The TALE PthA4 of <i>Xanthomonas citri</i> subsp. <i>citri</i> Indirectly Activates an Expansin Gene <i>CsEXP2</i> and an Endoglucanase <i>CsEG1</i> via CsLOB1 to Cause Citrus Canker Symptoms

Citrus canker caused by <i>Xanthomonas citri</i> subsp. <i>citri</i> (Xcc) is an important citrus disease worldwide. <i>PthA4</i> is the most important pathogenicity gene of Xcc and encodes a transcription activator-like effector (TALE) secreted by the type III secretion system. PthA4 is known to activate the expression of <i>CsLOB1</i>, the canker susceptibility gene and a transcription factor, to cause citrus canker symptoms. Extensive effort was made to identify downstream targets of CsLOB1 to investigate the mechanism underlying canker symptom development. However, none of the identified CsLOB1 target genes has been confirmed to be involved in citrus canker development. Here, we first identified the direct targets of CsLOB1 by generating a promoter-<i>uidA</i> (GUS) reporter fusion construct for the 13 genes highly induced by both PthA4 and CsLOB1 and monitored the reporter activity in <i>Nicotiana benthamiana</i> leaves co-expressing <i>CsLOB1</i>. <i>Agrobacterium tumefaciens</i>-mediated transient expression of <i>CsLOB1</i> activated seven gene promoters in <i>N. benthamiana</i>: Cs7g18460, Orange1.1t00600, Cs6g17190, Cs7g32410 (<i>CsEXP2</i>), Cs2g27100, Cs2g20750 (<i>CsEG1</i>), and Cs9g17380. Next, we constructed designer TALEs (dTALEs) to target unique sequences in the promoters of the seven direct target genes of CsLOB1 and transformed them into the Xcc<i>pthA4</i>::Tn<i>5</i> mutant. Our results indicated that a combination of five and seven dTALEs caused canker-like symptoms in the inoculated citrus leaves. In addition, dTALECsEXP2 and dTALECsEG1 caused water soaking and pustules, which are typical canker symptoms. Taken together, Xcc indirectly activates CsEXP2 and CsEG1 via PthA4-CsLOB1 to cause canker symptoms. Identification of direct targets of CsLOB1 provides alternative targets for genetic improvement of citrus against canker via genome editing. [Formula: see text] Copyright © 2025 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.