Klenow fragment

The template containing GRE sequences was extended using an Alexa647-labeled common primer and a Klenow fragment (TaKaRa) at 37 °C for 30 min. After extension, exonuclease treatment was used to digest the unreacted single-strand DNA. Alexa647-labeled DNA was purified using an Illustra AutoSeq G-50 column (GE Healthcare). The whole sequence of the Alexa647-labeled GRE constructs is shown below. The capital letter show the GREs.
PpGRE (36 bp); 5′-Alexa647-tcgagggatccgaattcaAGAACActgTGTTCTctc-3′
IpGRE (40 bp); 5′-Alexa647-tcgagggatccgaattcAGAACAgttTGTAACcaaaaact-3′
hGRE (40 bp); 5′-Alexa647-tcgagggatccgaattcAGAACAgttgcaaggactattga-3′.

Before the synthesis of dscDNA, 1 μL of RNase H (TaKaRa) was added to the obtained products to degrade the free RNA. To synthesize dscDNA, anchored random primers were added and incubated 75°C for 5 min and then on ice for 5 min for denaturation. Then, 1 μL of Klenow fragment (TaKaRa), 1 μL of 10 mM dNTPs (TaKaRa), 2 μL of 10 × Klenow buffer (TaKaRa), and 6 μL of dd H₂O (TaKaRa) were added and the samples were incubated at 37°C for 60 min, followed by 75°C for 10 min. Then, 0.5 μL of exonuclease I (TaKaRa), 1 μL of shrimp alkaline phosphatase (SAP, TaKaRa, Dalian, China), 5 μL of 10 × phosphatase buffer (TaKaRa), and 24 μL of DEPC H₂O (TaKaRa) were added and incubated at 37°C for 60 min followed by 75°C for 10 min to eliminate the phosphates and the free single-strand nucleic acid in the dscDNA reaction.

Total RNA was extracted from plant tissues using Trizolreagent (Life Technologies), separated on 1.5% formaldehyde-MOPS agarose gels and blotted onto N⁺ membranes (Millipore Corporation, http://www.millipore.com). Hybridization was performed at 68°C in Perfect HybTMPlus Buffer (Sigma-Aldrich). Probes were labeled with 32^P-dATP using the Klenow fragment (Takara).

Viral nucleic acid library was constructed by sequence-independent amplification using the viral RNA extracted by TIANamp virus RNA Kit. The first-strand cDNAs were synthesized with random reverse transcription primer of K-8N (GACCATCTAGCGACCTCCACNNNNNNNN) according to the previous description [10 (link)]. On the opposite strand of the cDNA, single round priming and extension was performed using Klenow fragment (TaKaRa Biotechnology, Dalian, China).
PCR of extension products was performed using 5 uL of the reaction described above in a total reaction volume of 50 uL containing 5U TaKaRa Ex Taq, 100 mM 10× Ex Taq Buffer (Mg2 + plus), 10 mM dNTPs (TaKaRa Biotechnology, Dalian, China), and 1 mM primer K (GAC CAT CTA GCG ACC TCC AC). Products were identified by agarose gel electrophoresis and the fragments larger than 250 bp were isolated and subcloned into pMD-19T vector for sequencing [10 (link),11 (link)].

The binding of OsARF19, preferentially expressed in LRP (Yamauchi et al., 2019 (link)), to auxin response elements (AuxREs) on OsWOX10 promoter was analyzed by performing an electrophoresis mobility shift assay (EMSA). The sequence encoding the N-terminal 265 amino acids of OsARF19, including the B3 DNA-binding domain (DB), that was optimized for the codon usage of E. coli, was fused to pET32a. The OsARF19-DB was expressed in E. coli BL21(DE3), followed by purification with TALON metal affinity resin (Clontech, United States) following the manufacturer’s instructions. To prepare DNA probes, the 59 or 60-bp oligonucleotides were labeled with Cy5 fluorescence dye using Klenow fragment (TaKaRa Bio, Japan) and purified on a column (NucleoSpin^® Gel and PCR Clean-up; Macherey-Nagel, Germany) following the manufacturer’s instructions. The DNA-binding reaction was performed at 4°C for 30 min in PBS (–) (137 mM NaCl, 8.10 mM Na₂HPO₄, 2.68 mM KCl, 1.47 mM KH₂PO₄; pH 7.4) + 1 mM 2-Mercaptoethanol with 25.3 nM probe with 0, 0.5, or 1 μM recombinant OsARF19-DB, and subjected to EMSA with 5% polyacrylamide gels in 0.5 × TBE buffer at 4°C. The Cy5-labeled probes were analyzed by Typhoon FLA9000 (GE Healthcare, United States).