Fig. 1
Effect of PthA5 and PthA6 in canker formation. A Grapefruit (Citrus paradisi) and kumquat (Fortunella margarita) leaves were infiltrated with Xcc003 and Xcc086 strains (OD600 = 1.0). Canker symptoms were recorded at 21 days post-inoculation (dpi). B Alignment of repeat-variable diresidues (RVDs) of PthA6, PthA5, PthA1, and PthA4. PthA6 (from Xcc003) and PthA5 (from Xcc086) were compared with PthA1 and PthA4 from X. citri strains. Black lines (-) indicate missing RVDs. Different RVDs are highlighted in gray and colored, corresponding to nucleotide association and DNA base specificities. Green: NI recognizes adenine (A); Red: NG recognizes thymine (T); Blue: HD recognizes cytosine (C); Orange: N* recognizes C and T; Purple: NS recognizes ACT and guanine (G). C Grapefruit leaves were infiltrated with Xcc suspensions (OD600 = 1.0) of tal-free deletion mutant Xcc049E/EV and complemented strains Xcc049E/pthA5 or Xcc049E/pthA6. Canker symptoms were evaluated at 21 dpi. Microscopic observation of thin cross-sections in the circled regions. D Effect of PthA5 and PthA6 on bacterial growth in grapefruit. Grapefruit leaves were infiltrated with Xcc suspensions (OD600 = 0.8). Bacterial growth assays of Xcc strains were monitored at 0, 1, 2, 3, 4, 8, and 12 dpi. Bacterial growth is presented in mean colony-forming units (CFU)/cm2, indicating PthA5 and PthA6 enhance bacterial growth in citrus host. Error bars represented standard deviations of three biological replicates. Asterisks denote statistically significant differences in bacterial-inoculated leaves, as determined by two-way ANOVA with Dunnett’s test: **p < 0.01, ***p < 0.001, and ****p < 0.0001. E Grapefruit leaves were infiltrated with Xcc suspensions (OD600 = 0.6) of Xcc049E/pthA5, Xcc049E/pthA6, and mixed suspensions of Xcc049E/pthA4 and Xcc049E/pthA5 or Xcc049E/pthA6 at a 1:1 ratio. Xcc049E/EV and Xcc049E/pthA4 were used as the negative and positive controls, respectively. Citrus canker symptoms were evaluated at 16 dpi. F Bacterial growth of Xcc strains in grapefruit leaves was monitored at 25 dpi. Error bars represent standard deviation of three biological replicates. Asterisks denote statistically significant differences, as determined by one-way ANOVA with Dunnett’s test: *p < 0.05, ****p < 0.0001