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

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