Supporting experimental procedures
sRNA Blot Assay
The total RNAs (20 μg) were isolated using Trizol reagent (invitrogen) and
separated on a 12% polyacrylamide gel containing 8 M urea. After electrophoresis,
gel was blotted to a Hybond-NX membrane (GE Healthcare,
http://www.gehealthcare.com), and then the membrane was UV cross-linked (Pall et
al., 2007). The blotted membranes were hybridized with the radio-labeled
gene-specific RNA probes, produced by in vitro transcription using T3 RNA
polymerase (Promega, http://www.promega.com). Single-stranded DNA fragments
containing T3 promoter with the sense sequences of sRNA8105 and the
IbMYB1-siRNA were synthesized to form T3 sRNA8105 and T3 IbMYB1siRNA,
individually (Table S3), and then annealed to T3 top strand (Table S3) to form DNA
templates for RNA probe synthesis. The procedure for in vitro transcription was
described (Jeng et al., 1990; Jeng et al., 1992). Prehybridization was undertaken in 6
x SSC (0.9 M sodium chloride, 0.09 M sodium citrate), 0.5% (w/v) SDS, 5 x
Denhard’s solution (0.1% [w/v] Ficoll, 0.1% [w/v] bovine serum albumin, and 0.1%
[w/v] polyvinyl pyrrolidine) with 100 mg/ml sheared and denatured salmon sperm
DNA at 50°C for more than 1 hour. After that, the radio-labeled probe was added in
prehybridization solution. Hybridization was performed under the same conditions for
overnight. Blots were washed twice in wash buffer 1 (2 x SSC and 0.1% (w/v) SDS)
at 55°C for 15 min and once in wash buffer 2 (0.2 x SSC and 0.1% (w/v) SDS) at
55°C for 15 min. Radioactive blots were displayed on the phosphorimager (Molecular
Dynamics, http://www.moleculardynamics.com). In addition, blots were stripped and
re-hybridized with the radio-labeled 5S rRNA probe. It severed as an internal control
for sRNA bolt assays.
Agrobacterium-Mediated Transient Expression in Tobacco
Transient expression of sRNA8105, mimic8105, and its target IbMYB1 in
tobacco leaves was performed (Kim et al., 2009). The fragments of IbMYB1 and
IbMYB1Δ8105 were obtained using PCR with primer pairs
XbaI-IbMYB1/SpeI-IbMYB1 (Table S3) from a gDNA and a cDNA library of sweet
potato, individually, and were inserted in pCambia2300. The fragment of
pre-sRNA8105 was obtained by PCR with primer pairs
XbaI-sRNA8105/SpeI-sRNA8105 (Table S3) from a cDNA library of sweet potato,
and was inserted into pCambia1300. The fragment of mimic8105 was obtained by
modifying the sequence of IPS1 gene (Franco-Zorrilla et al., 2007) using PCR with
primer pairs XbaI-mimic8105/SpeI-mimic8105 (Table S3), and were inserted in
pCambia1300. Then, Agrobacteria carrying pCambia1301, pCambia1301-
pre-sRNA8105, pCambia1301-mimic8105, pCambia2300-IbMYB1, or
pCambia2300-IbMYB1Δ8105 was generated to infiltrate mature leaves of tobacco
together or individually as indicated in each reaction. After 4 days, the gDNAs and
total RNAs of these leaves were isolated using Trizol reagent (Invitrogen). Then, total
RNAs were treated with DNase I (Ambion) and then reversely transcribed to be
cDNAs for gene expression assays. Quantitative RT-PCR was used to detect the
expression of IbMYB1, pre-sRNA8105, mimic8105, NPTII, and Actin, individually, in
tobacco. For DNA methylation assays, gDNAs were amplified by quantitative
McrBC-PCR with primer sets IbMYB1 F/ IbMYB1 R4 (Table S3).
Isolation of DCL and RDR genes
The Ipomoea EST and WGS databases from NCBI were used to blast the
conserve domain of DCL and RDR genes from Arabidopsis, tobacco, and rice. We
found that the sequence of jmsf28i20 is similar to that of DCL1, the sequence of
jmsf22h20 is similar to that of DCL2, the sequence of jm35j01 is similar to that of
RDR2, and the sequence of jm3a03 is similar to that of RDR6. The clones with the
inserted PCR products using primer pairs DCL1 F/DCL1 R, DCL2 F/DCL2 R, RDR2
F/RDR2 R, or RDR6 F/RDR6 R (Table S3) were analyzed to confirm IbDCL and
IbRDR sequences of sweet potato. Two IbDCL and two IbRDR genes were obtained.
One IbDCL proteins shares 95 and 96% identity with DCL1 from Arabidopsis and
rice, respectively; and another IbDCL protein shares 46 and 51% identity with DCL2
from Arabidopsis and tobacco, individually. These two IbDCL genes were then named
IbDCL1 and IbDCL2 based on their sequence similarities. Two IbRDR genes were
also named IbRDR2 and IbRDR6 based on their sequence similarities. One IbRDR
proteins shares 57 and 55% identity with RDR2 from Arabidopsis and rice,
respectively; and another IbRDR6 protein shares 61 and 67% identity with RDR6
from Arabidopsis and tobacco, individually.
Construction of Ibdcls-RNAi and Ibrdrs-RNAi
The sense and antisense DNA fragments of IbDCL1 were amplified using PCR with
primer pairs sIbDCL1 F/ sIbDCL1 R and asIbDCL1 F/ asIbDCL1 R (Table S3),
respectively. The sense and antisense DNA fragments of IbDCL2 were also amplified
by PCR with primer pairs sIbDCL2 F/ sIbDCL2 R and asIbDCL2 F/ asIbDCL2 R
(Table S3), respectively. The sense and antisense DNA fragments of IbRDR2 were
also amplified by PCR with primer pairs sIbRDR2 F/ sIbRDR2 R and asIbRDR2 F/
asIbRDR2 R (Table S3), respectively. The sense and antisense DNA fragments of
IbRDR6 were also amplified by PCR with primer pairs sIbRDR6 F/ sIbRDR6 R and
asIbRDR6 F/ asIbRDR6 R (Table S3), respectively. Then, these fragments were
inserted into their corresponding sites of pBI22178in sequentially. Plasmid
pBI22178in modified from pBI221 (Clontech, http://www.clontech.com/) contains the
intron 2 from OSAG78 of Oncidium (Lin et al., 2011). Finally, the RNAi cassette in
pBI22178in was digested and cloned into binary vector pCAMBIA1301.
Mapping of sRNA8105-Guided Cleavage Sites
The 5’ ends of the sRNA8105 cleavage products were amplified by PCR using the
primers 5-adapter and IbMYB1-RLM (Table S3). The 3’ ends of the sRNA8105
cleavage products were amplified by PCR using the primers 3-adapter and
IbMYB1-RLM2 (Table S3). The different size PCR fragments were isolated, cloned,
and sequenced to determine the cleavage sites in the RNAs of IbMYB1.
References:
Franco-Zorrilla, J.M., Valli, A., Todesco, M., Mateos, I., Puga, M.I.,
Rubio-Somoza, I., Leyva, A., Weigel, D., Garcia, J.A. and Paz-Ares, J.
(2007) Target mimicry provides a new mechanism for regulation of microRNA
activity. Nat. Genet. 39, 1033-1037.
Jeng, S.T., Gardner, J.F. and Gumport, R.I. (1990) Transcription termination by
bacteriophage T7 RNA polymerase at rho-independent terminators. J. Biol.
Chem. 265, 3823-3830.
Jeng, S.T., Gardner, J.F. and Gumport, R.I. (1992) Transcription termination in
vitro by bacteriophage T7 RNA polymerase. The role of sequence elements
within and surrounding a rho-independent transcription terminator. J. Biol.
Chem. 267, 19306-19312.
Kim, M.J., Baek, K. and Park, C.M. (2009) Optimization of conditions for transient
Agrobacterium-mediated gene expression assays in Arabidopsis. Plant Cell
Rep. 28, 1159-1167.
Lin, C.C., Chu, C.F., Liu, P.H., Lin, H.H., Liang, S.C., Hsu, W.E., Lin, J.S., Wang,
H.M., Chang, L.L., Chien, C.T. and Jeng, S.T. (2011) Expression of an
Oncidium gene encoding a patatin-like protein delays flowering in
Arabidopsis by reducing gibberellin synthesis. Plant Cell Physiol. 52,
421-435.
Pall, G.S., Codony-Servat, C., Byrne, J., Ritchie, L. and Hamilton, A. (2007)
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-e68.