Fig. 1
From: An effector from the Huanglongbing-associated pathogen targets citrus proteases
SDE1 interacts with citrus papain-like cysteine proteases. a Yeast-two-hybrid (Y2H) assays using the CLas effector SDE1 as the bait and full-length citrus papain-like cysteine proteases (CsPLCPs), representing different subfamilies as the prey. SDE1 was cloned into the vector pGBKT7 and individual CsPLCPs were cloned into the vector pGADT7. Growth of yeast cells on SD-3 selective media represents protein–protein interaction, growth of the same cells on SD-2 media confirms yeast transformation. Yeast transformed with the empty vectors served as negative controls. The initial PLCP found from Y2H screening (CsSAG12-1, XM_006495158) is indicated with an asterisk (*). The gene IDs of the other interacting PLCPs are CsSAG12-2 (XM_006470229), CsXCBP3 (orange1.1g012960), CsRD21a (XM_006473212), CsRD19 (orange1.1g017548), CsAALP (XM_006474664), and CsCTB (orange1.1g018568). b Phylogeny and subfamily classification of canonical PLCPs in the C. sinensis (sweet orange) genome. The phylogenetic tree was made with MEGA6.06 (100 bootstrap replicates, Maximum-Likelihood method, Jones–Taylor–Thornton model), using the Arabidopsis thaliana PLCP subfamily classification22. The asterisk (*) indicates the initially found CsSAG12-1. c Y2H assay examining the interaction of SDE1 with the cysteine protease domain of CsPLCPs. d In vitro pull-down assay using the GST-tagged cysteine protease domain of CsPLCPs to immunoprecipitate SDE1 protein. Input and immunoprecipitated proteins (output) were visualized by western blotting using anti-GST and anti-SDE1 antibodies. Asterisks (*) indicate the protein bands that correspond to individual CsPLCPs. GST-tagged Arabidopsis double-stranded DNA-binding protein 4 (AtDRB4) was used as a negative control
