Original data for Co-Immunoprecipitations from "The Arabidopsis TIR-NBS-LRR CSA1 guards BAK1 BIR3 homeostasis and mediates pattern- and effector-induced plant immune responses"

Description

Arabidopsis BAK1/SERK3, a co-receptor of leucine-rich repeat pattern recognition receptors (PRR), mediates pattern-triggered immunity (PTI). Genetic inactivation of BAK1 or BAK1-interacting receptors (BIR) causes cell death. We found that the TIR-NBS-LRR protein CONSTITUTIVE SHADE-AVOIDANCE 1 (CSA1) physically interacts with BIR3, but not with BAK1. Cell death in bak1-4 and bak1-4 bir3-2 mutants is dependent on CSA1 and on components of effector-triggered immunity-(ETI) pathways, including EDS1, PAD4, and the plant hormone, salicylic acid. Effector HopB1-mediated perturbation of BAK1 also results in CSA1-dependent cell death. Likewise, microbial pattern pg23-induced cell death, but not PTI responses, require CSA1. Thus, CSA1 integrates pattern- and effector-mediated cell death pathways downstream of BAK1. CSA1 guards BIR3 BAK1 homeostasis, and de-repression of CSA1 in the absence of intact BAK1 and BIR3 triggers ETI cell death. This suggests that PTI and ETI pathways are activated downstream of BAK1 for efficient plant immunity.

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Transient expression in Nicotiana benthamiana Agrobacterium tumefaciens GV3101 containing the respective constructs were grown 36 h at 28 °C in LB medium supplemented with appropriate antibiotics. Cultures were pelleted and resuspended in 10 mM MgCl2 to an optical density at 600nm of 1. Agrobacteria carrying different constructs were mixed 1:1 and infiltrated into 3-week-old N. benthamiana leaves. Samples were harvested 2 to 3 days after inoculation. Co-immunoprecipitations Leaves were ground in liquid nitrogen, and 250 µl extraction buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl, 1 % Nonidet P40, proteinase inhibitor cocktail (Roche) per 200 mg tissue powder was added. Samples were homogenized and incubated for 1 h at 4°C under gentle shaking. Samples were centrifuged two times at 4°C and 14,000 rpm for 10 min to obtain a clear protein extract. After washing with extraction buffer, GFP and V5-trap beads (Sigma or Chromotec) were used. Supernatants containing equal amounts of protein were incubated for 1 h at 4°C with the beads. Beads were washed two times with 50 mM Tris-HCl pH 8.0, 150 mM NaCl and one time with 50 mM Tris-HCl pH 8.0, 50 mM NaCl before adding SDS sample buffer and heating at 95°C for 5 min. SDS-PAGE and immunoblotting Proteins were separated, blotted, and incubated with antibodies as described by Schulze et al. (2010) but using 8% SDS gels and the following antibody dilutions: anti-GFP (Abcam), 1:3,000; anti-HA (Sigma), 1:2,000; anti-V5 (Sigma), 1:2,000; anti-BAK1 (Agrisera), 1:3,000; anti-luciferase (Sigma), 1:3,000; anti-VP16 (Santa Cruz), 1:1000; anti-ATPase (Agrisera), 1:5,000; anti-c-myc (Sigma), 1:5,000; anti-rabbit (Sigma),1:50,000; anti-goat (Sigma), 1:10,000; anti-mouse (Sigma), 1:10,000. Chemiluminescence was detected with the ECL Western blotting detection system (GE Healthcare) and a CCD camera (Amersham Imager 600). If figures were reconstituted from images of blots, lanes from the same blot are shown in one panel of a figure even if they were in a different order on the original blot (separated by dotted lines). Data from different blots are shown in separate figure parts. If panels of a figure are exposed differentially, all samples were exposed equally.

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