Computational and Biochemical Analysis of the Xanthomonas Effector AvrBs2 and Its Role in the Modulation of Xanthomonas Type Three Effector Delivery
Figure 2
The GDE catalytic sites are required for AvrBs2 virulence function.
A. The map of the AvrBs2 coding region with numbers representing amino acid positions. AvrBs2 mutations for the GDE catalytic site and the AvrBs2 mutations from Xe field strains isolated from diseased BS2 pepper. B. In planta pathogen growth assay for Xanthomonas strains GM98-38 Xe (w/o avrBs2), GM 98-38-1 Xe (avrBs2), and GM98-38-1 exchange mutants for the putative GDE catalytic site Xe (avrBs2-E304A/D306A) and Xe (avrBS2-H319A) and two previously published control exchange mutants Xe (avrBs2-R403P) and Xe (avrBs2-A410E). Host plants include the near-isogenic pepper (w/o Bs2) and pepper (Bs2) along with the tomato line VF36 (w/o Bs2) and the previously published transgenic line VF36 (Bs2). Student t-test was used to compare different growth assays with the most virulent case (Xe (avrBs2) on non-Bs2 plants) for both Pepper and Tomato hosts; p-values were <0.01 for all other combination when compared to (Xe (avrBs2) on non-Bs2 plants). The Xe strains carrying AvrBs2 mutations for the GDE catalytic site still activate full Bs2 resistance but do not maintain the full virulence in the absence of Bs2. There is a corresponding wild type HR brown necrosis phenotype for Bs2 pepper inoculated with high-density suspensions (2×108 CFU/ml) for these two Xe mutant strains (Supplemental Figure S1). The Xe strains carrying AvrBs2 mutations from Xe field strains isolated from diseased Bs2 pepper grow to similar levels in the presence or absence of Bs2. There is a corresponding loss of the HR brown necrosis phenotype for Bs2 pepper inoculated with high-density suspensions of Xe avrBs2-R403P and only a weak HR brown necrosis phenotype for Xe avrBs2-A410E (Supplemental Figure S1). C. Plot of amino acid substitution rate analysis using a sliding window calculation of non-synonymous (KA) and synonymous (Ks) changes between avrBs2 homologs from Xe and Xcc. The KA/Ks ratios less than 0.5 indicate that much of AvrBs2 is under purifying selection, including the region homologous to GDE that is required for AvrBs2 virulence function. The numbers below the plot represent amino acid positions.