Figure 1.
Disease symptoms in bean plants.
Plants were infiltrated with 106 cfu/ml, and symptoms documented at 7 days post-inoculation (dpi). A. Virulence symptoms caused by Pph ΔhopR1 are significantly reduced in relation to wild type (Pph)-induced symptoms. B. Pph ΔhopAY1 causes similar symptoms to those induced by wild type (Pph). The experiment was carried out thrice with similar results. Images show representative results.
Table 1.
Bacterial strains used in this work.
Table 2.
Analysis of symptom development after inoculation with different strains.
Figure 2.
Growth curves of Pph 1448a and Pph ΔhrpL.
Bacteria were inoculated either by infiltration with a bacterial suspension containing 5×104 cfu/ml or by dipping into a bacterial suspension containing 5×107 cfu/ml. Error bars represent standard error. Smallest error bars may be covered by the corresponding symbol.
Figure 3.
Competitive Index analysis of Pph 1448a effector mutants.
Mutant strains were co-infiltrated with the wild type strain, and samples were taken at different time points: A. 4, 7 and 14 days post-inoculation (dpi), or B. 4 and 7 dpi. Competitive indices correspond to the mean of two to four independent experiments with three replicates per experiment. A dashed line corresponding to a CI = 0.5 is included for reference. Error bars represent the standard error. Asterisks indicate results significantly different from one, as established by Student's t-test (P<0.05). Mean values marked with the same letter (a or b) indicate results not significantly different from each other, as established by One Way ANOVA and Holm-Sidak test for multiple comparison or One Way ANOVA on Ranks and Tukey test for multiple comparison when Equal Variance Test failed (P<0.05). Two asterisks indicate that attenuation was right on the limit of significance in each independent experiment or when results were pulled from independent experiments.
Figure 4.
Complementation analysis of ΔhopAB1 and ΔhopI1.
The mutant strains carrying a plasmid expressing the corresponding effector from their native promoter were co-inoculated with the wild type strain and samples were taken at different time points (4, 7 and 14 dpi). Competitive indices are the average of three replicates per experiment. The assays were repeated twice with very similar results. The CIs of the relevant mutants are included (grey) for comparison purposes. A dashed line corresponding to a CI = 0.5 is included for reference. Error bars represent the standard error. Asterisks indicate results significantly different from one, as established by Student's t-test (P<0.05). Mean values marked with the same letter (a or b) indicate results not significantly different from each other, as established by One Way ANOVA and Holm-Sidak test for multiple comparison (P<0.05).
Figure 5.
Effect on bacterial growth of overexpressing HopAB1 and HopI1.
Wild type strain Pph 1448a carrying plasmids expressing either of these effectors from the strong nptII promoter were co-inoculated with the wild type strain. Competitive index was analyzed either in bean leaves (at 7 dpi) or in rich medium (LB medium, at 24 hours post-inoculation). Competitive indices are the average of three replicates per experiment. A dashed line corresponding to a CI = 0.5 is included for reference. Error bars represent the standard error. Asterisks indicate results significantly different from one, as established by Student's t-test (P<0.05). Mean values marked with the same letter (a or b) indicate results not significantly different from each other, as established by One Way ANOVA and Holm-Sidak test for multiple comparison or One Way ANOVA on Ranks and Tukey test for multiple comparison when Equal Variance Test failed (P<0.05).
Figure 6.
Competitive Index analysis of Pph 1448a effector mutants following dip-inoculation.
Mutant strains were co-inoculated with the wild type strain, and samples were taken at different time points (4, 7 and 14 days post-inoculation, dpi) as indicated. Competitive indices correspond to the mean of two to three independent experiments with three replicates per experiment. A dashed line corresponding to a CI = 0.5 is included for reference. Error bars represent the standard error. Asterisks indicate results significantly different from one, as established by Student's t-test (P<0.05). Mean values marked with the same letter (a or b) indicate results not significantly different from each other, as established by One Way ANOVA and Holm-Sidak test for multiple comparison (P<0.05).
Figure 7.
Cancelled-out index (COI) epistasis analysis of Pph 1448a effectors.
Epistasis analysis of HopR1 (A) and HopI1 (B) on the contribution to virulence of HopAB1. Double mutants strains were co-infiltrated with each single mutant strain and the corresponding COI was determined at 7 dpi. All relevant CIs are included (grey) for comparison purposes. Each COI corresponds to the mean of at least three independent experiments with three replicates per experiment. A dashed line corresponding to an index value = 0.5 is included for reference. Error bars represent the standard error. Asterisks indicate results significantly different from one, as established by Student's t-test (P<0.05). Mean values marked with the same letter (a or b) indicate results not significantly different from each other, as established by One Way ANOVA and Holm-Sidak test for multiple comparisons (P<0.05).
Table 3.
Epistatic analysis of HopAB1 function on the contribution to virulence of HopR1 and HopI1.