1. Plant Biology

Decoys provide a scalable platform for the identification of plant E3 ubiquitin ligases that regulate circadian function

  1. Ann Feke
  2. Wei Liu
  3. Jing Hong
  4. Man-Wah Li
  5. Chin-Mei Lee
  6. Elton K Zhou
  7. Joshua M Gendron  Is a corresponding author
  1. Yale University, United States
  2. South China University of Technology, China
https://doi.org/10.7554/eLife.44558
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Cite this article
  1. Ann Feke
  2. Wei Liu
  3. Jing Hong
  4. Man-Wah Li
  5. Chin-Mei Lee
  6. Elton K Zhou
  7. Joshua M Gendron
(2019)
Decoys provide a scalable platform for the identification of plant E3 ubiquitin ligases that regulate circadian function
eLife 8:e44558.
https://doi.org/10.7554/eLife.44558
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  3. Download .RIS
12 figures, 5 tables and 8 additional files

Figures

Figure 1
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Construction of the Arabidopsis Decoy Library.

(A) Cloning and experimental workflow of the F-box and U-box decoy library. F-box decoys follow the same path as the N-terminal U-box decoys. (B) Distribution of protein recognition domains in the …

https://doi.org/10.7554/eLife.44558.003
Figure 2 with 1 supplement
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RAE distributions of F-box decoy plants.

Values presented are the RAE for each individual T1 seedling. The black line represents the standard RAE cutoff of 0.6. Genes are separated by protein recognition domain and ordered by closest …

https://doi.org/10.7554/eLife.44558.004
Figure 2—figure supplement 1
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RAE distributions of control plants in F-box experiments.

Values presented are the RAE of each individual control plant. The black line represents the standard RAE cutoff of 0.6. Only data from those experiments where the control CCA1p::Luciferase plants …

https://doi.org/10.7554/eLife.44558.005
Figure 3 with 1 supplement
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RAE distributions of U-box decoy plants.

Values presented are the RAE for each individual T1 seedling. The black line represents the standard RAE cutoff of 0.6. Genes are ordered by closest protein homology using Phylogeny.Fr, (Dereeper et …

https://doi.org/10.7554/eLife.44558.006
Figure 3—figure supplement 1
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RAE distributions of control plants in U-box experiments.

Values presented are the RAE of each individual control plant. The black line represents the standard RAE cutoff of 0.6.

https://doi.org/10.7554/eLife.44558.007
Figure 4 with 1 supplement
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Phase distributions of F-box decoy plants.

Values presented are the difference between the period of the individual decoy plant and the average period of the CCA1p::Luciferase control in the accompanying experiment. The grey line is at the …

https://doi.org/10.7554/eLife.44558.008
Figure 4—figure supplement 1
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Phase distributions of control plants in F-box experiments.

Values presented are the difference between the phase of the individual control plant and the average phase of the CCA1p::Luciferase control in the accompanying experiment. The grey line is at the …

https://doi.org/10.7554/eLife.44558.009
Figure 5 with 1 supplement
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Phase distributions of U-box decoy plants.

Values presented are the difference between the period of the individual decoy plant and the average period of the CCA1p::Luciferase control in the accompanying experiment. The grey line is at the …

https://doi.org/10.7554/eLife.44558.010
Figure 5—figure supplement 1
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Phase distributions of control plants in U-box experiments.

Values presented are the difference between the phase of the individual control plant and the average phase of the CCA1p::Luciferase control in the accompanying experiment. The grey line is at the …

https://doi.org/10.7554/eLife.44558.011
Figure 6 with 1 supplement
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Period distributions of F-box decoy plants.

Values presented are the difference between the period of the individual decoy plant and the average period of the CCA1p::Luciferase control in the accompanying experiment. The grey line is at the …

https://doi.org/10.7554/eLife.44558.013
Figure 6—figure supplement 1
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Period distributions of control plants in F-box experiments.

Values presented are the difference between the period of the individual control plant and the average period of the CCA1p::Luciferase control in the accompanying experiment. The grey line is at the …

https://doi.org/10.7554/eLife.44558.014
Figure 7
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Circadian Phenotypes for selected high-priority F-box decoy populations.

Period values and average traces for decoy plants with significant differences from the control across the entire population or a sub-population of plants greater than 1. Period values presented are …

https://doi.org/10.7554/eLife.44558.015
Figure 8 with 1 supplement
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Period distributions of U-box decoy plants.

Values presented are the difference between the period of the individual decoy plant and the average period of the CCA1p::Luciferase control in the accompanying experiment. The grey line is at the …

https://doi.org/10.7554/eLife.44558.017
Figure 8—figure supplement 1
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Period distributions of control plants in U-box experiments.

Values presented are the difference between the period of the individual control plant and the average period of the CCA1p::Luciferase control in the accompanying experiment. The grey line is at the …

https://doi.org/10.7554/eLife.44558.018
Figure 9
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Circadian Phenotypes for selected high-priority U-box decoy populations.

Period values and average traces for decoy plants with significant differences from the control across the entire population or a sub-population of plants greater than 1. Period values presented are …

https://doi.org/10.7554/eLife.44558.019
Figure 10
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qRT-PCR of clock gene expression in mac3a/mac3b mutants.

(A,C,E) CCA1 and (B,D,F)TOC1 expression was measured using quantitative RT-PCR in wildtype or homozygous (A-B) mac3a, (C-D) mac3b, and (E-F) mac3a/mac3b mutants under constant light conditions. …

https://doi.org/10.7554/eLife.44558.021
Figure 11
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qRT-PCR of PRR9 splicing in mac3a/mac3b mutants.

PRR9 isoform expression was measured using quantitative RT-PCR in mac3a/mac3b mutants. (A) PRR9a, (B) PRR9b, and (C) PRR9c isoforms were analyzed. Quantifications are the average of three biological …

https://doi.org/10.7554/eLife.44558.022
Figure 12
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Period analyses of MAC3B overexpression constructs.

Period was measured in T1 MAC3B full length, MAC3B decoy, and MAC3B WD insertion plants. Period values presented are raw period values measured by CCA1p::Luciferase expression. A schematic of which …

https://doi.org/10.7554/eLife.44558.023

Tables

Table 1
Publically available data for strong candidate F-box phase regulators.

Circadian expression data is from the Diurnal Project gene expression tool (Mockler et al., 2007). Tissue specific expression is from the Arabidopsis eFP browser (Klepikova et al., 2016; Winter et …

https://doi.org/10.7554/eLife.44558.012
Locus IDGene
name		Circadian expressionTissue specific expressionClosest homologPublications (PMID)
LDHCLL_LDHCLL12_LDHHLL23_LDHH
PhaseCorrelationPhaseCorrelationPhaseCorrelationPhaseCorrelationDevelopmental map (eFP)Klepikova atlasLocusE-Value
AT5G44980ACF1160.50230.38140.6940.43Global,
especially
senescent leaf		Global,
stamen		AT5G449601.60 × 10−146N/A
AT5G48980ACF2N/AN/AN/AN/AN/AN/AN/AN/AN/ASilique with
seed, seed		AT5G489901.10 × 10−124N/A
AT5G42350CFK1N/AN/AN/AN/AN/AN/AN/AN/AGlobal,
especially
pollen		GlobalAT5G423603.60 × 10−29823475998
Table 2
Publically available data for strong candidate F-box period regulators.

Circadian expression data is from the Diurnal Project gene expression tool (Mockler et al., 2007). Tissue specific expression is from the Arabidopsis eFP browser (Klepikova et al., 2016; Winter et …

https://doi.org/10.7554/eLife.44558.016
Locus IDGene
name		Circadian expressionTissue specific expressionClosest homologPublications (PMID)
LDHCLL_LDHCLL12_LDHHLL23_LDHH
PhaseCorrelationPhaseCorrelationPhaseCorrelationPhaseCorrelationDevelopmental map (eFP)Klepikova atlasLocusE-Value
AT1G20800ACF3180.5140.66150.65160.66N/AYoung flower,
stamen, ovule		AT1G208031.6 × 10−87N/A
AT2G44030ACF4180.7350.5460.62220.79Seed, pollenYoung
stamen,
flower buds		AT3G460502.5 × 10−59N/A
AT1G50870ACF5N/AN/AN/AN/AN/AN/AN/AN/AN/AAT1G477901.2 × 10−102N/A
Table 3
Publically available data for strong candidate U-box period regulators.

Circadian expression data is from the Diurnal Project gene expression tool (Mockler et al., 2007). Tissue specific expression is from the Arabidopsis eFP browser (Klepikova et al., 2016; Winter et …

https://doi.org/10.7554/eLife.44558.020
Locus IDGene nameCircadian expressionTissue specific expressionClosest homologPublications (PMID)
LDHCLL_LDHCLL12_LDHHLL23_LDHH
PhaseCorrelationPhaseCorrelationPhaseCorrelationPhaseCorrelationDevelopmental map (eFP)Klepikova atlasLocusE-Value
AT5G18340PUB48190.68170.86200.63140.86Global, especially
young flower
and senescent
leaf		Young flower
bud,
Stamen, mature
leaf mature
petiole		AT5G183203.8 × 10−12728077082
AT1G04510MAC3A200.84180.72230.48140.83GlobalGlobalAT2G333401.2 × 10−23329437988
28947490
19629177
AT2G33340MAC3B150.4980.56170.8080.73GlobalGlobalAT1G045107.2 × 10−22929437988
28947490
19629177
AT3G49810PUB3020.8710.5930.7600.91GlobalEverywhere
except young
seed and
young silique		AT5G659201.2 × 10−17128865087
25410251
Table 4
Selected IP-MS results from the MAC3B decoy.

MAC3B decoy peptide hits are from one IP-MS experiment using the MAC3B decoy as the bait. Combined control peptide hits are summed from the independent control experiments of wildtype Col-0 and 35S::…

https://doi.org/10.7554/eLife.44558.024
LocusProtein nameTotal spectral counts
MAC3B decoyCombined controls
AT2G33340MAC3B29415
AT1G04510MAC3A1290
AT1G09770CDC5360
AT1G07360MAC5A2444
AT3G18165MOS4350
AT4G15900MAC2/PRL11923
Key resources table
Reagent type
(species) or resource		DesignationSource or referenceIdentifiersAdditional
information
Genetic Reagent
(Arabidopsis thaliana)		CCA1p::LuciferasePruneda-Paz et al., 2009Dr. Pruneda-Paz
Genetic Reagent
(A. thaliana)		mac3aMonaghan et al., 2009Dr. Xin Li
Genetic Reagent
(A. thaliana)		mac3bArabidopsis Biological
Resource Center		SALK 144856C
Genetic reagent
(A. thaliana)		mac3a/mac3bMonaghan et al., 2009Dr. Xin Li
AntibodyMonoclonal mouse
anti-FLAG antibody		SigmaCat#: F3165
RRID:AB_259529		Conjugated to
Dynabeads M-270
Epoxy (Thermo
Fisher Scientific,
cat# 14311D)

Additional files

Source code 1

MATLAB script called by outlier_analysis that determines the number of subpopulations within a dataset.

https://doi.org/10.7554/eLife.44558.025
Source code 2

MATLAB script called by batch_outlier to compare individual datasets to the control dataset.

https://doi.org/10.7554/eLife.44558.026
Source code 3

MATLAB script used to batch-process circadian imaging data.

https://doi.org/10.7554/eLife.44558.027
Supplementary file 1

All generated data and publications which reference genes in our decoy library.

https://doi.org/10.7554/eLife.44558.028
Supplementary file 2

IP-MS results from the MAC3B decoy.

https://doi.org/10.7554/eLife.44558.029
Supplementary file 3

Primers used in this Study.

(Czechowski et al., 2004; Farré et al., 2005; Lee and Thomashow, 2012)

https://doi.org/10.7554/eLife.44558.030
Supplementary file 4

ABRC Stock numbers for pENTR-decoy vectors, 35S:HIS-FLAG-decoy vectors, and 35S:HIS-FLAG-decoy transgenic seed stocks.

https://doi.org/10.7554/eLife.44558.031
Transparent reporting form
https://doi.org/10.7554/eLife.44558.032

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