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. 2022 Jul 21;34(10):4088–4104. doi: 10.1093/plcell/koac209

Figure 3.

Figure 3

TCH3 and CAM1/4/6 contribute to PAMP-induced CBP60g phosphorylation and resistance to V. dahliae. A, Expression of TCH3 induces phosphorylation of CBP60g. Protoplasts isolated from WT plants were transfected with Pro35S:CBP60g-FLAG alone or together with HA fusions of TCH3, CML40, CML45, or CML46 as indicated. B, Expression of homologs of TCH3 induces phosphorylation of CBP60g. Protoplasts isolated from WT plants were transfected with Pro35S: CBP60g-FLAG alone or together with HA fusions of CAM1-7 or CML8 as indicated. C, CBP60g phosphorylation induced by flg22 is significantly compromised in the cmlquad mutant. Protoplasts isolated from WT or mutant plants were transfected with Pro35S:CBP60g-FLAG and treated with or without 2 µM flg22 for 5 min. Protein samples were separated by Phos-tag SDS-PAGE and subjected to anti-FLAG immunoblot. CBB staining of the filter indicates loading of the protein. D, TCH3 interacts with CBP60g. Nicotiana benthamiana leaves infiltrated with Agrobacterium strains carrying Pro35S:NLuc-TCH3, Pro35S:CLuc-CBP60g, Pro35S:CLuc-XLG2, or Pro35S:NLuc-BIK1 as indicated were subjected to luciferase complementation assay. Infiltrated N. benthamiana leaves were sliced into strips 2 d post-infiltration, and relative luminescence was determined by Microplate Luminometer. Error bars indicate SD of three biological repeats. Student’s t test was carried out to determine the significance of difference. Different letters indicate significant differences. NLuc stands for N-terminal fragment of firefly luciferase; CLuc stands for C-terminal fragment of firefly luciferase. E and F, The cmlquad mutant is compromised in resistance to V. dahliae. Disease symptoms (E) and index analyses (F) of Arabidopsis WT, cmlquad, cam1 cam6, tch3 cam4, or tch3 cam1 cam6 mutant plants infected with V. dahliae. The plants were photographed and subjected to disease index analyses 3–4 weeks post-inoculation. Disease indexes were evaluated with three replicates generated from 24 plants for each inoculum. Error bars indicate SD of three biological replicates. Student’s t test was carried out to determine the significance of difference. Different letters indicate significant differences.