A survey of FLS2 genes from multiple citrus species identifies candidates for enhancing disease resistance to Xanthomonas citri ssp. citri.

Pathogen-associated molecular patterns (PAMPs)-triggered immunity (PTI) is an important component of plant innate immunity. In a previous study, we showed that the PAMP flg22 from Xanthomonas citri ssp. citri (Xflg22), the causal agent of citrus canker, induced PTI in citrus, which correlated with the observed levels of canker resistance. Here, we identified and sequenced two bacterial flagellin/flg22 receptors (FLS2-1 and FLS2-2) from ‘Duncan’ grapefruit (Citrus paradisi, CpFLS2-1 and CpFLS2-2) and ‘Sun Chu Sha’ mandarin (C. reticulata, CrFLS2-1 and CrFLS2-2). We were able to isolate only one FLS2 from ‘Nagami’ kumquat (Fortunella margarita, FmFLS2-1) and gene flanking sequences suggest a rearrangement event that resulted in the deletion of FLS2-2 from the genome. Phylogenetic analysis, gene structure and presence of critical amino acid domains all indicate we identified the true FLS2 genes in citrus. FLS2-2 was more transcriptionally responsive to Xflg22 than FLS2-1, with induced expression levels higher in canker-resistant citrus than in susceptible ones. Interestingly, ‘Nagami’ kumquat showed the highest FLS2-1 steady-state expression levels, although it was not induced by Xflg22. We selected FmFLS2-1, CrFLS2-2 and CpFLS2-2 to further evaluate their capacity to enhance bacterial resistance using Agrobacterium-mediated transient expression assays. Both FmFLS2-1 and CrFLS2-2, the two proteins from canker-resistant species, conferred stronger Xflg22 responses and reduced canker symptoms in leaves of the susceptible grapefruit genotype. These two citrus genes will be useful resources to enhance PTI and achieve resistance against canker and possibly other bacterial pathogens in susceptible citrus types.