Pangenomic Approach for the Identification and Functional Characterization of Active GASA Antimicrobial Genes in Citrus Rootstocks for Resistance Breeding Against Bacterial Pathogens

The SNAKIN/GASA family comprises antimicrobial peptides with proven activity against bacteria and fungi, making them promising candidates for improving disease resistance in citrus rootstocks. In sixty-seven new GASA variants from a citrus germplasm collection, the presence of the characteristic 12-cysteine domain was confirmed and were classified into three subfamilies. The absolute expression levels of ten representative genes were analyzed in floral tissues, young leaves, and mature leaves from five citrus accessions with contrasting susceptibility to <i>Xanthomonas citri</i>. Expression profiling revealed tissue-specific patterns, with higher transcript abundance in juvenile and floral tissues of tolerant accessions. Meta-analysis of HLB-related RNA-seq datasets revealed the upregulation of specific GASA genes. Three genes from <i>Poncirus trifoliata</i>-<i>Pt</i>GASA6, <i>Pt</i>GASA8, and <i>Pt</i>GASA10-were then selected for functional validation in <i>Nicotiana benthamiana</i>. Transient overexpression of <i>Pt</i>GASA6 and <i>Pt</i>GASA10 significantly reduced disease symptoms caused by <i>Pseudomonas syringae</i> and heightened the hypersensitive response to <i>X. citri</i>, whereas <i>Pt</i>GASA8 showed no detectable effect. Notably, <i>Pt</i>GASA6 enhanced the hypersensitive response by 30% more than <i>Pt</i>GASA10, while <i>Pt</i>GASA10 delayed necrosis by 40% more than <i>Pt</i>GASA6, indicating distinct antimicrobial mechanisms. Together, these results identify <i>Pt</i>GASA6 and <i>Pt</i>GASA10 as strong candidates for breeding and biotechnological strategies aimed at improving broad-spectrum bacterial disease resistance in citrus.