CRISPR/Cas9-Mediated Disruption of <i>CsLIEXP1</i> Reveals Expansin as a Key Susceptibility Factor for Citrus Canker Disease

The <i>Citrus sinensis LATERAL ORGAN BOUNDERIES 1</i> (<i>CsLOB1</i>) gene, which is directly induced by the <i>Xanthomonas citri</i> subsp. <i>citri</i> effector PthA4, functions as a transcription factor and citrus canker susceptibility (S) gene. Genome editing of <i>CsLOB1</i> has been shown to confer resistance to citrus canker disease. Previous studies revealed that the citrus <i>CsLOB1-INDUCED EXPANSIN 1</i> gene (<i>CsLIEXP1</i>) is highly and directly upregulated by <i>CsLOB1</i> in <i>Xanthomonas citri</i> subsp. <i>citri</i>-infected plants. Because expansins are associated with cell wall loosening, potentially facilitating bacterial colonization, the <i>CsLOB1-</i>dependent activation of <i>CsLIEXP1</i> is thought to contribute to canker symptoms and disease progression. Thus, <i>CsLIEXP1</i> likely represents a critical canker susceptibility gene. In this study, we employed CRISPR/Cas9 to disrupt the function of <i>CsLIEXP1</i> by modifying its corresponding coding region in <i>Citrus sinensis</i> cultivar 'Hamlin' and evaluated the postinfection responses of edited plants. DNA sequencing confirmed the edition of the <i>CsLIEXP1</i>-edited plant, which exhibited 26.47% of <i>CsLIEXP1</i> edited sequences. Furthermore, <i>CsLIEXP1</i> protein accumulation was reduced in <i>CsLIEXP1</i>-edited plants compared with the wild type when infected with <i>X. citri</i>. Leaves of edited plants inoculated with <i>X. citri</i> showed significantly fewer canker symptoms, with lesions limited to the site of bacterial inoculation and less pronounced cellular hypertrophy compared with control plants. Our results show that <i>CsLIEXP1</i> is a citrus canker S gene that acts downstream of <i>CsLOB1</i>, thus providing new insights into plant-pathogen interactions. [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.