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 Xanthomonas citri. 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 Poncirus trifoliata—PtGASA6, PtGASA8, and PtGASA10—were then selected for functional validation in Nicotiana benthamiana. Transient overexpression of PtGASA6 and PtGASA10 significantly reduced disease symptoms caused by Pseudomonas syringae and heightened the hypersensitive response to X. citri, whereas PtGASA8 showed no detectable effect. Notably, PtGASA6 enhanced the hypersensitive response by 30% more than PtGASA10, while PtGASA10 delayed necrosis by 40% more than PtGASA6, indicating distinct antimicrobial mechanisms. Together, these results identify PtGASA6 and PtGASA10 as strong candidates for breeding and biotechnological strategies aimed at improving broad-spectrum bacterial disease resistance in citrus.

The SNAKIN/GASA family comprises antimicrobial peptides with proven activity against phytopathogens that may play a significant role in citrus rootstock breeding. We identified 67 new curated GASA variants present in a germplasm collection. They were checked for the diagnostic 12-cysteine-domain and classified into three GASA subfamilies. Absolute expression of ten representative GASA genes was analyzed in floral, young leaf and mature leaf tissues from five accessions with contrasting levels of disease tolerance against Xanthomonas citri. 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 candidate genes (PtGASA6, PtGASA8, and PtGASA10) derived from Poncirus trifoliata (a rootstock cultivar conferring disease tolerance), were selected and functionally assessed via transient overexpression in Nicotiana benthamiana. Overexpression of PtGASA6 and PtGASA10 significantly reduced disease symptoms caused by Pseudomonas syringae and enhanced hypersensitive response to Xanthomonas citri, whereas PtGASA8 showed no effect. Specific time-course dynamics and structural predictions suggest distinct antimicrobial mechanisms among these 2 genes. These findings provide new genetic targets for breeding and biotechnological strategies aimed at improving broad-spectrum bacterial disease resistance in citrus.