Genome-Wide Analysis of CsCAX Genes and Functional Characterization of CsCAX3 Revealing Its Negative Role in Citrus Bacterial Disease Resistance

Cation/proton antiporters (CAXs) are key membrane transporters involved in plant development and stress adaptation. In this study, five CsCAX genes (CsCAX1–CsCAX5) were identified from the Citrus sinensis genome through comprehensive bioinformatic analysis. All CsCAX proteins are hydrophobic with 10–11 transmembrane domains, predominantly composed of α-helices and random coils, and are localized to either the plasma membrane or vacuole. Phylogenetic analysis classified them into two subfamilies (IA and IB). Promoter prediction identified stress- and hormone-responsive cis-elements (e.g., LTR, SA, and ABA), implying transcriptional regulation under environmental cues. Tissue-specific expression profiling revealed the highest CsCAX3 transcript abundance in stems and leaves. CsCAX3 was upregulated upon Xanthomonas citri subsp. citri (Xcc) infection but downregulated by Candidatus Liberibacter asiaticus (CLas). Subcellular localization confirmed CsCAX3 targeting to the plasma membrane. Functional verification showed that CsCAX3 overexpression increased susceptibility to Xcc, accompanied by a six-fold rise in bacterial load and substantial repression of antioxidant genes (CAT2, POD). Furthermore, transient overexpression of CsCAX3 in tobacco (Nicotiana benthamiana) will eliminate reactive oxygen species (ROS) accumulation. These results indicate that CsCAX3 negatively affects disease resistance by reducing sensitivity to ROS-mediated defense responses. Overall, this study elucidates the structural and functional characteristics of the CsCAX gene family and provides new insights into ROS-mediated immune regulation in citrus.