, as represented in the logo representation in (C). In spite of this, all the 8-mers containing the element GTTGACY were very efficiently recognized by SlWRKY3.