Figure 1

Heterologous expression in citrus epicotyls, RVD sequences and phylogeny of TAL effectors PthA2, PthA4 and PthC1. (A) Western blot of protein extracts from sweet orange epicotyls transfected with A. tumefaciens EHA105 carrying pBI121-35S::pthA2 (PthA2), pBI121-35S::pthA4 (PthA4), pBI121-35S::pthC1 (PthC1), or the native plasmid pBI121-35S::uidA (GUS) for control of TAL effectors expression. Total protein from epicotyls expressing PthA2, PthA4 or, PthC1 proteins (~116 kDa) were separated by electrophoresis on 10% SDS-polyacrylamide gels, transferred to PVDF membranes, and detected with anti-PthA2 serum (left panel). The expression of uidA gene was assayed histochemically for β-glucuronidase (GUS) activity using 5-bromo-4-chloro-3-indolyl-β-D-glucuronic acid (X-Gluc) as substrate (right panel). (B) RVD sequence composition of X. citri isolate 306 TAL effectors PthA1, PthA2, PthA3 and PthA4, and PthC1 and PthC2 from X. aurantifolii ICMP 8435, aligned with the corresponding predicted DNA targets. According to the TAL effector code, only the first and second bases associated with higher frequency for each RVD are represented. (C) Phylogenetic tree of TAL effectors from different Xanthomonas strains that cause citrus canker disease and blight or leaf streak of rice. The maximum likelihood analysis was built with the PhyML tool using a bootstrapping procedure of 500 repetitions. Only the C-terminal domains (~278 residues) of TAL effectors from Xanthomonas spp. were used for the analysis. The four PthA“s” of Xc strain 306 belong into a group close to other TAL effectors from pathotype A strains; meanwhile, PthCs and PthBs from X. aurantifolii integrate a distinct group of pathotypes B and C strains, respectively. The tree is displayed with the TAL effectors from X. oryzae strains rooted as outgroup. Amino acid sequences were aligned using MUSCLE and analyzed on phylogenetic pipeline of Phylogeny.fr[41].