International Journal of Horticulture, 2024, Vol.14, No.3, 142-155 http://hortherbpublisher.com/index.php/ijh 147 basis of resistance mechanisms in ACP. For instance, research has identified several detoxification genes, such as cytochrome P450, glutathione-S-transferase, and esterase genes, which are overexpressed in imidacloprid-resistant ACP strains (Tian et al., 2019). Additionally, the differential gene expression analysis of ACP infected with Candidatus Liberibacter asiaticus (CLas) has revealed specific genes involved in the infection and circulation within the psyllid host, highlighting potential targets for genetic modification (He et al., 2023). By understanding the genetic basis of resistance, researchers can design CRISPR/Cas9 strategies to enhance these natural defense pathways or disrupt the interaction between ACP and the citrus plant, thereby reducing the incidence of HLB. 3.2 Successful case studies of CRISPR/Cas9-mediated gene editing in citrus The CRISPR/Cas9 technology has been successfully applied to various plant species, including citrus, to develop disease-resistant varieties. A example is the CRISPR/Cas9-mediated mutation of the Asian citrus psyllid (ACP) itself, demonstrating the feasibility of using gene editing to control the vector of HLB. The study conducted by Chaverra-Rodriguez et al. (2023) successfully introduced specific genetic modifications into the ACP genome. The experimental results showed that CRISPR-Cas9 technology could effectively introduce genetic mutations, thereby affecting gene expression in ACP. This indicates the feasibility of performing gene editing in this pest. Huang et al. (2022) reported an improved CRISPR/Cas9 system for generating canker-resistant mutants of Hamlin sweet orange (Citrus sinensis (L.) Osbeck) against Xanthomonas citri subsp. citri (Xcc). The research team enhanced the CRISPR/Cas9 system by optimizing the selection of Cas9 promoters, such as using the Cestrum yellow leaf curling virus (CmYLCV) or Citrus sinensis ubiquitin (CsUbi) promoter, and adjusting the culture temperature, significantly increasing gene editing efficiency (Figure 3). Consequently, this system achieved up to 89% biallelic mutation rates in sweet orange and tobacco. The study demonstrates that the improved CRISPR/Cas9 system exhibits high efficiency in gene editing in sweet orange and can also be applied to other dicot plants. This system provides new possibilities for gene editing research on citrus diseases and the development of disease-resistant varieties, offering critical technical support for disease control in the citrus industry. Figure 3 Induction of CsLOB1 by Xcc is abolished in a biallelic mutant line of Hamlin sweet orange (Adopted from Huang et al., 2022) Image caption: The figure shows the response of the biallelic mutant Hamlin sweet orange (Citrus sinensis (L.) Osbeck) to the induction of the CsLOB1 gene expression by the citrus canker pathogen (Xanthomonas citri subsp. citri, Xcc). A: RT-qPCR analysis indicates that in wild-type Hamlin sweet orange, the expression of the CsLOB1 gene significantly increased after Xcc inoculation, whereas there was no change in expression in the biallelic mutant Ham1. B: The Xcc pthA4dLOB2 strain induced the expression of the LOB2 gene, which was consistent in both the wild-type and mutant plants. C: The binding site of the dLOB2 effector with the LOB2 promoter, further confirming the resistance mechanism of the mutants against Xcc (Adapted from Huang et al., 2022)
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