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Engineering xopAG induced expression by Xanthomonas citri TALE confer resistance to citrus canker

Deepak Shantharaj: Auburn University, Department of Biological Sciences

<div><em>Xanthomonas citri</em> ssp. <em>citri</em> (Xcc), causal agent of citrus canker, delivers transcription activator-like effectors (TALEs) into plant cells via the type III secretion system. One group of TALEs targets effector binding elements (EBEs) in the host genome to activate expression of a susceptibility gene, essential for typical disease. Predictably, TALEs bind EBEs in host promoters. We introduced 14 EBEs, matching distinct Xcc TALEs, into the promoter of the pepper <em>Bs3</em> gene (ProBs3<span><sub>1EBE</sub></span>), and fused this engineered promoter (ProBs3<span><sub>14EBE</sub></span>) to the <em>X. fuscans </em>subsp. <em>aurantifolii</em> gene, <em>avrGf2</em>, which encodes a hypersensitive response (HR)-eliciting effector in grapefruit, to create ProBs3<span><sub>14EBE</sub></span>:<em>avrGf2</em>. This construct was placed in the binary vector, pTLAB21. <em>Agrobacterium</em> strain EHA101 carrying the binary vector pTLAB21 with ProBs3<span><sub>14EBE</sub></span>:<em>avrGf2</em> was used to produce genetically engineered grapefruit plants. The plant transformed with ProBs3<span><sub>14EBE</sub></span>:<em>avrGf2 </em>was evaluated for disease resistance. Infiltration of a bacterial suspension (10<sup>8</sup> CFU/ml) of Xcc into leaves elicited an HR in leaves of the transgenic grapefruit, but not wild-type grapefruit. Similarly, when a bacterial suspension (10<sup>5</sup> CFU/ml) of Xcc was infiltrated into leaves, bacterial populations were ten-fold lower in leaves of the transgenic plant than in the wild-type grapefruit, 5 days after infiltration. Grapefruit transformed with ProBs3<span><sub>14EBE</sub></span>:<em>avrGf2</em> offers resistance to a broad spectrum of citrus canker strains.</div>