Extended Data Fig. 2: MtLYK3 interacts with MtLICK1/2 on the cell embrane.
From: A kinase mediator of rhizobial symbiosis and immunity in Medicago
a, MtLICK1/2 is localized at the plasma membrane of N. benthamiana epidermal cells. RFP-OsRAC1 and GFP-MtLICK1/2 were transiently co-expressed in N. benthamiana leaves transformed with p35S:RFP-OsRAC1 and p35S:GFP-MtLICK1/2. RFP-OsRAC1 is plasma membrane localization marker. Scale bars, 20 µm. b, MtLICK1 and MtLICK2 are co-localized with MtLYK3 at the plasma membrane of N. benthamiana epidermal cell, MtLYK3-RFP is plasma membrane localization receptor-like kinase. MtLYK3-RFP and GFP-MtLICK1/2 were transiently co-expressed in N. benthamiana leaves transformed with pLjUBQ:MtLYK3-RFP and p35S:GFP-MtLICK1/2. Scale bars, 20 µm. c, MtLYK3-Flag or MtBRI1-Flag and GFP-MtLICK1 or GFP-MtLICK2 were transiently co-expressed in N. benthamiana leaves and Co-IP assay was performed with a-Flag. MtBRI1-Flag and GFP-MtLICK1/2, GFP-MtLICK1/2 and Flag or GFP and MtLYK3-Flag were transiently co-expressed in N. benthamiana leaves serve as control. MtBRI1 was used as a negative plasma membrane protein control. d, MtLICK1/2-Flag or Flag were expressed in Medicago stable transgenic plants (Mtlyk3 mutant transformed with pMtLYK3:gMtLYK3-GFP) hairy roots transformed with pMtLICK1:MtLICK1WT-Flag, pMtLICK2:MtLICK2WT-Flag, and pLjUBQ:Flag and Co-IP assay was performed with a-GFP antibodies. Flag was expressed in Medicago stable transgenic plants (Mtlyk3 mutant transformed with pMtLYK3:gMtLYK3-GFP) and MtLICK1/2-Flag were expressed in wild-type plants (A17) by hairy root transformation serve as control. e, Detection of protein-protein interactions in Medicago (A17) root protoplast by BiFC. MtLYK3-Yn (N-terminal 174 amino acids of YFP) and MtLICK1/2-Yc (C-terminal 66 amino acids of YFP) were transiently co-expressed in Medicago (A17) root protoplasts transformed with p35S:MtLYK3-Yn and p35S:MtLICK1/2-Yc. Protein interactions were not detected in the other two combinations: MtLYK3-Yn and Yc empty vector (EV) as well as Yn empty vector (EV) and MtLICK1/2-Yc. Scale bars, 10 µm. Data are representative of three independent experiments. f,g, qRT-PCR was used to detect mRNA levels of MtLICK1 and MtLICK2 in wild-type (WT) R108 1-week roots at 6-hour post inoculation (hpi) with Nod factor (NF) (3 biological repetitions per group). MtEF-1 was used as the reference gene. Data are mean ± SEM. Each biological replicate is plotted as black dot and triangle. Statistical analysis was performed using Two-sided Student’s t-test compared to Mock. h,i, qRT-PCR was used to detect mRNA levels of MtLICK1 and MtLICK2 in R108 roots at 1 hpi, 6 hpi, 1 dpi, 14 dpi and 21dpi with S. meliloti 1021 (3 biological repetitions per group). MtEF-1 was used as the reference gene. Data are mean ± SEM. Each biological replicate is plotted as black dot and triangle. Statistical analysis was performed using Two-sided Student’s t-test compared to Mock. j, Expression patterns of MtLICK1/2 and MtLYK3 in root hairs were determined by GUS reporter analysis. The roots of wild-type plants were transformed with pMtLICK1/2: GUS or pMtLYK3: GUS and GUS activity detected by treating with X-Gluc at 37 °C for 8 h (see Methods). Scale bars, 100 µm. k, Schematic representation of MtLICK1 genomic sequence and identification of Tnt1 insertion mutant of MtLICK1. Exons are shown as red boxes. Localization of Tnt1 insertion is represented as green lines with a green triangle. l, Schematic representation of MtLICK2 genomic sequence and identification of Tnt1 insertion mutant of MtLICK2. Exons are shown as red boxes. Localization of Tnt1 insertion is represented as green lines with a green triangle. For c, f-i and k, l the experiments were repeated three times with similar results. For d, the experiments were repeated two times with similar results.
