Supplemental Data. Shimoda et al. (2011). Plant Cell /tpc B C D E

Transcription

1 (kda) B C D E (0/15) (29/29) (0/29) (0/32) Supplemental Figure 1. Immunodetection of Myc-tagged CCaMK Protein Variants on ccamk-3 Roots. () Immunodetection of myc-tag fusion CCaMK WT, and EF1 EQ - 2 EQ -3 EQ proteins on the corresponding transformed hairy roots of ccamk-3 mutant. Left and right image show staining of total protein extracted from transformed hairy roots and immunodetection of CCaMK protein variants (arrowheads) with c-myc antibody, respectively. (B-E) Phenotype of ccamk-3 roots transformed with empty vector (B) and myc-tag fusion CCaMK WT (C), (D) and EF1 EQ -2 EQ -3 EQ (E). Numbers in parentheses indicate the number of plants that formed mature root nodules, out of total number of plants tested. Scale bars = 2 mm.

2 F K P EF1 EQ B G L Q EF2 EQ C H M R EF3 EQ D I N S EF0 Q E J O T EF1 IQ Supplemental Figure 2. Complementation of Nodulation of ccamk-3 Mutant by CCaMK Variants Containing Point Mutation in Single EF-hand Motif. (-E) Root hairs of ccamk-3/ef-hand mutation CCaMKs, shown as merged images of bright-field and fluorescence-field of DsRed. Scale bars = 100 μm. Nodulation of CCaMK-transformed hairy roots was observed at (F-J) bright-field and fluorescent-field of (K-O) GFP and (P-T) DsRed. Scale bars = 1 mm. Normal infection threads and effective nodules were observed on all CCaMK variants.

3 T265D B ar rh Supplemental Figure 3. Mycorrhization on ccamk-3/1-340 and ccamk-3/1-340 T265D Roots. Mycorrhizal fungi infection of () ccamk-3/1-340 and (B) ccamk-3/1-340 T265D roots, visualized using trypan blue staining. ar, arbuscule;, intracellular hyphae; rh, running hyphae. Scale bars = 200 μm.

4 ap rh D rh ap B E rh C F ab ab Supplemental Figure 4. G. intraradices Infection on ccamk-3/ccamk WT and ccamk-3/ccamk FN-ED Roots. Epidermal and cortical infection of G. intraradices on ccamk-3/ccamk WT (- C) and ccamk-3/ccamk FN-ED roots (D-F). (, D) appressorium formation on root epidermis. (B, E) internal hyphae that path through intra- intercellularly fashion. (C, F) arbuscule formation in cortical cells. rh; running hyphae, ap; appressorium, ; intracellular hyphae, ar; arbuscule. Scale bars = 100 μm.

5 Ca 2+ CaM B MBP-P MBP-P C D MBP-P MBP-P Supplemental Figure 5. in vitro Kinase ctivity of Recombinant CCaMK Proteins with Myelin Basic Protein (MBP) as a substrate. () utophosphorylation () and substrate phosphorylation (MBP-P) activity of CCaMK WT in the presence (+) and absence (-) of Ca 2+ and CaM. (B-D) Kinase activity of deletion CCaMK proteins (B), CCaMK EF EQ proteins (C) and CCaMK proteins containing the mutation of ccamk mutant alleles (D). In all experiments, myelin basic protein (MBP) was used as a phosphorylation substrate and loading of equivalent molar of CCaMK proteins used for kinase assay is shown by staining (). in vitro kinase assays in (B-D) were carried out in the presence of Ca 2+ and CaM.

6 CYCLOPS-P B CYCLOPS-P Supplemental Figure 6. in vitro Kinase ctivity of Recombinant CCaMK Proteins with CYCLOPS as a Substrate. Kinase activity of deletion CCaMK proteins () and CCaMK EF EQ proteins (B). Recombinant protein of CYCLOPS was used as a phosphorylation substrate. Closed and open arrowheads in the upper images of () and (B) indicate CYCLOPS phosphorylation (CYCLOPS-P) and autophosphorylation of CCaMK (), respectively. Loading of equivalent molar of CYCLOPS and CCaMK proteins used for kinase assay is shown by staining, indicated by closed and open arrowheads, respectively. in vitro kinase assays were carried out in the presence of Ca 2+ and CaM.

7 CYCLOPS-P B Bait Prey C Prey Bait CCaMK WT CCaMK WT CCaMK G30E CCaMK T265D CCaMK S25F CCaMK FN-ED CCaMK G124D ps2-1 CCaMK G204R SD-LW SD-LWH CCaMK T265D ps2-1 SD-LW SD-LWH Supplemental Figure 7. in vitro Kinase ssay with CCaMK Variants and Their Interaction with CYCLOPS. () Kinase activity of CCaMK proteins containing the mutation of ccamk mutant alleles. Recombinant protein of CYCLOPS was used as a phosphorylation substrate. Closed and open arrowheads in the upper image indicate CYCLOPS phosphorylation (CYCLOPS-P) and autophosphorylation of CCaMK (), respectively. Loading of equivalent molar of CYCLOPS and CCaMK proteins used for kinase assay is shown by staining, indicated by closed and open arrowheads, respectively. in vitro kinase assays were carried out in the presence of Ca 2+ and CaM. (B, C) Interaction between CCaMK variants and CYCLOPS analyzed by yeast twohybrid analysis. Representative yeast growth on the selection medium (SD-LWH) is shown. Growth on non-selective medium (SD-LW) is shown for successful yeast mating of bait and prey clones. ps2-1 and pct2 was used as empty clone for bait and prey, respectively.

8 B C G D E F H I CYCLOPS-P MBP-P Supplemental Figure 8. Complementation of ccamk-3 Mutant by CCaMK T265 and in vitro Kinase ctivity of Gain-of-Function CCaMK Proteins. (-G) Symbiotic phenotypes of ccamk-3/ccamk T265, with (-C) or without (D- F) DsRed-labelled M.loti inoculation (visualized with bright field [, D] and fluorescence of GFP [B, E] and DsRed [C, F]). Upon G. intraradices inoculation, normal mycorrhization occurred (G). Scale bar, 1mm (-F), 200 μm (G). (H, I) Kinase activity of CCaMK proteins containing the gain-of-function mutation with MBP (H) and CYCLOPS (I) as a phosphorylation substrate. Closed and open arrowheads in upper image of (I) indicate CYCLOPS phosphorylation (CYCLOPS- P) and autophosphorylation of CCaMK (), respectively. Loading of equivalent molar of CYCLOPS and CCaMK proteins used for kinase assay is shown by staining, indicated by closed and open arrowheads, respectively. in vitro kinase assays were carried out in the presence of Ca 2+ and CaM.

9 B CCaMK WT : 310 MDPEIVSRLQSFNRRKLRISVWSSTIFLRTKKLRSLVGTYDLKEEE-360 ********************* CCaMK FN-ED : 310 MDPEIVSRLQSEDRRKLRISVWSSTIFLRTKKLRSLVGTYDLKEEE 360 Supplemental Figure 9. Comparison of Predicted Protein Structure of CCaMK WT and CCaMK FN-ED () Homology model of CCaMK structure shown with hydrogen bond network connected K264, T265, S327, E313 and R317. The region colored in pink (I314- S320) is a helical region that is predicted in CCaMK WT but not in CCaMK FN-ED. (B) Predicted secondary structure of the region of aa of CCaMK WT and CCaMK FN-ED. Helical region predicted by Jpred3 program is shown in red. In CCaMK WT, helix including CaMBD is predicted to start at I314, whereas it starts at E321 in CCaMK FN-ED. sterisks under the amino acid sequence indicate possible CaM binding region predicted by Calmodulin Target Database ( CaM binding motif were predicted in CCaMK WT, but not in CCaMK FN-ED. Underlined sequence indicates the mutated amino acid residues in CCaMK FN-ED.

10 CCaMK G30E CCaMK T265D _EF1 EQ -3 EQ B rh ar Supplemental Figure 10. Mycorrhization on ccamk-3/ccamk G30E and ccamk-3/ccamk T265D _EF1 EQ -3 EQ. Mycorrhizal fungi infection on the root of ccamk-3/ccamk G30E () and ccamk-3/ccamk T265D _EF1 EQ -3 EQ (B), visualized using trypan blue staining. ar; arbuscule, ; intracellular hyphae, rh; running hyphae. Scale bars = 200 μm.

11 Supplemental Table 1 Results of Complementation nalysis of ccamk-3 Mutant by Point Mutated CCaMK The numbers in table indicate the number of plants that are complemented rhizobial infection, spontaneous nodulation and M fungi infection, out of the total number of plants tested. Functional domains Results of complementation of ccamk-3 mutant Rhizobial infection and nodulation M fungi infection Construct Mutation Promoter Kinase CaM BD E F 0 E F 1 E F 2 E F 3 Infection thread Infected mature nodule Spontaneou s nodule rbuscule formation Root colonization d CCaMK WT - CaMV 35S 41/41 41/41 0/12 31/ (4.4) Kinase domain mutation CCaMK G30E G30E CaMV 35S 0/18 0/18 0/17 0/24 0 CCaMK T265D T265D CaMV 35S 37/39 37/39 68/75 44/ (3.2) CCaMK T265 T265 CaMV 35S 29/31 29/31 28/31 34/ (2.0) CCaMK R317H R317H CaMV 35S 47/50 47/50 45/46 26/ (3.0) CaMBD mutation CCaMK FN-ED F321E, N322D CaMV 35S 9 a /85 3/85 (43/85) b 27/41 39/ (3.3) EF-hand mutation (mutation in single EF-hand motif) EF0 Q 378Q CaMV 35S 17/17 17/17 N.T. N.T. N.. EF1 IQ I414Q CaMV 35S 22/24 21/24 N.T. N.T. N.. EF1 EQ E418Q CaMV 35S 16/16 16/16 N.T. 24/ (3.8) EF2 EQ E454Q CaMV 35S 14/15 13/15 N.T. 32/ (5.6)

12 EF3 EQ E496Q CaMV 35S 16/17 11/17 N.T. 17/ (4.4) EF1 EQ E418Q CCaMK native 10/10 9/10 N.T. N.T. N.. EF2 EQ E454Q CCaMK native 14/14 13/14 N.T. N.T. N.. EF3 EQ E496Q CCaMK native 13/13 8/13 N.T. N.T. N.. (mutations in two EF-hand motifs) EF0 Q -1 IQ 378Q, I414Q CaMV 35S 30/60 9/60 N.T. 33/ (5.5) EF1 EQ -2 EQ E418Q, E454Q CaMV 35S 41/41 37/41 0/18 45/ (4.7) EF2 EQ -3 EQ E454Q, E496Q CaMV 35S 0/60 0/60 0/17 2 c / (2.7) e EF1 EQ -3 EQ E418Q, E496Q CaMV 35S 0/65 0/65 0/18 9 c / (1.3) (mutations in three EF-hand motifs) EF1 EQ -2 EQ -3 EQ E418Q, E454Q, E496Q CaMV 35S 0/46 0/46 0/28 0/51 0 Combined mutation CCaMK T265D _EF1 EQ -2 EQ T265D, E418Q, E454Q CaMV 35S N.T. N.T. 14/40 10/10 N.. CCaMK T265D _EF2 EQ -3 EQ T265D, E454Q, E496Q CaMV 35S 44/51 44/51 0/69 35/ (3.7) CCaMK T265D _EF1 EQ -3 EQ T265D, E418Q, E496Q CaMV 35S 39/41 38/41 0/66 36/ (6.5) CCaMK T265D, FN-ED T265D, F321E, N322D CaMV 35S 0/52 0/52 (27/52) b 21/33 31/ (6.0) CCaMK G30E, T265D G30E, T265D CaMV 35S 0/42 0/42 0/52 0/ FN-ED F321E, N322D, deletion of 341 to CaMV 35S 0/60 0/60 (35/60) b 23/36 52/ (4.8) 518 aa

13 1-340 T265D T265D, deletion of 341 to 518 aa CaMV 35S 0/38 0/38 (24/38) b 25/48 35/ (4.9) CCaMK G30E, FN-ED G30E, F321E, N322D CaMV 35S 0/43 0/43 0/31 0/42 0 Empty vector pcmbi-gfp - CaMV 35S 0/35 0/35 0/24 0/33 0 a Infection threads were arrested in root epidermis. b Empty nodule without rhizobial invasion were observed. (number of plants that formed empty nodules are shown in parenthesis). c Formation of few arbuscules were observed in limited regions. d Mean arbuscular colonization (%) per mycorrhizal root (n=10) shown with standard error in parenthesis. e Four mycorrhizal roots were analyzed. N. T. not tested N.. not analyzed

14 Supplemental Table 2 Sequences of Primers Used in This Study Primers used for the introduction of point mutations into CCaMK Mutation Name of primer Sequence (5'- 3') S25_Fw GGTTCTTTGTTGTCGGGCC S25F S25_Rv GCCGTCCCCTCTTCCTG G30E G124D G204R T265D T265 R317H F321E, N322D (FN-ED) 378Q I414Q E418Q G30E_Fw G30E_Rv G124D_Fw G124D_Rv G204R_Fw G204R_Rv T265D_Fw T265D_Rv T265_Fw T265_Rv R317H_Fw R317H_Rv FN-ED_Fw FN-ED_Rv 378Q_Fw 378Q_Rv I414Q_Fw I414Q_Rv E418Q_Fw E418Q_Rv GGCCTCGGCT TTTGGTTTCTTTTCTGCCGG TGTTCTGTGGGGGCTGTTTGTGG CTCCCCTCGCGCTCCGCC TTGTTTGTCCTTGTTTGTTTC TGGTCTCCCCCCGG GGGGTTGGGGGCTTCCC CTTCCTCCTTCTCTGCTG GGGGCTTGGGGGCTTCCC CTTCCGCCTTCTCTGCTG GTCTCCCCTGCGGCTTTTGC CTGCGGTGTGGCTCTCGGGTC CGGCGGTGCGCGCCTCCGC TCTTGCTCTTCGCTCTGCGCCTTGGC GCTCCTCTGTCTGGTTTGTG CGGTTTGTTGTCTCCTTTCCC GGCCGCTGGGGTCT CTGTCTTGTGTTCCTCCGGTTGTT TGGCTCTTGTGGGTTTTCT TGTTTTGTCTCTGTCTTGTTCC

15 E454Q E496Q E454Q_Fw E454Q_Rv E496Q_Fw E496Q_Rv GGCGTGCTCCTGCTCTGT TGCTCTTGTTCCTTGGTGTGCCCC TTTGCTTCGCTGCTTGCG TTTGTTGTTCTGTCTTTTCC Primers used for cloning of full length and deletion CCaMKs into pentr-d-topo vector Name of primer caccccamk_fw Sequence (5'- 3') CCCTGGGTTGTCCCG CCaMK_Rv CTTGTGGCGGGG 314_Rv CTTCTCGGGTCCTTTGCTC 340_Rv CTGTTGTGCTGCTCCCC 429_Rv CTGGGTTCTTGGGCTG 471_Rv CTGTTCGCTGGGCTT Primers used for cloning of CCaMK and CYCLOPS into protein expression vector (pcold-gst) Name of primer NdeI_CCaMK_Fw Sequence (5'- 3') GCGCTTGGGTTGTCCCG NdeI_334_Fw GCGCTTGGTGTTTGGGCGCCT XhoI_CCaMK_Rv CGCCTCGGCTTGTGGCGGGG XhoI_340_Rv CGCCTCGGCTGTTGTGCTGCTCCCC XhoI_429_Rv CGCCTCGGCTGGGTTCTTGGGCTG XhoI_471_Rv CGCCTCGGCTGTTCGCTGGGCTT NdeI_CYCLOPS_Fw GCGCTTGGGGGGGGGGTTTTC XhoI_CYCLOPS_Rv CGCCTCGGTCTTCTTTTTTCGTTT