Supplemental Data. Xie et al. (2014). Plant Cell …...2014/06/17  · Xie et al. (2014). Plant Cell...

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Supplemental Figure 1. The phylogenetic tree of rice and Arabidopsis Group A/B/C MAPKs that contain the TEY motif in their activation loop. Tobacco SIPK and WIPK, human ERK2 and P38 were also included in the tree. The numbers at the nodes indicate the bootstrap value. Rice MAPK nomenclature and accession numbers were the same as described in (Reyna and Yang, 2006). Arabidopsis MAPK accession numbers were the same as described in (MAPK-group, 2002). The Group D MAPKs, which contain the TDY motif in their activation loop, are schematically illustrated as triangles at the bottom. At, Arabidopsis; Os, rice. The alignment used to generate the phylogeny is shown in Supplemental Dataset 1. Supplemental Data. Xie et al. (2014). Plant Cell 10.1105/tpc.114.126441 1

Transcript of Supplemental Data. Xie et al. (2014). Plant Cell …...2014/06/17  · Xie et al. (2014). Plant Cell...

Page 1: Supplemental Data. Xie et al. (2014). Plant Cell …...2014/06/17  · Xie et al. (2014). Plant Cell 10.1105/tpc.114.126441 5 Supplemental Figure 6. Mapping CPK18 phosphorylated sites

Supplemental Figure 1. The phylogenetic tree of rice and Arabidopsis Group A/B/C MAPKs that contain the TEY motif in their activation loop. Tobacco SIPK and WIPK, human ERK2 and P38 were also included in the tree. The numbers at the nodes indicate the bootstrap value. Rice MAPK nomenclature and accession numbers were the same as described in (Reyna and Yang, 2006). Arabidopsis MAPK accession numbers were the same as described in (MAPK-group, 2002). The Group D MAPKs, which contain the TDY motif in their activation loop, are schematically illustrated as triangles at the bottom. At, Arabidopsis; Os, rice. The alignment used to generate the phylogeny is shown in Supplemental Dataset 1.

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Supplemental Figure 2. Phylogenesis of rice (Os) and Arabidopsis (At) CDPKs. CDPKs were divided into four groups (Group I-IV). CRK, PEPRK and SnRK are the three plant protein kinase families closely related to CDPKs. CRK, CDPK-related kinase; PERPK, phosphoenolpyruvate carboxylase kinase-related kinase; SnRK, SNF1-related kinase. The numbers at the nodes indicate the bootstrap value. The LOC_Os indicates the locus ID of rice gene annotations (http://rice.plantbiology.msu.edu/). At, Arabidopsis; Os, rice. The alignment used to generate the phylogeny is shown in Supplemental Dataset 2.

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Supplemental Figure 3. The MPK5 TEY motif is phoshorylated by MKK4 and autophosphorylation, but not by CPK18. (A) MKK4 was capable of phosphorylating MPK5KR but not MPK5KR-AEF in which the TEY motif was substituted by AEF (MPK5KR-AEF). (B) CPK18 was capable of equally phosphorylating MPK5KR and MPK5KR-AEF. (C) Detection of the phos-TEY level of MPK5 after autophosphorylation (in the absence of CPK18) and phosphorylation by CPK18. MPK5KR and MPK5-AEF were used as negative controls in immunoblotting.

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Supplemental Figure 4. Expression of CPK18, CPK4, MKK4 and MKK6 in three CPK18-RI lines (9, 10, and 13). The relative expression level of these genes was measured by RT-qPCR. Data presented as Mean ± SD (n = 3). Asterisks indicate statistically significant differences (* p<0.05; **p<0.01, Student’s t-test).

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Supplemental Figure 5. Prediction of CPK18 phosphorylated sites on MPK5. Potential CPK18 phosphorylated residues on MPK5 were labelled with red arrows (predicted according to CPK18-MAPK phosphorylation specificity, see Figure 3) or blue arrows (predicted according to common CDPK phosphorylated motifs). The T-x-Y motif and ATP binding pocket (glycine-rich loop) were labelled with rectangles. The numbers and asterisks on the top indicate coordinates of aligned sequence. At, Arabidopsis; Os, rice.

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Supplemental Figure 6. Mapping CPK18 phosphorylated sites on MPK5. MPK5-KR-M2 possessed five substitutions including S211A, T212A, S215A, T283A, and T304A. The amount of mutated His-MPK5 proteins was shown by Coomassie Brilliant Blue (CBB) staining. The loadings of MPK5KR-T117A and MPK5KR-S339A were lower than others because of their poor solubility. The relative phosphorylation level (% to MPK5KR) of MPK5 mutant proteins was shown at the bottom.

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Supplemental Figure 7. Native CPK18 activities in WT and MPK5-RI lines. An in-gel kinase assay was performed to measure native CPK18 activities in WT and two MPK5-RI lines (#3 and #5). His-MPK5KR was used as substrate and embedded in SDS-PAGE.

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Supplemental Table 1 List of genes and DNA oligos used in this study

Genes and primers used in cloning Gene Primer Name Primer Sequence (5’->3’) Comment

MPK5

(AF479883)

MPK5-ENTR-F CACC GAATTCATGGACGGGGCGCCGGTG TOPO Cloning

MPK5-ENTR-R CTAGTACCG GAT GTT TGG GTT CAT With stop codon

MPK5-CF-R CCGGATGTTTGGGTTCA Without stop codon

CPK18

(AK121471)

CPK18-ENTR-F157 CACC CAG CCG ACG ACG ATG GGA CT TOPO cloning

CPK18-ENTR-R1703 CGATCTGTGAACACTCCTCG Full length

CPK18-ENTR-R1121 TGC CTG TCC TCC TTC TCT CAC OsCPK18AC

constructs

CPK18 CPK18-ENTR-F1364 CACC GATTGTTGAGGCAATTGACAG RNAi

constructs CPK18-ENTR-R GGATCC AATGACTGCCCTTTGTTCA

CPK4

(AK060738)

CPK4-ENTR-F CACC ATGGGCGCGTGCTTCTCATC TOPO cloning

CPK4-ENTR-R TCA CAG GGGTTGTGGATTTGGAG

CPK7

(AK066500)

CPK7-ENTR-F CACC ATGGGGAATCAGTGCCAGAA TOPO cloning

CPK7-ENTR-R TCA ATG TACTTGAGGTGCGTCTC

MPK4

(AK071376)

MPK4-ENTR-F CACC ATGGCGATGATGGTGGACCC TOPO cloning

MPK4-ENTR-R TCA CATATTCACTCCTGCAACAA

MPK6

(AK111579)

MPK6-ENTR-F CACC ATGGATTCCTCCTCCGGCGG TOPO cloning

MPK6-ENTR-R TTA AATGCCAAGGATTCCC

MKK4

(AK120525)

MKK4-ENTR-F99 CACC GCCGTCGCG ATGCGACCGG TOPO cloning

MKK4-ENTR-R1292 TCCCACGTCTCAATGCCAAAC

PR5

(AK241419)

promoter

Pro-PR5-F CACCATGGAAGTTATATTACGTCTAC TOPO cloning

Pro-PR5-R TAACAATTTACTCTACTATA

Hin1

(AK068115)

promoter

Pro_Hin1-F CACC GCATGCATGTGGACCC TOPO cloning

Pro_Hin1-R GATCGTAGCTAGTTGTGACAATT

Genes and primers used in qRT-PCR Gene Primer Primer Sequence (5’->3’)

CPK4

(AK060738)

CPK4-qF1942 TGCTGTTCTAGTCTCCCATTCTCC

CPK4-qR2032 CCCAGGCAACTTTATTGCGA

CPK18

(AK121471)

CPK18-qF1686 AGGAGTGTTCACAGATCGTAGC

CPK18-qR1802 TTCTGGCAACAATTTTGATTACAC

UBQ10 UBQ10-qF1447 TGGTCAGTAATCAGCCAGTTTG

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(AK101547) UBQ10-qR1521 CAAATACTTGACGAACAGAGGC

MPK4

(AK071376)

MPK4-qF1605 GGCCACGTTTACACATATATTTGC

MPK4-qR1666 CCGCCCTAAAACCAAGCA

MPK5

(AF479883)

MPK5-F TCGAGCAGAAGGCTCTAAACG

MPK5-R CCGGATGTTTGGGTTCATCT

MPK6

(AK111579)

MPK6-qF1102 TTTGACCCAAGCAGACGGATA

MPK6-qR1163 AGAGAAGCCAAGTATGGGTGATG

MKK4

(AK120525)

MKK4-qF1266 ACGGTGGTTTGGCATTGAG

MKK4-qR1327 GAATACCCAAAATGGCTAGGAAGA

MKK6

(AK059461) MKK6-qF1344 TCTTGGTAGCGGCACATGTTC

MKK6-qR1407 CCTCCCGAATTTTCAACATCA

PR5

(AK241419)

PR5-qF TACAACGTCGCCATGAGCTTCT

PR5-qR TGGGCAGAAGACGACTTGGTAGTT

PR10

(D38170)

PR10-qF TGGCATGCTCAAGATGATCGAGGA

PR10-qR TTACTCTCACGGACTCAAACGCCA

Chitinase

(AK104397)

CHI-qF830 GTTCATCTGGTCAGCGGATAGC

CHI-qR896 CTGAGCCTTGGTCTCGTACTCA

Hin1

(AK068115)

Hin1-qF ATTGACGTGTTCGTGTCACTGAT

Hin1-qR GTTCCCAGCCGAGGAGTTC

DNA oligos used for site-directed mutangenesis

Gene Oligo Name

(Gene-mutation)

Oligo for mutagenesis

MPK5 MPK5-T12A AGTTCAGGCCG GCG ATGACGCACGG MPK5-T14A GGCCGACGATG GCG CACGGCGGC MPK5-T32A GAACAAGTTCGAGGTGGCG AACAAGTACCAGCC MPK5-T117A ACGTCTACATCGCC GCG GAGCTCATGGAC MPK5-S187A TGGCGCGGCCG GCG TCGGAGAGC

ACCCGCGC CGG GCG TTCGCGAGC

MPK5-S211A-T212A-S215A

TGCTGCTCAACGCCGCCGACTACGCCGCCGCCATC

MPK5-S339A GCCTGGAGCCCTTCGCCTTCGACTTCGAG MPK5-T250A GATGCGCCTCATCGCCGAGGTGATCGG MPK5-S190A GGCCGTCGTCGGAGGCCGACATGATGACGG MPK5-S188A CGCGGCCGTCGGCGGAGAGCGAC MPK5-T304A TCGAGAGGATGCTCGCCTTCAACCCGCTG MPK5-K65R GAGATGGTGGCGATAAGGAAGATCGCCAACGC MPK5-T194A GAGAGCGACATGATGGCGGAGTACGTGGTCA

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MPK5-T194A-E-Y196F

GAGCGACATGATGGCGGAGTTCGTGGTCACCCGG

MPK5-T14D TTCAGGCCGACGATGGATCACGGCGGCCGGTAC

MPK5-T32D GGGAACAAGTTCGAGGTGGATAACAAGTACCAGCCGCCC

CPK18 CPK18 (D178A) GGTTTGGTTCATCGGGCCATGAAGCCTGAGAAC

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(WT) PC V SC SC - The Plant Cell · 1 Supplemental Data. Huang et al. Plant Cell (2015) 10.1105/tpc.15.00015 Supplemental Table 1. List of the TFs Coexpressed with Secondary Wall

(WT) PC V SC SC - The Plant Cell · 1 Supplemental Data. Huang et al. Plant Cell (2015) 10.1105/tpc.15.00015 Supplemental Table 1. List of the TFs Coexpressed with Secondary Wall

Supplemental Data. Gong et al. (2012). Plant Cell 10.1105 ... · 9/11/2012  · Plant Cell 10.1105/tpc.111.100511 . Supplemental Figure 2. Fine structure of the functional core of

Supplemental Data. Gong et al. (2012). Plant Cell 10.1105 ... · 9/11/2012  · Plant Cell 10.1105/tpc.111.100511 . Supplemental Figure 2. Fine structure of the functional core of

Data. Bedewitz et al. Cell (2014) 10.1105/tpc.114.130534...Supplemental Data. Bedewitz et al. Plant Cell (2014) 10.1105/tpc.114.130534 Supplemental Figure 5. The Impact of Silencing

Data. Bedewitz et al. Cell (2014) 10.1105/tpc.114.130534...Supplemental Data. Bedewitz et al. Plant Cell (2014) 10.1105/tpc.114.130534 Supplemental Figure 5. The Impact of Silencing

Supplemental Data. Xia et al. Plant Cell. (2013). …...2013/05/09  · Supplemental Data. Xia et al. Plant Cell. (2013). 10.1105/tpc.113.110957 2& & & Supplemental Figure 2. The miRNA-TASL-PPR-siRNA

Supplemental Data. Xia et al. Plant Cell. (2013). …...2013/05/09  · Supplemental Data. Xia et al. Plant Cell. (2013). 10.1105/tpc.113.110957 2& & & Supplemental Figure 2. The miRNA-TASL-PPR-siRNA

Supplemental Data. Park et al. (2012). Plant Cell 10.1105 ......NPB Supplemental Data. Park et al. (2012). Plant Cell 10.1105/tpc.111.105429. ... 170 130 70 40 100 55 35 KDa RING Finger

Supplemental Data. Park et al. (2012). Plant Cell 10.1105 ......NPB Supplemental Data. Park et al. (2012). Plant Cell 10.1105/tpc.111.105429. ... 170 130 70 40 100 55 35 KDa RING Finger

GFP-Rx-CC-NB-final - Plant Cell · 2010-12-13 · Supplemental Data. Tameling et al. (2010). Plant Cell 10.1105/tpc.110.077461 Supplemental Table 1. Primers used in plasmid construction

GFP-Rx-CC-NB-final - Plant Cell · 2010-12-13 · Supplemental Data. Tameling et al. (2010). Plant Cell 10.1105/tpc.110.077461 Supplemental Table 1. Primers used in plasmid construction

pam71-1 pam71-2 - The Plant Cell · 2016-03-28 · 1 Supplemental Data. Schneider et al. Plant Cell (2016) 10.1105/tpc.15.00812 Supplemental Figure 1. Complementation of pam71-1 and

pam71-1 pam71-2 - The Plant Cell · 2016-03-28 · 1 Supplemental Data. Schneider et al. Plant Cell (2016) 10.1105/tpc.15.00812 Supplemental Figure 1. Complementation of pam71-1 and

A B - The Plant Cell · 10/06/2010  · Supplemental Data. Je et al. (2010). Plant Cell 10.1105/tpc.109.069575 . Supplemental Table 1. The list of primers used for real-time RT-PCR

A B - The Plant Cell · 10/06/2010  · Supplemental Data. Je et al. (2010). Plant Cell 10.1105/tpc.109.069575 . Supplemental Table 1. The list of primers used for real-time RT-PCR

Supplemental Data. Adachi et al. (2015). Plant Cell …...2015/09/02  · Supplemental Data. Adachi et al. (2015). Plant Cell 10.1105/tpc.15.00213 Supplemental Figure 1. Promoter Activity

Supplemental Data. Adachi et al. (2015). Plant Cell …...2015/09/02  · Supplemental Data. Adachi et al. (2015). Plant Cell 10.1105/tpc.15.00213 Supplemental Figure 1. Promoter Activity

SUPPLEMENTAL FIGURES - The Plant Cell · 3/14/2014  · Supplemental Data. Morreel et al. Plant Cell (2014). 10.1105/tpc.113.122242. 1 SUPPLEMENTAL FIGURES Supplemental Figure 1.

SUPPLEMENTAL FIGURES - The Plant Cell · 3/14/2014  · Supplemental Data. Morreel et al. Plant Cell (2014). 10.1105/tpc.113.122242. 1 SUPPLEMENTAL FIGURES Supplemental Figure 1.

Supplemental Information - Plant Cell · Supplemental Data. Elliott et al. (2014). Plant Cell 10.1105/tpc.114.123620 1 Supplemental Information Supplemental Figures Supplemental Fig.

Supplemental Information - Plant Cell · Supplemental Data. Elliott et al. (2014). Plant Cell 10.1105/tpc.114.123620 1 Supplemental Information Supplemental Figures Supplemental Fig.

Data. Mbengueet al. (2010). Plant Cell 10.1105/tpc.110.075861...Supplemental Figure 6. PUB1-RNAi Construct Efficiency. The PUB1-RNAi construct efficiently down-regulates PUB1 gene

Data. Mbengueet al. (2010). Plant Cell 10.1105/tpc.110.075861...Supplemental Figure 6. PUB1-RNAi Construct Efficiency. The PUB1-RNAi construct efficiently down-regulates PUB1 gene

A B {FAFA} ncc1-ncc2 - The Plant Cell · Supplemental Data. Boulouis et al. (2015). Plant Cell 10.1105/tpc.15.00010 1 Supplemental Figure 1: Phenotype of the double mutant ncc1 ncc2

A B {FAFA} ncc1-ncc2 - The Plant Cell · Supplemental Data. Boulouis et al. (2015). Plant Cell 10.1105/tpc.15.00010 1 Supplemental Figure 1: Phenotype of the double mutant ncc1 ncc2

Supplemental Data. Balcke et al. (2017). Plant Cell 10.1105/tpc.17 · Supplemental Data. Balcke et al. (2017). Plant Cell 10.1105/tpc.17.00060 Supplemental Figure 2. Total GC-MS and

Supplemental Data. Balcke et al. (2017). Plant Cell 10.1105/tpc.17 · Supplemental Data. Balcke et al. (2017). Plant Cell 10.1105/tpc.17.00060 Supplemental Figure 2. Total GC-MS and

Supplemental Data. Guo et al. (2015). Plant Cell 10.1105 ... · 11/2/2015  · Supplemental Data. Guo et al. (2015). Plant Cell 10.1105/tpc.15.00829 Supplemental Figure 7. The Possible

Supplemental Data. Guo et al. (2015). Plant Cell 10.1105 ... · 11/2/2015  · Supplemental Data. Guo et al. (2015). Plant Cell 10.1105/tpc.15.00829 Supplemental Figure 7. The Possible

Boter et al-YAB supplemental figures - Plant Cell · FBE2 G-box FBE1 -463 -1 Supplemental Data. Boter et al. (2015). Plant Cell 10.1105/tpc.15.00220 6. Ler fil yab3 Coler fil yab235

Boter et al-YAB supplemental figures - Plant Cell · FBE2 G-box FBE1 -463 -1 Supplemental Data. Boter et al. (2015). Plant Cell 10.1105/tpc.15.00220 6. Ler fil yab3 Coler fil yab235

Supplemental Data. Golczyk et al. (2014). Plant Cell …...2014/03/14  · Supplemental Data. Golczyk et al. (2014). Plant Cell 10.1105/tpc.114.122655. 6 Supplemental Figure 5. Structure

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Supplemental Material - The Plant Cell...2016/12/16  · Supplemental Data. Grimm et al. (2016). Plant Cell 10.1105/tpc.16.00551 2 Supplemental Figure 2. Data Repository and Detailed

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Supplemental Data. Galka et al. Plant Cell. (2012). 10 ... · Supplemental Data. Galka et al. Plant Cell. (2012). 10.1105/tpc.112.100339 2 The second band of PSI found near B12 could

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Supplemental Data. Geiser et al. (2016). Plant Cell …...2015/12/14  · Supplemental Data. Geiser et al. (2016). Plant Cell 10.1105/tpc.15.00791 AK2 D E P O Q R AK6 L M N K AK5 tPCK

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