OsSHI1 Regulates Plant Architecture Through Modulating theTranscriptional Activity of IPA1 in Rice[OPEN]

ErchaoDuan,a,1 YihuaWang,a,1 Xiaohui Li,a Qibing Lin,b Ting Zhang,c YupengWang,b Chunlei Zhou,a Huan Zhang,a

Ling Jiang,a Jiulin Wang,b Cailin Lei,b Xin Zhang,b Xiuping Guo,b HaiyangWang,b and Jianmin Wana,b,2

a State Key Laboratory for Crop Genetics and Germplasm Enhancement, Jiangsu Plant Gene Engineering Research Center, NanjingAgricultural University, Nanjing 210095, ChinabNational Key Facility for Crop Gene Resources and Genetic Improvement, Institute of Crop Science, Chinese Academy ofAgricultural Sciences, Beijing 100081, ChinacCollege of Horticulture, Nanjing Agricultural University, Nanjing 210095, China

ORCID IDs: 0000-0003-1193-4995 (E.D.); 0000-0003-1945-1277 (Y.W.); 0000-0002-9590-6109 (X.L.); 0000-0002-3494-286X (Q.L.);0000-0003-4839-6297 (T.Z.); 0000-0003-1427-833X (Y.W.); 0000-0002-0137-3314 (C.Z.); 0000-0002-0257-0248 (H.Z.); 0000-0003-1464-5881 (L.J.); 0000-0003-4375-4892 (JL.W.); 0000-0002-8473-1948 (C.L.); 0000-0002-2519-4441 (X.Z.); 0000-0002-4936-7562(Z.G.); 0000-0002-1302-5747 (H.W.); 0000-0002-7813-4362 (JN.W.)

Tillering and panicle branching are important determinants of plant architecture and yield potential in rice (Oryza sativa).IDEAL PLANT ARCHITECTURE1 (IPA1) encodesSQUAMOSA PROMOTER BINDING PROTEIN-LIKE14, which acts as a keytranscription factor regulating tiller outgrowth and panicle branching by directly activating the expression of O. sativaTEOSINTE BRANCHED1 (OsTB1) and O. sativa DENSE AND ERECT PANICLE1 (OsDEP1), thereby influencing grain yield inrice. Here, we report the identification of a rice mutant named shi1 that is characterized by dramatically reduced tiller number,enhanced culm strength, and increased panicle branch number. Map-based cloning revealed that O. sativa SHORTINTERNODES1 (OsSHI1) encodes a plant-specific transcription factor of the SHI family with a characteristic family-specificIGGH domain and a conserved zinc-finger DNA binding domain. Consistent with the mutant phenotype, OsSHI1 ispredominantly expressed in axillary buds and young panicle, and its encoded protein is exclusively targeted to the nucleus.We show that OsSHI1 physically interacts with IPA1 both in vitro and in vivo. Moreover, OsSHI1 could bind directly to thepromoter regions of both OsTB1 and OsDEP1 through a previously unrecognized cis-element (T/GCTCTAC motif). OsSHI1repressed the transcriptional activation activity of IPA1 by affecting its DNA binding activity toward the promoters of bothOsTB1 and OsDEP1, resulting in increased tiller number and diminished panicle size. Taken together, our results demonstratethat OsSHI1 regulates plant architecture through modulating the transcriptional activity of IPA1 and provide insight into theestablishment of plant architecture in rice.

INTRODUCTION

As a major staple crop worldwide, the yield of rice is multiplica-tivelydeterminedby threemajor agronomic traits: paniclenumber,grain number per panicle, and grain weight. The numbers ofpanicles and grains per panicle are mainly determined by theability of the plant to produce tillers, the primary and secondarybranches of the panicle. These traits are also important deter-minants of overall plant architecture in rice (Oryza sativa; Wangand Li, 2008; Xing and Zhang, 2010; Wang et al., 2018a).

The formation of a tiller can be divided into two consecutivesteps: tiller bud formation and outgrowth, both of which are reg-ulatedbyelaboratecrosstalkamonghormonal,developmental,andenvironmental factors (Domagalska and Leyser, 2011). Recentmolecular and genetic studies have revealed much insight into the

control of bud formation in both dicots and monocots. RiceMONOCULM1 (MOC1) encodes a transcription factor of theGRASfamily orthologous to Lateral suppressor (LS) of tomato (Solanumlycopersicum) and LATERAL SUPPRESSOR (LAS) of Arabidopsis(Arabidopsis thaliana; Schumacher et al., 1999; Greb et al., 2003; Liet al., 2003). The ricemoc1mutant has no tillers due to the defect inaxillarymeristem formation, which is similar to the phenotypeof thetomato lsmutant, implying that LS/LAS/MOC1 plays a conservedrole inmaintainingthepotential foraxillarymeristeminitiation inbothmonocots and dicots. Further studies demonstrated that Tillering andDwarf 1 (also named TE) acts as a component of the APC/CTAD1/TE

E3 ligase to modulate tiller bud formation by facilitating the degra-dation of MOC1 (Lin et al., 2012; Xu et al., 2012).Studiesof variousbranchingmutants, suchas thedwarfmutants

in rice (Ariteet al., 2007, 2009;Gaoet al., 2009; Lin et al., 2009; Jianget al., 2013; Zhou et al., 2013), more axillary growth mutants inArabidopsis (Sorefan et al., 2003; Booker et al., 2004, 2005;Stirnberg et al., 2007), ramosus mutants in pea (Pisum sativum;Sorefan et al., 2003; Johnson et al., 2006) and decreased apicaldominance mutants in petunia (Petunia hybrida; Snowden et al.,2005) revealedanessential roleof theplanthormonestrigolactones(SLs) in bud outgrowth. Deficiencies in both SL production andsignaling leadtoexcessiveoutgrowthof theaxillarybuds (Smithand

1 These authors contributed equally to this work.2 Address correspondence to: wanjm@njau.edu.cn.The author responsible for distribution of materials integral to the findingspresented in this article in accordance with the policy described in theInstructions for Authors (www.plantcell.org) is: Jianmin Wan (wanjm@njau.edu.cn).[OPEN]Articles can be viewed without a subscription.www.plantcell.org/cgi/doi/10.1105/tpc.19.00023

The Plant Cell, Vol. 31: 1026–1042, May 2019, www.plantcell.org ã 2019 ASPB.

Li, 2014). Recent studies have demonstrated that SLs repress tilleroutgrowththroughpromoting thedegradationofacentral repressorprotein, D53 (Jiang et al., 2013; Zhou et al., 2013).

SQUAMOSA PROMOTER BINDING PROTEIN-box genes(SBP-box genes) encode plant-specific transcription factors thatshare a highly conserved DNA binding domain, the SBP do-main. At least 18 putative SQUAMOSA PROMOTER BINDINGPROTEIN-LIKE genes exist in the rice genome, and most ofthem are regulatory targets of microRNA (miRNA) OsmiR156(Xie et al., 2006). Several members of the SPL family, OsSPL7,OsSPL13, OsSPL14, OsSPL16, and OsSPL17, have beenshown to regulate vegetative and inflorescence architecture inrice (Jiao et al., 2010; Miura et al., 2010;Wang et al., 2012,2015a, 2015b; Liu et al., 2016; Si et al., 2016; Wang and Wang,2017). Among these factors, OsSPL14,also known as IDEALPLANT ARCHITECTURE1 (IPA1) or WEALTHY FARMER’SPANICLE, is the best studied so far (we use “IPA1” hereafter). Itis predominantly expressed in the shoot apex at both thevegetative and reproductive stages. A C-to-T Single Nucleo-tide Polymorphism that escapes OsmiR156 targeting or in-creasing IPA1 expression via epigenetic regulation confers anideal plant architecture to rice, including reduced tiller number,stronger culm, enlarged panicle and, ultimately, enhancedgrain yield (Jiao et al., 2010; Miura et al., 2010). IPA1 bindsdirectly to the promoter regions of several important regulatorsof rice plant architecture, including O. sativa TEOSINTEBRANCHED1 (OsTB1), O. sativa DENSE AND ERECT PANI-CLE1 (OsDEP1), LONELY GUY, SLENDER RICE1, and PIN-FORMED (Lu et al., 2013), as well as WRKY45 to promoteboth yield and immunity in rice (Wang et al., 2018b). OsTB1, atranscription factor of the TEOSINTE BRANCHED1/CYCLO-IDEA/PCF family, is also referred to as FINE CULM1 and wasinitially identified as a counterpart of maize (Zeamays; ZmTB1),which is involved in inhibiting lateral branching in maize

(Doebley et al., 1995; LukensandDoebley, 2001;Donget al., 2017).Later studies confirmed that OsTB1 also acts to suppress axillarybuds outgrowth in rice (Takeda et al., 2003; Minakuchi et al., 2010).OsDEP1 encodes the g-subunit of the heterotrimeric G proteincomplex.Gain-of-functionmutationofOsDEP1 results in increasedprimary and secondary branches and number of grains per panicleand consequently, increased grain yield (Huang et al., 2009). Inaddition, recent studies implicatedOsDEP1 in regulating nitrogen-use efficiency and grain size determinacy in rice (Sun et al., 2014,2018; Liu et al., 2018).TheArabidopsis SHI family contains 10members referred to as

SHORT INTERNODES (SHI), STYLISH1 and 2 (STY1, STY2), SHI-RELATED SEQUENCE3 to SHI-RELATED SEQUENCE8, andLATERAL ROOT PRIMORDIUM1. This gene family encodes plant-specifictranscriptionfactorscharacterizedbyaconservedzincfingerdomainofCys/HisconsensussequenceC3HC3Handaunique IGGHdomain, which was named after the four highly conserved residueswithin this region (Fridborget al., 2001).Genetic studiesof shi-relatedmutants implied thatSHI familymembersplay indispensable roles forgynoecium and leaf development and photomorphogenesis inArabidopsis,probablybyregulatingauxinhomeostasisorexpressionofHY5,BBX21, andBBX22 (SmithandFedoroff,1995;Fridborgetal.,1999, 2001; Kuusk et al., 2002, 2006; Sohlberg et al., 2006; Eklundet al., 2010; Baylis et al., 2013; Yuan et al., 2018). In addition,SHORTAWN2 and SIX-ROWED SPIKE2, encoding two SHI family tran-scription factors, regulate awn elongation, pistil morphology,and inflorescence patterning in barley (Hordeum vulgare; Yuoet al., 2012; Youssef et al., 2017). However, the precise roles andsignificance of this gene family in regulating rice development andplant architecture establishment are still not well characterized.In thisstudy,wecharacterizea ricemutantnamedshi1,whichhas

significantly reduced tiller number, enhanced culm strength, andincreasedpanicle branchnumbers.Molecular cloning revealed thatthe mutant defects are caused by deletion of the SHI family gene

OsSHI1 Regulates Plant Architecture in Rice 1027

OsSHI1. We show that OsSHI1 regulates tillering and paniclebranching throughphysical interactingwith IPA1andmodulating itstranscriptional activity on downstream target genes.

RESULTS

Characterization of the shi1 Mutant Phenotype

In a screen for regulators of plant architecture in rice, we iden-tified the shi1 mutant from a 60Co-g irradiation-induced mutantpopulation of the indica cultivar 9311. Compared with the wildtype, shi1 exhibited dramatically reduced tiller number from the4th-leaf stage to themature stage (Figures1A to1H).Histologicalanalysis revealed that axillary bud initiation was largely normal;however, theoutgrowthof axillarybudswasobviously delayed inthe shi1 mutant (Supplemental Figure 1). Notably, shi1 hada more compact plant architecture with significantly reducedtiller number at the reproductive developmental stage, com-pared with the wild-type plant (Figures 1H and 1I). Panicles ofshi1 were also more compact and erect with slightly increasedprimary branch number and substantially increased secondarybranch and spikelet numbers (Figures 1J to 1N). However, due tothe trade-off between spikelet number andgrain size and variousdefects in floral organ development, the grain size, 1,000-grainweight, and the seed setting rate of shi1, were significantly re-duced (Supplemental Figures 2A to 2C). Leaves of shi1 wereshorter but wider, especially for the flag leaves (SupplementalFigures 2D to 2F), and more dark-green with increased chlo-rophyll contents (Supplemental Figure 2G). More strikingly, theculm diameters of shi1 were greatly increased due to the in-creased parenchyma tissue layers and vascular bundles(Supplemental Figures 2H to 2M). These observations suggestthatOsSHI1playsapleiotropic role in regulatingplant architectureestablishment in rice.

Map-Based Cloning of OsSHI1

Genetic analysis of two F2 populations derived from the reciprocalcrosses between shi1 andwild type (9,311) indicated that the shi1phenotype is controlled by a single recessive nuclear locus, giventhat the numbers of wild-type and mutant individuals approxi-mately fit the expected 3:1 ratio (Supplemental Table 1).

To identify the causal gene, an F2 population was generatedfrom a cross between shi1 and 02428 (O. sativa ssp japonica).Linkageanalysis revealed that theOsSHI1 locus isassociatedwiththe simple sequence repeat markers RM107 and RM189 on thelong arm of chromosome 9. Subsequent fine-mapping using7,547 progeny from the F2 population delimited theOsSHI1 locusto a 50-kb region with four predicted Open Reading Frames(ORFs; Figure 2A). Sequencing and PCR analysis revealed thata ;18-kb genomic region covering ORF2 is deleted in the shi1mutant (Figures2Aand2B),butnoDNAsequencealterationswerefound in the promoter and coding regions of ORF1, ORF3, andORF4. Further, RT-PCR analysis and immunoblot analysis usinganti-OsSHI1–specific polyclonal antibodies confirmed no ex-pression of ORF2 in the shi1 mutant (Figures 2C and 2D). Theseresults suggest that ORF2 likely corresponds to OsSHI1.

To verify whether ORF2 is indeed responsible for the shi1phenotype, an ;5-kb genomic fragment of ORF2 (full-lengthORF2 including a 3-kb promoter, two exons, one intron, anda 400-bp downstream region) was transformed into shi1. Asexpected, positive transgenic plants displayed normal plantdevelopment with recovered tiller numbers (Figures 2E and2F). Moreover, ORF2 knockout transgenic plants generatedby clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR associated protein 9 (Cas9) genome-editingapproach (in Kitaake background, O. sativa ssp japonica) showedreduced tiller number, but increased panicle branch number,compared with the wild-type plants (Kitaake; SupplementalFigure 3). On the contrary, the ORF2 overexpression lines hadreduced plant height (Supplemental Figures 4A and 4B) andectopic tillers usually formed at the upper internodes (Supple-mental Figures 4C and 4D). At the reproductive developmentalstage, the primary and especially secondary branch numbers ofORF2 overexpression lines were remarkably decreased (Supple-mental Figures 4E to 4G). Taken together, these molecular andgenetic lines of evidence confirmed thatORF2 indeed representsOsSHI1.

Expression Pattern of OsSHI1

Sequence analysis revealed that OsSHI1 encodes a tran-scription factor homologous to the Arabidopsis SHI familywith the intrinsic C3HC3H zinc finger domain and the SHIfamily-specific IGGH domain (Figure 3A; Supplemental Fig-ure 5). Consistent with its function as a transcription factor,transient expression analysis in rice protoplasts showed thatthe OsSHI1-GFP fusion protein was exclusively localized tothe nucleus (Figure 3B). The transactivation activity assayindicated that OsSHI1 exhibited weak transcriptional acti-vation activity in yeast cells (Supplemental Figure 6) and wascapable of forming homodimer via its C terminus (Figure 3C).RT-quantitative (q)PCR analysis revealed that OsSHI1 wasexpressed in various tissues, with higher transcript abun-dance being detected in root, young panicle, and axillarybuds (Figure 3D). Histochemical staining analysis of thepOsSHI1:GUS (b‑glucuronidase) transgenic plants showedstrong GUS staining in root, young panicle, and axillary buds,but not in the culm, leaf blade, or leaf sheath, further con-firming that the OsSHI1 promoter is active in these tissues(Supplemental Figure 7). Moreover, immunoblot analysisusing anti-OsSHI1–specific antibodies (Supplemental Fig-ure 8) validated that OsSHI1 protein was mainly accumulatedin young panicle and axillary buds, consistent with its role inregulating tiller and panicle development (Figure 3E).

OsSHI1 Physically Interacts with IPA1

To elucidate the regulatorymechanismofOsSHI1on rice tiller andpanicle development, a yeast two-hybrid (Y2H) screening wasperformed to identify the interacting partners of OsSHI1(Supplemental Table 2). Intriguingly, two positive clones con-taining the coding region of IPA1 were isolated. Considering thatIPA1 plays a critical role in the establishment of rice plant

1028 The Plant Cell

Figure 1. Phenotypic Characterization of the shi1 Mutant.

(A) to (G)Tilleringphenotypesofwild type (9311) and shi1at 2weeksafter germination (WAG) (A), 3WAG (B)and (C), 4WAG (D), 5WAG (E), 6WAG (F), and7WAG (G). (C) is the enlarged image of the dotted box in (B). White arrows indicate the tillers. Bars = 2 cm (A) and (B), 1 cm (C), 5 cm (D) to (G). WT, wild type.(H) Plant architectures of wild type and shi1 at the grain-filling stage. Bar = 20 cm. WT, wild type.(I)Statistical analysis of tiller numbers ofwild type and shi1. Values are presented asmean6 SD, and the statistically significant differenceswere determinedby Student’s t test (n = 15, **P < 0.01). WT, wild type.(J) Panicle morphologies of wild type and shi1 at the mature stage. Bar = 5 cm. WT, wild type.(K) Panicle branch architectures of wild type and shi1 with grains removed. Bar = 5 cm. WT, wild type.(L) to (N)Statistical analysis of the primary branch numbers (L), secondary branch numbers (M), and spikelet numbers per panicle (N) of wild type and shi1.Values are presented as mean 6 SD, and the statistically significant differences were determined by Student’s t test (n = 10, *P < 0.05, **P < 0.01). WT,wild type.

OsSHI1 Regulates Plant Architecture in Rice 1029

architecture, we pursued their interaction and physiological sig-nificance further. Dissection of their interactive domains showedthat both theN- andC-terminal regionsofOsSHI1 could interactwiththe C terminus, but not the SBP domain of IPA1 (Figures 4A and 4B).The interaction between IPA1 and OsSHI1 was further confirmed by

in vitro pull-down assay (Figure 4C; Supplemental Figure 9),in vivo bimolecular fluorescence complementation (BiFC) assay inleaf epidermal cells of Nicotiana benthamiana (Figure 4D), andcoimmunoprecipitation (CoIP) assay in axillary buds of wild-typeseedlings (Figure 4E).

Figure 2. Map-Based Cloning of OsSHI1.

(A)OsSHI1was narrowed down to an;50-kb region of chromosome 9 containing fourORFs. A genomic region of;18 kb is deleted in shi1. The markers,BACs and numbers of recombinants are indicated. WT, wild type. Rec, recombinants. BACs, Bacterial Artifical Chromosomes.(B)PCRamplifications by primer pairs located at the flankingboundaries of thedeleted region. NoPCRproduct could be amplified fromwild type due to thelarge size of genomic region using the primer pair F48 and R56. WT, wild type.(C) RT-PCR analysis of the four ORFs. Expression of ORF2 was not detected in shi1. Actin2 was used as an endogenous control. WT, wild type.(D) Protein levels of OsSHI1 in the panicle tissues of wild type and shi1 detected by immunoblot using anti-OsSHI1–specific polyclonal antibodies. HSPantibody was used as the loading control. Molecular masses of proteins (kDa) are shown on the left. WT, wild type.(E) Plant phenotypes of wild-type, shi1, and two independent complementation lines before the heading stage. Bar = 10 cm. WT, wild type.(F) Tiller numbers of the complemented transgenic lines compared with the wild-type and mutant levels. Values are presented as mean 6 SD, and thestatistically significant differences were determined by Student’s t test (n = 5, **P < 0.01). WT, wild type.

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OsSHI1 Directly and Negatively Regulates the Expression ofOsTB1 and OsDEP1

Previous reports revealed that IPA1 directly activates the ex-pression of OsTB1 and OsDEP1, two key regulators for tiller andpanicle development in rice (Jiao et al., 2010; Lu et al., 2013). The

SBP-box of IPA1 functions as the conserved DNA binding domainand its C terminus confers transcriptional activation activity (Luet al., 2013). As the shi1 mutants exhibited reduced tillers and in-creased panicle branch number, we speculated that OsSHI1 mayact together with IPA1 to coregulate the expression of OsTB1 andOsDEP1. In support of this notion, RT-qPCR analysis revealed thatindeed, the expression levels of both OsTB1 and OsDEP1 weresignificantly increased in the shi1 background (Figures 5A and 5B).Sequenceanalysis identified twoandoneT/GCTCTACmotifs in thepromoter regions of OsTB1 and OsDEP1, respectively. Notably,these elements are quite similar to the binding motif (ACTCTAC) ofthe Arabidopsis AtSTY1 homologous protein (Eklund et al., 2010).Intriguingly, these T/GCTCTAC motifs are located near the IPA1recognition sites (GTAC) in the promoter regions of bothOsTB1 (59bp) andOsDEP1 (113 bp; Supplemental Figures 10 and 11), hintingthat OsSHI1 and IPA1may coordinately regulate the expression ofOsTB1 and OsDEP1 to affect plant architecture.To test this, we first performed yeast one-hybrid (Y1H) assay

to test for direct binding of OsSHI1 to the OsTB1 and OsDEP1promoters. As shown in Figures 5C and 5D, OsSHI1 bound directly tothe F4 (21;2336) and F3 (21274;21578) promoter regions ofOsTB1 and OsDEP1, respectively, where the three OsSHI1 recog-nition cis-elements reside. We further used electrophoretic mobilityshift assay (EMSA) to verify thebindingspecificity ofOsSHI1 to thesemotifs. Full-length recombinant OsSHI1 proteins (fused with gluta-thione S-transferase [GST] or maltose-binding protein [MBP] tags)were expressed in Escherichia coli BL21 (DE3) (TransGen Biotech)andaffinity-purified (Supplemental Figure 9).We found that theGST-or MBP-OsSHI1 fusion proteins could bind DNA probes containingthe T/GCTCTAC motifs. Moreover, nonlabeled competing probescould effectively reduce the binding ability of OsSHI1 in a dosage-dependentmanner andmutationof thecore sequence (T/GCTCTACmutated to T/GAAAAAC) abolished the binding (Figures 5E to 5G).Furthermore, chromatin immunoprecipitation assay (ChIP) usinganti-OsSHI1 specific polyclonal antibodies verified that OsSHI1could be specifically recruited to the P3 promoter regions ofOsTB1andOsDEP1, adjacent to the IPA1 recognition sites (Figures5H and 5I). Moreover, ChIP-reChIP analysis with chromatin im-munoprecipitated sequentially by OsSHI1- and IPA1-specificpolyclonal antibodies showed that OsSHI1 and IPA1 co-occupycommon target promoters (OsTB1andOsDEP1) in vivo (Figure5J;Supplemental Figure 12). Further domain dissection analysisrevealed that the N-terminal region of OsSHI1 (containing theconserved C3HC3H zinc finger domain) confers the DNA bindingability (Supplemental Figure 13).

OsSHI1 Represses the DNA Binding Activity of IPA1 to thePromoters of OsTB1 and OsDEP1

Previous studies demonstrated that IPA1 acts as a transcriptionalactivator, promoting the accumulation of OsTB1 and OsDEP1transcripts (Lu et al., 2013). The upregulation of OsTB1 andOsDEP1 in the shi1 background indicates that OsSHI1 and IPA1act antagonistically in regulating the expression of OsTB1 andOsDEP1. We thus performed transient dual-luciferase (LUC) as-say in rice protoplasts to evaluate the transcriptional regulatoryrelationship betweenOsSHI1 and IPA1. As shown in Figures 6A to6C, IPA1 alone greatly enhanced the expression of the luciferase

Figure 3. Expression Analysis ofOsSHI1 and Functional Characterizationof OsSHI1 Protein.

(A) Schematic representation of the OsSHI1 protein. The conserved zincfinger domain and IGGH domain are indicated.(B) Subcellular localization of the OsSHI1-GFP fusion protein in riceprotoplast. OsD53-mCherry was used as the nuclearmarker. Bar = 20mm.(C) OsSHI1 is capable of forming homodimer through the C terminus inyeast cells. Transformed yeast cells were spotted on the control mediumDDO (SD/-Trp/-Leu) andselectivemediumQDO (SD/-Trp/-Leu/-His/-Ade).The empty pGADT7 was used as the negative control. OsSHI1-N” and“OsSHI1-C indicate the N- andC-terminal regions of OsSHI1 including thezinc finger domain and IGGH domain, respectively.(D) RT-qPCR analysis of the expression pattern of OsSHI1 in varioustissues. R, root; C, culm; LB, leaf blade; LS, leaf sheath; YP, young panicle;MP, mature panicle; AB, axillary buds. Values are presented as mean6 SD

(n = 3).(E) Immunoblot analysis showing the accumulation of OsSHI1 protein invarious tissues. HSP antibody was used as the loading control. Molecularmasses of proteins (kDa) are shown on the left. R, root; C, culm; LB: leafblade; LS, leaf sheath; YP, young panicle; MP, mature panicle; AB,axillary buds.

OsSHI1 Regulates Plant Architecture in Rice 1031

(LUC) reporter gene driven by theOsTB1 andOsDEP1 promoters,while OsSHI1 alone had no significant effect. However, whencoexpressedwithOsSHI1, the transcriptional activation activity ofIPA1 was significantly attenuated. Moreover, mutations of theT/GCTCTAC motifs did not compromise the effect of OsSHI1-mediated repression of the transcriptional activation of OsTB1conferred by IPA1 (Figure 6D). To test whether OsSHI1 affects theDNA binding affinity of IPA1, in vitro EMSAs were performed. Aspreviously reported, IPA1 could bind directly to the GTACmotifs inthe OsTB1 and OsDEP1 promoter regions, and no shifted bandswere observed for OsSHI1 to the GTACmotifs (Figures 6E and 6F).However, the presence of increasing amounts of OsSHI1 protein inthe reactions significantly reduced the binding ability of IPA1 to thetarget probes, independent of OsSHI1 binding (Figures 6E and 6F;SupplementalFigure14), indicatingthatOsSHI1could interferewiththe DNA binding ability of IPA1. Moreover, immunoblot analysisusing anti-IPA1–specific polyclonal antibodies (SupplementalFigure 12) revealed that no differences of IPA1 proteinabundance were observed in either the young seedling or

young panicle tissues of wild-type and shi1 (Figures 6G and 6H).However, in vivoChIP-qPCRassay performedwithDNAprecipitatedbyanti-IPA1antibodies revealed that theP3 andP4promoter regionsof OsTB1 and OsDEP1 were significantly more enriched in the shi1mutant (Figures6I and6J),which isconsistentwith theupregulationofexpression levels of OsTB1 and OsDEP1 in shi1 mentioned above.To further investigate the biological significance of the OsSHI1

and IPA1 interaction, we generated 35S:IPA1-Flag transgenicplants, which displayed significantly repressed tiller developmentas expected (Figures 7A and 7B). We further overexpressedOsSHI1under thecontrolof theACTIN1promoter in the35S:IPA1-Flag transgenic background (Figure 7C). Immunoblot analysisshowed that OsSHI1 accumulation did not obviously affect thestability or abundance of IPA1 protein (Figure 7C). In vivo ChIP-qPCR performed with DNA precipitated by anti-Flag antibodyfrom the 35S:IPA1-Flag transgenic plants revealed that the P3promoter region of OsTB1 (where the two IPA1 recognition sitesreside) was predominantly enriched (Figure 7D). However, theenrichment of the P3 promoter regionwas significantly reduced in

Figure 4. OsSHI1 Physically Interacts with IPA1.

(A) Schematic representation of the various truncated visions of OsSHI1 and IPA1 proteins. The conserved zinc finger, IGGH, and SBP domains areindicated.(B)Both theN- andC-terminal regions of OsSHI1 interact with the C terminus of IPA1 in yeast cells. Transformed cells were spotted on the control medium(DDO, SD/-Leu/-Trp) and selective medium (QDO, SD/-Leu/-Trp/-His/-Ade). The empty pGADT7 was used as the negative control.(C) Invitropull-downassayconfirms thatOsSHI1-GST,butnotGST itself, couldprecipitate IPA1asdetectedbyanti-MBPantibody.Thesymbols “–”and “+”indicate the absence and presence of the corresponding proteins.(D)BiFCassay verifies the interaction betweenOsSHI1 and IPA1 in the nuclei of epidermal cells ofN.benthamiana. OsSHI1 and IPA1were fusedwith theN-and C terminus of YFP, respectively. eYNE and eYCE were used as the negative controls. OsSPL16, a homologous protein of IPA1, was also used asanegative control to demonstrate the specific interactionbetweenOsSHI1 and IPA1.DIC, differential interference contrast;Merged,merged images of YFPchannel and DIC. Bar = 30 mm.(E) In vivo CoIP assay shows that OsSHI1 interacts with IPA1 in the axillary buds of wild-type seedlings. Total protein extracts were immunoprecipitated bythe anti-IPA1–specific polyclonal antibodies and analyzed by immunoblot probed with the anti-IPA1 and anti-OsSHI1 polyclonal antibodies. Immuno-globulin G was used as the negative control.

1032 The Plant Cell

the Actin1:OsSHI1/35S:IPA1-Flag transgenic plants, whencompared with the scenario of 35S:IPA1-Flag transgenic plants(Figure 7D). Consistent with this, the Actin1:OsSHI1/35S:IPA1-Flag transgenic plants displayed obviously reduced plant heightand somewhat recovered tiller development, as well as

significantly reduced expression levels ofOsTB1, compared withthe 35S:IPA1-Flag parental plants (Figure 7E and 7F). Taken to-gether, these results support the conclusion that OsSHI1 nega-tively regulates the transcriptional activation activity of IPA1 onOsTB1 and OsDEP1 by repressing its DNA binding activity.

Figure 5. OsSHI1 Binds Directly to the Promoter Regions of OsTB1 and OsDEP1.

(A) and (B) RT-qPCR analysis of OsTB1 (A) and OsDEP1 (B) expression levels in wild-type and shi1 axillary buds or young panicle tissues, respectively.Valuesarepresentedasmean6 SD, and thestatistically significantdifferencesweredeterminedbyStudent’s t test (n=4, *P<0.05, **P<0.01).WT,wild type.(C) and (D) Y1H assays to dissect the binding regions of OsSHI1 in the promoter regions of OsTB1 (C) and OsDEP1 (D). Series of promoter fragments ofOsTB1 and OsDEP1 were fused to the upstream region of the LacZ reporter gene and tested for OsSHI1 binding.The empty pB42AD was used as thenegative control.(E) to (G) EMSAs to test OsSHI1 binding to the two TCTCTAC (E) and (F) and oneGCTCTAC (G)motifs in theOsTB1 andOsDEP1 promoters, respectively.The T/GCTCTACmotif wasmutated into T/GAAAAAC to test for sequence specificity. The triangles indicate increased amounts of competing probes. GSTor MBP proteins were used as the negative controls. The symbols “–” and “+” indicate the absence and presence of the corresponding proteins or probes.(H) and (I)ChIP-qPCR analyses of the P3 promoter regions ofOsTB1 (H) andOsDEP1 (I) in ChIP samples precipitated by anti-OsSHI1–specific polyclonalantibodies. The fold enrichmentwas calculated as IP/Input. Values arepresented asmean6 SD, and the statistically significant differenceswere determinedby Student’s t test (n = 4, **P < 0.01).(J)ChIP-reChIPanalysisof IPA1andOsSHI1co-occupycommon targetpromoters. Thechromatinofwild-typeplantswasfirst immunoprecipitatedbyanti-OsSHI1–specific polyclonal antibodies and then by anti-IPA1–specific polyclonal antibodies, and the precipitated DNA was quantified by qPCR analysis.The fold enrichment was calculated as IP/Input and normalized to that of the UBIQUITIN promoter region as an internal control. Values are presented asmean 6 SD, and the statistically significant differences were determined by Student’s t test (n = 4, **P < 0.01).

OsSHI1 Regulates Plant Architecture in Rice 1033

IPA1 Acts Downstream of OsSHI1 to Regulate PlantArchitecture in Rice

Todetermine thegenetic relationshipofOsSHI1and IPA1, aseriesof shi1, ipa1, tb1, dep1, ipa1 shi1, tb1 shi1, and dep1 shi1mutantswere generated in the same genetic background (Kitaake) usingCRISPR/Cas9-mediated genome-editing approach (SupplementalFigure 15). As expected, shi1mutant exhibited reduced tillering and

increased panicle branching (Supplemental Figure 3). In contrastwith the phenotype of shi1mutant, the tiller number of ipa1mutantwas significantly increased, accompanied with diminished paniclesize. Tiller development was greatly intensified in the tb1 mutant.Panicle branching was compromised in both the tb1 and dep1mutants, similar to ipa1 mutant (Figures 8A and 8B). Further phe-notypicobservationsofthe ipa1shi1, tb1shi1, anddep1shi1doublemutants revealed their similarity to the ipa1, tb1, and dep1 single

Figure 6. OsSHI1 Represses the DNA Binding Activity of IPA1.

(A) Schematic representation of the effector and reporter constructs. Full-length coding regions of OsSHI1 and IPA1 under control of the double 35Spromoterwereusedas theeffectors. TheFirefly luciferasegeneLUCdrivenby theOsTB1orOsmTB1andOsDEP1promotersand theRenilla luciferasegeneRen driven by the 35S promoter were used as the reporter and internal control, respectively. d35S, double 35S promoter.(B) to (D) OsSHI1 represses the transcriptional activation activities of IPA1 on OsTB1 (B), OsDEP1 (C), and OsmTB1 (D) promoters in rice protoplasts.Relative LUC activity was calculated by LUC/Ren and normalized to that of vector control which was set as 1. Values are presented as mean6 SD, and thestatistically significant differences were determined by Student’s t test (n = 3, **P < 0.01).(E) and (F) EMSAs show that OsSHI1-IPA1 interaction attenuates the DNA binding activity of IPA1 to theGTACmotifs in the promoter regions ofOsTB1 (E)and OsDEP1 (F). The triangles indicate increased amounts of OsSHI1 proteins. MBP proteins were used as negative controls. The symbols “–” and “+”indicate the absence and presence of the corresponding proteins.(G) and (H) Immunoblot analyses of IPA1 protein accumulation in young seedling (G) and youngpanicle (H) tissues of wild type and shi1. HSPantibodywasused as the loading control. The molecular masses of proteins (kDa) are shown on the left. WT, wild type.(I) and (J)ChIP assays of the P3 and P4 promoter regions ofOsTB1 (I) andOsDEP1 (J) in shi1 compared with the wild type. DNAs precipitated from axillarybuds or young panicle tissues of wild type and shi1 by anti-IPA1–specific polyclonal antibodies were subjected into ChIP-qPCR analysis. Values arepresented as mean 6 SD, and the statistically significant differences were determined by Student’s t test (n = 4, **P < 0.01).

1034 The Plant Cell

mutant, respectively (characterized by enhanced tillering andcompromised panicle branching; Figures 8A and 8B). These ob-servations support the conclusion thatOsSHI1 functions upstreamof IPA1, OsTB1 and OsDEP1 to regulate plant architecture in rice.

DISCUSSION

OsSHI1 Acts Antagonistically with IPA1 in Regulating PlantArchitecture in Rice

Tiller number and panicle size are critical determinants of plantarchitecture and crop yield. To meet the constantly increasing

demand for food productivity, a new breeding approach of theNewPlantTypeor IPAstrategyhasbeenproposed (Khush,2001;Wang and Li, 2008). The IPA traits characterized by fewer steriletillers, larger panicles, and stronger culms are closely correlatedwith theaccumulationof IPA1protein,which further activates theexpression ofOsTB1 andOsDEP1 to regulate plant architecturein rice (Jiao et al., 2010; Miura et al., 2010; Lu et al., 2013).Notably, several aspects of the shi1 mutant phenotype (re-markably reduced tiller number, enlarged panicles, and en-hanced culm strength; Figures 1H to 1N; Supplemental Figures2H to 2M) are reminiscent of the effects of IPA1 overexpression.Furthermore, OsSHI1 overexpression lines exhibit significantly

Figure 7. OsSHI1 Acts Antagonistically with IPA1 to Regulate Tillering in Rice.

(A) Plant morphologies of Kitaake, 35S:IPA1-Flag and Actin1:OsSHI1/35S:IPA1-Flag transgenic lines at the heading stage. Bar = 10 cm.(B) Determination of IPA1-Flag protein accumulation in the 35S:IPA1-Flag transgenic young seedlings. IPA1-Flag protein was detected using anti-Flagantibody. HSP antibody was used as the loading control. The molecular masses of proteins (kDa) are shown on the left.(C) Immunoblot analyses showing the accumulation of OsSHI1 and IPA1-Flag proteins in theActin1:OsSHI1/35S:IPA1-Flag transgenic seedlings. OsSHI1and IPA1-Flag proteinswere detectedwith anti-OsSHI1–specificpolyclonal antibodies andanti-Flag antibody, respectively. HSPantibodywasused as theloading control. The molecular masses of proteins (kDa) are shown on the left.(D) OsSHI1 reduces the enrichment of the promoter region of OsTB1 immunoprecipitated by IPA1. DNAs precipitated from 35S:IPA1-Flag andActin1:OsSHI1/35S:IPA1-Flag transgenicseedlingsbyanti-Flagantibodyweresubjected intoChIP-qPCRanalysis.Valuesarepresentedasmean6SD,andthe statistically significant differences were determined by Student’s t test (n = 4, *P < 0.05).(E)Overexpression ofOsSHI1 in the 35S:IPA1-Flag transgenic background results in increased tiller number. Values are presented as mean6 SD, and thestatistically significant differences were determined by Student’s t test (n = 5 independent plants, *P < 0.05, **P < 0.01).(F) RT-qPCR analysis showing OsTB1 expression levels in Kitaake, 35S:IPA1-Flag, and Actin1:OsSHI1/35S:IPA1-Flag transgenic lines. Values arepresented as mean 6 SD, and the statistically significant differences were determined by Student’s t test (n = 4, **P < 0.01).

OsSHI1 Regulates Plant Architecture in Rice 1035

reduced plant height, ectopically formed tillers, and diminishedpanicle size (Supplemental Figure 4), similar to the previouslyreported IPA1 transgenic RNAi plants (Wang et al., 2015a).In addition, OsSHI1 is predominantly expressed in axillarybuds and young panicle (Figures 3D and 3E; SupplementalFigure 7) and its expression pattern partially overlaps with thatof IPA1 (Jiao et al., 2010; Miura et al., 2010; Lu et al., 2013).These observations hint that OsSHI1 and IPA1 may antago-nistically regulate plant architecture in rice. This notion isfurther supported by the observation that the expressionlevels of OsTB1 and OsDEP1, two positively regulated targetsof IPA1, are significantly upregulated in shi1 (Figures 5A and5B). Moreover, the Actin1:OsSHI1/35S:IPA1-Flag double-overexpression plants exhibit significant dwarfism and some-what increased tiller number, in comparison with the 35S:IPA1-Flag parental plants (Figures 7A and 7E). Further, in contrastwith the repressed tiller development and enlarged panicle ar-chitecture of shi1, the tiller and panicle branch numbers of ipa1shi1, tb1 shi1, and dep1 shi1 double mutants are significantlyincreased or reduced, similar to the scenario of ipa1, tb1, or dep1single mutants (Figure 8). These results together suggest thatOsSHI1 acts antagonistically and upstream of IPA1 to regulateplant architecture in rice.

OsSHI1 Represses the Transcriptional Activity of IPA1 byInterfering with Its DNA Binding Activity

Recent studies have identified three IPA1-interacting proteins,IPA1 INTERACTING PROTEIN1 (OsIPI1), ovarian tumor domain-containing ubiquitin aldehyde-binding protein 1 (OsOTUB1), andOsD53, and demonstrated their roles in regulating plant archi-tecture in rice (Jiang et al., 2013; Zhou et al., 2013; Song et al.,2017; Wang et al., 2017a, 2017b). OsIPI1 acts as a RING-type E3ligase to promote the degradation of IPA1 in panicles by addingK48-linked poly-ubiquitin chains while stabilizing IPA1 in shootapexes by mediating K63-linked poly-ubiquitin modification(Wang et al., 2017a). OsOTUB1 is a deubiquitinating enzyme withboth K48- and K63-linked poly-ubiquitin cleavage activities, andOsOTUB1-IPA1 interaction limits the K63-linked ubiquitination ofIPA1, which in turn promotes K48-linked ubiquitination -de-pendent proteasomal degradation of IPA1 (Wang et al., 2017b).Thus, both OsIPI1 and OsOTUB1 are enzymes involved in theposttranscriptional poly-ubiquitin modification of IPA1. Recentstudies also showed that OsD53 represses the transcriptionalactivation activity of IPA1 by interacting with the TOPLESStranscriptional corepressors, which in turn recruit histonedeacetylases complexes to modulate local chromatin status,

Figure 8. OsSHI1 Acts Upstream of IPA1 to Regulate Plant Architecture in Rice.

(A) Plant morphologies of Kitaake, shi1, ipa1, tb1, dep1, ipa1 shi1, tb1 shi1, and dep1 shi1 mutants at the heading stage. Bar = 10 cm.(B) Panicle architectures of Kitaake, shi1, ipa1, tb1, dep1, ipa1 shi1, tb1 shi1, and dep1 shi1 mutants. Grains were removed to show the primary andsecondary branch patterns of the panicles. Bar = 2 cm.

1036 The Plant Cell

thereby influencing downstream target gene expression. Thedegradation of OsD53 by the 26S proteasome system releasesIPA1 to proceed with the activation of downstream target genes(like OsTB1), allowing tiller development (Jiang et al., 2013; Zhouet al., 2013; Song et al., 2017). Notably, OsIPI1, OsOTUB1, andOsD53 all interact with IPA1 through the conserved SBP domainand they do not affect the DNA binding ability of IPA1 (Song et al.,2017; Wang et al., 2017a, 2017b).

In this study, we used various assays to show that OsSHI1 is aninteracting partner of IPA1 (Figure 4; Supplemental Table 2). Incontrast with the previous identified IPA1 interacting partners, weshowed that OsSHI1 interacts with IPA1 through the C-terminalregion but not theSBPdomain (Figure 4B). In addition,weshowedthat the levels of IPA1 transcript and IPA1 protein are not affectedin the shi1 mutant (Figures 6G and 6H; Supplemental Figure 16),suggesting that OsSHI1 regulates IPA1 through a previouslyuncharacterized mechanism. In support of this notion, our tran-sient analysis revealed that OsSHI1 represses the transcriptionalactivation activity of IPA1 in protoplasts (Figures 6B and 6C).Further, both in vitro EMSAs and in vivoChIP assays revealed thattheOsSHI1-IPA1 interaction reduces thebindingaffinityof IPA1 tothe promoter regions of bothOsTB1 andOsDEP1 (Figures 6E, 6F,6I, and 6J, and 7D), but no obvious effect of IPA1 on the DNAbinding ability of OsSHI1was observed (Supplemental Figure 17).Compromised DNA binding affinity of IPA1 conferred by OsSHI1was partially independent of its binding to its own cis-element(Figure 6D; Supplemental Figure 14).

Based on our findings, we propose a model to illustrate howOsSHI1 and IPA1 act cooperatively to regulate plant architecturein rice (Supplemental Figure 18). Inwild-type plants, OsSHI1/IPA1heterodimer formation reduces the DNA binding ability of IPA1 tomodulate the expression of downstream target genes and plantarchitecture. However, in shi1, the absence of OsSHI1 enhancesthebindingof IPA1 to thepromoter regionsofOsTB1andOsDEP1to upregulate their expression levels, consequently altering plantarchitecture. Taken together, our results suggest that OsSHI1regulates IPA1activity throughaffecting its transcriptional activity.However, other possibilities exist, such as that the formation ofa OsSHI1-IPA1 complex may alter the protein/DNA conformationof target genes thus reducing the access of IPA1 or block thetranscriptional activation activity conferred by the C-terminalregion of IPA1 or interfere with the interaction of IPA1 withother proteins (such as chromatin remodeling factors). Given thedemonstration that plant architecture could be improved throughfine-tuning the tissue-specificexpressionor protein accumulationpattern of IPA1 (Wang andWang, 2017), our findingsmay providenewalternative approaches tomodify the activity of IPA1 and thusbear important implications for genetic improvement of rice plantarchitecture in future breeding.

METHODS

Plant Materials and Growth Conditions

The shi1mutant was initially isolated from a mutant library of 9,311 (Oryzasativa ssp indica) mutagenized by 60Co-g irradiation. Two F2 populationsderived from the reciprocal crossesbetween shi1andwild type (9311)wereutilized for genetic analysis of segregation. For map-based cloning, an F2

population was generated from the cross between shi1 and cv 02,428(O. sativa ssp japonica). Plants were grown in the paddy fields at theChinese Academy of Agricultural Sciences and Nanjing Agricultural Uni-versity under natural conditions with conventional management.

5-ethynyl-2�-deoxyuridine (EdU) Staining Observation

Wild-type and shi1 young seedlings were incubated in the Murashige andSkoog solution with the 5-ethynyl-2�-deoxyuridine substrate overnight.Shoot bases with axillary buds were carefully dissected and fixed in theformaldehyde - acetic acid - alcohol (FAA) fixative solution (50% (v/v)ethanol, 5%(v/v)aceticacid, and10%(v/v) formaldehyde) at4°Covernight.Sampleswere rehydrated through the70% (v/v), 50% (v/v), 30% (v/v), 15%(v/v), and 0% (v/v) ethanol series (each for 15 mins) and incubated in 1%(v/v) Triton X-100 for 2 h. Then the samples were incubated in a stainingsolution (invitrogen) (13Click-iT reaction buffer, 100mMofCuSO4, 10mMof Alexa Fluor azide, and 13 Reaction buffer additive) for 3 h in darkness.The samples were subsequently washed three times in water and dehy-drated through the 15% (v/v), 30% (v/v), 50% (v/v), 70% (v/v), 85% (v/v),and 95% (v/v) ethanol series (each for 20 min) followed by incubating with100% (v/v) ethanol for 2 h. After sufficient dehydration, samples werehyalinized through the 2:1, 1:1, 1:2, and0:1 ethanol:methyl salicylate series(each for 1 h). Fluorescence signals were observed using a confocal laserscanning microscope (LSM 700; Zeiss).

Paraffin Section Analysis

Shoot bases with axillary buds were carefully dissected and fixed in theformaldehyde - acetic acid - alcohol (FAA) solution at 4°C for 24 to 72 h.Samples were dehydrated through the 70% (v/v), 80% (v/v), 90% (v/v) and100% (v/v) ethanol series (each for 60 mins). After sufficient dehydration,samples were hyalinized through the 1:2, 1:1, 2:1, and 1:0 xylene:ethanolseries (each for 45 min). Samples were then incubated in the 1:1 ethanol:Paraplast Plus solution (Sigma-Aldrich) at 42°C for 24hand repeatedonce,afterwhich sampleswere embedded inParaplast Plus at 60°C for 4d. Thenthe samples were sectioned into 8-mm–thick sections using a model no.RM2245 Rotary Microtome (Leica). After the removal of Paraplast Plus byseries of xylene and ethanol solutions, sectionswere stainedwith toluidinebluebeforepictures takenusingamodel no. ICC50HDMicroscope (Leica).The images were processed using the software suite ACDSee (https://www.acdsee.com/en/index).

Map-Based Cloning of OsSHI1

A total of 137 genome-wide primer pairs that exhibit polymorphisms be-tween 9311 and 02428 were identified from our primer library. TheOsSHI1locus was initially mapped to an interval between the simple sequencerepeatmarkersRM107andRM189on the longarmof chromosome9using180 F2 mutant plants. Subsequent fine-mapping based on 7,547 progenydelimited the mutant locus to a ;50-kb genomic region with additionalnewly developedmolecular markers (Supplemental Table 3). cDNAs of thefourORFs in thefine-mappedregionwereamplified frombothwild typeandthe shi1mutant (primer pairs listed in Supplemental Table 3), and the PCRproducts were confirmed by sequencing.

RT-PCR and RT-qPCR Analyses

Total RNA was extracted from various tissues using the ZR Plant RNAMiniPrep Kit (Zymo Research) following the manufacturer’s recom-mendations. The first-strand cDNA was synthesized based on theQuantiTectReverse TranscriptionKit (Qiagen). RT-PCRwith 28cycleswasperformed to amplify the four ORFs in the fine-mapped region and24 cycles for ACTIN2 (which was used as the endogenous control).

OsSHI1 Regulates Plant Architecture in Rice 1037

Real time-qPCR was performed on an ABI7500 Real-Time PCR Systemusing SYBR Premix Ex Taq (Takara) with riceUbiquitin as the endogenouscontrol. Relative changes in gene expression levels were quantitatedbased on three biological replicates via the 22OOCt method (Livak andSchmittgen, 2001). All primer pairs used for RT-PCR and Real time-qPCRare listed in Supplemental Tables 3 and 4.

Vector Construction and Plant Transformation

A;5-kb genomic DNA fragment (consisting of a 2.9-kb upstream region,the entire OsSHI1 coding region including two exons, one intron, anda 400-bp downstream region) was amplified with the primer pair OsSHI1-complementation (Supplemental Table 5) and inserted into the HindIII/BamHI restriction sites of the pCUbi1390 binary vector to generate theproOsSHI1:OsSHI1 construct, which was introduced into the calli of shi1via Agrobacterium-mediated transformation using a method described inHiei et al. (1994).

To knock out the OsSHI1, IPA1, OsTB1, and OsDEP1 genes, 20-bpgene-specific spacer sequenceswere cloned into the sgRNA-Cas9 vector(Miao et al., 2013) and subsequently introduced into the calli of Kitaake (orOsSHI1-CRISPR-#1), a japonica variety suitable for transformation, viaAgrobacterium-mediated transformation. Positive transgenic individualswere identified by sequencing or immunoblot analyses.

To verify the expression pattern of OsSHI1, an ;2-kb promoter frag-ment upstream of the ATG start codon was amplified using the primer pairOsSHI1-GUS (Supplemental Table 5) and the PCR product was fused intothe EcoRI/NcoI restriction sites of the binary vector pCAMBIA1305. ThegeneratedpOsSHI1:GUS constructwas introduced into the calli of Kitaakevia Agrobacterium-mediated transformation to generate the pOsSHI1:GUS reporter lines.

To generate the IPA1-overexpressing plants, full-length cDNA of IPA1was amplified using the primer pair IPA1-Flag (Supplemental Table 5) andfused into the XbaI restriction site of the 1300-221-33Flag binary vector.The generated 35S: IPA1-Flag construct was introduced into the calli ofKitaake via Agrobacterium-mediated transformation.

To determine the effect of OsSHI1 overexpression, full-length cDNAof OsSHI1 was amplified using the primer pair OsSHI1-overespression(Supplemental Table 5). The PCR product was inserted into the SmaIrestriction site of the pCAMBIA2300 binary vector to generate the Actin1:OsSHI1-overexpression construct, which was subsequently introducedinto the calli of Kitaake or 35S:IPA1-Flag transgenic plants viaAgrobacterium-mediated transformation.

Subcellular Localization

For subcellular localization of OsSHI1 protein, the 945-bp coding region ofOsSHI1 was inserted into the XbaI restriction site upstream of GFP in thetransient expression vector pAN580 driven by the double CaMV35Spromoter to generate theOsSHI1-GFP construct (primer pairOsSHI1-GFPin Supplemental Table 5). The OsSHI1-GFP plasmid was introduced intothe riceprotoplasts according toprotocols described inZhanget al. (2011).Fluorescence of GFP was observed using a confocal laser scanning mi-croscope (LSM 700; Zeiss).

Transactivation Activity Assay

The full-length OsSHI1 coding region was cloned into the pGBKT7(Clontech) vector at theEcoRI/PstI restriction sites (primer pair OsSHI1-BDin Supplemental Table 5) to generate theOsSHI1-BD construct which wasthen transformed into the Saccharomyces cerevisiae strain AH109, to-gether with empty pGADT7. Transactivation activity assay was performedon selective solid medium followed with quantitative b-gal assay of theLacZ reporter gene using chlorophenolred-beta-D-galactopyranoside

(CPRG) as the substrate. Early heading date1 (Cho et al., 2016) and theempty pGBKT7 vector were used as the positive and negative control,respectively. All procedures were performed according to the manu-facturer’s recommendations (Clontech).

Y2H

The coding region ofOsSHI1was fused to theGAL4 binding domain of the“bait” pGBKT7 vector (Clontech; primer pairs listed in Supplemental Ta-ble 5). A cDNA library prepared from rice young inflorescenceswas used toperform the Y2H screening and positive clones were identified by se-quencing. Full-length and various truncated versions of IPA1were insertedinto theEcoRI/XhoI restrictionsitesof the “prey”pGADT7vector (Clontech;primer pairs listed in Supplemental Table 5). Series of combined bait andprey constructs were cotransformed into the yeast strain AH109 (Clon-tech). After growing on SD-Trp/-Leu plates for 3 d at 30°C, interactionsbetween baits and preys were examined on the selectivemediumSD-Leu/-Trp/-His/-Ade. Yeast strain containing OsSHI1-BD in combination withthe empty pGADT7 vector was used as the negative control. Detailedprocedures were performed according to the manufacturer’s recom-mendations (Clontech).

GUS Staining Assay

GUS histochemical staining was performed according to the methoddescribed inDuanet al. (2016). Briefly, various tissuesof thepOsSHI1:GUStransgenic plants were detached and incubated in the GUS staining bufferovernight. Tissueswere then transferred into alcohol solution to extract thechlorophyll before pictures taken using amodel no. ICC50HDmicroscope(Leica). The images were processed using the software suite ACDSee.

In Vitro Pull-Down Assay

Full-length coding sequences of OsSHI1 and IPA1 were cloned into theexpression vectors pGEX4T-1 and pMAL-c2x, respectively (primer pairslisted in Supplemental Table 5), to generate GST or MBP tag fusion pro-teins. Expression of GST, GST-OsSHI1, andMBP-IPA1 in Escherichia coliBL21 (DE3) cells (TransGen) was induced by 0.4-mM isopropyl-b-D-thiogalactoside at 18°C for 16 h. Fusion proteins were purified usingthe GST Bind Resin (Novagen) or Amylose Resin (New England BioLabs)according to the manufacturer’s protocols, and protein concentrationswere determined by the BSA quantitative assay.

For pull-down assay, roughly equal amounts of purified GST and GST-OsSHI1 proteins were incubated with 30 mL of GST Bind Resin in 1 mL ofPBSsolution for30minwithgentle rotation, afterwhich;2mgof IPA1-MBPfusionproteinwasadded.Aftera further incubation for60min, the resinwaswashed five times with PBS, diluted in 50 mL 13 Protein Loading Buffer,and denatured at 100°C for 10 min before separation on 10% SDS-PAGEgel. Proteins were then transferred to the nitrocellulose (NC) membrane,detected with horseradish peroxidase conjugated anti-GST (PM013-7;MBL International) or anti-MBP (E8032L;NewEnglandBioLabs) antibodiesat 1:5000 dilutions, and visualized with an enhanced chemiluminescencereagent (GE Healthcare).

BiFC

Full-length coding region of OsSHI1 was amplified (primer pair listed inSupplemental Table 5) andcloned into theEcoRI/SalI restriction sitesof thepSPYNE173 (for split YFP N-terminal fragment expression) expressionvector to generate theOsSHI1-eYNE construct. Full-length coding regionsof IPA1 and OsSPL16 were amplified (primer pair listed in SupplementalTable 5) and inserted into theEcoRI/SalI restriction sites of thepSPYCE (forsplit YFP C-terminal fragment expression) expression vector to generate

1038 The Plant Cell

the IPA1-eYCE or OsSPL16-eYCE constructs. For transient expression,Agrobacterium tumefaciens strain EHA105 carrying the combinedOsSHI1-eYNE and IPA1- or OsSPL16-eYCE constructs was coinfiltratedwith the p19 strain into leaves of 5-week–old Nicotiana benthamiana. Theyellow fluorescent protein (YFP) fluorescent signals were monitored 48 to72hafter infiltrationusinga laser confocal scanningmicroscope (model no.LSM 700; Zeiss).

Preparation and Determination of OsSHI1- andIPA1-Specific Antibodies

The synthetic peptides of OsSHI1 (Os09g0531600, SRDPTKRPRARP-SATTP) and IPA1 (Os08g0509600; RIDPGSGSTFDQTSNTMD) wereinjected into rabbits togenerate thecorrespondingpolyclonal antibodiesatABclonal Technology. The specificities of anti-OsSHI1 and anti-IPA1polyclonal antibodiesweredeterminedby immunoblot analysis usingwild-type young panicle tissues. Sampleswere ground into fine powder in liquidN, suspended in 23 volumes of Protein Extraction Buffer (50 mM ofTris–HCl at pH 8.0, 150mMof NaCl, 10mMofMgCl2, 1mMof EDTA, 10%glycerol, and 1 6 Protease inhibitor cocktail) and incubated at 4°C for30 min with rotation. After centrifuging at 12,000g at 4°C for 10 min, thesupernatant was boiled in 13 Protein Loading Buffer before separation on10%SDS-PAGE gel. Proteins were then transferred to the NCmembrane,detectedwith anti-OsSHI1 or anti-IPA1 antibodies at 1:1,000 dilutions andvisualized with enhanced chemiluminescence reagent (GE Healthcare).HSP antibody (AbM51099-31-PU; Beijing Protein Innovation) was used asthe endogenous control.

In Vivo CoIP Assay

Axillary buds of wild-type seedlings were carefully detached and groundinto fine powder in liquid N. Samples were suspended in 43 volumes ofProtein Extraction Buffer (50 mM of Tris–HCl at pH 8.0, 150 mM of NaCl,10 mM of MgCl2, 1 mM of EDTA, 10% (v/v) glycerol, and 13 Proteaseinhibitor cocktail) and incubated at 4°C for 30 min with rotation. Aftercentrifuging at 12,000g for 10 min at 4°C, the supernatant was mixed with30-mLProtein Abeads (Millipore) and incubatedwith rotation at 4°C for 1 h.Beads were pelleted and the cleaned supernatant were incubated with10mLof anti-IPA1–specificpolyclonal antibodies for at least 2 h at 4°Cwithgentle rotation. Thirty-microliter Protein A beadswere added to precipitatethe protein complex with rotation at 4°C for 1 h. Beads were pelleted andwashed four times with prechilled Protein Extraction Buffer. The boundproteins were eluted from the beads with 13 Protein Loading Buffer byboiling at 100°C for 10 min. Protein samples were then separated by 10%SDS-PAGE gel, transferred to NC membrane, and immunoblotted withanti-IPA1 and anti-OsSHI1–specific polyclonal antibodies.

Y1H

Y1Hanalysiswasperformedaccording to themethoddescribed inLinet al.(2007). Briefly, the full-length coding region ofOsSHI1was cloned into thepB42AD vector at the EcoRI restriction site to generate the AD-OsSHI1construct (primer pair OsSHI1-42AD in Supplemental Table 5). The full;2 kb and various truncated versions of promoter regions of OsTB1 orOsDEP1 were amplified (primer pairs listed in Supplemental Table 5) andligated into the XhoI restriction site of the pLacZi reporter vector. Con-structs were then cotransformed into the yeast strain EGY48. Trans-formants were grown on SD-Trp/-Ura plates for 3 d at 30°C and thentransferred onto 5-bromo-4-chloro-3-indolyl-b-d-galactopyranoside platesfor blue color development. Yeast strains containing the empty pB42ADin combination with the LacZ reporter constructs were used as negativecontrols.

Transient Expression Assay in Protoplasts and LUCActivity Determination

Approximately 1-kb and 2-kb promoter regions of OsTB1 and OsDEP1 wereamplified using the primer pairs OsTB1-LUC or OsmTB1-LUC and OsDEP1-LUC (primerpairs listed inSupplemental Table 5) andcloned into theupstreamof LUC reporter gene to generate the OsTB1-LUC or OsmTB1-LUC andOsDEP1-LUC reporter constructs. The luciferase gene fromRenilla reniformis(Ren) under thecontrol ofCaMV35Spromoterwasusedas the internal control.Full-length cDNAs of OsSHI1 and IPA1 were amplified (primer pairs listed inSupplemental Table 5) and inserted into theBamHI/PstI restriction sites of thepAN580 vector to generate the d35S:OsSHI1 and d35S:IPA1 effector con-structs, respectively. The combined reporter and effector plasmids were co-transformed into the rice protoplasts according to a protocol described inZhang et al. (2011). The LUC activity was quantified with a Dual-LuciferaseAssay Kit (Promega) following the manufacturer’s recommendations and therelative LUC activity is calculated as the ratio of LUC/Ren.

ChIP Assay

To assess the enrichment of OsSHI1 or IPA1 at the promoter regions ofOsTB1 and OsDEP1 in vivo, ChIP assays were performed using anti-OsSHI1 or anti-IPA1–specific polyclonal antibodies. Approximately 4 gof wild-type axillary buds or young panicle tissues (1 to 2 g for transgenicplants) were ground into fine powder in liquid N, resuspended in 20 mLExtraction buffer 1 (0.4 M of Suc, 10 mM of Tris-HCl at pH 8.0, 10 mMof MgCl2, 5 mM of b-mercaptoethanol [b-ME], 0.1 mM of phenyl-methylsulfonyl fluoride [PMSF], 13 proteinase inhibitor, and 1% (v/v)formaldehyde), and thoroughlymixed to release thenuclei. After incubationunder vacuum conditions for 30 min, 0.125 M of Gly was added and in-cubated for another 5 min to stop the crosslink reaction. The solution wasfiltered by two layers of Miracloth (Millipore) and centrifuged at 3000g at 4°Cfor 20 min to remove the supernatant. The pellet was resuspended in 1-mLExtraction buffer 2 (0.25 M of Suc, 10 mM of Tris-HCl at pH 8.0, 10 mM ofMgCl2, 1% (v/v) Triton X-100, 5 mM of b-ME, 0.1 mM of PMSF, and 13proteinase inhibitor) and centrifuged at 12,000g at 4°C for 10min. The pelletwas resuspended in 300 mL of Extraction buffer 3 (1.7 M of Suc, 10 mM ofTris-HCl at pH8.0, 2mMofMgCl2, 0.15% (v/v) TritonX-100, 5mMofb-ME,0.1 of mMPMSF, and 13 proteinase inhibitor), laid on top of another clean300mL of Extraction buffer 3 and centrifuged at 15,000g at 4°C for 1 h. Thechromatinpelletwas resuspended in200mLofNuclei lysisbuffer (50mMofTris-HCl at pH 8.0, 10 mM of EDTA, 1% (w/v)SDS, and 13 proteinaseinhibitor) andsonicated to200;500bpwith3-sburst/7s interval frequencyat 2-W power for 33 min. After centrifugation at 12,000g at 4°C for 10 min,the chromatin supernatant was diluted with Dilution buffer (0.01 (w/v)%SDS, 1.1% (v/v) Triton X-100, 1.2 mM of EDTA, 16.7 mM of Tris-HCl at pH8.0, and 167 mM of NaCl) and 1 mL of the diluted chromatin sample wasprecleared with 20-mL Protein A beads (Millipore) for 1 h at 4°C with ro-tation. Then 10 mL of anti-OsSHI1 or anti-IPA1–specific polyclonal anti-bodies together with 10 mL of BSA (10 mg/mL) were added to theprecleared sample and incubated overnight with gentle rotation. Onemicroliter of salmon sperm DNA (10 mg/mL) was added as the blockingreagent and the immune complexes were collected by 30-mL Protein Abeads for 1 h at 4°C with rotation. The Protein A beads were washedstepwisewitha lowsaltwashbuffer (0.1% (w/v)SDS,1%(v/v) TritonX-100,2 mM of EDTA, 20 mM of Tris-HCl at pH 8.0, and 150 of mM NaCl), a highsalt wash buffer (0.1%(w/v) 1% (v/v) Triton X-100, 2mMof EDTA, 20mMofTris-HCl at pH 8.0, and 500 mM of NaCl), the LiCl wash buffer (0.25 M ofLiCl, 1% (v/v) Nonidet P-40, 1% (w/v) deoxycholic acid sodium, 1 mM ofEDTA, and20mMofTris-HCl atpH8.0), andTEbuffer (10mMofTris-HCl atpH 8.0 and 1 mM of EDTA) each two times. The immune complexes wereeluted from the Protein A beads by incubating with 250mL of Elution buffer(0.1M of NaHCO3 and 1% (w/v)SDS) at 65°C for 20min with agitation. Thesupernatant was transferred to another tube to repeat elution and the two

OsSHI1 Regulates Plant Architecture in Rice 1039

eluateswere combined. Twentymicroliters of NaCl (5M), 10mLof RNaseA(10mg/mL), and 2.5mL of protease K (10mg/mL) were added to the elutedsolution (for the input sample, two volume amounts were added) and in-cubatedat65°C forat least6hwithagitation.The immunoprecipitatedDNAwasextractedwith isopropyl alcoholprecipitation.The recoveredDNAwasused as the template forChIP-qPCRand the enrichmentwas calculated asthe ratio of immunoprecipitation (IP) to Input. The ChIP-reChIP assay wasperformedaccording toprotocol reported in Furlan-Magaril et al. (2009). Allprimer pairs are listed in Supplemental Table 6.

EMSA

A quantity of 39-digoxigenin-labeled probes containing the putative OsSHI1binding sites were synthesized by Invitrogen (primer pairs listed inSupplemental Table 7). EMSAs were performed with the DIG Gel Shift Kit(Roche, 03353591910) following the manufacturer’s recommendations.Briefly, equal amounts of complementary oligonucleotides were incubated at95°C for 10min, cooleddownslowly to15°C (0.1°C/1s), anddiluted to50 fmol/mLfinalconcentrations.TheDNAbindingreactionwasperformedwith100-fmolprobe, 2-mg poly (dI-dC), and 100-ng purified MBP or MBP-OsSHI1proteins and incubated at room temperature for 30 min. Then the sam-ples were immediately applied to the pre-run native polyacrylamide gelcontaining 6.5% acrylamide in 0.53 Tris-Borate-EDTA buffer. Afterelectro-blotting onto a nylon membrane (Millipore), the oligonucleotideswere crosslinked using UV-light. The membrane was incubated ina blocking solution for 30 min, followed by incubating in a DIG antibodysolution for another 30 min. After intensive washing with a washing buffer,CSPD working solution was applied to the membrane to visualize thesignal.

Accession Numbers

Sequence data from this article can be found in the EMBL/GenBank datalibraries under the following accession numbers: OsSHI1, Os09g0531600;IPA1, Os08g0509600; OsSPL16, Os08g0531600; OsTB1, Os03g0706500;OsDEP1, Os09g0441900; UBIQUITIN, Os03g0234200; and ACTIN2,Os03g0718100.

Supplemental Data

Supplemental Figure 1. Normal initiation of axillary buds in shi1.

Supplemental Figure 2. Grain, leaf, and culm morphologies of wildtype and shi1.

Supplemental Figure 3. Phenotypes of ORF2 (OsSHI1) knockoutplants.

Supplemental Figure 4. Phenotypes of ORF2 (OsSHI1) overexpres-sion plants.

Supplemental Figure 5. Characterization and sequence analysis ofOsSHI1.

Supplemental Figure 6. OsSHI1 exhibits weak transcriptional activa-tion activity in yeast cells.

Supplemental Figure 7. Histochemical staining of the pOsSHI1:GUStransgenic plants.

Supplemental Figure 8. Specificity tests of the anti-OsSHI1 poly-clonal antibodies used for ChIP and immunoblot analyses.

Supplemental Figure 9. Protein induction and purification used forimmunoblot and EMSAs.

Supplemental Figure 10. Schematic depictions of the cis-elements inthe promoter regions of OsTB1 and OsDEP1.

Supplemental Figure 11. Sequence analysis of the promoter regionsof OsTB1 and OsDEP1.

Supplemental Figure 12. Specificity tests of the anti-IPA1 polyclonalantibodies used for ChIP and immunoblot analyses.

Supplemental Figure 13. The zinc finger domain is indispensable forthe DNA binding ability of OsSHI1.

Supplemental Figure 14. OsSHI1 represses the DNA binding ability ofIPA1 independent of OsSHI1 binding.

Supplemental Figure 15. Generation and identification of mutantsgenerated by CRISPR/Cas9 genome-editing approach.

Supplemental Figure 16. OsSHI1 does not affect the expression levelof IPA1.

Supplemental Figure 17. IPA1 does not interfere with the DNAbinding ability of OsSHI1.

Supplemental Figure 18. A proposed working model for OsSHI1 inthe regulation of plant architecture in rice.

Supplemental Table 1. Phenotypic segregation identified by test forgoodness-of-fit.

Supplemental Table 2. Putative interacting partners of OsSHI1identified by Y2H screening.

Supplemental Table 3. Primers used for map-based cloning.

Supplemental Table 4. Primers for detection of the RNA levels ofselected genes.

Supplemental Table 5. Primers used for vector constructions.

Supplemental Table 6. Primers used for ChIP-qPCR analyses.

Supplemental Table 7. DIG-labeled oligonucleotides used for EMSAs.

ACKNOWLEDGMENTS

This researchwassupportedbygrants fromtheNationalKeyResearchandDevelopment Program of China (2016YFD0100901), the National NaturalScience Foundation of China (31671769), the Guangdong Province-National Natural Science Foundation of China (U1701232), and the Na-tional Transgenic Science and Technology Program (2016ZX08001004-002).

AUTHOR CONTRIBUTIONS

JM.W. supervised the project; L.J., H.W., and JM.W. designed the re-search; E.D., X.L., Q.L., T.Z., Y.W., C.Z., andH.Z. performed research; E.D.,X.L., T.Z., and JM.W. analyzed data; Y.W. provided the plant material; C.L.and JL.W. cultivated the transgenic plants in the field; X.Z. and X.G.generated the transgenic plants; E.D. drafted the manuscript; H.W. andJM.W. revised the manuscript.

Received January 17, 2019; revised February 27, 2019; accepted March24, 2019; published March 25, 2019.

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DOI 10.1105/tpc.19.00023; originally published online March 25, 2019; 2019;31;1026-1042Plant Cell

Zhang, Ling Jiang, Jiulin Wang, Cailin Lei, Xin Zhang, Xiuping Guo, Haiyang Wang and Jianmin WanErchao Duan, Yihua Wang, Xiaohui Li, Qibing Lin, Ting Zhang, Yupeng Wang, Chunlei Zhou, Huan

RiceOsSHI1 Regulates Plant Architecture Through Modulating the Transcriptional Activity of IPA1 in

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