Supplemental material

Supplementary Table 1. List of primer pairs used for qPCR analysis

Tagret genes
(GenBank accession no.) / Expected
size (bp) / Forward primer (5'-3') / Reverse primer (5'-3')
DCL1 (NM_099986) / 209 / CTTTTGGTTTGGGAGACTGC / CCCTTCTGCTTGTTGCTGGC
HYL1 (NM_100842) / 227 / GAGGCAGCATTTGGGAGTGTG / GGTTCCAGATTTGGCGTTGT
SE (NM_128268) / 337 / GATGATAGAGGTGGACGCAG / GAGACAACAGAAAAGGGGCAG
TGH (NM_001036849) / 397 / CAAGTCCAGAAGCAAACCCGAAG / GCTATACCGTGAAGAAGAAG
DDL (NM_112947) / 208 / CAGCGAAGAAAAAGGAAGAAC / CGTCCAAAGAGGTAGCAGC
DCL2 (NM_111200) / 345 / CAAACCTGCACAAACACATCC / CAACCGTGAAAGAATCGAAATC
DCL3 (NM_001161190) / 346 / CAATCATAGCGACAGAGAAAC / CTCATAAAGCAATTCCACGAC
HEN1 (NM_001203853) / 357 / GACTCTCTACTTGACTATCC / GTTGTTTTCTTCTTGGGTTTC
HASTY (NM_111376) / 250 / GGACAGATGGAGAAGCGAC / CACGGGAGCGAAAGCAAAAC
AGO1 (NM_001198240) / 304 / GCCACCCTACAGAGTTTGAC / CTTCTACCAGCCATTCCACC
AGO4 (NM_128262) / 246 / TGTATGATGAGATTGGTTTTTC / CGTTGTCTTTGAGTCTTGGG
RDR2 (NM_117183) / 122 / GAAACCAACATACATCTCTG / CAAGTGTCACATCATAAGCTAC

Supplementary Fig. 1. The subnetwork regulating miRNA biogenesis in Arabidopsis.

The subnetwork was constructed using the PAIR database and the program Cytoscape. The proteins, the genes of which were overexpressed in rolB-transformed cells, are marked by a bright orange color. Solid lines represent the interactions presented in PAIR; dashed lines represent the interactions taken from BioGRID and TAIR. The dash-dotted line connecting VIP2/NOT2b-NRPB1 presents the interaction published by Wang and co-workers (2013). This interaction connects two separate modules, the DCL1-HYL1-SE module and the AGO4-based module. The dotted line connecting HEN1 and DCL1 represents a functional interaction, though a physical interaction is not known for these proteins. RDR2 and HST are not presented here because the physical interactions of these proteins with the components of the miRNA subnetwork have not been uncovered.

DRBs, dsRNA-binding proteins; CBP20 (CAP-BINDING PROTEIN 20); CPB80/ABH1 (NUCLEAR CAP-BINDING PROTEIN 80); VIP2/NOT2b (VIRE2-interacting protein 2); NRPB1 (DNA-directed RNA polymerase II subunit RPB1); RDM1 (RNA-DIRECTED DNA METHYLATION 1); DRM2 (DOMAINS REARRANGED METHYLTRANSFERASE 2); DMS3 (DEFECTIVE IN MERISTEM SILENCING 3); DRD1 (DEFECTIVE IN RNA-DIRECTED DNA METHYLATION 1); NRPBs and NRPEs (DNA-directed RNA polymerase II subunits); NRPD2A (nuclear RNA polymerase D2A); GRP23 (glutamine-rich protein 23); KTF1 (KOW DOMAIN-CONTAINING TRANSCRIPTION FACTOR 1).

Network visualization

The network was constructed using the program Cytoscape, version 2.8.3 (Shannon et al. 2003). The data loaded into the program were obtained from the latest version of PAIR [PAIR-V3.3 (Lin et al. 2011); http://www.cls.zju.edu.cn/pair/]. The protein-protein interactions presented in PAIR were compared with the databases BioGRID (Stark et al., 2006; http://thebiogrid.org/) and TAIR (Swarbreck et al. 2008; http://www.arabidopsis.org./). The interactions added to the PAIR-based Arabidopsis Interactome from BioGRID and TAIR are presented here as dashed lines. New recently published interactions absent in any databases were also integrated in the Interactome and are shown as dashed lines.

Literature

Lin M,Zhou X,ShenX,Mao C,Chen X (2011) The predicted Arabidopsis interactome resource and network topology-based systems biology analyses. Plant Cell23:911-922

Stark C, Breitkreutz BJ, Reguly T, Boucher L, Breitkreutz A, Tyers M (2006) BioGRID: A general repository for interaction datasets. Nucleic Acids Res 34 (Database issue): D535–D539

Shannon P, Markiel A, Ozier O, Baliga NS, Wang JT, Ramage D, Amin N, Schwikowski B, Ideker T (2003) Cytoscape: A software environment for integrated models of biomolecular interaction networks. Genome Res 13:2498-2504

Swarbreck D,Wilks C,Lamesch P,Berardini TZ,Garcia-Hernandez M,Foerster H,Li D,Meyer T,Muller R,Ploetz L,Radenbaugh A, Singh S,Swing V,Tissier C,Zhang P,Huala E (2008) The Arabidopsis Information Resource (TAIR): Gene structure and function annotation. Nucleic Acids Res 36 (Database issue): D1009–D1014

Wang L,Song X,Gu L,Li X,Cao S,Chu C,Cui X,Chen X,Cao X (2013) NOT2proteinspromotepolymeraseII-dependenttranscriptionandinteractwith multiple microRNA biogenesis factors in Arabidopsis. Plant Cell25:715-727