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. 2018 Oct 23;14(10):e1007708. doi: 10.1371/journal.pgen.1007708

Fig 1. GTL1 associates with MPK4 and allelic gtl1 mutants show higher susceptibility to various Pseudomonas syringae strains.

Fig 1

A) In-vitro pull-down assays of MBPHis-tagged GTL1 show interaction with MPK4, unlike to MPK3, MPK6 and single GST. Pull-down assays were performed by incubating bacterial lysates of GST (lane 1), MPK3-GST (lane 2), MPK4-GST (lane 3), and MPK6-GST (lane 4) with GST beads followed by the incubation with bacterial lysate of MBPHis-tagged GTL1 (lane 6, 10% INPUT). The pull-downs and 10% INPUT were probed with an anti-His antibody (WB: anti-His). Proteins were stained with Ponceau-S. B) Nuclear interaction of GTL1 with MPK4. Nicotiana benthamiana epidermal cells were analyzed by Bimolecular Fluorescence Complementation. MPK3 and empty YFPn/ YFPc-vector (EV) serve as a negative control, scale bar = 25 μm. C) LC-MS/MS analysis of co-immunoprecipitated MPK4-GTL1 complex. A genomic TAP-tagged MPK4 construct was generated, stably introduced in Arabidopsis thaliana, and used for tandem affinity purification of MPK4 protein without treatment and 15 min after flg22 application. GTL1 was reproducibly identified via the GTL1-specific peptide EETLALLR whose relative abundance was normalized to MPK4 protein abundance. Box plots are depicted for GTL1 protein abundance associated with MPK4, boxes showing the interquartile range (IQR) 25st to 75th percentiles, inner ellipse representing the median, whiskers show the SEM. D-F) The allelic GTL1 mutants gtl1-2 and gtl1-5, as well as two GTL1-overexpressing lines (GTL1ox1, GTL1ox) were challenged by the use of PstDC3000, PstDC3000 Δ avrPto/avrPtoB and PstDC3000 hrcC- (GTL1ox, see under S2C Fig). Plants, of three biological replicates (n = 30), were spray-inoculated with a bacterial suspension at OD600 0.2, the density of colony-forming units (cfu) was analyzed 2 and 72 hours post inoculation (hpi). Error bars, mean ± SEM, statistical significance was analyzed by Student’s test, asterisks indicate significant differences compared to treated WT, * p≤ 0.05, ** p≤ 0.01, *** p≤ 0.001.