SEARCH

SEARCH BY CITATION

Figure S1. Measurements of shoot and root length in har1-1 and sup11. Data represent mean values ± SE for n = 10. Asterisks (*) denote statistically significant differences between genotypes within corresponding categories as determined using a Student’s t-test (P < 0.05). The measurements were performed 21 dai on plants grown in soil.

Figure S2. Kinetics of nodule development in har1-1 parental line and sup11. M.  loti strain NZP2235 tagged with constitutive hemA::LacZ reporter gene fusion was used for inoculation and roots were stained for β-galactosidase activity (blue) prior to nodule counting. Note that all nodule-associated cortical cell division events that have not yet emerged from the epidermis were categorized as nodule primordia. Un-colonized and partially colonized nodules were categorized following examples shown in Figure 3. Data represent mean values ± SE for n = 10. Asterisks (*) denote statistically significant differences between genotypes within corresponding nodulation categories, as determined using a Student’s t-test (P < 0,05).

Figure S3. sup11 aborts normal root colonization by M. loti and AM fungus. Plants were inoculated with M.  loti strain NZP2235 tagged with a constitutive hemA::LacZ reporter gene fusion (a-d). Roots were stained (see Experimental Procedures) and the symbiotic phenotypes were evaluated 7 dai. (a) The parental har1-1 line; note that M.  loti (blue) migrated through a root hair infection thread (IT), which then ramified within the underlying nodule primordium (NP). (b) Unsuccessful root colonization by M.  loti in sup11; note that block of infection has occurred, which led to the accumulation of bacteria within swollen IT in the subepidermal cortical cell. (c) Enlarged microcolonies of M.  loti formed at the sup11 root epidermis; notice that IT was initiated from one of the microcolonies but its normal progression was halted, such that it became swollen upon entry into the subtending cortical cell. (d) Scores of infection events in har1-1 and sup11 mutant. Data represent mean values ± SE for n = 10. Asterisks (*) denote statistically significant differences between the har1-1 and sup11 genotypes for a given category as determined using a Student’s t-test (P < 0,05). (e and f) Representative fragments of L.  japonicus har1-1 and sup11 roots 8 weeks after inoculation (wai) with Rhizophagus irregularis; note that unlike in har1-1 (e), the fungus failed to penetrate sup11 roots (f). IH: intraradical hypha; A: arbuscule; EH: extraradical hypha; V: vesicle.

Figure S4. Map-based cloning and next generation sequencing identify two linked mutations. (a) A schematic representation of the L.  japonicus chromosome 3. (b) A portion of the chromosome 3, as represented by large insert clones (TM or BM) and the corresponding sequence contigs (CM) or sequenced genomic clones (LjT). Frequency of recombination events at the given position is provided in parentheses. (c) A mutant ccamk-14 allele (gray box), containing G3922 to A nucleotide substitution, as identified by map-based cloning and sequencing. Next generation sequencing has confirmed the presence of the ccamk-14 mutation and also revealed an additional mutant allele (black box), called nph3-1 (C272 to T). (d) A schematic representation of the predicted L. japonicus NPH3 protein with main domains indicated. Arrow indicates an approximate position of the predicted amino-acid substitutions in nph3-1 (T91 to I).

Figure S5. The genotypes. The HAR1 locus specific MvaI-CAPS marker and BsrBI- and BsrDI-dCAPS markers for the CCaMK and nph3-1 loci, respectively, were used (see Experimental Procedures. The following size (bp) of PCR fragments for wild-type and mutant alleles were obtained, as predicted by the position of gene-specific primers and digestion products: (1) HAR1: 369, har1-1: 463; (2) CCaMK: 125, ccamk-14: 150; (3) NPH3: 244; nph3-1: 216. Note that “sup11” reflects the original mutant line carrying har1-1, ccamk-14 and nph3-1 mutant alleles.

Figure S6. nph3-1 single mutant phenotypes. M. loti strain NZP2235 was used for plant inoculation. (a) Wild-type L.  japonicus Gifu and the nph3-1 single mutant, 14 dai. Note that the wild-type Gifu and the nph3-1 develop only pink nodules (see inserts). (b) Measurements of shoot and root length in wild-type and nph3-1 14 dai on plants grown in soil. (c) Counts of nodules at 14 dai. Data shown in B and C represent mean values ± SE for n = 10. Asterisk (*) denotes statistically significant difference between genotypes within the corresponding category as determined using a Student’s t-test (P < 0,05). (d-e) Representative fragments of L.  japonicus wild-type (d) and nph3-1 mutant roots (e) showing successful colonization by R.  irregularis.

Figure S7. The L.  japonicus CCaMK gene restores AM development in sup11. A genomic fragment containing the entire CCaMK locus was introduced by A.  rhizogenes (AR12)-mediated transformation (see Experimental Procedures) to generate transgenic hairy roots on non-transgenic shoots. The resulting hairy roots were stained with 5% ink in 5% acetic acid eight weeks after inoculation to reveal the location of R.  irregularis. (a) har1-1 transformed with the AR12 strain; (b) sup11 transformed with the AR12 strain; (c) sup11 transformed with the AR12 strain containing the CCaMK gene.

Figure S8. The L.  japonicus CCaMK gene restores wild-type nodulation and AM development in ccamk-14. A genomic fragment containing the entire CCaMK locus was introduced by A.  rhizogenes (AR12)-mediated transformation to generate transgenic hairy roots on non-transgenic shoots. The resulting plants were inoculated with the DsRED-containing M.  loti or R.  irregularis and their nodulation (a, b, d, e, g, h) and mycorrhiza (c, f, i) phenotypes were evaluated 11 dai or 8 wai, respectively. (a-c) wild-type L.  japonicus transformed with the AR12 strain; notice the formation of pink nodules (a), presence of DsRED fluorescence inside a small nodule primordium (b) and the wild-type AM symbiotic phenotype (c). (d-f) ccamk-14 transformed with the AR12 strain; notice that white or pale-pink nodules are present (d) and clumps of fluorescence are visible at the surface of a small nodule primordium (e). Fungal hyphae failed to penetrate the ccamk-14 roots (f). (g-i) ccamk-14 transformed with the AR12 strain containing the CCaMK genomic DNA. The wild type nodulation (g and h) and AM symbiotic phenotype are restored.

Figure S9. MS/MS spectrum of CCaMK phosphopeptide 329-AAAIASVWpSSTIFLR-343 containing pS337. The mass difference of 167 Da between fragment ions y6 (mass: 736.435 Da) and y7 (mass: 903.434 Da) indicates specific phosphorylation of serine 337 (S*) resulting in a mass increment of serine (87 Da) of 80 Da by the attached phosphate group (pS = 167 Da).

Figure S10. Expression of CCaMKS337D does not rescue the ccamk-13 null symbiotic phenotype. The wild-type CCaMK cDNA (cDNACCaMK) and a cDNA encoding the CCaMKS337D variant (cDNACCaMKS337D) under the control of the cognate CCaMK promoter (b, d, e and f) or polyubiquitin promoter (g and h) were introduced by A. rhizogenes AR1193 mediated transformation to either wild-type L.  japonicus Gifu (b) or ccamk-13 mutant (c-h) to generate transgenic hairy roots on non-transgenic shoots. The resulting chimeric plants along with those carrying control transgenic hairy roots (a and c) were inoculated with M.  loti or R.  irregularis and their nodulation (a–e) and mycorrhiza (g and h) phenotypes were evaluated 11 dai and 8 wai, respectively. (a) wild-type Gifu transformed with the control AR1193 strain; (b) wild-type Gifu transformed with AR1193 containing cDNACCaMKS337D; (c) ccamk-13 transformed with the control AR1193 strain; (d and g) ccamk-13 transformed with AR1193 containing cDNACCaMK (note the complementation of nodulation and mycorrhization) (e and h) ccamk-13 transformed with AR1193 containing cDNACCaMKS337D (f) ccamk-13 transformed with AR1193 containing cDNACCaMKS337D and grown in the absence of either M.  loti or R.  irregularis for 6 weeks (note lack of spontaneous nodules). Although the mycorrhiza phenotypes in g and h are shown for ccamk-13 plants transformed with constructs driven by the polyubiquitin promoter, the same results were obtained with ccamk-13 plants expressing the corresponding constructs from the endogenous promoter.

Figure S11. Quantitative yeast two-hybrid interaction analysis of CCaMK, kinase dead CCaMKG30E, CCaMKS337N and CCaMKS337D with CYCLOPS. The interaction was assayed by the quantitative analysis of ß-galactosidase activity obtained from Saccharomyces cerevisiae HF7c cells transformed with the indicated constructs. Compared to the kinase inactive mutant CCaMKG30E which is impaired in the interaction with CYCLOPS, CCaMK-S337 mutant variants (S337D, S337N) show wild type-like interaction. Presented are mean values and standard deviations obtained from three biological replicates. One Miller unit of ß-galactosidase is defined as the amount which hydrolyzes 1 μmol of ONPG to o-nitrophenol and D-galactose per min per cell. BD: fusion to the Gal4 DNA binding domain, AD: fusion to the Gal4 activation domain.

Figure S12. In vitro kinase activity of CCaMK, CCaMKS337N and CCaMKS337D in the presence of MBP. Maltose-binding protein-tagged CCaMK and the mutant variants CCaMKS337D and CCaMKS337N were tested for in vitro kinase activity in the presence of either 4 mM EGTA (-), 0.1 mM CaCl2 (Ca2+), or 0.1 mM CaCl2 and 1 μM calmodulin (CaM). Each reaction contained 2 μg of CCaMK protein and 10 μg of myelin basic protein (MBP) as substrate. Incorporation of radioactive phosphate was visualised using a Typhoon phosphorimager. (a) Auto-P: Autophosphorylation of CCaMK, CCaMKS337D, or CCaMKS337N. (b) MBP-P: Phosphorylation of the CCaMK artificial phosphorylation substrate, MBP. (a, b) Quantitation of autophosphorylation (Auto-P) and MBP phosphorylation (MBP-P). Incorporation of radioactive phosphate was quantified using a Typhoon phosphorimager and band intensities (auto-, or MBP phosphorylation) were normalized to values determined for CCaMK wild type in the presence of EGTA (1-fold incorporation). Graph represents mean values ± standard deviation of kinase activity from two independent experimental set-ups. The same letter denotes lack of significant differences between activity values, as determined using the Kruskal-Wallis multiple comparison with the Bonferroni correction; alpha = 0.05.

Figure S13. In vitro autophosphorylation activity of CCaMK, CCaMKS337N and CCaMKS337D in the absence of substrate. Maltose binding protein-tagged CCaMK, CCaMKS337D and CCaMKS337N were tested for in vitro autophosphorylation in the presence (+) of either 4 mM EGTA, 0.1 mM CaCl2 (Ca2 + ), or 0.1 mM CaCl2 and 1 μM calmodulin (CaM). Each reaction contained 2 μg of CCaMK protein. Auto-P: Autophosphorylation of CCaMK, CCaMKS337D, or CCaMKS337N. Incorporation of radioactive phosphate was quantified using a Typhoon phosphorimager and band intensities were normalized to values determined for CCaMK wild type in the presence of EGTA (1-fold incorporation). Graph represents mean values ± 1 standard deviation from two technical replicates of one experimental set-up.

As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer-reviewed and may be re-organized for online delivery, but are not copy-edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.

FilenameFormatSizeDescription
tpj5098_sm_FigS1-S13.pdf1992KSupporting info item
tpj5098_sm_Supporting-Information-legends.docx26KSupporting info item

Please note: Wiley Blackwell is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.