advances.sciencemag.org/cgi/content/full/6/21/eaaz1622/DC1

Supplementary Materials for

RALF1-FERONIA complex affects splicing dynamics to modulate stress responses

and growth in plants

Long Wang, Tao Yang, Bingqian Wang, Qinlu Lin*, Sirui Zhu, Chiyu Li, Youchu Ma, Jing Tang, Junjie Xing, Xiushan Li, Hongdong Liao, Dorothee Staiger, Zhiqiang Hu, Feng Yu*

*Corresponding author. Email: linql0403@126.com (Q.L.); feng_yu@hnu.edu.cn (F.Y.)

Published 20 May 2020, Sci. Adv. 6, eaaz1622 (2020)

DOI: 10.1126/sciadv.aaz1622

The PDF file includes:

Figs. S1 to S12 Other Supplementary Material for this manuscript includes the following: (available at advances.sciencemag.org/cgi/content/full/6/21/eaaz1622/DC1)

Tables S1 and S2

Fig. S1: PCA and GO term analysis of RALF1-triggered splicing change.

A, Principal component analysis plot of gene expression and alternative splicing of

seven-day-old Arabidopsis seedlings in the presence/absence of RALF1. PC1

indicated the difference between RALF1-treated and mock, and PC2 indicated the

difference within RALF1-treated and mock. B, Gene Ontology (GO) enrichment of

genes with significant splicing changes after RALF1 treatment compared with those

in the mock control (n=3). The black dotted line indicates P=0.05.

Fig. S2: Validation of AS events in RALF1-treated as determined by RT-PCR

analysis.

A, AS patterns and P values for the genes analyzed in B from the RNA-seq data. B,

Validation of selected AS events in RALF1-treated roots of Col-0 WT plants relative

to mock-treated roots. The annotated gene structure of the representative isoforms was

based on NCBI and ARAPORT; IS1 and IS2 indicate the two isoforms of the splicing

event studied. Three independent experiments were performed. C-D, Relative

amounts of the isoforms calculated from the semi-qPCR shown in B by Image J. Data

are shown as the mean±SD, n=3. Student’s t-test, *P<0.05, **P<0.01; n.s. means not

significant.

Fig. S3: RALF1/FER responsive splicing in Arabidopsis (Photo Credit: Long

Wang, Hunan university).

A, Summary of differentially expressed genes in RALF1-treated plants vs

mock-treated plants. The number above the bar indicates the number of genes. B, GO

term distribution of differentially expressed genes in RALF1-treated plants (n=3). The

black dotted line indicates P=0.05. C, Root lengths of different genotypes

mock-treated or treated with 1 μM RALF peptides. Four-day-old seedlings were

transferred to liquid ½ MS medium with or without 1 μM RALFs for 2 days, bar =1

cm. D, Root lengths of different genotypes grown in mock-treated medium and

medium with 1 μM RALF1, 1 μM RALF23 and 1 μM RALF17 (n=20 for each group).

n=20 roots per experimental group, n=20 roots per control group. Statistical analysis

indicates a difference between inhibition rates. One-way ANOVA with Tukey’s test,

**P<0.01. E, Phenotypic analysis of Col-0 and fer-4 with different concentrations of

RALF1. Bar=1 cm. F, Root lengths of Col-0 and fer-4 grown with different

concentrations of RALF1. n=20 roots per experimental group, n=20 roots per control

group. One-way ANOVA with Tukey’s test were used to determine the statistical

significance, **P<0.01. G, Semi-qPCR analyses of splicing patterns. The results

shown are representative of three independent experiments.

Fig. S4: Interaction analysis between GRP7, GRP8 and CrRLK1L subfamily

members. A, Representative images of the protein interaction assay in Fig. 1C,

SD/-Ade/-Leu selection medium (left) and SD/-Ade/-Leu/-His selection medium

containing 20 mM 3-AT (right) were used for screening yeast growth. All assays were

performed in three independent experiments, and similar results were obtained. 100

indicates the OD600=1, 10-1 indicates 1:10 dilution, and 10-2 indicates 1:100 dilution. B,

Y2H analysis of the interaction between GRP7 and multiple CrRLK1L subfamily

members’ kinase domains. Cells were grown on medium with (top) or without

(bottom) histidine (His). C, Y2H analysis of interactions between FER kinase domain

and two GRP subfamily members. Cells were grown on medium with (left) or without

His (right). D, Purification of GRP7-GST, GRP71-86-GST, and GRP787-176-GST

proteins. M indicates marker. E, GST pull-down assay. The eluted proteins were

separated by SDS-PAGE and probed with a 6-His-tag antibody. All assessments were

independently repeated three times with similar results. F, Protein expression in the

BiFC assay. Arabidopsis protoplasts in the BiFC assay were collected, and total

protein was extracted for SDS-PAGE-western blot analysis. The proteins expressed in

the BiFC assay were detected by GFP antibody. FER-nVenus, CVY1-nVenus,

GRP7-cCFP and GRP6-cCFP proteins are indicated. β-actin was used as loading

control.

Fig. S5: ESI mass spectrometric spectra analysis of GRP7 phosphorylated

residues. The identified GRP7 phosphorylation residues were Tyr111 (A), Ser112 (B),

Ser132 (C), Tyr138 (D), Ser139 (E), and Ser140 (F). The identified peptide sequences are

displayed. The y-ion and b-ion are shown above the sequences.

Fig. S6: Phylogenetic analysis and plant identification of GRP7. A, Phylogenetic

tree of GRP7 in diverse plant species based on protein sequence. B, Alignment of

GRP7 homologs from different plant species created with DNAMAN and BioEdit.

The blue frames represent the phosphorylation residues identified, and the yellow

circles show other, nonconserved phosphorylation residues. The red background

indicates homology >75%. C, Relative mRNA levels of GRP7. ACTIN2 was used as

an internal control. The error bars represent the SD of three technical replicates and

three independently repeated experiments; One-way ANOVA with Tukey’s test,

**P<0.01. D, Immunoblot analyses of GRP7 protein using GRP7 antibody. β-actin is

shown in the lower panel as a loading control. Different lines are indicated as

“-number”, and three independent experiments were performed. E, qPCR analysis of

the expression level of GRP7. ACTIN2 was used as the internal control. Data are

means ± SD based on three technical replicates and three independently repeated

experiments; One-way ANOVA with Tukey’s test; n.s. means not significant. F, PCR

identification of fer mutations and GRP7-GFP in genomic DNA from WT and

GRP7-GFP/fer-4 mutants. PCR with the primer set fer-4 F and fer-4 R was used to

amplify the T-DNA insert for fer-4, the primer set FER LP and FER RP was used to

amplify FER DNA in WT, and the primer set GRP7 F and 2300R was used to amplify

the GRP7-GFP DNA.

Fig. S7: Global AS patterns in fer-4 and grp7-1 8i. A-B, Functional categorization

(biological process) of genes with significant splicing changes in fer-4 and grp7-1 8i

mutants (n=3). The y-axis indicates gene numbers. C, Validation of alternatively

spliced transcripts by semi-qPCR. The gene model is based on NCBI and ARAPORT.

Red triangles indicate the positions of the PCR primers. D, AS patterns and P values

from the RNA-seq data. E, Bar chart showing the relative amounts of the isoforms

calculated from the semi-qPCR by ImageJ. Data shown as the mean±SD, n=3.

Student’s test, *P<0.01, **P<0.01; n.s. means not significant.

Fig. S8: GRP7 functions downstream of RALF1/FER to modulate RNA splicing

(Photo Credit: Long Wang, Hunan university). a-b, Heatmap of differential

splicing changes in RALF1-treated and fer-4 (A) or grp7-1 8i (B) compared with

mock. Sig. indicates P<0.05 for either of mutant and an exon inclusion level

difference >0.05. C-D, Density figure showing a comparison between

RALF1-triggered splicing changes and upon FER (C) or GRP7 knockout (D) (n=3).

Spearman correlations were used in the correlation analysis. E, flg22-triggered ROS

burst levels in different genotypes without or with 1 μM RALF1 treatment (n=2).

Values are means±SD. At least three biological replicates were performed. One-way

ANOVA with Tukey’s test, **P<0.01. F, Phenotypic analysis of different genotypes

on 1/2 MS medium with or without ABA. The pictures were taken 5 days after

germination. G, Statistical analysis of seedling greening with or without ABA

treatment in different genotypes (n=3). Different lines are indicated as “-number”. All

assessments were independently repeated three times with similar results. Data are

shown as the mean±SD. One-way ANOVA with Tukey’s test, **P<0.01.

Fig. S9: FER-induced GRP7 phosphorylation relays RALF1 signaling to the

nucleus. A, Immunofluorescence labeling assay. The GRP7 and DAPI signals are

shown. Preimmune serum (“Preim”) was used as a negative control. The images are

representative of three independent experiments. B, Statistical analysis of the nuclear

localization ratio of cells treated or not treated with 1 μM RALF1 for 3 h in Col-0 and

fer-4 roots. Values are mean±SD (n=10). One-way ANOVA, **P<0.01. C, WB

analysis of GRP7. Proteins were extracted from the cytoplasm- and nucleus-enriched

fractions of ten-day-old Col-0 and fer-4 plants treated with RALF1 for 3 h or

mock-treated. GAPC is shown as a loading control for the cytoplasmic fractions, and

H3 shows loading for the nuclear fractions. The experiments were repeated three

times independently. D, Subcellular localization of the GFP signal in root cells of

different genotypes with or without 1 μM RALF1 treatment for 3 h (top); partially

enlarged picture (bottom; red-framed area from top). The depicted imaging

experiments were repeated at least three times. In addition to the RALF1-induced

GRP7 nucleus accumulation, RALF1 also induced GFP speckles via a still unknown

mechanism. E, Statistical analysis of the nuclear localization ratios of cells treated or

not treated with 1 μM RALF1 for 3 h. Data of 10 individual roots are shown as the

mean±SD. One-way ANOVA, **P<0.01. F, Subcellular localization of the GFP signal

in root cells of GRP7-GFP/grp7-1 8i-3 and GRP7mut6D-GFP/grp7-1 8i-3 plants,

bar=10 μm. G, Statistical analysis of the nuclear localization ratios of cells in

GRP7-GFP/grp7-1 8i-3 and GRP7mut6D-GFP/grp7-1 8i-3, One-way ANOVA,

**P<0.01.

Fig. S10: GRP7 directly binds ABA-related genes transcripts. A, Gene Ontology

(GO) enrichment of GRP7 binding genes identified by both iCLIP and RIP-seq at

LL36. The black dotted line indicates P=0.05. B, Schematic diagram of the positions

of putative GRP7-binding motifs in the genomic sequence of PUB9, ABF1 and

LRK10L1.1. Red arrowheads indicate the positions of the PCR primers for RIP-qPCR

in Fig. 4G, and arrowheads indicate the direction of gene transcription. C-E, GRP7

bound to PUB9 sense (C), ABF1 sense (D) and LRK10L1.1 sense RNA probes (E) in

RNA-EMSA. Competitors A were the unlabeled RNA fragments. Competitors B were

nonspecific unlabeled RNA fragments. Probes used for the EMSA were also amplified

with the primers F and R in A. Similar data were obtained from three independent

experiments.

Fig. S11: Genetic reversal of ABF1 mRNA splicing rescues ABA response defects

of grp7-1 8i (Photo Credit: Long Wang, Hunan university). A, Schematic diagram

outlining the organization of the ABF1.1 and ABF1.2 variants. Blue boxes indicate

exons, bZIP indicates basic leucine zipper domain, and C4 indicates C4 domain. B,

Semi-qPCR analysis of the ABF1 transcript isoform levels in 7-day-old seedlings. The

primers used for the PCR are listed in table S1. Three independent experiments were

performed with similar results. A bar chart represents the relative amounts of the

isoforms calculated from the semi-qPCR with ImageJ. C, Phenotypic analysis of WT

(Col-0), ABF1.1-OE-1, ABF1.2-OE-2 plants grown on 1/2 MS medium with or

without 0.4 μM ABA. The experiment was repeated three times. D, Seedling greening

ratio in C. The SD of three independent experiments is shown using error bars.

One-way ANOVA; n.s. means not significant, **P<0.01. E, Seed germination and the

development of different genotypes on 1/2 MS medium with or without 0.2 μM ABA.

F, Seedling greening ratio in E (n=3). Data are presented as the means ± SD. One-way

ANOVA with Tukey’s test, **P<0.01. G, Root architectures of different seedling

genotypes with or without 10 μM ABA. Three independent experiments were

conducted, each with three replicates. H, Bar graph of root lengths with or without

ABA treatment in G. n=15 roots per experimental group, n=15 roots per control group.

The bar represents means±SD, and three independent experiments were performed

with similar results. One-way ANOVA, **P<0.01. Different lines are indicated as

“-number”.

Fig. S12: GRP7 is subject to NMD control; working model of

RALF1-FER-GRP7 pathway in pre-mRNA splicing. A, GRP7 protein level. The

GRP7/β-actin ratio is displayed below the gel. The experiments were performed at

least three times with similar results. B, Total RNA levels of GRP7. Seven-day-old

Col-0 seedlings were treated with 100 μM ABA for 1, 2, or 3 h, starting at ZT 12. The

error bars represent the SD of three biological replicates. One-way ANOVA with

Tukey’s test, **P<0.01; n.s. means not significant. C, ABA-induced AS of GRP7.

Semi-qPCR analysis of GRP7 transcripts in the presence or absence of 100 μM ABA.

The as_GRP7/PP2A ratio is displayed below the gel. The band ratio was measured

with ImageJ. D, Semi-qPCR analysis of GRP7 transcripts on different genotype

background. The as_GRP7/PP2A ratio is displayed below the gel. Three independent

experiments were conducted with similar results in panels A-D. E, Working model.

Upon RALF1 stimulation, FER phosphorylates GRP7. In parallel, the phosphorylated

GRP7 translocates into the nucleus and displays increased RNA-binding activity to its

target mRNAs. GRP7 thus fine-tunes the splice site selection during pre-mRNA

splicing, further affecting the ABA and RALF1 responses together with other factors.

In turn, AS-induced NMD of the GRP7 mRNA feeds back on the RALF1-FER-GRP7

pathway.