www.sciencemag.org/content/348/6230/120/suppl/DC1

Supplementary Materials for

Suppression of endogenous gene silencing by bidirectional cytoplasmic

RNA decay in Arabidopsis

Xinyan Zhang, Ying Zhu, Xiaodan Liu, Xinyu Hong, Yang Xu, Ping Zhu, Yang Shen,

Huihui Wu, Yusi Ji, Xing Wen, Chen Zhang, Qiong Zhao, Yichuan Wang, Jian Lu,

Hongwei Guo*

*Corresponding author. E-mail: hongweig@pku.edu.cn

Published 3 April 2015, Science 348, 120 (2015)

DOI: 10.1126/science.aaa2618

This PDF file includes:

Materials and Methods

Figs. S1 to S19

Captions for Tables S1 to S4

Full Reference List

Other Supplementary Material for this manuscript includes the following:

(available at www.sciencemag.org/content/348/6230/120/suppl/DC1)

Tables S1 to S4 (Excel file)

2

Materials and Methods

Plant Materials and Growth Conditions

Commercially-available Murashige and Skoog (MS) medium and nitrogen-depleted

MS salt (PhytoTechnology Laboratories; Catalog: M524, M531) were used in preparing

the full-nutrition MS medium and nitrogen-depleted medium (pH 5.7-5.8, 1% sucrose, 10

g/L agar), respectively. Seeds were surface-sterilized and plated on the medium. Seeds

pretreated with stratification for 2-4 days at 4°C were kept in the greenhouse for another

5-7 days (22°C, 16 h/8 h photoperiod) before transferring the seedlings to the soil or

phenotyping. Ethylene response assays and phenotypic analysis were performed as

described previously (26, 27).

Genetic Screen and Map-Based Cloning

Map-based cloning for each mutant was done with publicly available markers (28).

EMS-mutagenized M2 seeds in EIN3ox background were pooled in 50 families and 3000

M2 seeds in each family were screened on MS medium supplemented with 10 μM ACC.

Large-cotyledon seedlings were selected as suppressor mutants and phenotypically

confirmed in the next generation. The suppressor mutants with stable large-cotyledon

phenotypes were designated according to the original family number; for example, s28

was a suppressor mutant from the pool 28.

To identify the causal genes associated with the EIN3ox suppressor phenotypes in

s28, s37 and s40 mutants, map-based cloning strategy was used. Genomic DNA was

extracted using the simplified CTAB method described previously (29). Rough mapping

was performed using about 50 large-cotyledon seedlings selected from the F2 mapping

populations that phenocopied each mutant on Murashige and Skoog medium

supplemented with ACC. The s28 mutation was mapped to an interval around T6H20 on

chromosome 3 (17123 kb, 2 recombinants/758; 17294 kb, 0 recombinants/758; 17312 kb,

2 recombinants/758). We sequenced the coding sequences of 22 genes (At3g46960-

At3g47160) and found one point mutation in At3g46960 as described in the main text.

3

The s37 mutant was mapped ~0.5 cM on the upper side of a marker (28837 kb) on

chromosome 1. However, no recombinants were found in 468 individual plants within the

large interval further on the upper side (27944-28724 kb), because the T-DNA insertion

of EIN3ox resided at 27885 kb on chromosome 1, and we found the large-cotyledon

phenotype manifested by s37 and s40 was dependent on the existence of two copies of

the EIN3 transgene. We sequenced the candidate AtSKI3 gene (AT1G76630) in both s37

and s40, and found two distinct nonsense mutations described in the main text (Fig. 1D).

Genetic Analysis and Genotyping

Mutants and transgenic materials used in this study were either maintained in our

laboratory or purchased from ABRC. The embryo defects were analyzed by examining

both the ratio of aborted seeds within heterozygous siliques and the segregation ratios in

the filial generation of the heterozygous individuals (30). The T-DNA insertional Salkline

mutants ski2-2 (Salk_129982) and ski2-3 (Salk_063541) were obtained from ABRC and

verified by PCR amplification (31). The EIN3ox transgene were genotyped by PCR, and

the point mutations such as rdr6-11 and ago1-45 were genotyped as described previously

(19, 32).

Homozygous double and triple mutants were generated by genetic crosses and were

identified from the F2 or F3 population. Each mutation was confirmed by PCR-based

genotyping and phenotypic analysis or by using antibiotic-resistant markers. To generate

the ein5-1 ski2-2 dcl4-2 dcl2-1 and ein5-1 ski2-3 dcl4-2 dcl2-1 quadruple mutants, we

genotyped the F2 and F3 plants propagated from the ein5-1 ski2-2 (ski2-3) hemizygote

and dcl4-2 dcl2-1 cross. In the filial generation of ein5-1 (+/-) ski2-2 (+/-) dcl4-2 (+/-)

dcl2-1 (+/-), no ein5-1 ski2-2 double mutant, ein5-1 ski2-2 dcl2-1 or ein5-1 ski2-2 dcl4-2

triple mutants were verified, while the combinations of other genotypes were viable.

Similarly, no ein5-1 ski2-3 dcl4-2 plant was verified from the segregating population

derived from the ein5-1 ski2-3 hemizygote and dcl4-2 dcl2-1 cross. In these experiments,

the ski2-2, ski2-3, dcl4-2, dcl2-1 loci were genotyped by PCR, and the ein5-1 mutation (1

bp deletion, frame shift) was identified by ethylene-related phenotyping (27) and

confirmed by Sanger sequencing.

4

Gene Expression Analysis by RT-PCR

Total RNA extracted with the TRIzol reagent (Invitrogen) was used for the reverse

transcription reaction (M-MLV reverse transcription system, Promega) after Dnase I

(Promega) digestion. Real-time PCR was performed using SYBR GreenMix (Takara)

with the UBQ10 gene as a reference on the Roche realtime PCR platform

(LightCycler®480). The gene-specific primers used in real-time PCR were designed

using the QuantPrime tool (33) and listed in Supplement Table S4 online.

Transformation Vectors and Construction of Transgenic Plants

To generate the 35S:GFP-AtSKI2 transgenic lines, the full-length AtSKI2 coding

region was amplified and constructed into the pEGAD binary vector in-frame with an

upstream GFP. The primers for making these constructs are listed in Supplement Table

S4 online. The floral-dipping transformation method was performed using Agrobacterium

tumefaciens strain GV3101. Transgenic plants were selected on MS medium

supplemented with 20 μg/L glufosinate and then transferred to soil. Homozygous lines

were used in this study.

Microscopy

GFP fluorescence was acquired using a confocal laser microscope (LSM 710; Carl

Zeiss) at an excitation wavelength of 488 nm. Live seedlings were stained for 15 min in

DAPI solution (1 μg/mL in water) before mounting and inspection.

In Vitro Pull-Down Assay

GST pull-down assays were performed following the procedure described

previously (34). The coding sequences corresponding to the 344 amino-acids of the SKI2

amino terminus (SKI2N) and 240 amino-acids of the SKI3 carboxyl terminus (SKI3C)

were constructed in frame into the pGEX-6p-1 (GE Healthcare) and pCold-TF (Takara

Bio) vectors, respectively. The GST-tagged SKI2N and His-tagged SKI3C proteins were

expressed in the E.coli BL21 (DE3) strain with isopropylbeta-D-thiogalactopyranoside-

induction at 0.1 mM and 0.5 mM, respectively, and purified using glutathione Sepharose

5

4B (GE Healthcare) and Ni-NTA Agarose (Qiagen) following the manufacturer’s

instructions.

Immunoprecipitation Assay and Mass Spectrometry

Tissues (6-day-old light-grown seedlings of homozygous 35S:GFP-SKI2 transgenic

plants) were ground into a fine powder in liquid nitrogen. Two grams of powder was

homogenized in 4 mL lysis buffer [10 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.5 mM

EDTA, 0.5% NP-40, 1× complete protease inhibitor cocktail (Roche)] and centrifuged at

12,000 g for 10 min after 30 min incubation at 4°C. GFP-Trap_A (50 μL; Chromotek)

beads were added into the supernatant. The following steps of immunoprecipitation were

carried out according to the manufacturer’s instructions. Precipitates were eluted by

adding 50 μL 4×LDS sampling buffer (Invitrogen) with 50 mM DTT (Invitrogen) and

heated at 65°C for 10 min followed by SDS-PAGE separation. Silver staining and mass

spectrometry analysis were performed as described previously (35, 36). Silver-stained gel

pieces were excised and then digested following the procedure described previously (37).

Peptides were further extracted from the trypsin-digested gel pieces and separated with

the EASYnLCIITM

integrated nano-HPLC system (Proxeon, Denmark) and the eluate was

used in the Thermo LTQ-Orbitrap velos mass spectrometer as described previously (36).

MS/MS analysis was performed with a spray voltage of 2.8 kV. The range of the MS

scan was 350-20000 m/z.

RNA-seq and Small RNA-seq

Both total RNA (for mRNA-seq) and small RNA were extracted from the shoot

tissues of 16-day-old plants. For mRNA-seq, total RNA was extracted using the RNeasy

Plant Mini Kit (QIAGEN). Enriched mRNA using the Epicentre Ribo-Zero™ rRNA

Removal Kits (Plant Leaf) from 10 μg total RNA was sheared before random-primed

cDNA synthesis and amplification using the TruSeq mRNA preparation kit (Illumina)

following the standard protocol. The size-selected 100-bp single-ended libraries were

sequenced with Illumina HiSeq 2000. Deep sequencing data were generated on the

Illumina Hiseq 2000 platform and filtered reads were aligned to the Col-0 transcriptome

(main gene models, TAIR 9.0 release) using the BWA program (38) with a seed length of

6

32 and up to 4% mismatches and 1 gap allowed. The clustering was run with the log2

ratios of 15663 genes using Cluster 3.0 software (39) and visualized with the Java

Treeview program (40). The small RNAs (< 200 nt) were isolated using the miRNeasy

Mini Kit (Qiagen) and then used in constructing the sequence library. The small RNA seq

was performed with Illumina HiSeq 2500. The 21-22 nt clean reads after adaptor-removal

and filtering were mapped to the genome (Tair 10.0) using the Bowtie program (41).

Reads mapped to each gene model were counted, then followed by normalization and

differential expression analysis as described previously (IHGSC, 2004). Manual

correction was done for the reads mapped to miRNA target genes over which some

miRNA reads piled up at the miRNA target sites. Visualization of the mapped reads was

done using the Integrated Genome Browser, version 6.7.2 (42). Geneset enrichment

analysis was performed using an online webserver PlantGSEA (43) and the KOBAS 2.0

program (44). The primers for validation of the RNA seq results are listed in Supplement

Table S4 online.

Small RNA Northern Blotting

Small RNA was extracted through TRIzol-isolation method followed by a PEG-NaCl

enrichment [0.5 M NaCl, 10% polyethylene glycol 8000 (w/v)] and precipitated by

isopropanol. For each sample, 10 μg small RNA was separated in a denaturing 17%

polyacrylamide gel and then transferred to a neutral nylon membrane (Hybond-NX, GE

Healthcare) and immobilized by EDC fixation method as previously described (45). We

used the PerfectHyb™ plus Hybridization Buffer (Sigma) for small RNA hybridization.

End-labled DNA oligo probes were used in the detection of U6, and in vitro transcribed

RNA probes were used in the detection of siRNAs except for the EIN3-siRNAs with

DNA probes from random primed labeling.

7

Fig. S1

Fig. S1. Overexpression of EIN3 induced siRNA-mediated gene silencing in the ein5

mutant.(A) The cotyledon phenotype of 6-day-old seedlings grown on MS medium

supplemented with or without 10 μM ACC, an ethylene biosynthetic precursor. s53 was

generated in the EIN3ox background. Bar, 1 mm. (B) Identification of the causal gene of

s53 by map-based cloning. The amino acid mutation is indicated. (C) The cotyledon

phenotype of 6-day-old light-grown seedlings. Bar, 1 mm. (D) Relative expression levels

of EIN3 in 6-day-old seedlings. (E) Northern blot detection of the EIN3-derived siRNAs.

8

Fig. S2

Fig. S2. Identification of the SKI complex in Arabidopsis by map-based cloning. (A)

Positional cloning of AtSKI2 and AtSKI3. Mutations of s28 (ski2-1), two T-DNA

insertional mutants (ski2-2 and ski2-3), and s37, s40 (ski3) are indicated. The ski2-1

mutation resulted in a T389I amino-acid substitution in the well-defined motif-I in the

AtSKI2 protein, which is essential for its helicase activity. The two ski3 mutants resulted

in a non-sense mutation at Q20 and W158, respectively. (B) Schematic diagram of the

SKI complex (SKI2/3/8), SKI7 protein and the exosome engaged in 3’-5’ RNA decay in

the cytoplasm.

9

Fig. S3

Fig. S3.Transgenic expression of GFP-AtSKI2 complements the mutant phenotype of

s28. (A) The cotyledon phenotype of 6-day-old seedlings. Bar, 1 mm. Three independent

transgenic lines were shown. (B) Relative expression levels of EIN3 in 6-day-old

seedlings.

10

Fig. S4

Fig. S4. Verification of two T-DNA insertional ski2 mutants. (A) PCR-verification of the

T-DNA insertions in ski2-2 and ski2-3. The ACT2 gene was used as an internal control.

LB was a T-DNA specific primer while LP and RP were designed according to the

genomic sequences. LB+RP was used to detect the T-DNA insertions while LP+RP was

used to amplify the genomic DNA fragments that harbor the T-DNA insertions. (B)

Relative expression levels of AtSKI2 in 16-day-old plants indicating that ski2-2 failed to

express AtSKI2 RNA while ski2-3 had reduced expression, so ski2-3 seemed a weak

allele while ski2-2 could be a null allele.

11

Fig. S5

Fig. S5. The insertional ski2 mutants show normal ethylene response. (A) The phenotype

of 3-day-old etiolated seedlings treated with or without 10 μM ACC. Bar, 2 mm. (B-C)

Quantification of the hypocotyl (B) and root (C) lengths of 3-day-old etiolated seedlings.

Mean ±SD, n > 20.

12

Fig. S6

Fig. S6. Immunoprecipitation-mass spectrometry detection of proteins associated with

GFP-AtSKI2. (A)Silver staining of the proteins that immunoprecipitated with GFP

antibody. Key proteins in the SKI complex or associated with GFP-AtSKI2 protein

detected by mass spectrometry are indicated. (B) Peptide counts of the SKI complex

proteins detected by mass spectrometry.

13

Fig. S7

Fig. S7. Overexpression of APT1 in the ski2 mutant phenocopies the apt1 loss-of-

function mutant. The phenotype of five-day-old light-grown seedlings treated with or

without 200 μM kinetin (KT, an active form of plant hormone cytokinin), which caused

marked growth and greening suppression. It is noted that overexpression of APT1 in

wild-type background (Col-0) results in a hypersensitive response to toxic dose of KT

manifested by retarded germination. In contrast, overexpression of APT1 in ski2-2

background results in KT hyposensitivity manifested by enhanced growth and green

cotyledons, reminiscent of apt1 mutant. Bar, 2 mm.

14

Fig. S8

Fig. S8. Rescue of the embryo-lethality of ein5-1 ski2-2 by the rdr6 mutation. (A) The

phenotypes of immature siliques. Colorless seeds segregated in the siliques of ein5-1-/-

ski2-2+/- hemizygous parents are indicated by arrows. About one quarter (80/354, 2 =

0.666, p > 0.41) of seeds aborted in the ein5-1-/- ski2-2+/- siliques, and the filial

generation conformed to a 1:2 ratio between wild type and ski2-2 heterozygous plants

(25:45; 2 = 0.179, p > 0.67), indicating a recessive lethal segregation of the embryos

homozygous ein5-1 ski2-2 (see supplemental Methods). The ein5-1 ski2-2 rdr6-11 line

was fertile and was maintained homozygously. Bar, 0.5 mm. (B) Rosette morphology of

18-day-old plants of indicated genotypes. Bar, 1 cm.

15

Fig. S9

Fig. S9. Restoration of the disturbed transcriptome of ein5-1 ski2-3 by the rdr6 mutation.

(A) Venn diagram of differentially-expressed genes (2-fold cutoff, RPKM>3) among

indicated comparisons identifies 596 genes that are defined as EIN5/AtSKI2-coregulated

genes. C: Col-0; e: ein5-1, s: ski2-3; es: ein5-1 ski2-3; res: rdr6-11 ein5-1 ski2-3. (B)

Comparisons of the expression levels of EIN5/AtSKI2-coregulated genes (111 up-

regulated and 485 down-regulated in ein5-1 ski2-3 ) between ein5-1 ski2-3 (es) and rdr6-

11 ein5-1 ski2-3 (res) (2-fold cutoff).

16

Fig. S10

Fig. S10. The developmental defects of ein5 ski2 are rescued by the PTGS mutations.

Rosette morphology of adult plants of indicated genotypes. Plants were photographed at

different times (16-day-old in panel A-B and 19-day-old in C). The ein5-1 ski2-2 double

mutants were lethal. The ein5-1 ski2-2 ago1-45 triple mutants were viable but showed

various degree of growth retardation. Bar, 1 cm.

17

Fig. S11

Fig. S11. Restoration of select gene expression in ein5-1 ski2-3 by ago1-45.(A) Relative

expression levels of anthocyanin biosynthesis genes in 16-day-old plants.(B) Relative

expression levels of ARF6/8 and HD-ZIPIII family genes in 16-day-old plants.(C)

Relative expression levels of NIA1/2 and nitrogen-responsive genes in 16-day-old plants.

18

Fig. S12

Fig. S12. Statistics of small RNA profiling. (A) Statistics of the small RNA sequencing

data and detected ct-siRNA reads. (B-C) tasiRNA reads detected in small RNA

sequencing (B) and normalized tasiRNA abundance in each genotype (C).

19

Fig. S13

Fig. S13. Relative expression levels of TAS4 in 16-day-old plants.

20

Fig. S14

Fig. S14. Northern blot detection of small RNAs derived from ARF6 and PHB in 16-day-

old plants.

21

Fig. S15

Fig. S15. Distribution of ct-siRNA biogenesis along the transcripts that are cleaved by

miRNA. (A) and (B) 21-22 nt siRNA reads mapped over the 5’ and 3’ miRNA cleavage

fragments of 39 miRNA target genes. RPM: reads per million (21-22 nt) reads mapped to

all genes.

22

Fig. S16

Fig. S16. The ct-siRNA loci are significantly enriched in the genes that are either up- or

down-regulated in the ein5-1 ski2-3 mutant. The expression levels of the genes were

normalized and the up- and down-regulated genes were defined with a two-fold cutoff.

RDR6-dependent differentially expressed gene sets (up- and down-regulated genes) were

obtained by comparing the ein5-1 ski2-3 transcriptome with that of rdr6-11 ein5-1 ski2-3.

χ2 test was used to calculate the significance of enrichment.

23

Fig. S17

Fig. S17. The top-scoring 20 of 441 ct-siRNA-generating genes ranked by the fold-

change of ct-siRNA abundance between Col-0 (C) and ein5-1 ski2-3 (es).

24

Fig. S18

Fig. S18. The 441 ct-siRNA loci have significantly higher expression levels than the rest

of the genome in ein5-1 ski2-3. 23700 transcripts that have expression level of RPKM >

0.5 were considered. The difference is highly significant (p < 10-16

, Kolmogorov-Smirnov

test).

25

Fig. S19

Fig. S19. A proposed model depicting that bidirectional cytoplasmic RNA decay

suppresses endogenous PTGS. The cytoplasmic aberrant mRNAs are efficiently

processed by 5’-3’ (EIN5) and 3’-5’ (SKI-Exosome) RNA decay machineries. Upon

RNA decay deficiency, aberrant mRNAs are channeled into the SGS3/RDR6-dependent

double-strand synthesis and the production of 21-22 nt ct-siRNAs. The DCL4-mediated

21 nt ct-siRNA pathway could serve as an decoy to compete with the more destructive

DCL2-mediated 22 nt ct-siRNA pathway that is capable of triggering amplified PTGS.

26

Table S1 to S4

These tables are provided as a separate Excel document.

Table S1. Geneset enrichment analysis with the gene set whose expression was co-

regulated by EIN5 and SKI2.

Table S2. Quantitation of 21-22 nt smRNA reads arising from the 441 genes from which

EIN5- and SKI2-affected smRNAs are produced.Three biological repeats for

each genotypes: c1-c3, Col-0; es1-es3, ein5-1 ski2-3; r1-r3, rdr6-11; res1-res3,

rdr6-11 ein5-1 ski2-3.

Table S3. A list of 39 ct-siRNA-generating miRNA target genes.

Table S4. Primers used in this research.

References and Notes

1. C. Cogoni, G. Macino, Post-transcriptional gene silencing across kingdoms. Curr. Opin.

Genet. Dev. 10, 638–643 (2000). Medline doi:10.1016/S0959-437X(00)00134-9

2. R. C. Wilson, J. A. Doudna, Molecular mechanisms of RNA interference. Annu. Rev. Biophys.

42, 217–239 (2013). Medline doi:10.1146/annurev-biophys-083012-130404

3. M. Ghildiyal, P. D. Zamore, Small silencing RNAs: An expanding universe. Nat. Rev. Genet.

10, 94–108 (2009). Medline doi:10.1038/nrg2504

4. M. Incarbone, P. Dunoyer, RNA silencing and its suppression: Novel insights from in planta

analyses. Trends Plant Sci. 18, 382–392 (2013). Medline

doi:10.1016/j.tplants.2013.04.001

5. N. Pumplin, O. Voinnet, RNA silencing suppression by plant pathogens: Defence, counter-

defence and counter-counter-defence. Nat. Rev. Microbiol. 11, 745–760 (2013). Medline

doi:10.1038/nrmicro3120

6. A. B. Moreno, A. E. Martínez de Alba, F. Bardou, M. D. Crespi, H. Vaucheret, A. Maizel, A.

C. Mallory, Cytoplasmic and nuclear quality control and turnover of single-stranded

RNA modulate post-transcriptional gene silencing in plants. Nucleic Acids Res. 41, 4699–

4708 (2013). Medline doi:10.1093/nar/gkt152

7. S. Gazzani, T. Lawrenson, C. Woodward, D. Headon, R. Sablowski, A link between mRNA

turnover and RNA interference in Arabidopsis. Science 306, 1046–1048 (2004). Medline

doi:10.1126/science.1101092

8. Q. Chao, M. Rothenberg, R. Solano, G. Roman, W. Terzaghi, J. R. Ecker, Activation of the

ethylene gas response pathway in Arabidopsis by the nuclear protein ETHYLENE-

INSENSITIVE3 and related proteins. Cell 89, 1133–1144 (1997). Medline

doi:10.1016/S0092-8674(00)80300-1

9. H. Guo, J. R. Ecker, Plant responses to ethylene gas are mediated by SCF(EBF1/EBF2)-

dependent proteolysis of EIN3 transcription factor. Cell 115, 667–677 (2003). Medline

doi:10.1016/S0092-8674(03)00969-3

10. G. Olmedo, H. Guo, B. D. Gregory, S. D. Nourizadeh, L. Aguilar-Henonin, H. Li, F. An, P.

Guzman, J. R. Ecker, ETHYLENE-INSENSITIVE5 encodes a 5′→3′ exoribonuclease

required for regulation of the EIN3-targeting F-box proteins EBF1/2. Proc. Natl. Acad.

Sci. U.S.A. 103, 13286–13293 (2006). Medline doi:10.1073/pnas.0605528103

11. R. Solano, A. Stepanova, Q. Chao, J. R. Ecker, Nuclear events in ethylene signaling: A

transcriptional cascade mediated by ETHYLENE-INSENSITIVE3 and ETHYLENE-

RESPONSE-FACTOR1. Genes Dev. 12, 3703–3714 (1998). Medline

doi:10.1101/gad.12.23.3703

12. E. Dorcey, A. Rodriguez-Villalon, P. Salinas, L. Santuari, S. Pradervand, K. Harshman, C. S.

Hardtke, Context-dependent dual role of SKI8 homologs in mRNA synthesis and

turnover. PLOS Genet. 8, e1002652 (2012). Medline doi:10.1371/journal.pgen.1002652

13. J. T. Brown, X. Bai, A. W. Johnson, The yeast antiviral proteins Ski2p, Ski3p, and Ski8p

exist as a complex in vivo. RNA 6, 449–457 (2000). Medline

doi:10.1017/S1355838200991787

14. X. Zhang, Y. Chen, X. Lin, X. Hong, Y. Zhu, W. Li, W. He, F. An, H. Guo, Adenine

phosphoribosyl transferase 1 is a key enzyme catalyzing cytokinin conversion from

nucleobases to nucleotides in Arabidopsis. Mol. Plant 6, 1661–1672 (2013). Medline

doi:10.1093/mp/sst071

15. T. Dalmay, A. Hamilton, S. Rudd, S. Angell, D. C. Baulcombe, An RNA-dependent RNA

polymerase gene in Arabidopsis is required for posttranscriptional gene silencing

mediated by a transgene but not by a virus. Cell 101, 543–553 (2000). Medline

doi:10.1016/S0092-8674(00)80864-8

16. E. Grotewold, The genetics and biochemistry of floral pigments. Annu. Rev. Plant Biol. 57,

761–780 (2006). Medline doi:10.1146/annurev.arplant.57.032905.105248

17. N. G. Bologna, O. Voinnet, The diversity, biogenesis, and activities of endogenous silencing

small RNAs in Arabidopsis. Annu. Rev. Plant Biol. 65, 473–503 (2014). Medline

doi:10.1146/annurev-arplant-050213-035728

18. F. Vazquez, T. Hohn, Biogenesis and biological activity of secondary siRNAs in plants.

Scientifica 2013, 783253 (2013). Medline doi:10.1155/2013/783253

19. A. Peragine, M. Yoshikawa, G. Wu, H. L. Albrecht, R. S. Poethig, SGS3 and

SGS2/SDE1/RDR6 are required for juvenile development and the production of trans-

acting siRNAs in Arabidopsis. Genes Dev. 18, 2368–2379 (2004). Medline

doi:10.1101/gad.1231804

20. E. Allen, Z. Xie, A. M. Gustafson, J. C. Carrington, microRNA-directed phasing during

trans-acting siRNA biogenesis in plants. Cell 121, 207–221 (2005). Medline

doi:10.1016/j.cell.2005.04.004

21. C. F. Hwang, Y. Lin, T. D’Souza, C. L. Cheng, Sequences necessary for nitrate-dependent

transcription of Arabidopsis nitrate reductase genes. Plant Physiol. 113, 853–862 (1997).

Medline doi:10.1104/pp.113.3.853

22. G. Rubin, T. Tohge, F. Matsuda, K. Saito, W. R. Scheible, Members of the LBD family of

transcription factors repress anthocyanin synthesis and affect additional nitrogen

responses in Arabidopsis. Plant Cell 21, 3567–3584 (2009). Medline

doi:10.1105/tpc.109.067041

23. C. Diaz, V. Saliba-Colombani, O. Loudet, P. Belluomo, L. Moreau, F. Daniel-Vedele, J. F.

Morot-Gaudry, C. Masclaux-Daubresse, Leaf yellowing and anthocyanin accumulation

are two genetically independent strategies in response to nitrogen limitation in

Arabidopsis thaliana. Plant Cell Physiol. 47, 74–83 (2006). Medline

doi:10.1093/pcp/pci225

24. D. Schubert, B. Lechtenberg, A. Forsbach, M. Gils, S. Bahadur, R. Schmidt, Silencing in

Arabidopsis T-DNA transformants: The predominant role of a gene-specific RNA

sensing mechanism versus position effects. Plant Cell 16, 2561–2572 (2004). Medline

doi:10.1105/tpc.104.024547

25. C. A. Beelman, R. Parker, Degradation of mRNA in eukaryotes. Cell 81, 179–183 (1995).

Medline doi:10.1016/0092-8674(95)90326-7

26. F. An, Q. Zhao, Y. Ji, W. Li, Z. Jiang, X. Yu, C. Zhang, Y. Han, W. He, Y. Liu, S. Zhang, J.

R. Ecker, H. Guo, Ethylene-induced stabilization of ETHYLENE INSENSITIVE3 and

EIN3-LIKE1 is mediated by proteasomal degradation of EIN3 binding F-box 1 and 2 that

requires EIN2 in Arabidopsis. Plant Cell 22, 2384–2401 (2010). Medline

doi:10.1105/tpc.110.076588

27. P. Guzmán, J. R. Ecker, Exploiting the triple response of Arabidopsis to identify ethylene-

related mutants. Plant Cell 2, 513–523 (1990). Medline doi:10.1105/tpc.2.6.513

28. X. Hou, L. Li, Z. Peng, B. Wei, S. Tang, M. Ding, J. Liu, F. Zhang, Y. Zhao, H. Gu, L. J. Qu,

A platform of high-density INDEL/CAPS markers for map-based cloning in Arabidopsis.

Plant J. 63, 880–888 (2010). Medline doi:10.1111/j.1365-313X.2010.04277.x

29. J. Doebley, A. Stec, Inheritance of the morphological differences between maize and

teosinte: Comparison of results for two F2 populations. Genetics 134, 559–570 (1993).

Medline

30. D. W. Meinke, “Seed development in Arabidopsis thaliana.” in Arabidopsis, E. M.

Meyerowitz, C. R. Somerville, Eds. (Cold Spring Harbor Laboratory Press, Cold Spring

Harbor, 1994); pp. 253–295.

31. J. M. Alonso, A. N. Stepanova, T. J. Leisse, C. J. Kim, H. Chen, P. Shinn, D. K. Stevenson,

J. Zimmerman, P. Barajas, R. Cheuk, C. Gadrinab, C. Heller, A. Jeske, E. Koesema, C.

C. Meyers, H. Parker, L. Prednis, Y. Ansari, N. Choy, H. Deen, M. Geralt, N. Hazari, E.

Hom, M. Karnes, C. Mulholland, R. Ndubaku, I. Schmidt, P. Guzman, L. Aguilar-

Henonin, M. Schmid, D. Weigel, D. E. Carter, T. Marchand, E. Risseeuw, D. Brogden,

A. Zeko, W. L. Crosby, C. C. Berry, J. R. Ecker, Genome-wide insertional mutagenesis

of Arabidopsis thaliana. Science 301, 653–657 (2003). Medline

doi:10.1126/science.1086391

32. M. R. Smith, M. R. Willmann, G. Wu, T. Z. Berardini, B. Möller, D. Weijers, R. S. Poethig,

Cyclophilin 40 is required for microRNA activity in Arabidopsis. Proc. Natl. Acad. Sci.

U.S.A. 106, 5424–5429 (2009). Medline doi:10.1073/pnas.0812729106

33. S. Arvidsson, M. Kwasniewski, D. M. Riaño-Pachón, B. Mueller-Roeber, QuantPrime – a

flexible tool for reliable high-throughput primer design for quantitative PCR. BMC

Bioinformatics 9, 465 (2008). Medline doi:10.1186/1471-2105-9-465

34. C. Fankhauser, K. C. Yeh, J. C. Lagarias, H. Zhang, T. D. Elich, J. Chory, PKS1, a substrate

phosphorylated by phytochrome that modulates light signaling in Arabidopsis. Science

284, 1539–1541 (1999). Medline doi:10.1126/science.284.5419.1539

35. E. Mortz, T. N. Krogh, H. Vorum, A. Görg, Improved silver staining protocols for high

sensitivity protein identification using matrix-assisted laser desorption/ionization-time of

flight analysis. Proteomics 1, 1359–1363 (2001). Medline doi:10.1002/1615-

9861(200111)1:11<1359::AID-PROT1359>3.0.CO;2-Q

36. H. Tabara, E. Yigit, H. Siomi, C. C. Mello, The dsRNA binding protein RDE-4 interacts with

RDE-1, DCR-1, and a DExH-box helicase to direct RNAi in C. elegans. Cell 109, 861–

871 (2002). Medline doi:10.1016/S0092-8674(02)00793-6

37. A. Shevchenko, M. Wilm, O. Vorm, M. Mann, Mass spectrometric sequencing of proteins

silver-stained polyacrylamide gels. Anal. Chem. 68, 850–858 (1996). Medline

doi:10.1021/ac950914h

38. H. Li, R. Durbin, Fast and accurate short read alignment with Burrows-Wheeler transform.

Bioinformatics 25, 1754–1760 (2009). Medline doi:10.1093/bioinformatics/btp324

39. M. J. L. de Hoon, S. Imoto, J. Nolan, S. Miyano, Open source clustering software.

Bioinformatics 20, 1453–1454 (2004). Medline doi:10.1093/bioinformatics/bth078

40. A. J. Saldanha, Java Treeview—Extensible visualization of microarray data. Bioinformatics

20, 3246–3248 (2004). Medline doi:10.1093/bioinformatics/bth349

41. B. Langmead, C. Trapnell, M. Pop, S. L. Salzberg, Ultrafast and memory-efficient alignment

of short DNA sequences to the human genome. Genome Biol. 10, R25 (2009). Medline

doi:10.1186/gb-2009-10-3-r25

42. J. W. Nicol, G. A. Helt, S. G. Blanchard Jr., A. Raja, A. E. Loraine, The Integrated Genome

Browser: Free software for distribution and exploration of genome-scale datasets.

Bioinformatics 25, 2730–2731 (2009). Medline doi:10.1093/bioinformatics/btp472

43. X. Yi, Z. Du, Z. Su, PlantGSEA: A gene set enrichment analysis toolkit for plant community.

Nucleic Acids Res. 41, W98–W103 (2013). Medline doi:10.1093/nar/gkt281

44. C. Xie, X. Mao, J. Huang, Y. Ding, J. Wu, S. Dong, L. Kong, G. Gao, C. Y. Li, L. Wei,

KOBAS 2.0: A web server for annotation and identification of enriched pathways and

diseases. Nucleic Acids Res. 39 (suppl. 2), W316–W322 (2011). Medline

doi:10.1093/nar/gkr483

45. G. S. Pall, C. Codony-Servat, J. Byrne, L. Ritchie, A. Hamilton, Carbodiimide-mediated

cross-linking of RNA to nylon membranes improves the detection of siRNA, miRNA and

piRNA by northern blot. Nucleic Acids Res. 35, e60 (2007). Medline

doi:10.1093/nar/gkm112