Supplementary Material for: Escherichia colimazEF Toxin-Antitoxin System as a Tool to Target Cell Ablation in Plants

<p><b><i>Background/Aims:</i></b> The <i>Escherichia coli</i> MazF is an endoribonuclease that cleaves mRNA at ACA sequences, thereby triggering inhibition of protein synthesis. The aim of this study is to evaluate the efficiency of the <i>mazEF</i> toxin-antitoxin system in plants to develop biotechnological tools for targeted cell ablation. <b><i>Methods:</i></b> A double transformation strategy, combining expression of the <i>mazE</i> antitoxin gene under the control of the <i>CaMV 35S</i> promoter, reported to drive expression in all plant cells except within the tapetum, together with the expression of the <i>mazF </i>gene under the control of the <i>TA29</i> tapetum-specific promoter in transgenic tobacco, was applied. <b><i>Results:</i></b> No transgenic <i>TA29-mazF</i> line could be regenerated, suggesting that the <i>TA29</i> promoter is not strictly tapetum specific and that MazF is toxic for plant cells. The regenerated<i> 35S-mazE</i>/<i>TA29-mazF</i> double-transformed lines gave a unique phenotype where the tapetal cell layer was necrosed resulting in the absence of pollen. <b><i>Conclusion:</i></b> These results show that the <i>E. coli</i><i>mazEF</i> system can be used to induce death of specific plant cell types and can provide a new tool to plant cell ablation.</p>