Volume 10, Issue 11, Pages 1417-1433 (November 2017) Hairy Leaf 6, an AP2/ERF Transcription Factor, Interacts with OsWOX3B and Regulates Trichome Formation in Rice  Wenqiang Sun, Dawei Gao, Yin Xiong, Xinxin Tang, Xiongfeng Xiao, Chongrong Wang, Sibin Yu  Molecular Plant  Volume 10, Issue 11, Pages 1417-1433 (November 2017) DOI: 10.1016/j.molp.2017.09.015 Copyright © 2017 The Author Terms and Conditions

Figure 1 Map-Based Cloning of HL6. (A and B) Morphology of adaxial leaf epidermis in W39 (A) and ZS97 (B) using light microscopy. (C and D) Morphology of adaxial leaf epidermis in W39 (C) and ZS97 (D) using scanning electron microscopy (SEM). Bar, 100 μm. (E) Primary mapping of HL6 in an F2 population of W39/ZS97. The numbers of recombinants between HL6 and the markers are indicated under the map. (F) Fine mapping of HL6 to a 55-kb region. The most likely candidate gene is shown. The phenotype of each recombinant (Rec.) was investigated by progeny test. (G) Schematic gene structure of HL6 with sequence variation. The horizontal lines represent promoter and introns. Blank boxes represent untranslated regions; black boxes represent exons. Triangles and vertical lines on the gene model represent indel and SNP between W39 and ZS97. The relative positions of the variations are given with respect to the start codon (ATG). (H and I) SEM of the trichome phenotype of WT (H) and the complementary transgenic line CPL1 (I). Bar, 500 μm. (J) Macro-hair length in WT and the CPL1 lines. Asterisks indicate significant difference compared with WT at P < 0.01 by Student's t-test. Data are means ± SD (n = 15). Molecular Plant 2017 10, 1417-1433DOI: (10.1016/j.molp.2017.09.015) Copyright © 2017 The Author Terms and Conditions

Figure 2 HL6 Transcriptionally Regulates Macro-Hair Elongation. (A) Schematic of the overexpression vectors (pOX) used for HL6 functional investigation. The common ubiquitin promoter (ProUbi) from maize and the cDNA of HL6 from W39, ZS97, and ZH11 with amino acid variations indicated. (B) HL6 expression in three overexpression lines, OX1, OX2, and OX3. WT represents ZH11. Asterisks indicate significant difference compared with WT at P < 0.01 by Student's t-test. Error bars represent SD with triplicates. (C) Macro-hair length in overexpression lines. Asterisks indicate significant difference against WT at P < 0.01. Error bars represent SD (n = 15). (D) Phenotype of the overexpression line OX1. OX1+ and OX1− represent the positive and negative transgenic plants using pOX1 vector. (E) SEM of trichome length, OX1-5 and OX1-7, two independent overexpression HL6 lines using pOX1 vector. Bar, 500 μm. (F) Expression of HL6 in OX1-5, OX1-7, and ZH11 (WT). Different letters indicate significant difference by Duncan's test at P < 0.01. Error bars represent SD with triplicates. Molecular Plant 2017 10, 1417-1433DOI: (10.1016/j.molp.2017.09.015) Copyright © 2017 The Author Terms and Conditions

Figure 3 OsWOX3B Regulates Trichome Initiation. (A) Schematic of the mutant gene oswox3b showing the location of the T-DNA insertion. (B and C) Expression analysis of OsWOX3B in the mutant and WT (B), the overexpression line (OsWOX3B-OX1+), negative control (OsWOX3B-OX1−) (C). Asterisks indicate significant difference at P < 0.01. Error bars represent SD (N = 3). (D) Macro-hair number in oswox3b, WT, and the OsWOX3B-OX1 line. Asterisks indicate significant difference at P < 0.01 by t-test. Error bars represent SD (n = 10). (E and F) SEM of trichomes of negative control (E) and OsWOX3B-OX1+ (F). (G–J) SEM of trichomes of four genotypes (HL6/OsWOX3B, HL6/oswox3b, HL6-OX/OsWOX3B, and HL6-OX/oswox3b). HL6/OsWOX3B is the WT genotype. HL6-OX indicates overexpressed HL6. Arrows mark macro-hairs. Bar, 100 μm. Molecular Plant 2017 10, 1417-1433DOI: (10.1016/j.molp.2017.09.015) Copyright © 2017 The Author Terms and Conditions

Figure 4 HL6 Physically Interacts with OsWOX3B. (A) Bimolecular fluorescence complementation in rice protoplasts shows HL6 interacting with OsWOX3B. Representative protoplasts transfected with OsWOX3B:CFPn and HL6:CFPc, OsWOX3B:CFPn and CFPc, CFPn and HL6:CFPc. Co-localization of two signals is indicated in merged images by confocal laser scanning microscopy. Bar, 10 μm. NLS:RFP, the marker with red fluorescence localized in the nucleus. CFPn, the N-terminal region for CFP. CFPc, the C-terminal region for CFP. (B) Schematic of the conserved domains in HL6 and OsWOX3B. The truncated proteins each containing the AP2 domain (AP2), homeobox domain (HD), and WUS box (WB) were used for yeast two-hybrid analyses. (C) Yeast two-hybrid analysis (also see Supplemental Figure 8). AD/BD represent activation domain/binding domain. CK+ and CK− indicate the positive and negative control, respectively. The yeast clones were cultured on SD/-Leu/-Trp or SD/-Leu/-Trp/-His/-Ade medium. (D) Pull-down assays using anti-MBP or anti-GST antibodies. In the pull-down anti-MBP, the presence of a band indicates that the pulled-down proteins contain MBP-HD or MBP-AP2, suggesting that the MBP fusion proteins could be pulled down by GST-AP2 or GST-HD. GST protein was used as a negative control. Protein size (kDa) is provided on the right of the gel. Molecular Plant 2017 10, 1417-1433DOI: (10.1016/j.molp.2017.09.015) Copyright © 2017 The Author Terms and Conditions

Figure 5 OsWOX3B Regulates HL6 Expression via Binding to Its Promoter. (A) Relative expression of OsWOX3B in the shoots of the HL6-overexpression line and hl6 mutant. OX1− and OX1+ represent the negative and positive lines; WT represents ZH11. Error bars represent SD (n = 3). (B) Relative expression of HL6 in the shoots of the OsWOX3B-overexpression line and oswox3b mutant. Error bars represent SD (n = 3). Asterisks indicate significant difference at P < 0.01 by t-test. (C) The relative position of a fragment (p1) in the HL6 promoter used for EMSA. The fragment p1 contains the putative WOX-binding motifs (TTAATAG/TTAATTG/TTAATCG) underlined. (D) EMSAs. The recombinant protein GST-HD retarded the probe p1 shifting but GST did not, indicating that the homeobox domain (HD) binds to the HL6 promoter. 50× and 100× indicate excess amounts of non-labeled probes as competitor. (E) Yeast one-hybrid analysis showing direct binding of OsWOX3B to the promoter of HL6 from W39 or ZS97. AD and HIS2 represents the pGADT7-Rec2 vector, the p53HIS2 vector. CK+ and CK− represent the positive and negative control, respectively. Yeast clones were grown in SD/-Leu/-Trp/-His medium containing 0 mM 3-AT (as control) or 40 mM 3-AT. Molecular Plant 2017 10, 1417-1433DOI: (10.1016/j.molp.2017.09.015) Copyright © 2017 The Author Terms and Conditions

Figure 6 HL6 and OsWOX3B Regulate the Expression of Auxin-Related Genes. (A and B) Relative expression of auxin biosynthesis genes OsYUCCA and transport genes OsPIN (A), and signaling genes OsARF and OsIAA (B) in the shoots of HL6 NILs (NIL-W39 and NIL-ZS97), the HL6-overexpression line (HL6-OX1), mutant hl6, and ZH11(WT). (C and D) Relative expression of the auxin biosynthesis and transport genes (C), and the signaling genes (D) in the shoots of the overexpression line OsWOX3B-OX1 and mutant oswox3b. OX− and OX + indicate the transgenic negative and positive lines, respectively. Error bars represent SD (n = 3). Significant differences between pairs (NIL-ZS97 versus NIL-W39, OX1+ versus OX1–, mutant versus WT) at **P < 0.01 or *P < 0.05 by t-test. Molecular Plant 2017 10, 1417-1433DOI: (10.1016/j.molp.2017.09.015) Copyright © 2017 The Author Terms and Conditions

Figure 7 A Proposed Molecular Regulation Network for Trichome Development in Rice. OsWOX3B interacts with HL6 and activates the auxin-related genes such as OsYUCCA5 transcriptions. Arrows indicate activation. The bottom panel depicts trichome initiation and elongation. Molecular Plant 2017 10, 1417-1433DOI: (10.1016/j.molp.2017.09.015) Copyright © 2017 The Author Terms and Conditions