Confocal Laser Scanning Microscopy
Intracellular fluorescence was observed using a Leica SP8 upright confocal microscope with high sensitivity hybrid detectors. Intra-
cellular membrane organelle markers used were described previously (Brkljacic et al., 2009; Gu and Innes, 2011, 2012; Nelson et al.,
2007 ). Three-dimensional image reconstruction and co-localization statistics were carried out using IMARIS 8.0 (Bitplane). FRAP
experiment was performed using Leica SP8 FRAP Wizard. Fluorescence intensity was normalized to the average expression level
defined by random sampling before photobleaching. Recovery curves were plotted with 50 frames (1 frame/sec) recorded after
photobleaching.
Immunoelectron Microscopy and Electron Tomography
Root tip samples were dissected fromArabidopsisseedlings expressing GFP-CPR5 and cryofixed by an HPM100 (Leica Microsys-
tems). The frozen specimens were freeze-substituted at 80 C and embedded in HM20 resin (Electron Microscopy Sciences) at
45 C. After polymerization at 45 C, the root tip samples were sectioned and immunolabeled with a GFP antibody (Santa Cruz)
as described previously (Kang, 2010).
Co-immunoprecipitation
All tagged proteins for in vitro pull-down assays were synthesized using a wheat germ-based translation system (BioSieg).
Synthesized proteins were mixed and incubated with GFP-TrapA (Chromo Tek) or Pierce anti-HA agarose beads (Thermofisher)
overnight at 4C in the pull-down buffer (50 mM Tris [pH 7.5], 150 mM NaCl, 0.1% Triton X-100, 0.2% Nonidet P-40, plant protease
inhibitor cocktail (Sigma) and 40mM MG115). Following immunoprecipitation (IP), beads were precipitated and washed five times
with the pull-down buffer before eluted with the SDS sample buffer. For in vivo co-immunoprecipitation, leaf tissues of four-week-
old transgenic plants were collected. Total protein was extracted using IP buffer with high concentration of detergents to completely
solubilize membrane protein (50 mM Tris [pH 7.5], 150 mM NaCl, 0.5% Triton X-100, 0.5% Nonidet P-40, 0.25% Na-deoxycholate,
plant protease inhibitor cocktail, and 40 mM MG115) followed by immunoprecipitation with GFP-TrapA beads overnight at 4C.
After IP, beads were washed five times with the IP buffer. Samples were boiled with loading buffer for 10 min before separated by
SDS-PAGE.
LC-MS/MS and Data Analysis
A total of 5 g leaf tissues from four-week-old transgenic plants were collected. Total protein was extracted with IP buffer (50 mM
Tris [pH 7.5], 150 mM NaCl, 0.5% Triton X-100, 0.5% Nonidet P-40, 0.25% Nadeoxycholate, plant protease inhibitor cocktail,
and 40 mM MG115) and immunoprecipitated with GFP-TrapA beads overnight at 4C. After IP, samples were washed five times
with the IP buffer and three times with 50 mM NH 4 HCO 3 before on-bead trypsin digestion. Following immunoprecipitation, on-
bead trypsin digestion, peptide lyophilization and LC-MS/MS were performed by the Duke Proteomics Core Facility. MS/MS samples
were analyzed using Mascot (Matrix Science, London, UK; version 2.5.0) and searched with a fragment ion mass tolerance of
0.020 Da and a parent ion tolerance of 5.0 PPM. Scaffold (version 4.4.1.1, Proteome Software, Portland, OR) was used to validate
MS/MS based peptide and protein identifications. Peptide/protein identifications were accepted if they could be established at
greater than 95% probability by the Peptide/Protein Prophet algorithm, which yielded a false-discovery rate of 0.1% and 0.5% on
the peptide and protein match level, respectively. Data from35S:GFPsamples ran in parallel were used as controls for a statistical
model-based selection of CPR5-specific interactors. The resulting list was shortened to 28 by further excluding proteins that are
abundant in chloroplasts and mitochondria. Predicted interactions between CPR5 interactors were based on an interolog method
(Geisler-Lee et al., 2007).
qPCR
ArabidopsisRNA was extracted using TRIzol Reagent (Thermofisher), and cDNA was synthesized using the SuperScript III cDNA
Synthesis (Thermofisher). qPCR was performed using FastStart Universal SYBR Green Master Kit (Roche) in Mastercycler ep real-
plex (Eppendorf).
Microarray Procedure and Data Analysis
ForRPS2-dependent ETI response, four-week-old WT andrps2mutant plants were inoculated withPsm/AvrRpt2. At 0, 6 and 10 hr
post inoculation, leaf tissues were collected for RNA preparation and microarray. The resulting dataset was deposited to GEO:
GSE72742. For transient interference of the CPR5 function, four-week-old WT and T3 homozygousDex:YFP-CPR5-Ctransgenic
plants were sprayed with water or 50mM dexamethasone (Sigma). After 24 hr, leaf tissues were collected for RNA preparation
and microarray. The resulting dataset was deposited to GEO: GSE72743. RNA quality control, cDNA synthesis, aRNA purification
and fragmentation, hybridization, washing, and scanning ofArabidopsisATH1 Genome Arrays (Affymetrix) chips were performed
by the Duke Microarray Facility. For CPR5-C interference microarray, differentially expressed genes were further filtered by subtract-
ing genes whose expression are affected by the empty vector control upon dexamethasone induction (GEO: GSE8741). The resulting
gene list was provided inTable S1. TheRPS4-dependent ETI genes,RPS2-dependent ETI genes and basal immunity genes were
determined by microarray analysis of dataset GEO: GSE50019, GSE73742 and GSE17464, respectively. Statistical analysis and nat-
ural language processing-based network regulator discovery were performed using GeneSpring 13.0 (Agilent, 2014) and R 3.0.1
Cell 166 , 1526–1538.e1–e4, September 8, 2016 e3