1. Volume 10, Issue 6, Pages 821-833 (June 2017)
DNA Topoisomerase 1 Prevents R-loop Accumulation to Modulate Auxin-Regulated Root Development in Rice 
Sarfraz Shafiq, Chunli Chen, Jing Yang, Lingling Cheng, Fei Ma, Emilie Widemann, Qianwen Sun 
Molecular Plant 
Volume 10, Issue 6, Pages (June 2017)
DOI: /j.molp
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2. Figure 1 OsTOP1 Is Highly Expressed in Rice Root.
(A) Domain structure of OsTOP1 based on protein sequence comparisons. NCBI Reference sequences: AtTOPIα (NP_ ), AtTOPIβ (NP_ ), Oryza sativa (NP_ ), Saccharomyces cerevisiae (NP_ ), Homo sapiens (NP_ ), Mus musculus (NP_ ). The active site (Y) and TPT/CPT inhibition sites (R-D-N) of OsTOP1 are indicated in red and black, respectively.
(B) Expression pattern of the OsTOP1::GUS reporter in the root of 3-day-old rice seedlings: (a) seedling (scale bar, 2 mm), (b) stele (scale bar, 50 μm), (c) meristematic zone (scale bar, 50 μm), (d) coleoptiles (scale bar, 2 mm), (e–h) 7-day-old lateral roots (scale bars, 50 μm). (i) Lateral root primordium. Primary root segments were embedded in 5% agarose and cross-sections of 50 μm were cut with a vibratome, 2–4 cm from the root tip. Scale bar, 200 μm.
(C) RNA in situ hybridization of OsTOP1 in 3-day-old rice root tip. (a) Longitudinal section of meristematic zone; (b) cross-section of stele; (c) longitudinal section of meristematic zone and stele. Scale bars, 50 μm.
Molecular Plant  , DOI: ( /j.molp )
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3. Figure 2 Plant Development Is Affected in OsTOP1-RNAi Lines.
(A) Phenotypes of 10-day-old seedlings from wild-type ZH11 and OsTOP1-RNAi lines.
(B) Plant phenotype of 80-day-old ZH11 and OsTOP1-RNAi lines.
(C) Root and shoot phenotypes of 30-day-old ZH11 and OsTOP1-RNAi lines.
(D) Real-time qPCR analysis of the expression level of OsTOP1 in the OsTOP1-RNAi lines C15 and C16. Mean results from three biological replicates are shown. OsGAPDH was used as an internal reference. Error bars indicate SEM.
(E) Western blot analysis of OsTOP1 levels in ZH11 and the OsTOP1-RNAi lines C15 and C16 using antibodies against the C terminus (left panel) and N terminus (right panel) of OsTOP1. The red arrows represent OsTOP1 protein.
Molecular Plant  , DOI: ( /j.molp )
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4. Figure 3 OsTOP1 Is Essential for Root Growth and Gravitropism.
(A) Primary root growth of wild-type ZH11 and OsTOP1-RNAi line C15 after 72 h of treatment with DMSO or CPT. Arrowheads indicate the wavy root phenotype due to the defective gravitropic response.
(B) The frequency of root waving was calculated by dividing the number of waves per root by root length in ZH11, C15, and ZH11 treated with CPT (30 nM). Mean results from 15 plants are shown. Error bars represent SEM.
(C) Primary root length of ZH11 and OsTOP1-RNAi C15 after 72 h of treatment with different concentrations of CPT. Data shown are the mean of two independent replicates and each replicate consisted of about 15 plants.
(D) After excision of the primary root, lateral root growth was checked with ZH11, ZH11 plants treated with CPT, and OsTOP1-RNAi lines C15 and C16. The primary root was cut off 3 days after germination, after which the seedlings were grown for another 1 day.
(E) Root development of ZH11 plants after 72 h of treatment with DMSO or CPT. White arrowheads point to the boundaries of the different regions. DZ, differential zone; EZ, elongation zone; MZ, meristematic zone; Scale bars, 50 μm.
(F) State of ZH11 Root cap regeneration on DMSO or CPT 24, 48, and 72 h after root cap excision. Scale bars, 2 mm.
Molecular Plant  , DOI: ( /j.molp )
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5. Figure 4 OsTOP1 Is Essential for Symmetric Auxin Distribution.
(A) Expression of the OsDR5::GUS reporter in the root tip after 72 h of DMSO or different concentrations of CPT treatment. Scale bars, 50 μm.
(B) Plants were treated with DMSO or CPT for 72 h, then rotated to 90°. Expression of the OsDR5::GUS reporter in the root was analyzed after 12 h of gravi-stimulation. GUS expression in the root regions marked by dotted boxes and numbered middle panels are shown enlarged in the right panels. Scale bars represent 1 cm (left), 500 μm (middle), and 50 μm (right).
(C) Expression of the OsDR5::GUS reporter in the sites of root curvature after 72 h of DMSO or CPT treatment. White arrowheads point to the regions that presented for GUS staining in the right. Scale bars, 50 μm. The graph at the top represents the intensity of blue color from GUS staining across the root (calculated by ImageJ).
Molecular Plant  , DOI: ( /j.molp )
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6. Figure 5
OsTOP1 Differentially Regulates the Expression of Auxin-Related Genes.
(A) Real-time qPCR of ARFs and ABCB/PIN transporters in ZH11, ZH11 treated with CPT (ZH11-CPT) (30 nM), and OsTOP1-RNAi lines C15 and C16. Root samples were harvested after 24 or 48 h of treatment with DMSO or CPT. OsUBQ5 was used as an internal reference. The mean of three independent biological replicates is presented. Error bars indicate SEM.
(B and C) The GUS signal from the OsARF19::GUS and OsABCB14::GUS reporters is shown in root tips after 72 h of DMSO or CPT treatment. Scale bar, 2 mm. Bar graphs represent real-time qPCR of GUS transcripts by using the OsARF19::GUS and OsABCB14::GUS reporter lines. The mean of two independent biological replicates is presented. Error bars indicate SEM.
Molecular Plant  , DOI: ( /j.molp )
Copyright © 2017 The Author Terms and Conditions

7. Figure 6
OsTOP1 Prevents Inherent R-loop Accumulation over Auxin-Related Genes.
(A) Illustrations of the selected genes are shown. Amplicons from the regions a to e were used in qPCR for DRIP/LM-PCR (Figure 7B–7D).
(B–D) DNA immunoprecipitation using the DNA:RNA hybrid-specific antibody S9.6. Root samples were harvested after 48 h of treatment with DMSO or CPT (30 nM) and the percentage relative to input was calculated and normalized to the region where the highest R-loop levels were detected in DMSO-treated plants. The mean of three independent biological replicates is presented. Error bars indicate SEM.
Molecular Plant  , DOI: ( /j.molp )
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8. Figure 7
Knocking Down OsTOP1 Gene or Blocking OsTOP1 Protein Activity with its Inhibitor CPT Causes More ssDNA to Accumulate at Auxin-Related Genes.
(A) Schematic of KMnO4 treatment followed by LM-PCR to identify the single-stranded DNA levels in R-loops. Nuclear DNA was treated with KMnO4 to oxidize the thymine residues (T). NaOH was used to induce ssDNA breakage at the oxidized T, followed by extension with an antisense gene-specific primer (GSP1). The adaptors were annealed, phosphorylated, and ligated to the GSP1 product, which was then purified using streptavidin beads. Template enrichment was done using a nested gene-specific primer (GSP2) and linker primers. qPCR was then performed to amplify each region using gene-specific primers along the gene of interest.
(B–D) KMnO4 treatment followed by LM-PCR to identify the levels of ssDNA. Regions and primers used to quantify the ssDNA levels by LM-PCR are the same as in Figure 6. The position of GSP1 and GSP2 is shown in Figure 6A. Root samples were harvested after 48 h of treatment with DMSO or CPT (30 nM) and the percentage relative to input was calculated and normalized to the same region as in DRIP. The mean of three independent biological replicates is presented. Error bars indicate SEM.
Molecular Plant  , DOI: ( /j.molp )
Copyright © 2017 The Author Terms and Conditions

9. Figure 8
A Proposed Model Illustrating How OsTOP1 Regulates Root Growth and Gravitropism in Rice.
OsTOP1 is required for proper transcription of genes, including those related to auxin function. Compromised OsTOP1 activity will cause R-loops to accumulate over auxin-related genes and block transcription, resulting in impaired polar auxin transport and root gravitropism. The blue color represents the auxin gradient in the root.
Molecular Plant  , DOI: ( /j.molp )
Copyright © 2017 The Author Terms and Conditions