Figure 7
Characterization of the HAT and Radical SAM Domain Mutants of AtELP3. (A) Schematic representation of the HAT and radical SAM domain mutations. The radical SAM and HAT domains of AtELP3 are aligned with those of the yeast (ScELP3) and mouse ELP3 (MmELP3). Only sequences that are part of the alignment are shown. The conserved amino acid residues are labeled in red. Arrows indicate the mutations created in the SAM and HAT mutants. (B) Ten-day-old seedlings of WT, npr1, Atelp3-10 npr1, HAT(n), and SAM(n) grown on half-strength MS agar medium containing 0.26 mM SA. HAT(n) and SAM(n), transgenic lines in the Atelp3-10 npr1 genetic background expressing the HAT and the SAM mutant, respectively. (C) Free SA levels in Psm ES4326-infected WT, npr1, Atelp3-10 npr1, HAT(n), and SAM(n) plants. Plants were inoculated with Psm ES4326 (OD600 = 0.001). Twenty-four hours later, the inoculated leaves were collected for SA measurement. Data represent the mean of three independent samples with SD. Different letters above the bars indicate significant differences (p < 0.05, Student’s t-test). (D) Total SA levels in Psm ES4326-infected WT, npr1, Atelp3-10 npr1, HAT(n), and SAM(n) plants. Experiment was performed as in (C). (E) Expression of eight defense genes in Pst DC3000/avrRpt2-infected WT, Atelp3-10, HAT(C), and SAM(C) plants. HAT(C) and SAM(C), transgenic lines in the Atelp3-10 genetic background expressing the HAT and the SAM mutant, respectively. Data represent the mean of three independent samples with SD. An asterisk (*) indicates that the expression level of the gene in the WT was significantly higher than that in Atelp3-10, HAT(C), and SAM(C) (p < 0.05, Student’s t-test). (F) Growth of Psm ES4326 in WT, Atelp3-10, HAT(C), and SAM(C) plants. Cfu, colony-forming units. Data represent the mean of eight independent samples with standard deviation. Different letters above the bars indicate significant differences (p < 0.05, Student’s t-test).