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. 2017 May 15;114(22):5749–5754. doi: 10.1073/pnas.1614468114

Fig. 3.

Fig. 3.

LLG1 interacts with FLS2. (A) Co-IP of LLG1 and FLS2 from N. benthamiana transiently expressing YFP–LLG1, YFP–LLG1m (carrying the llg1-3 mutation), and FLS2-YFP-HA. Plants were treated with 0 (−) or 1 μM (+) flg22 for 10 min. Total protein was extracted and subjected to IP by HA antibody, followed by immunoblot analysis with anti-GFP or anti-HA antibody. Plants transiently expressing both BAK1 and FLS2 were used as a positive (treated with flg22) or negative control (not treated with flg22), respectively. (B) Co-IP of LLG1 and FLS2 in Arabidopsis. Three-week-old transgenic Arabidopsis plants were treated with 0 (−) or 1 μM (+) flg22 for 10 min. Total protein was subjected to IP by anti-HA antibody, followed by immunoblot analysis with anti-HA or anti-GFP antibody. (C) Co-IP assay showing that LLG1 associated with EFR in N. benthamiana. Plants were treated with 0 (−) or 1 μM (+) elf18 for 10 min. The proteins were analyzed by immunoblot analysis with anti-GFP or anti-HA antibody after IP with HA antibody. (D) Co-IP of LLG1 and BAK1 in Arabidopsis. Three-week-old Col-0 plants and 35S:YFP-LLG1 (in the llg1-3 background) plants were treated with 0 (−) or 1 μM (+) flg22 for 10 min. YFP-LLG1 and BAK1 were detected by immunoblot analysis with anti-GFP or anti-BAK1 antibody after IP with anti-GFP antibody. (E) Yeast two-hybrid assay showing that LLG1 interacted with the extracellular juxtamembrane domains of FLS2 and EFR. exJM: extracellular juxtamembrane domain. Yeast cells containing the indicated plasmids were plated on dropout medium (-Leu-Trp) or on dropout medium (-Leu-Trp-His-Ade) supplemented with 10 mM 3-amino-1,2,4-triazole. AD represents yeast GAL4 activation domain. BD represents the GAL4 DNA binding domain. All experiments were repeated at least three times with similar results.