The alteration in the architecture of a T‐DNA insertion rice mutant osmtd1 is caused by up‐regulation of MicroRNA156f

Abstract Plant architecture is an important factor for crop production. Some members of microRNA156 (miR156) and their target genes SQUAMOSA Promoter‐Binding Protein‐Like (SPL) were identified to play essential roles in the establishment of plant architecture. However, the roles and regulation of miR156 is not well understood yet. Here, we identified a T‐DNA insertion mutant Osmtd1 (Oryza sativa multi‐tillering and dwarf mutant). Osmtd1 produced more tillers and displayed short stature phenotype. We determined that the dramatic morphological changes were caused by a single T‐DNA insertion in Osmtd1. Further analysis revealed that the T‐DNA insertion was located in the gene Os08g34258 encoding a putative inhibitor I family protein. Os08g34258 was knocked out and OsmiR156f was significantly upregulated in Osmtd1. Overexpression of Os08g34258 in Osmtd1 complemented the defects of the mutant architecture, while overexpression of OsmiR156f in wild‐type rice phenocopied Osmtd1. We showed that the expression of OsSPL3, OsSPL12, and OsSPL14 were significantly downregulated in Osmtd1 or OsmiR156f overexpressed lines, indicating that OsSPL3, OsSPL12, and OsSPL14 were possibly direct target genes of OsmiR156f. Our results suggested that OsmiR156f controlled plant architecture by mediating plant stature and tiller outgrowth and may be regulated by an unknown protease inhibitor I family protein.


INTRODUCTION
Plant architecture is important for plant morphology and crop production. Branching and plant height are key factors affecting plant architecture. As specialized branches bearing grains, rice tillers are formed on the un-elongated basal internodes and grow independently from the mother culm with its own adventitious roots (Li 1979;Wang and Li 2005). Rice tillering is important for rice ideal plant architecture (IPA) which indicates a rice variety without unproductive tillers, with more grains per panicle, and with thick and sturdy stems. Because IPA plants have more potential for higher yields in rice (Jiao et al. 2010;Miura et al. 2010), it is feasible to increase rice yield by reducing the unproductive tillers in plant breeding.
MicroRNAs (miRNAs) are 21-24 nucleotide RNAs binding to target genes by imperfect base pairing and cause cleavage of target mRNA or repression of translation (Chen 2008). Being regarded as universal regulators of gene expression, many miRNAs have been proved to be essential in regulating plant architecture (Schwab et al. 2005;Xie et al. 2006;Chen 2008;Wang et al. 2011a;Fu et al. 2012;Wei et al. 2012). Among them, the microRNA156 (miR156) family is a class of miRNAs playing pivotal roles in plant architecture. The miR156 genes are conserved and highly expressed miRNAs in the plant kingdom. Computational and experimental analysis indicated that miR156s directly regulate the expression of SQUAMOSA-PROMOTER BINDING LIKE (SPL) genes (Rhoades et al. 2002;Xie et al. 2006;Guo et al. 2008). SPL genes encode plant-specific transcription factors containing a DNA-binding motif highly conserved in SQUA promoter-binding protein (SBP) (Shikata et al. 2009). The miR156 and its target SPL genes are involved in various developmental processes. In Arabidopsis, miR156 controls proper pattern formation during early embryogenesis by mediating SPL10 and SPL11 (Nodine and Barte 2010). The miR156, miR172, and SPL genes (including SPL3/4/5, SPL9, SPL10, SPL15) determine plant development by promoting juvenile/ adult phase transition (Chuck et al. 2007;Wu et al. 2009;Jung et al. 2011;Willmann and Poethig 2011;Yang et al. 2011). The miR156-targeted SPL factor SPL9 affects leaf plastochron length and organ size ) and regulates the anthocyanin accumulation during the transition from the leaf to flower formation (Gou et al. 2011). Enhanced miR156b expression leads to branching, altered trichome morphology, and increased seed carotenoid levels through the suppression of SPL15 and other SPL target genes (Wei et al. 2012). In addition, it is also reported that miR156 is a phloem-mobile signal regulating the process of tuberization in potato by targeting StSPL3, StSPL6, StSPL9, and StSPL13 (Bhogale et al. 2014). In switchgrass, overexpression of miR156 genes improves biomass by changing apical dominance and floral transition through suppressing target SPL genes (Chuck et al. 2011;Fu et al. 2012).
Although miR156s have been elucidated to regulate diverse processes in plants, the function and regulation of OsmiR156 family genes in rice remain elusive. In this paper, we identified a rice T-DNA insertion mutant Osmtd1 (Oryza sativa multi-tillering and dwarf mutant). The Osmtd1 produced many more tillers and reduced plant height. We located one T-DNA insertion in Os08g34258, an unknown gene encoding a putative protease inhibitor, in Osmtd1. Overexpression of Os08g34258 complemented the multi-tillering and dwarf phenotypes of Osmtd1. We demonstrated that OsmiR156f was upregulated and OsSPL3, OsSPL12, and OsSPL14 were downregulated in Osmtd1. Our findings provided a new layer of understanding on the function and regulation of OsmiR156f in rice architecture establishment.

RESULTS
The Osmtd1 mutant exhibited dwarf and bushy phenotypes We identified a mutant from a rice T-DNA insertion mutant collection and named it Osmtd1 because the mutant displayed multi-tillering and dwarfism phenotypes. Although no differences between Osmtd1 and wild-type plants were observed at the seedling stage, Osmtd1 produced plant architecture distinct from wild-type at the tillering stage. The phenotypes of Osmtd1 include decreased plant height and increased tiller number ( Figure 1A). To further illustrate Osmtd1 phenotypes, we investigated the plant height and tiller number in a time course during plant development. Osmtd1 was shorter than wild-type rice from 45 to 104 d after sowing (DAS). At 75 DAS, the Osmtd1 height was no longer increased, while the wildtype rice further grew to a higher stature ( Figure 1B). The analysis of tiller number indicated that there were no obvious differences between wild-type rice and Osmtd1 found at 45 DAS. However, Osmtd1 produced more tillers than wildtype rice after 62 DAS ( Figure 1C). Usually, rice tillers are only formed on the un-elongated basal internodes of rice main culm in wild-type rice. However, we observed the outgrowth of tiller buds at the elongated higher internodes in Osmtd1 ( Figure 1D). In addition, the grain number was reduced and the primary panicle branches were shorter in Osmtd1 ( Figure 1D).
It has been suggested that dwarfism may be the secondary effect of the formation of more tillers, because removal of tillers rescues plant height in some rice mutants (Zou et al. 2006). To evaluate whether dwarf phenotype is caused by the excessive tillers in Osmtd1, we removed the new axillary tiller buds of wild-type or Osmtd1 every day from tillering stage to reproductive stage, and measured the plant height. The results showed that removal of tillers did not increase the height of Osmtd1, but did increase the height of wild-type plants ( Figure 1E), indicating that the dwarf trait of Osmtd1 was independent from multi-tillering and the mutation caused the high tillering and dwarfism simultaneously.
The phenotypes of Osmtd1 were caused by a single T-DNA insertion As Osmtd1 was obtained from a T-DNA mutant collection, we first test whether the phenotypes of Osmtd1 could be resulted from T-DNA insertion. We crossed Osmtd1 with the wildtype rice cultivar Zhonghua 11 (ZH11) and then analyzed the F 1 plants. We found that the heterozygous Osmtd1 displayed moderate phenotypes in terms of the plant height, tiller number, length of main panicles, and the total and filled grain number (Table 1), indicating that Osmtd1 was a semi-dominant mutant. The T-DNA insert harbor a bar resistance gene conferring the transgenic plants resistant to the herbicide phosphinothricin (PPT). We then investigated whether the PPT resistance could be cosegregated with the phenotypes of Osmtd1. We generated an F 1 B 1 population by crossing F 1 plants to ZH11. The progenies in the F 1 B 1 population were either PPT-resistant or PPT-sensitive. We found that all the PPTresistant plants displayed moderate dwarf and multi-tillering phenotypes, while all the PPT-sensitive plants had no difference from wild-type ZH11. We further generated the F 2 populations. We analyzed the segregation ratio of PPTresistant plants to PPT-sensitive ones using the F 2 populations and found that the ratio is statistically 3:1 (Table 2). Phenotype analysis showed that all the PPT-resistant plants in the F 2 populations displayed strong or moderate phenotypes in plant architecture, while the PPT-sensitive plants showed no phenotypes (Table 2), indicating that the phenotypes The influence of rice plant height after removing axillary tiller buds in Osmtd1 and ZH11. Plant height of Osmtd1 did not increase after removing the tillers while the wild-type rice ZH11 did. Bar, 10 cm. All data and pictures were examined using the plants grown in the pots.
of Osmtd1 may be cosegregated with PPT-resistance conferred by T-DNA insertion. Furthermore, we observed that the ratio of the plants without phenotypes, the ones with moderate phenotypes and the ones with strong phenotypes, is approximately 1:2:1 ( Table 2), suggesting that the phenotypes of Osmtd1 were possibly caused by a single mutated locus.
The T-DNA insertion was located in an unknown gene To reveal the molecular base of the phenotypes in Osmtd1, we first identified the T-DNA flanking sequence by thermal asymmetric interlaced polymerase chain reaction (TAIL-PCR) . A BLAST search on the Rice Genome Annotation Project website showed that the T-DNA insertion was localized in the middle of an unknown gene, Os08g34258. Os08g34258 has no introns and OsmiR156f was located in the 3.3 kb downstream of it (Figures 2A, S1). A database search showed that Loc_Os08g34249 had the same sequence as Os08g34258 in rice genome. Reverse transcription (RT)-PCR analysis revealed that Os08g34258 was possibly knocked out in Osmtd1 because the T-DNA was inserted in the exon of Os08g34258 and the trace of product may be derived from Os08g34249 ( Figure 2B). Os08g34258 encodes a putative protein containing only 67 amino acid residues and a conserved domain shared by the protease inhibitor I family (potato inhibitor type I). This protein family contains a class of small protease inhibitors widely spreading in plants and has been found in potato, tomato, barley, pumpkin, buckwheat, Solanum nigrum, and so on (Melville and Ryan 1972; Svendsen et al. 1980;Plunkett et al. 1982;Krishnamoorthi et al. 1990;Hartl et al. 2010;Wang et al. 2011). No functions of this kind of protease inhibitor have been reported in rice. To first test whether Os08g34258 could encode a protein, we cloned Os08g34258 and generated the construct 35Spro-Os08g34258-GFP in which Os08g34258 was fused with GFP and driven by the CaMV 35 S promoter. We transformed 35Spro-Os08g34258-GFP into tobacco leaves. Western blotting showed that a clear Os08g34258-GFP band was observed in the isolates from leaves transformed with 35Spro-Os08g34258-GFP but not in the control, indicating that Os08g34258 is a real gene ( Figure 2C). We then generated the construct 35Spro-Os08g34258 in which Os08g34258 was driven by the CaMV 35 S promoter. We transformed 35Spro-Os08g34258 into Osmtd1 and obtained 31 transgenic rice plants which recovered to the architecture of wild-type plants ( Figure 2D), indicating that disruption of Os08g34258 by T-DNA insertion caused the defects of plant architecture in Osmtd1. To further demonstrate the roles of Os08g34258 in rice architecture, we performed RNA interference (RNAi) to knockdown Os08g34258 in wild-type ZH11. In the RNAi plants, we indeed observed the dwarfism and multi-tillering phenotypes similar to those observed in Osmtd1 ( Figure S2), indicating that Os08g34258 may play important roles in plant architecture.
OsmiR156f was upregulated in Osmtd1 The OsmiR156 family gene OsmiR156d and OsmiR156h play essential roles in plant architecture (Xie et al. 2006). We found that Osmtd1 displayed similar phenotypes as those observed   in the OsmiR156d or OsmiR156h overexpressing rice plants.
Considering that the Os08g34258 gene is located in the 3.3 kb upstream of OsmiR156f (Figure 2A), we hypothesized that OsmiR156f may be regulated in Osmtd1. To test the hypothesis, we analyzed the expression level of OsmiR156. Interestingly, the transcripts of the mature OsmiR156 were increased significantly in Osmtd1 ( Figure 3A). We further determined that the transcripts of the OsmiR156f precursor were increased in Osmtd1 (Figure 3B), suggesting that the upregulation of OsmiR156f may be the molecular base of multi-tillering and dwarfism in Osmtd1.
To further confirm that the excessive production of OsmiR156f accounts for the phenotypes of Osmtd1, we generated the construct UBQpro-OsmiR156f in which the OsmiR156f precursor was driven by the UBIQUITIN1 promoter from maize (Cornejo et al. 1993). We transformed UBQpro-OsmiR156f into ZH11. Among the 217 UBQpro-OsmiR156f transgenic lines, more than 180 plants displayed dwarfism and multi-tillering similar to those observed in Osmtd1. Some lines showed severe phenotypes with more than 100 tillers ( Figure 3C). Similar to Osmtd1, the axillary tiller buds in higher internodes were also observed in UBQpro-OsmiR156f transgenic lines ( Figure 3C). We then analyzed the expression level of OsmiR156f in the UBQpro-OsmiR156f transgenic line, Osmtd1, or wild-type ZH11. The results showed that the OsmiR156f expression level was highly increased in the UBQpro-OsmiR156f transgenic line and in Osmtd1, when compared with that in wild-type ZH11. These results demonstrated that OsmiR156f was critical for rice tillering and plant height.
OsmiR156f regulated SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) genes at a post-transcriptional level It has been suggested that SPL genes are the direct targets of miR156 (Rhoades et al. 2002;Xie et al. 2006;Guo et al. 2008).

DISCUSSION
Plant architecture is crucial for yield in rice. The development of IPA has been proved to facilitate rice yield increase (Jiao et al. 2010;Miura et al. 2010). Plant height and tiller number are the two most important agronomic traits for IPA in rice. In this study, we identified a new semi-dominant rice mutant, Osmtd1, that displayed dwarfism, multi-tillering, late flowering, and shortened panicle size. Molecular analysis suggested that Os08g34258 encoding a protease inhibitor I family protein and the miRNA gene OsmiR156f were affected in Osmtd1. Overexpression of Os08g34258 complemented the phenotypes of Osmtd1, while overexpression of OsmiR156f in the wild type recapitulated abnormal phenotypes of Osmtd1. Our findings established the important roles of Os08g34258 and OsmiR156f in control of plant architecture in rice.
Rice tillering is affected by various factors including the ambient temperature, the plant density, the axil position, and the plant age. Genetic analysis has identified important genes essential for tiller development. The temporal and spatial accumulation of these regulators determined the proper development of rice tillers. MOC1 initiates axillary buds and promoted tiller outgrowth. MOC1 encodes a putative GRAS family nuclear protein regulated by 26 S proteasome degradation (Li et al. 2003;Lin et al. 2012;Xu et al. 2012). OsPUP7 has a dominant effect on rice tiller number by regulating the transport of cytokinins (Qi and Xiong 2013). Whereas, OsTB1 encoding a TCP transcription factor represses axillary bud activity (Takeda et al. 2003). Several factors involved in strigolactone (SL) signaling and biosynthesis have been found to be important for controlling rice architecture. D3 is a homolog of Arabidopsis MAX2/ORE9 possessing leucinerich repeat (LRR) and F-box domain and is an important component in SL signaling in control of rice tillering (Ishikawa et al. 2005). D53 functions as a repressor of SL signaling and affects axillary bud outgrowth Zhou et al. 2013). D14 encodes a proposed SL receptor for perception in SL signaling and inhibits rice tillering (Arite et al. 2009;Nakamura et al. 2013). HTD1, D27, and D10 all function in SL biosynthesis and control rice tillering (Zou et al. 2006;Arite et al. 2007;Lin et al. 2009). Some factors including LAZY1, LPA1, and PROG1 regulate the angle of rice tillering (Li et al. 2007;Jin et al. 2008;Tan et al. 2008;Wu et al. 2013). In this paper, we identified a new gene, Os08g34258, that may also participate in the regulation of rice tillering and plant height. Os08g34258 encodes a protease inhibitor I family protein which serves as a defensive compound against pathogens. The protease inhibitor inactivates the hydrolytic cleavage ability of proteases which are important pathogenic substances secreted by insects and pathogenic microorganisms. We first discovered the roles of the protease inhibitor I family protein in the development of plant architecture. This protein is completely different from the above-mentioned known factors which are transcription factors, signaling components, and enzymes in SL biosynthesis, indicating that Os08g34258 may use a new mechanism to control plant architecture.
The miRNA miR156 family contains 12 members in rice and is highly conserved in plants. Some members of this gene family have been identified to play essential roles in plant development. Constitutive expression of miR156 homologous genes in different plants causes similar morphological changes including increased number of axillary buds and dwarfism, late flowering, and low fertility (Schwab et al. 2005;Xie et al. 2006;Chuck et al. 2007Chuck et al. , 2014. In this paper, we demonstrated that the mutant Osmtd1 and OsmiR156f overexpression lines showed dramatic phenotypes similar to those observed in the overexpression lines of OsmiR156d or OsmiR156h (Xie et al. 2006). However, we found that OsSPL3, OsSPL12, and OsSPL14, but no other OsSPLs, were targeted by OsmiR156f, indicating that different OsmiR156 genes may have differentiated functions in the regulation of rice development. In addition, we unexpectedly found that the expression level of OsSPL9 was significantly increased in the OsmiR156f overexpression lines, suggesting that the regulation between OsSPL family genes and OsmiR156 family may be complicated.
In the Osmtd1 mutant, the expression level of Os08g34258 was possibly knocked out, while the expression level of OsmiR156f was increased. Interestingly, when we searched the rice database, we found that Loc_Os08g34249 had the same sequence as Loc_Os08g34258. So, it is possible that Loc_Os08g34249 may also be expressed and may have redundant function with Loc_Os08g34258. However, the expression level of Loc_Os08g34249 is rather low, so it is reasonable that disruption of Loc_Os08g34258 may lead to obvious phenotypes in rice. The generation of double knockout mutants would be very important for ultimately revealing the function of Loc_Os08g34258 and Loc_ Os08g34249 in the future. The fact that either knockout of Os08g34258 or overexpression of OsmiR156f led to multitillering and short stature in Osmtd1 implied that Os08g34258 may regulate the abundance of OsmiR156f, although we cannot provide enough evidence to connect them at present. Especially, OsmiR156f is only located in approximately 3.3 kb downstream of Os08g34258. It is possible that the T-DNA insertion may cause both knockout of Os08g34258 and overexpression of OsmiR156f. Further experiments will be needed to verify the possible relationship between OsmiR156f and Os08g34258. We hypothesize that two mechanisms may exist if OsmiR156f were regulated by Os08g34258 during rice tillering. First, Os08g34258 may control the OsmiR156f transcriptions by indirectly regulating unknown transcription factors which control the transcripts of OsmiR156f. Second, as Os08g34258 encodes a putative protease inhibitor, it may regulate the OsmiR156f abundance by negatively affecting the process of miRNA biogenesis. In plants, there are at least two important and critical steps involved in cleavage or dicer processing during miRNA biogenesis: the processing of pri-miRNAs to pre-miRNAs, and further processing to miRNA-miRNA* duplex by DCL1 and its interacting partners (Kurihara and Watanabe 2004;Qi et al. 2005;Kurihara et al. 2006;Chen 2008). Based on the fact that both protease and DCL1 perform a similar role in cleavage of their targets, the protease inhibitor encoded by Os08g34258 may affect the activities of DCL1 or other enzymes during miRNA biogenesis. The investigation of the interactions between Os08g34258 and the components in the miRNA pathway including DCL1, HYL1 (Kurihara et al. 2006), SE (Lobbes et al. 2006;Yang et al. 2006), HEN1 (Yu et al. 2005), AGO (Baumberger and Baulcombe 2005;Qi et al. 2005Qi et al. , 2006, and HASTY (Bollman et al. 2003;Park et al. 2005) would provide further evidence to prove the possible implications of Os08g34258 in regulation of miRNA biogenesis in the future.

Plant materials
A multi-tillering and dwarf rice mutant Osmtd1 was obtained by a T-DNA insertion mutant collection. The plasmid including maize Ds sequence and a bar gene (Wang et al. 2000) was induced in Oryza Sativa spp. japonica cv. ZH11. Agronomic traits were investigated in detail between Osmtd1 and ZH11 at different developmental stages. Genetic analysis was carried The expression level of OsSPL genes in the young culms from an UBQpro-OsmiR156f transgenic line, Osmtd1, or Zhonghua 11 (ZH11) was revealed by quantitative reverse transcription polymerase chain reaction. The expression levels of the genes in ZH11 were set to 1.0. The error bars represent the standard deviation of three biological replicates. out using Osmtd1 or ZH11 as pollen donors to obtain F 1 hybrids of Osmtd1 Â ZH11 or ZH11 Â Osmtd1. Reciprocal crossing experiments were carried out between F 1 hybrids and Osmtd1 or ZH11. The agronomic traits of plant height, tiller number, and panicle length were examined in different hybrid populations.

Constructs and transformation
To generate 35Spro-Os08g34258 in which the Os08g34258 gene was driven by the CaMV 35 S promoter, the coding region of Os08g34258 was amplified from spp. japonica (cv. Nipponbare) genomic DNA using primers 5 0 -ATG AGC CAG AAG TCG TGG C-3 0 and 5 0 -ACA CAT GAA CGT ACA CGG CGC C-3 0 . The fragment was cloned into the EcoR V site of pBluescript SK þ (pBS) (designated pBS-inh) and was sequenced. The overexpression construct of 35Spro-Os08g34258 was generated by ligation of the Kpn I/Xba I digested fragment from pBS-inh and vector pQG111 digested by the same restriction enzymes (Tao et al. 2013). 35Spro-Os08g34258 was then transformed into the mutant Osmtd1.
To generate 35Spro-Os08g34258-GFP, the coding region of Os08g34258 was amplified using the primers 5 0 -CAC CAT GAG CCA GAA GTC GTC G-3 0 and 5 0 -ACC GAT GAC GGG AAT TTT GA-3 0 . The fragment was cloned into the vector pENTR/D-TOPO (Invitrogen, San Diego, CA, USA) to generate pENTRY-Os08g34258. The 35Spro-Os08g34258-GFP was generated by LR reaction between pENTRY-Os08g34258 and pK7FWG2. 35Spro-Os08g34258-GFP was infiltrated into the leaves of tobacco by Agrobaterium tumefaciens-mediated transformation. The tobacco plants were grown in the dark for 24 h and then in a greenhouse under normal conditions for 48 h. Protein isolates were mixed with sample buffer and separated in 12% sodium dodecylsulfate polyacrylamide gel electrophoresis minigel and then were blotted onto nitrocellulose membranes for western blotting.
To generate UBQpro-Os08g34258-RNAi to knockdown Os08g34258 in ZH11, the fragment was amplified from pBSinh plasmid DNA using the primers 5 0 -ATG AGC CAG AAG TCG TGG C-3 0 and 5 0 -ACG GAC GCG CTT GTC GTT GA-3 0 . The fragment was cloned into the EcoR V site of pBluescript SK þ to obtain the sense insert named as pBS-4inh and antisense insert named pBS-A4inh. The intron-containing construct pBS-A4inh-GUS-4inh was first generated by ligating four fragments including pBS vector digested by Cla I/Pst I, A4inh fragment from pBS-A4inh by Hind III/Pst I digestion, the 1 kb fragment GUS fragment from pBS-GUS by EcoR I/Hind III digestion, and 4inh fragment from pBS-4inh Cla I/EcoR I. The UBQpro-Os08g34258-RNAi were generated by ligating the pWM101 vector digested by Hind III/Sal I, the ubiquitin promoter from pBS-pUBQ with Hind III/BamH I digestion, and A4inh-GUS-4inh fragment from pBS-A4inh-GUS-4inh digested by Sal I/BamH.
To generate UBQpro-OsmiR156f in which the OsmiR156f gene was driven by a UBIQUITIN1 promoter, the OsmiR156f precursor was amplified from the Nipponbare genomic DNA using primers 5 0 -CGC CCA CCT TTC TCC CA-3 0 and 5 0 -AAG GAG CAG TTA GAT AAT GGA G-3 0 . The fragment was cloned into the EcoR V site of pBS (designated pBS-miR156) and sequenced. The Ubiquitin promoter was released by digesting the pBS-pUbq construct  with Hind III/BamH I. The pBS-miR156 was digested by Sal I/BamH I, and the fragments including OsmiR156f, the pWM101 vector digested by Hind III/ Sal I, and the Ubiquitin promoter released from pBS-pUBQ with Hind III/BamH I digestion were ligated to obtain UBQpro-OsmiR156f. UBQpro-Os08g34258-RNAi and UBQpro-OsmiR156f were then transformed into ZH11. The A. tumefaciensmediated transformation was employed as previously reported (Liu et al. 1998).

Polymerase chain reaction analysis
The T-DNA flanking sequence in Osmtd1 was isolated by TAIL-PCR method . Three specific primers (5 0 -CGA TTA CCG TAT TTA TCC CGT TCG-3 0 , 5 0 -GTT ACC GGT ATA TCC CGT TTT CG-3 0 , and 5 0 -GAG GTA TTT TAC CGA CCG TTA CCG-3 0 ) complementary to the Ds sequence were used as described ). The T-DNA insertion site was obtained by searching the T-DNA flanking sequence in the rice genome using BLAST (Basic Local Alignment Search Tool) software.
To perform RT-PCR, qRT-PCR, and Stem-loop RT-PCR, total RNA was isolated using Trizol reagent (Invitrogen) and removed DNA contamination with RNase-free DNase I (Invitrogen). M-MLV kit (Invitrogen) was used according to the manufacturer's instructions to synthesize first-strand cDNA for PCR.

SUPPORTING INFORMATION
Additional supporting information may be found in the online version of this article at the publisher's web-site. Figure S1. The T-DNA flanking sequence in Osmtd1 mutant Figure S2. RNA interference (RNAi) showed that disruption of Os08g34258 caused dwarfism and multi-tillering phenotypes similar to those observed in Osmtd1.