Supplementary Information for Partial maintenance of organ-specific epigenetic marks during plant asexual reproduction leads to heritable phenotypic variation Anjar Wibowo, Claude Becker, Julius Durr, Jonathan Price, Stijn Spaepen, Sally Hilton, Hadi Putra, Ranjith Papareddy, Quentin Saintain, Sarah Harvey, Gary D. Bending, Paul Schulze-Lefert, Detlef Weigel and Jose Gutierrez-Marcos Detlef Weigel Email:
[email protected] This PDF file includes: Supplementary Material and Methods Figures S1 to S15 Tables S1 to S4 Description of Datasets S1 to S4 References for SI reference citations Other supplementary materials for this manuscript include the following: Datasets S1 to S4
www.pnas.org/cgi/doi/10.1073/pnas.1805371115
S1
Supplementary Material and Methods Oligonucleotide primers Given in Table S1. Plant material and growth For the targeted hypermethylation of RSM1-DMR, we synthesized a 173 bp genomic fragment (Chr2; 9,262,579...9,262,751), which was used to generate an inverted repeat transgene (1). This construct was transformed into non-transgenic Col-0 plants to generate homozygous lines that were employed for molecular and phenotypic analysis. For the overexpression of RSM1 we chemically synthesized a codon optimized gene that was inserted into the pOpON2.1 vector (2). Plants were selected for the presence of single transgene insertions to generate homozygous lines. Leaf infection assays Infections with Hyaloperonospora arabidopsidis NoksI were performed on 2-week-old seedlings by spray-inoculation with a fresh suspension of 40,000 conidiospores/ml. Five days after infection, development hyphal growth was monitored by conidiospore counting (3). As a positive control for enhanced susceptibility, we used a transgenic line over-expressing the HaRxL14 effector (4). Bacterial infection with Pseudomonas syringae pv tomato strain DC3000 was carried out by infiltrating leaves of 4week-old plants with a bacterial suspension in 10 mM MgSO4 at a density of 100 colony-forming units (cfu)/cm2 of leaf area. Four days later, bacterial growth was assayed (5). Bacterial growth in leaves was determined by collecting leaf disks that were macerated in plastic tubes. Serial dilutions were plated on standard medium and bacterial titers were determined after overnight growth. As control for success of bacterial infection we used the fls2-1 mutant (6). RNA expression analysis Leaves and roots were collected from 4-week-old plants and total RNA was extracted using the RNeasy Plant Mini Kit (Qiagen) according to the manufacturer’s instructions. For quantitative RT-PCR analysis, RNA was treated with TURBO DNA-free (Promega, Madison, WI). cDNA was synthesized from 1 µg of extracted RNA using the RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific). All RT-qPCR analyses were performed using a MyiQ System (BIO-RAD). Using Primer3 software (7), specific primers were designed (Table S1). PCR fragments were analyzed using a dissociation protocol to ensure that each amplicon was a single product. Amplicons were also sequenced to verify PCR specificity. The amplification efficiency was calculated from raw data using LingRegPCR software (8). All RT-qPCR experiments were performed using five biological replicates, with a final volume of 25 µl containing 5 µl of cDNA template (diluted beforehand 1:10), 0.2 µM of each primer, and 12.5 µl of 2×MESA Blue qPCR MasterMix (Eurogentec). The following thermal cycling profile was used: 95°C for 10 min, followed by 40 cycles of 95°C for 10 s, 60°C for 15 s, and 72°C for 15 s. Following cycling, the melting curve was determined in the range of 60–95°C, with a temperature increment of 0.01°C/sec. Each reaction was run in triplicate (technical replicates). Negative controls included in each run were a reaction conducted in absence of reverse transcriptase and a reaction with no template (2 µl of nuclease-free water instead of 2 µl of cDNA). No signals were observed in the negative controls. Raw Ct data were analyzed using GeneExPro software (9). Analysis of expression data was performed according to the ddCT method (10) using GADPH (At1g13440), PDF2 (At1g13320) and UBQ5 (At3g62250) as housekeeping genes for normalization (11).
S2
Transcriptome analysis For mRNA-seq analysis, total RNA (10 µg) for each sample was used to purify polyA+ mRNA that was used synthesis and amplification of cDNA. The RNA-seq libraries were prepared using TruSeq RNA Sample Preparation Kit from Illumina (San Diego, CA). RNA libraries were sequenced on an Illumina HiSeq2000 instrument (100 bp single-end). Read quality was assessed using FastQC and trimming of low quality bases at the 3’ end of reads and adapter removal was done using Trimmomatic. Reads were mapped to the TAIR10 reference genome using Tophat (parameters -i 20 -I 30,000) with on average 85.4% unique mappings (min = 82.2, max = 88.8). The read counts for these libraries are given in Dataset S1. We used the R package DESEQ2 (version 1.10.1) (14). Genes were classified as significantly differentially expressed at either an FDR 0.05).
NR LO RO
0.5 0.0 −0.5 −0.6
−0.4
−0.2 0.0 CPCoA 1 (79.4%)
0.2
PC 1 [4.6 %]
Col-0 leaf Col-0 root LO G0 leaf LO G1 leaf LO G2 leaf RO G0 leaf RO G1 leaf RO G2 leaf LO G0 root LO G1 root LO G2 root RO G0 root RO G1 root RO G2 root Col-0 x RO leaf RO x Col-0 leaf
PC 3 [3.2 %]
PC 3 [3.2 %]
PC 2 [3.4 %]
Fig. S6. Leaf colonization of non-regenerated and regenerated plants by SynCom root bacterial communities. Canonical analysis of principal coordinates (based on Bray-Curtis distances) reveals distinct SynCom bacterial communities in leaves of non-regenerated and regenerated plants (n=12).
PC 1 [4.6 %]
PC 2 [3.4 %]
Fig. S7. Background variation of DNA methylation Principal component (PC) analysis of DNA methylation at methylated positions that were not identified as differentially methylated in pairwise comparisons. Numbers in brackets indicate the fraction of overall variance explained by the respective PC.
S8
0.7
MRs DMRs
Relative frequency
0.6 0.5 0.4 0.3 0.2
0.0
2 kb upstr. 5'UTR CDS Intron 3'UTR 2 kb downstr. as−lncRNA lncRNA miRNA pri−miRNA ncRNA snoRNA tRNA pseudogene TE gene TE Intergenic
0.1
Fig. S8. Annotation of methylated and differentially methylated regions. Each basepair contained in an MR or a DMR was annotated to features from the Araport11 annotation. No hierarchy of features was applied, a position could be assigned to several categories when features coincided at that position, leading to a sum of all fractions >1. The “intergenic” category was defined as the genome space that did not overlap with any element of any of the other categories.
B
root 2
−15 0 15 Methylation rate difference (%)
root 1
Seedlings: shoot vs. root
shoot 2
root 2
root 1
leaf 2
leaf 1
−15 0 15 Methylation rate difference (%)
Adult plants: leaf vs. root
shoot 1
A
Fig. S9. Methylation differences between roots and shoots. (A) Methylation at DMR loci between roots and leaves of adult plants. Methylation is expressed as the difference to the mean methylation of each DMR across all four samples. (B) Methylation at DMR loci between roots and shoots of two-week-old seedlings; strategy as in (A).
−30
0
30
root Col-0
leaf RO G2
leaf RO x Col-0
leaf Col-0 x RO
Fig. S10. DMRs present in RO plants are meiotically stable. Gains and losses of DNA methylation (in %) in relation to leaves of dexamethasone-treated Col-0 plants. Methylation was analyzed in control root samples, RO-regenerated plants (G2), and reciprocal crosses S9
CHG only
CG only LO root LO root
LO root
rdr1
rdd
rdd
LO root LO root LO root NR root
LO root
NR root
1 RO root
RO root RO leaf RO leaf RO root RO leaf RO root RO leaf
PC2 (4.7% explained var.)
0
ddm1
1
vim123 met1 met1 cmt3
suvh6 ago3 Col-0 LO leaf LO leaf Col-0 mom1 drd1 suvr3 LO leaf Col-0 LO leaf rpa2 ago4 dcl234 bru1 sdg2 dcl3 rdr2 suvh10 fas2 ago5 ago9 hda6-6suvh5 drm3 suvh3 fcafpavim1 ago2 suvr12345 ros3ago7 idnl12 ddc NR leaf cmt3 ago6 NR leaf ago1 hda6-7 idn2sde3 dms4 hda6-6 vim2 suvh456 cmt2 nrpb2 suvh7 drm12 rdm1 dcl24 ibm1dms3 ktf1 sde5 met2 suvh8 suvr2 rdr6 dcl4 fld ref6 clsy1 suvh9fpasuvr5 sgs3 vim3 dcl2 fca dnmt2 drm12 suvr1 cmt1 fve hen1 ago8 ago10 msi2 kyp suvh1 atxr56 nrpd1 suvh2 nrpe1 dnmt2 cmt3 sdg8 idn2 idnl12
1
PC2 (9.9% explained var.)
NR root NR root
0
NR rootLO root dnmt2 cmt3 cmt3 RO root RO root RO leaf NR root RO root RO root RO leaf RO leaf RO leaf kyp hda6-6 hda6-7 hda6-6 ddm1
cmt2 suvh6ago7Col-0 vim1 ago2ago1 suvr3 suvh5 ros3 sdg2 rpa2 suvh10 sde3 ago9 Col-0 mom1ago3 Col-0 ago5bru1
rdr1 LO leafnrpb2 LO leaf LO leaf LO leaf NR leaf NR leaf
met2
fas2
suvh7 vim2 drm3 hen1 cmt1suvh1 suvr5 vim3 fcafpa msi2 ago10 sde5 atxr56 dcl2 ref6 sgs3 fld suvr1 ibm1 idnl12 dcl4 ago8 fcasuvh8 dcl24 suvr12345 suvh9 fpa ago6 rdr6 dcl3 suvh3
suvh456 met1 cmt3 ddc
idn2 ago4 sdg8
1 rdr2
dcl234 clsy1
suvr2
fve ktf1 idn2 idnl12vim123 suvh2 dnmt2drm12 rdm1 drm12drd1 dms3 x WTdms4 nrpd1 met1met1 het met1
2
2
nrpe1
2
1
0
1
PC1 (42.6% explained var.) 3
Groups
met1het met1 x WT
3
2
1 0 PC1 (57.9% explained var.)
1
AGO
mCG
NR leaf
mCHG
NR root
mCHG mCHH
Demethylation
mCHH
H3K27me
RdDM medium
H3K36me
RdDM strong
H3K4me
RdDM weak
H3K9me
RNAi
HxAc
RO leaf (G2)
LO leaf (G2)
RO root (G2)
LO root (G2) mC
Unknown Col-0 (Stroud et al.)
(F1) of RO to Col-0; at coordinates of DMRs identified between leaf tissue of RO and LO plants. Fig. S11. Methylation of DMR coordinates in methylation-deficient mutants. PCA using methylation levels at LO vs. RO DMR loci in 80 methylation-deficient mutants, using data from (30). Color code is based on descriptors from (30) and indicates association with DNA methylation pathways in A. thaliana.
S10
6
*
*
log10[Distance (bp)]
4 DMR
2
MR
0
TE
24 nt-siRNA
Fig. S12. Distance of DMRs to the nearest transposable element (TE) or 24 nt-siRNA locus (Rajagopalan et al., 2007) (FDR
