Phylogeny of RIN4 protein homologs from model legume species and a novel-RIN4-motif within soybean RIN4

(A) Phylogeny of soybean RIN4 closest homologs including GmRIN4a, GmRIN4b, GmRIN4c, GmRIN4d, L. japonicus RIN4 (Lj3g3v0730080), two M. truncatula RIN4 (Mtr8g012960, Mtr7g056147), two P. vulgaris RIN4 (Pvul010G021200, Pvul008G130600), and nodulating non-legume P. andersonii (Pan P5HM0) and its non-nodulating relative Trema orientale (Tor P5CEA4). Tree was rooted using A. thaliana RIN4 (At3g25070). (B) Novel-RIN4-motif within RIN4 protein sequences (red box). One of the identified phosphorylation sites, S143 is located within this motif (red arrow) within a “GRDSP” core sequence that is highly conserved among legume species and species of Rosales. Here, we show the sequence alignment of Arabidopsis RIN4 with soybean (Gma), L. japonicus (Lj), M. truncatula (Mtr), P. vulgaris (Pvul) and nodulating non-legume P. andersonii (Pan) and its non-nodulating relative T. orientale (Tor) RIN4 proteins. Grey underline indicates Nitrate-induced domain (NOI). Red underline indicates motif for proteolytic cleavage targeted by pathogenic effector proteins, while blue line shows cleavage site. The characteristic feature of the NOI-domain is that it harbors the PXFGXW motif which is target site for effector protein. Tryptophan (W) is a crucial residue within the motif. RIN4c and RIN4d is missing W (blue arrow), and SMART analysis also predicted only one Pfam: AvrPt2 cleavage site.

RIN4a and RIN4b are required for proper symbiosis formation

(A) Micrographs showing representative transgenic roots of gene silencing, visualized by green fluorescence originating from GFP marker carried by all vectors. Scale bars represent 2 mm. (B) Significantly reduced nodule numbers were observed on soybean transgenic roots carrying RNAi constructs targeting RIN4a and RIN4b. Roots were phenotyped 5 wpi. Representative data of one biological replicate, experiment was done in 3 biological replicates. Student’s t-test * p<0.05. (C) Quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis confirmed reduced transcript levels of RIN4a and RIN4b in RNAi transgenic roots, strongly suggesting that the individual constructs targeted both genes. (D) Roots of wild-type RIN4b (WT) and mutant rin4b (in Bert background) homozygous mutant plants. Scale bars represent 2 cm. (E) Reduced nodule numbers in Bert soybean roots carrying a CRISPR-Cas9 edited 2 bp deletion in RIN4b (rin4b) in comparison to plants expressing wild-type RIN4b (WT) which out segregated as non-transgenic and non-edited. Experiments were done twice. Student’s t-test * p<0.05.

Nodulation-related S143 phosphorylation contributes to symbiosis development

(A) Phosphor-minus (Ala; S143A) and phosphomimic (Asp; S413D) mutations were introduced into RIN4a and RIN4b to replace S143 residue, and the constructs were expressed in soybean transgenic roots. RIN4aS143A and RIN4bS143A displayed significantly reduced nodule numbers in comparison to transgenic roots carrying empty vector (EV) and the wild-type RIN4a and b protein. RIN4aS143D and RIN4bS143D did not have an effect on nodulation. Roots were phenotyped 5 wpi. Experiment was repeated three times with similar results. Student’s t-test * p<0.05. (B) Micrographs showing representative transgenic roots expressing the respective constructs, visualized by green fluorescence originating from GFP marker carried by all vectors. Scale bars represent 2 mm.

Nodulation-related S143 is phosphorylated by SymRKβ in planta

(A) Phosphorylation of RIN4a by SymrkβΔMLD is demonstrated using α-pRIN4-S143, while no phosphorylation can be observed when RIN4a was co-expressed with the kinase inactive version of SymRKβ in planta, in Arabidopsis protoplasts. (B) shows the expression of all components co-expressed in protoplasts: HA-tagged RIN4a, HA-RIN4aS143A, HA-tagged wild-type SymrkβΔMLD and kinase-dead SymrkβΔMLD. (C) Phosphorylation of RIN4b by SymrkβΔMLD is demonstrated using α-pRIN4-S143, while no phosphorylation can be observed when RIN4b was co-expressed with the kinase inactive version of SymRKβ (D) shows the expression of all components co-expressed in protoplasts: HA-RIN4b, HA-RIN4bS143A, wild-type HA-SymrkβΔMLD and HA-SymrkβΔMLD-D734N. *: non-specific band shows equal loading of the protein extracts as well as CBB staining of the membranes displays equal protein extract loading.

Symbiotic signaling is affected in the rin4b-CRISPR/Cas9 mutant

Expression of transcription factors (TF) involved in the symbiotic signaling pathway was investigated in rin4b mutant background by qRT-PCR. Induction of the closest homolog of nodulation-specific transcription factor NIN1a can be observed in wild type (wt) and it does not change in rin4b in response to B. japonicum (Bj) 12 hpi. While two other homologs (NIN2a and NIN2b) respond to Bj in rin4b, their induction is significantly lower in comparison to Bert wt. Two soybean NF-YA TFs were tested as they are activated by NIN, therefore their lower expression in response to Bj in rin4b was expected. ERN1 is another TF downstream of NIN. Its induction in rin4b in response to Bj is significantly lower than the response in Bert wt. Student’s t-test * p<0.05.

Phylogenetic tree of RIN4 proteins shows a nodulation-specific subclade

Phylogenetic tree of 149 closest RIN4 homologs from 66 species, from non-legumes such as Arabidopsis thaliana, A. lyrata, Brassica rapa, Brachypodium distachyon, Setaria viridis, Zea mays, Oryza sativa, Nicotiana benthamiana, N. silvestris, Solanum lycopersicum. and from the phylogenetic FaFaCuRo clade: Fabales (nodulating species: Abrus precatorius, Arachis ipaensis, A. duranensis, Cajanus cajan, Chamaecrista fasciculata, Cicer arietinum, Faidherbia albida, Glycine max, Lablab purpureus, Lotus japonicus, Lupinus angustifolius, Medicago truncatula, Mimosa pudica, Mucuna pruriens, Phaseolus vulgaris, Spatholobus suberectus, Trifolium pratense, T. subterraneum, Vigna radiata, V. angularis, V. unguiculata, V. subterranean; non-nodulating species: Chastanospermum australe, Cercis canadensis, Nissolia schottii), Rosales (Parasponia andersonii nodulating with rhizobia, its non-nodulating relative Trema orientale; Dryas drummondii nodulating with Frankia, and non-nodulating Cannabis sativa, Ceanothus thyrsiflorus, Fragaria vesca, Fragaria x ananassa, Humulus lupulus, Morus notabilis, Prunus persica, P. mume, P. avium, Pyrus bretschneideri, Rubus occidentalis, Rosa chinensis, Ziziphus jujube), Fagales (nodulating Alnus glutinosa, Casuarina glauca and non-nodulating Junglans regia, Quercus robur, Q. loboa), and Cucurbitales (nodulating Datisca glomerata, non-nodulating Begonia fuchsioides, Citrullus lanatus, Cucumis sativus, C. pepo, C. melo, Lagenaria siceraria, Momordica charantia) (37, 52). Two sub-subclades formed: the blue-highlighted contain all the legume RIN4 homologs, whereas the green contains RIN4 homologs from Rosales. The tree was build using Average linkage (UPGMA) method and JTT substitution model.

Alignment of representative RIN4 homologs from the FaFaCuRo clade

The FaFaCuRo clade contains species which are able to form symbiotic nitrogen fixation. Here, we show that the 15 amino acid novel-RIN4-motif (NRM) sequence (red box) and its “GRDSP” core motif (green box) – which is present in soybean (Gma03G084000/RIN4a, Gma16G090700/RIN4b), L. japonicus (Lj3g3v0730080), N. schottii (Nsch1371S14229) and Parasponia (PanPON36036) RIN4 proteins – is not conserved in nodulating species from Fagales (Alnus glutinosa – Agl160713S33275; Casuarina glauca – Cgl1230S11910) and Cucurbitales (Datisca glomerata – Dgl127393S26490). In addition, non-Fabales species: rice (Oryza sativa – Os03g63140), maize (Zea mays – ZmB84.05G005000) and poplar (Populus trichocarpa – Potri.002G245400) were included in the alignment.

Soybean RIN4a and RIN4b are highly expressed in root hair and stripped root

(A) Relative gene expression levels of soybean RIN4 homologs (RIN4a-d) and a RIN4-like gene (Glyma09G008700). RIN4a and RIN4b display higher expression level than RIN4c, RIN4d and the RIN4-like gene in root hair (RH). All four have lower expression levels in roots. No difference could be observed between mock and rhizobial (Bj wt) treatment in root hairs or in roots. qRT-PCR analysis was done on 3 biological replicates, data show the mean of 2 technical replicates of 1 biological replicate. (B) Immuno-blot analysis, RIN4 proteins detected using RIN4-specific antibody, shows higher protein levels in root hairs than in roots (upper panel). No response was observed to treatment with B. japonicum (Bj) in comparison to mock treatment (M). Lower panel: Coomassie Brilliant Blue (CBB) staining of the same membrane showing equal loading. Immuno-blot analysis was performed on three biological replicates. (C) Custom-made anti-RIN4 antibody was tested on recombinantly expressed his-epitope tagged RIN4a, RIN4b, RIN4c and RIN4d proteins. Anti-RIN4 can recognize only RIN4a and RIN4b proteins. Upper panel: Immuno-blot showing RIN4a, b, c, d proteins detected using anti-His antibody. Bottom panel: Immuno-blot detecting RIN4 proteins using anti-RIN4 antibody.

Rin4b-CRISPR-Cas9 deletion and reduced mRNA and protein level in rin4b-CRISPR-Cas9 mutant

(A) Two base pairs deletion was introduced in the second exon of RIN4b (in Bert background) using CRISPR-Cas9 technology which led to a pre-mature stop codon in rin4b mutant. (B) qRT-PCR analysis performed on roots shows that RIN4b mRNA levels were reduced in rin4 mutant in comparison to wild-type roots (RIN4b), while RIN4a, RIN4c and RIN4d levels were not affected in the rin4b mutant background. Error bars represent standard error. Student t-test ** p<0.005. (C) RIN4 protein abundance was reduced in rin4b-CRISPR-Cas9 mutant roots (rin4b) in comparison to wild type roots (RIN4b). Immuno-blot detection was performed using anti-RIN4 antibody on total protein extracted from roots (upper panel). Lower panel: Membrane stained with Coomassie Brilliant Blue (CBB) to show loading.

Nodulation-related S143 phosphorylation site is upregulated in response to rhizobium

(A) Table describing the sequence of the phosphopeptide identified in our previous study (Nguyen et al., 2012). The peptide sequence carrying the S143 phosphorylation sites is identical in RIN4a and RIN4b proteins. The right column shows the sequence of the anti-phosphopeptide against the sequence carrying the S143 phosphorylation site. A cysteine “C” is automatically added to a peptide during synthesis. (B) Phosphorylation of RIN4S143 is up-regulated in soybean root hairs in response to soybean symbiont B. japonicum. Left panel: Immune-blot detection using pS143 peptide antibody performed on root hairs treated with H2O (mock) and soybean symbiont B. japonicum (Bj), 1 hpi. Right panel: Coomassie Brilliant Blue (CBB) staining of protein gel previously run to determine equal loading. (C) Quantification of phosphorylation using ImageJ software.

Protein expression of RIN4a and RIN4b and their mutated versions in soybean transgenic roots

Expression of HA-epitope tagged RIN4a and RIN4b and their phosphor-minus (S143A) and phosphor-mimic (S143D) versions in soybean transgenic roots. Immuno-blot analysis confirmed the expression of each version of RIN4a and b. Upper panel: HA antibody detecting RIN4a and b native and mutated proteins. Lower panel: detecting free GFP (27kDa) used as a marker to detect transgenic roots.

Phosphor-negative RIN4bS143A does not complement rin4b mutant phenotype

(A) rin4b-CRISPR/Cas9 mutant was transformed with RIN4b, RIN4bS143A, RIN4bS143D and empty vector. Only RIN4b and RIN4bS143D could rescue the phenotype caused by rin4b mutation (micrographs on the right). RIN4bS143A and empty vector could not restore nodule numbers on the transgenic roots (micrographs on the left). Micrographs showing representative transgenic roots expressing the respective constructs, visualized by green fluorescence originating from GFP marker. Scale bar represent 1 mm. (B) Graphical representation of the results from 2 biological replicates. Error bars represent standard error. Student t-test, * p<0.05. (C) Western-blot analysis showed that the transgenic roots expressed the HA-tagged RIN4b proteins, as well as the GFP marker carried by the vector.

RIN4a and RIN4b interacts with symbiotic receptor-like kinases NFR1α and SymRKß in planta

(A) Bimolecular Fluorescence Complementation assay where the RIN4a and RIN4b interaction was used as a positive control. (B) RIN4a interacts with SymRKßΔMLD (C) and with NFR1α. (D) No fluorescence signal was observed when RIN4a was co-expressed with the Arabidopsis P2K1 receptor-like kinase, used as a negative control. (E) RIN4b interaction with SymRKßΔMLD (F) and with NFR1α. (G) No fluorescence signal was observed when RIN4b was co-expressed with P2K1. Left panels: BiFC, middle left panels FM4-64 staining of the plasma membrane (PM) to show that interaction occurs at the (PM). Middle right panels: bright field (BF). Right panels: merge of YFP (interaction signal) and red PM signal. Scale bars represent 10 μm. Leica SP8 confocal microscope was used to take images. At least three biological replicates were performed with similar results.

Split-Luciferase assay confirmed interaction of RIN4a and RIN4b with symbiotic receptors

Upper panel shows RIN4a interaction with SymRKßΔMLD and NFR1α. Upon interaction between the co-expressed proteins fused to split domains of Luciferase, Luciferase activity is restored, and bioluminescence is detected (right panels). Lower panel shows RIN4b interaction with SymRKßΔMLD and NFR1α. In both cases Arabidopsis P2K1 receptor was used as negative control and no bioluminescence could be observed.

Soybean SymRKβ is an active kinase Soybean

SymRKβ kinase domain fused to GST was recombinantly expressed and kinase activity was detected in vitro using radioactive ATP. SymRKβ-KD trans-phosphorylates MBP substrate (left side). A point mutation was introduced to create a kinase inactive version, SymRKβD734N, and it resulted in abolished kinase activity (right side, upper panel). Lower panel: CBB staining to show that both native and mutant version were equally loaded.

In vitro phosphorylation of nodulation-related S143 by SymRKβ.

(A) Recombinantly expressed SymRKß and NFR1α kinase domains phosphorylate RIN4a and RIN4b in vitro. Myelin basic protein (MBP) was used as a positive control for SymRKβ and NFR1α kinase activity, and GST was used (left side) as a negative control. (B) Quantitative mass spectrometry (MS-MRM) was performed to identify the phosphorylation site triggered by the 2 kinases. S143 phosphorylation was phosphorylated only by SymRKß and quantified using heavy-labeled peptides generated against native peptides carrying S143. In both RIN4a and RIN4b S143 nodulation-related phosphorylation site is phosphorylated by SymRKβ whereas another phosphorylation site T173, used as negative control was not phosphorylated neither by SymRKß nor by NFR1α. MS run was performed in 2 biological and 3 technical replicates. Phosphorylation is expressed in percentage as mean of all replicates.

Calibration curves and correlation coefficients of RIN4a and RIN4b established for MS-SRM

(A, C, E, G) Calibration curves of S143 and T173 containing native peptides at different protein concentration of recombinantly expressed RIN4a and RIN4b proteins were established at fixed amount (100 fmol S143; 50 fmol T173) of heavy-labeled AQUA peptide. Both S143 and T173 carrying peptides are 100% identical between RIN4a and RIN4b, therefore the experiment was done in RIN4a and RIN4b recombinantly expressed protein background separately, resulting in similar values, therefore suggesting that the proteins behave in a similar way when LC-MS was run. Left panels show calibration curves expressing peak area ratio of native peptide normalized to heavy-labeled peptide. As expected, concentration gradient led to growth curve. (B, D, F, H) Scatter plots expressing relationship between protein concentrations loaded per injection and peak area ratio normalized to heavy obtained per concentration gradient. Correlation coefficient (R2 was 0.99 in both RIN4a and RIN4b) values strongly support correlation between the protein concentration injected and the native peptide amount detected (normalized to heavy-labeled peptide).

List of RIN4 proteins used for the phylogenetic tree

Summary of L. japonicus rin4 mutant screen

Soybean RIN4 native and AQUA peptides