RpfF Transgenic citrus unveil diffusible signal factor as a broad-spectrum defense elicitor

Brazil is the largest producer of sweet oranges and the leading exporter of orange juice, but bacterial diseases threaten the citrus industry. To address this, our research group developed a transgenic sweet orange overexpressing rpfF, a gene involved in synthesizing diffusible signaling factor (DSF), which regulates quorum sensing (QS) in Xylella fastidiosa (causal agent of citrus variegated chlorosis, CVC) and Xanthomonas citri (causal agent of citrus canker, CC). DSF-overexpressing plants disrupted bacterial communication, reducing CC and CVC under greenhouse. In the field, these genetically modified plants (GMO) also exhibited a significant reduction in HLB symptoms. Since Candidatus Liberibacter asiaticus (Clas), the causal agent of HLB, lacks a QS system regulated by DSF, we hypothesized that DSF might act as an elicitor. Lipid extracts from GMO and wild-type (WT) plants were infiltrated into Nicotiana benthamiana, showing a hypersensitive response (HR) only in DSF-expressing samples. We then performed transcriptomic and lipidomic analyses to identify the genetic and lipid profiles associated with resistance in GMO plants. Differential gene expression revealed 35 upregulated genes unique to GMOs, with several associated with plant defense against biotic stress. Lipidomic analysis identified 20 distinct lipids, with several linked to plant defense signaling. Overall, our results suggest that rpfF generates a small unsaturated fatty acid, priming resistance against HLB.