FIGURE 5.

XaFliC is the main elicitor of canker resistance in sweet oranges. (a) Western blot analysis showing the expression of XaFliC (arrow) in wild‐type Xanthomonas aurantifolii (Xa) and Xanthomonas citri (Xc), fliC mutants XaΔfliC and XcΔfliC, and corresponding fliC mutants complemented with XaFliC (XaΔfliC + XaFliC, XcΔfliC + XaFliC). Blots were probed with anti‐XaFliC serum. (b) Motility assays performed on soft agar showing that disruption or deletion of FliC in Xa and Xc significantly reduces bacterial motility. Complementation of these mutants with XaFliC only partially restored Xa motility, whereas in Xc, XaFliC expression significantly increased cell motility. (c) Leaves of Natal, Pera, and Valencia plants infiltrated with Xc (control) or with a mixed suspension (OD₆₀₀ = 0.1) of Xc + Xa (Xa) or Xc + XaΔfliC transformed with empty vector or vector expressing XaFliC (at a 1:1 ratio), showing that Xa but not XaΔfliC blocks canker development when simultaneously inoculated with Xc. (d) Canker symptoms developed on Natal, Pera, Valencia, and Sorocaba sweet oranges and in Mexican lime leaves 10 days after they were infiltrated with a water suspension (OD₆₀₀ = 0.1) of Xc or the corresponding XcΔfliC mutants (areas surrounded by dashed lines). Expression of XaFliC in the XcΔfliC mutant was sufficient to trigger a robust defence response in all sweet orange varieties tested, but not in Mexican lime. Three leaves of each citrus variety were infiltrated and the images presented are representative of the phenotype observed in most infiltrated leaves. (e) Bacterial counts on Natal and Mexican lime leaves infiltrated with wild‐type Xc or XcΔfliC transformed with XaFliC or the empty vector (EV), used as control, determined at 0, 3, 7, and 14 days post‐infection (dpi). While in Natal leaves the XcΔfliC mutant expressing XaFliC showed a sharp reduction in growth within 14 days of bacterial inoculation, relative to Xc or the XcΔfliC mutant carrying the empty vector, this complemented strain reached much higher titres in Mexican lime leaves at 3, 7, and 14 dpi. Error bars represent standard deviations of three independent biological samples, and asterisks indicate statistically significant differences between the means at α = 0.05 relative to Xc