Short tandem target mimic rice lines uncover functionsof miRNAs in regulating important agronomic traitsHui Zhanga,b,c,1, Jinshan Zhanga,b,d,1, Jun Yana,b,c, Feng Goua,b,d, Yanfei Maoa,b, Guiliang Tange, José Ramón Botellaf,and Jian-Kang Zhua,b,c,2

aShanghai Center for Plant Stress Biology, Chinese Academy of Sciences, Shanghai 201602, People’s Republic of China; bCenter of Excellence in MolecularPlant Sciences, Chinese Academy of Sciences, Shanghai 201602, People’s Republic of China; cDepartment of Horticulture and Landscape Architecture,Purdue University, West Lafayette, IN 47907; dUniversity of Chinese Academy of Sciences, Shanghai 201602, People’s Republic of China; eDepartment ofBiological Sciences, Michigan Technological University, Houghton, MI 49931; and fPlant Genetic Engineering Laboratory, School of Agriculture and FoodSciences, University of Queensland, Brisbane, QLD 4072, Australia

Contributed by Jian-Kang Zhu, March 31, 2017 (sent for review March 6, 2017; reviewed by Shou-Wei Ding and Ramanjulu Sunkar)

Improvements in plant agricultural productivity are urgently neededto reduce the dependency on limited natural resources and produceenough food for a growing world population. Human interventionover thousands of years has improved the yield of important crops;however, it is increasingly difficult to find new targets for geneticimprovement. MicroRNAs (miRNAs) are promising targets for cropimprovement, but their inactivation is technically challenging andhas hampered functional analyses. We have produced a largecollection of transgenic short tandem target mimic (STTM) linessilencing 35 miRNA families in rice as a resource for functionalstudies and crop improvement. Visual assessment of field-grownmiRNA-silenced lines uncovered alterations in many valuable agro-nomic traits, including plant height, tiller number, and grain number,that remained stable for up to five generations. We show thatmanipulation of miR398 can increase panicle length, grain number,and grain size in rice. In addition, we discovered additional agronomicfunctions for several known miRNAs, including miR172 and miR156.Our collection of STTM lines thus represents a valuable resource forfunctional analysis of rice miRNAs, as well as for agronomic improve-ment that can be readily transferred to other important food crops.

small RNA | microRNA | silencing | yield | crop

Population growth worldwide is skyrocketing and expected tocontinue, making food security one of the most important

challenges facing humanity in the 21st century. To meet an in-creasing demand for food, crop production may need to increaseby 100–110% between 2005 and 2050 (1). Unfortunately, thisdemand for increased food production cannot be met by simplyexpanding the area of cultivation because most arable land is al-ready in use (2). Climate change will add an additional level ofuncertainty to agriculture in the future. The need to increase foodproduction necessitates the development of vastly superior cropsthat not only require less water, chemical fertilizers, and pesticidesbut also deliver higher yields. Rice is a staple food for nearly halfof the world’s population and provides almost one-quarter of theglobal per capita dietary energy supply. Many quantitative traitloci (QTLs) and genes have been identified and shown to controlimportant agronomic traits in rice (3, 4); however, our knowledgeof the gene networks controlling such traits is still limited.New strategies are needed to unlock the full potential of the rice

genome. The use of microRNAs (miRNA) as targets for cropimprovement is promising, although it has not yet been thoroughlyexplored (5). Homology-mediated pairing of plant miRNAs, pre-dominantly 21 nt in length, with their mRNA targets triggersmRNA cleavage or translational inhibition and, ultimately, post-transcriptional gene repression (6). The first plant miRNA wasreported in 2002 (7), and there are currently in excess of 7,000confirmed or putative mature miRNAs from 73 plant species(miRBase, version 21; www.mirbase.org/). Some rice miRNAs arethought to be at the center of complex gene regulatory networksthat control crop development and architecture, with specificmiRNAs affecting yield by regulating tiller number, grain size, and

panicle branching (8–13). These miRNAs also regulate responsesto the different stresses encountered by crops in the field, suchas drought, salinity, heat, cold, nutrient, and oxidative stress(reviewed in refs. 14, 15). Given their involvement in the control ofso many valuable agronomic traits, miRNAs are emerging assuitable but underexplored targets for crop improvement. Un-fortunately, their small size and extensive genetic redundancy havehampered approaches for functional analyses. Most of the avail-able data have been generated by miRNA overexpression and thegeneration of miRNA-resistant target genes, rather than by loss offunction. Some of the phenotypes from overexpression studiesmay be a consequence of temporal or spatial misexpression, andthus can be misleading. The target mimicry technologies, such astarget MIMICs (MIMs) (16) and short tandem target MIMICs(STTMs) (17), have provided effective tools to block endogenousmature miRNA activity, making it technically possible to un-dertake large-scale genome-wide studies.Target mimicry technology was based on the discovery of an

endogenous regulatory mechanism used by INDUCED BYPHOSPHATE STARVATION1 (IPS1) to modulate miR399 ac-tivity in Arabidopsis thaliana (16). IPS1 encodes a noncoding

Significance

Plant microRNAs (miRNAs) control intricate gene regulatorynetworks and have been implicated in important develop-mental switches and stress responses. Plant miRNAs have re-cently emerged as promising targets for crop improvementbecause they can control complex agronomic traits; however,functional studies using reverse genetics have been hamperedby practical difficulties. We have silenced 35 miRNA families inrice to generate a resource for discovering new functions ofmiRNAs and targets of agronomic improvements. As a proof ofconcept, we show that manipulation of a promising miRNA,miRNA398, leads to important yield improvements. Our find-ings also reveal important agronomic roles for several miRNAs.

Author contributions: H.Z. and J.-K.Z. designed research; H.Z., J.Z., J.Y., F.G., and Y.M.performed research; H.Z., J.Z., G.T., J.R.B., and J.-K.Z. analyzed data; and H.Z., J.Z., J.R.B.,and J.-K.Z. wrote the paper.

Reviewers: S.-W.D., University of California; and R.S., Oklahoma State University.

The authors declare no conflict of interest.

Data deposition: Sequence data from this article have been deposited in the miRBasedatabase or Rice Genome Annotation Project website (rice.plantbiology.msu.edu/) (Mich-igan State University) under the following accession numbers: miR398a (MI0001051),OsSPL2 (LOC_Os01g69830), OsSPL3 (LOC_Os02g04680), OsSPL4 (LOC_Os02g07780),OsSPL7 (LOC_Os04g46580), OsSPL11 (LOC_Os06g45310), OsSPL12 (LOC_Os06g49010),OsSPL13 (LOC_Os07g32170), OsSPL14 (LOC_Os08g39890), OsSPL16 (LOC_Os08g41940),OsSPL17 (LOC_Os09g31438) , OsSPL18 (LOC_Os09g32944), and OsGAMYB(LOC_Os01g59660).1H.Z. and J.Z. contributed equally to this work.2To whom correspondence should be addressed. Email: jkzhu@purdue.edu.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1703752114/-/DCSupplemental.

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RNA that contains a motif complementary to miR399 except fora central stretch of three nucleotides. When miR399 pairs withIPS1, the duplex contains a central bulge that prevents cleavageof IPS1, thereby sequestering miR399 and reducing its bio-availability (16). MIMs have been used to sequester miRNAfamilies in Arabidopsis, although this approach is not equally ef-fective for all targeted miRNAs (18). STTMs were developed asan improvement on target mimicry and contain two imperfectmiRNA target sites with a CTA trinucleotide bulge in the centerlinked by a 31- to 96-nt short spacer (17). STTMs can trigger ef-ficient degradation of the targeted miRNAs in Arabidopsis andmice (17, 19). Here, we report an extensive functional study of ricemiRNAs using STTM technology. Our results revealed importantfunctions of rice miRNAs in the regulation of key agronomic traits.

Results and DiscussionSTTM-Mediated Silencing of miRNA Families in Rice Is Highly Specificand Stable for Multiple Generations. We designed 35 STTM con-structs, containing two noncleavable miRNA-binding sites linkedby a spacer (48–88 nt) (SI Appendix, Table S1), that target either asingle mature miRNA or two different mature miRNAs of thesame family in rice (SI Appendix, Table S2). We generated manyindependent transgenic rice lines (n = 21–46) for each construct inthe japonica variety Nipponbare (SI Appendix, Table S2). Most ofthe targeted miRNA families were chosen due to their potentialroles in plant development/architecture or in response to variousabiotic and biotic stresses (SI Appendix, Dataset S1). Visual in-spection of the transgenic lines (T0–T4) grown in the field in twodifferent locations (Shanghai and Lingshui, China) revealed a widerange of obvious above-ground phenotypic alterations. Impor-tantly, the phenotypes generated by each STTM were consistentand detected in multiple independent transgenic lines (SI Appen-dix, Table S2). We observed strong transgenerational stability ofSTTMs in rice, with phenotypes being stable for up to five gen-erations thus far (SI Appendix, Fig. S1). Our results also showedhigh STTM specificity; for instance, miR156, but not miR160, ex-pression levels were dramatically decreased in the STTM156 lines,and vice versa (discussed below). Rice STTM lines displayed a widerange of phenotypic alterations distinct from those phenotypic al-terations reported in Arabidopsis, suggesting substantial evolu-tionary functional diversifications of miRNA functions betweendicots and monocots.

New Agronomic Targets: miR398 Inhibition Increases Rice Yield. Ourinitial characterization of the STTM transgenic rice lines re-vealed alterations in agronomic traits such as seed size, tillernumber, plant height, and flowering time, emphasizing the im-portance of miRNAs in rice productivity.We further investigated miR398, which has been extensively

studied and is thought to regulate the response to abiotic andbiotic stresses by promoting cleavage of its target genes, Cu/Zn-superoxide dismutases (CSD1 and CSD2) (20–23). Although nodevelopmental or architectural phenotypes have been associatedwith this miRNA in Arabidopsis, we found that silencing ofmiR398 resulted in rice lines (STTM398) that were significantlyshorter than wild-type (WT) plants (62.7 ± 4.1 cm vs. 86.7 ±2.4 cm, respectively) (Fig. 1 A and E). STTM398 also displayedsmaller panicles, containing a reduced number of grains, than WT(77.8 ± 10.1 vs. 96.31 ± 1.3; P < 0.001) (Fig. 1 B, F, and G) andflowered about 2 wk later than WT plants (87.4 ± 1.6 d vs. 71.8 ±1.4 d; P < 0.001) (SI Appendix, Fig. S2). Importantly, STTM398lines showed a significant decrease in grain length (12.9%) andwidth (19.5%) compared with WT, resulting in a 40% decrease in1,000-grain weight (Fig. 1C, D, and H). Northern blot analysisshowed that the expression of miR398 was almost undetectable inthe STTM398 transgenic plants (Fig. 1I), whereas several pre-dicted miR398 target genes showed significantly increased ex-pression levels in the STTM398 transgenic lines relative to WT

(Fig. 1J). Our results show that miR398 plays a significant role inrice growth and seed development, identifying it as a promisingtarget for improvement.To validate our hypothesis, we generated transgenic lines over-

expressing miR398a (OX-miR398a). Analysis of two independentfield-grown T1 transgenic OX-miR398a lines showed an overallincrease in height compared with WT, accompanied by biggerpanicles and an increased number of grains per panicle (Fig. 1K–O).Trade-offs between grain number and size are commonly observed

Fig. 1. miR398 controls rice panicle and seed development and can be ma-nipulated for rice improvement. (A) Gross morphologies of WT (Nipponbare)and STTM398 plants at maturity. (Scale bar, 10 cm.) (B) Panicle morphologies ofWT and STTM398 plants. (Scale bar, 5 cm.) (C) Grain width of WT andSTTM398 plants. (Scale bar, 1 cm.) (D) Grain length of WT and STTM398 plants.(Scale bar, 1 cm.) Plant height (E), main panicle length (F), grain number perpanicle (G), and 1,000-grain weight (H) of WT and STTM398 plants are shown.(I) Expression levels of miR398 inWT and STTM398 plants detected using Northernblotting. U6 was used as a loading control. (J) Expression levels of miR398predicted targets inWT and STTM398 plants determined by qRT-PCR. (K) Grossmorphologies of WT, OX-miR398a, and mOs07g46990 plants at maturity. (Scalebar, 10 cm.) (L) Panicle morphologies of WT, OX-miR398a, and mOs07g46990plants at maturity. (Scale bar, 5 cm.) Plant height (M), main panicle length (N),grain number per panicle (O), and 1,000-grain weight (P) of WT, OX-miR398a,and mOs07g46990 plants are shown. Data are presented as mean ± SD [n =15 replicates in E andM; n = 30 replicates in F,G,N, andO; n = 10 (>120 seeds foreach biological replicate) replicates in H and P; n = 3 in replicates in J]. *P < 0.05,**P < 0.01; two-tailed, two-sample t test. NS, not significant.

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in both natural (24) and domesticated systems (25); however, OX-miR398a lines displayed increased seed length, seed width, and1,000-grain weight compared withWT (Fig. 1P and SI Appendix, Fig.S3 A and B). To confirm our hypothesis further, we producedtransgenic lines carrying a resistant version of one of themiR398 targets, Os07g46990, that had shown increased levels inSTTM398 lines (Fig. 1J). The modified Os07g46990 gene(mOs07g46990) contained five mismatches in the miR398 targetsite and was expressed in transgenic rice under its native promoter(SI Appendix, Fig. S4). The mOs07g46990 lines showed some of thephenotypes of STTM lines, such as decreased plant height, paniclelength, and grain number per panicle, compared with WT (Fig. 1K–O). However, mOs07g46990 plants showed WT phenotypes forseed length, seed width, and 1,000-grain weight (Fig. 1P and SIAppendix, Fig. S3 C and D), confirming that the phenotypes ob-served in STTM lines result from the compounded effects ofmiR398 on multiple target genes. The strong developmentalphenotypes observed in the STTM and overexpression lines in riceare in clear contrast to the lack of visible phenotypes reported forArabidopsis, which may reflect different experimental approachesor evolutionary differences between monocots and eudicots.

Previously Undiscovered Functions for miR172 in Culm and PanicleDevelopment. miR172 has been the focus of multiple studies us-ing different approaches, such as overexpression, expression ofresistant target genes, or mutations in individual loci encoding themiRNA (26–35). These previous studies suggested that miR172mainly controls flowering time and floral organ identity. For in-stance, loss of function of one locus, miR172a, and overexpressionof miR172 targets resulted in slow vegetative development andlate flowering in Arabidopsis (30), whereas overexpression ofmiR172 caused an early flowering phenotype and defective floralorgan identity (9, 26, 30). Overexpression of miR172 in rice causedloss of spikelet determinacy and multiple abnormalities in floralorgans, including an increased number of bract-like structures andmultiple layers of lemma and palea, ultimately resulting in de-creased fertility or even complete sterility (31, 32).Unexpectedly, we found that STTM172 lines targeting miR172

showed defects mainly in culm (main stem) and panicle devel-opment. STTM172 plants had very short culms compared withWT, resulting in enclosed panicles and dwarf plants (Fig. 2 A, B,D, and F). Whereas WT culms have either four (63.9%) or five(34.7%) elongated internodes at maturity, 75.8% of STTM172culms had only three elongated internodes (Fig. 2 D, E, and J). In

addition, the internodes in STTM172 plants were shorter, espe-cially the top internode (internode I: 7.5 ± 2.4 cm in STTM172 vs.33.7 ± 2.2 cm in WT, n = 25), which also showed a wavy phe-notype (Fig. 2 D, E, and I). Natural variation in miR172 and themiR172 target site in maize and barley have been linked to panicledensity (29, 34). In agreement with this finding, our STTM172lines displayed increased density and shorter panicles comparedwith WT (Fig. 2 C and G). The grain number per panicle wasidentical in WT and STTM172 plants (Fig. 2H), and no abnor-malities were observed in STTM172 flowers (SI Appendix, Fig. S5A and B), in contrast to previous observations that overexpressionof miR172 affected these features (31, 32). STTM172 plantsproduced normal fertile pollen (SI Appendix, Fig. S5 C and D),although the seed setting rate was significantly lower than WT,perhaps due to the enclosed panicle (SI Appendix, Fig. S6). Ex-pression of miR172 was strongly inhibited in STTM172 lines, andexpression of the predicted target gene was consequently in-creased (Fig. 2 K and L). Our results reinforce the limitations ofrelying on overexpression as a tool to perform miRNA functionalstudies. The different phenotypes arising from single-locusknockouts of miR172 in Arabidopsis and maize (29, 30) and thecomplete inhibition of miR172 in rice suggest that this miRNA orspecific miRNA loci might have undergone functional specializa-tion during evolution, perhaps associated with distinct tissue- orcell-specific expression patterns.

New Roles for Old miRNAs: miR156 Controls Root Development at theSeedling Stage. Some agronomically important QTLs have beenlinked to differential regulation of individual genes by specificmiRNAs, but the effects of manipulating the miRNA instead of asingle target gene are still unknown. In rice, regulation ofOsSPL14 by miR156 is associated with “ideal plant architecture,”because increased expression of OsSPL14 reduces the number oftillers, increases grain number per panicle, and promotes a moresturdy stem. These observations suggest that miR156 is a potentialtarget for rice improvement (8, 9). Our results from silencingmiR156 confirmed the published data but also revealed previouslyunknown functions. STTM156 plants have fewer tillers than WT(4.1 ± 1.5 vs. 17.8 ± 4.7) (Fig. 3 A, B, and E) and thicker culms(4.3 ± 0.4 mm vs. 3.2 ± 0.4 mm) (Fig. 3 C and F). STTM156 plantsalso show a slight increase in grain length and 1,000-grain weight(P < 0.001) (Fig. 3 D, G, and H) but show no difference in grainwidth (SI Appendix, Fig. S7 A and B). In addition to the expectedphenotypes, analysis of STTM156 plants revealed a previously

Fig. 2. New roles for miR172 in rice shoot and panicle development. (A) Gross morphologies of WT (Left) and STTM172 (Right) plants at maturity. (Scale bar,10 cm.) (B) Panicle and upper part of stem of WT (Upper) and STTM172 (Lower) plants at maturity. (Scale bar, 10 cm.) (C) Panicle morphologies of WT (Upper)and STTM172 (Lower) plants. (Scale bar, 5 cm.) (D) Elongated internodes of WT (Left) and STTM172 (Right) plants. (Scale bar, 10 cm.) (E) EnlargedSTTM172 internodes from D. (Scale bar, 5 cm.) Plant height (F), panicle length (G), and grain number per panicle (H) of WT and STTM172 plants are shown.(I) Length of top four internodes (I to IV, where I is the uppermost) of WT and STTM172 plants. (J) Percentage of different internode number in WT andSTTM172 plants. (K) Expression levels of miR172 in WT and STTM172 plants detected using Northern blotting. U6 was used as a loading control. (L) Expressionlevels of predicted miR172 target in WT and STTM172 plants determined by qRT-PCR. Data are presented as mean ± SD (n = 15 replicates in F and G, n =25 replicates in H and I, n = 66 replicates in J, n = 3 replicates in L). **P < 0.01; two-tailed, two-sample t test.

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unknown role for miR156 in the regulation of shoot and root de-velopment at the seedling stage. Both 7- and 18-d-old STTM156seedlings grown hydroponically or in soil showed a substantial de-crease in root number and an increase in root width compared withWT seedlings (Fig. 3 I, J, and K and SI Appendix, Fig. S8). Shootsfrom the STTM156 seedlings were also shorter than shoots fromWTseedlings (Fig. 3I and SI Appendix, Fig. S8); however, WT andSTTM156 plants showed similar height at maturity (Fig. 3A).Northern blot analysis showed that miR156 was efficiently down-regulated in STTM156 plants (Fig. 3L); however, quantitative RT-PCR (qRT-PCR) revealed that only five of the 11 predicted targetswere up-regulated in the STTM156 plants (Fig. 3M), suggesting thatthe regulatory role of this miRNA is quite complex and goes beyondmere sequence conservation, even within a single protein family.

Identification of Multiple Rice miRNAs with Effects on AgronomicTraits. In addition to the examples discussed above, we observedalterations in important agronomic traits in other STTM lines.miR159 is highly conserved in eudicots and monocots, and regu-lates the expression of a family of GAMYB or GAMYB-like genesat the posttranscriptional level (36, 37). In Arabidopsis, themiR159 gene family has three members: MIR159a, MIR159b, andMIR159c. The Arabidopsis mir159ab double mutant has pleiotropicmorphological defects, including reduced height, curled leaves,shorter siliques, and smaller and irregularly shaped seeds (37). Wefound that rice STTM159 lines exhibited very similar phenotypes tothe Arabidopsis mir159ab double mutant (Fig. 4 A–D). STTM159plants were dwarf (36.0 ± 3.1 cm vs. 86.7 ± 2.4 cm for WT) anddisplayed curled leaves and small, irregularly shaped seeds com-pared with WT (Fig. 4 A–D). Grain length and width in STTM159lines showed a reduction of 21.3% and 9.1%, respectively, com-pared with WT, and 1,000-grain weight was decreased by 47.2% (SIAppendix, Fig. S9 A–C). Northern blot analysis showed thatmiR159 was efficiently silenced in the STTM transgenic lines, andqRT-PCR revealed that the expression of two predicted miR159target genes, OsGAMYB and GAMYB-like gene Os06g40330, wasup-regulated in the STTM159 plants, as expected (Fig. 4 N and R).

miR165 and miR166 have almost identical sequence except fora C/U substitution at position 17, and the two miRNAs havefunctional redundancy in Arabidopsis (38). Down-regulation ofboth miRNA families by STTM and MIM in Arabidopsis suggeststhat they function in multiple developmental processes via nega-tive regulation of class III homeodomain-leucine zipper (HD-ZIPIII) transcriptional factors (17, 18, 39). We found that STTM166lines displayed a wide tiller angle (Fig. 4E), which is a key agro-nomic trait for ideal plant architecture (40), and upwardly curledleaves (Fig. 4F), indicating loss of leaf polarity. Expression ofmiR166 was drastically down-regulated in the transgenic lines, andthe expression of two predicted miR166 targets, HD-ZIP IIIfamily genes Os03g43930 and Os12g41860, was up-regulated in theSTTM166 plants (Fig. 4 Q and T), as expected.miR160 is a highly conserved miRNA family that controls root,

embryo, leaf, and floral organ development as well as seed ger-mination in Arabidopsis by negatively regulating auxin responsefactor (ARF) family genes (41–44). Rice STTM160 transgeniclines shared some phenotypic alterations with Arabidopsis, such asreduced plant height (Fig. 4G). Although down-regulation ofmiR160 in Arabidopsis resulted in reduced fertility, it producedcomplete male sterility in rice (Fig. 4H). Other phenotypes ob-served in Arabidopsis, such as serrated leaves and aberrant floralorgans, were not observed in the rice transgenic lines.Overexpression of miR171 results in multiple and, on many

occasions, opposite phenotypes. Barley plants overexpressingmiR171 were dwarf and had a reduced number of tillers comparedwith WT, whereas rice miR171 overexpression lines were tallerand had increased tiller numbers compared with WT (45, 46). InArabidopsis, miR171 targets the scarecrow-like (SCL) transcrip-tion factors and is thought to regulate meristematic cell pro-liferation, polar organization, and chlorophyll synthesis (47, 48).Silencing of miR171 in Arabidopsis using MIM produced roundand pale leaves. In contrast, we found that rice STTM171 plantsshowed semidwarf stature, semienclosed panicles, and droopingflag leaves (Fig. 4 I and J), as well as no obvious changes in leafshape or color, again suggesting a diversification in the function ofmiRNAs between monocots and eudicots.Very little is known about the roles of miR441 and miR1428. In

our study, STTM441 plants were taller than WT (Fig. 4K and SIAppendix, Fig. S10), whereas STTM1428 plants showed abortedpollen at the pollen maturation stage (Fig. 4 L andM). In line withthe phenotypic effects, STTM lines showed almost complete si-lencing of the targeted miRNAs, which was accompanied by de-regulation of the predicted target genes (Fig. 4N–T). Interestingly,many miRNAs with reported phenotypes upon overexpression orpartial suppression, such as miR169, miR393, miR397, andmiR167, did not produce any obvious above-ground phenotypes inour STTM transgenic rice lines.

STTM Is a Powerful Tool for Functional Characterization of miRNAsand Manipulating Agronomic Traits. There are very few functionalstudies available for rice miRNAs, largely due to the technicaldifficulties in down-regulating mature miRNA expression. Here,we used STTM to silence 35 different miRNA families, nine ofwhich produced obvious above-ground phenotypes (SI Appendix,Table S2). Our results have unveiled a number of characteristicsthat make STTM the technique of choice for miRNA functionalstudies compared with other available methods, such as MIM. First,the phenotypes, when available, were observed in multiple in-dependent transgenic lines and were consistent among lines. Sec-ond, the levels of down-regulation achieved by STTM were vastlysuperior to the levels of down-regulation reported using MIM inrice. For example, miR156 levels in transgenic lines carrying MIMconstructs were reduced about twofold compared with WT (49),whereas the expression levels of all of the STTM-targeted miRNAswere reduced by up to 100-fold in transgenic lines compared withWT (Figs. 1I, 2K, 3L, and 4 N–Q and SI Appendix, Fig. S11 A–C).

Fig. 3. miR156 affects rice seedling development and plant architecture.(A) Gross morphologies of WT (Left) and STTM156 (Right) plants at maturity.(Scale bar, 10 cm.) (B) Magnification of A highlighting differences in tillernumbers. (C) Comparison of third internodes between WT (Left) and STTM156(Right) plants. (Scale bar, 5 mm.) (D) Grain length of WT (Left) and STTM156(Right) plants. (Scale bar, 1 cm.) Tiller number per plant (E), third internodediameter (F), grain length (G), and 1,000-grain weight (H) of WT and STTM156plants are shown. (I) Hydroponically grown 7-d-old seedlings of WT (Left) andSTTM156 (Right) plants. (Scale bar, 1 cm.) Transverse sections of 7-d-old rootsof WT (J) and STTM156 (K) plants are shown. (Scale bars, 100 μm.) (L) Ex-pression levels of miR156 and miR160 in WT and STTM156 plants detectedusing Northern blotting. U6 was used as a loading control. (M) Expressionlevels of predicted miR156 targets in WT and STTM156 plants determined byqRT-PCR. Data are presented as mean ± SD [n = 20 replicates in E, n = 43 rep-licates in F, n = 10 (>120 seeds for each biological replicate) replicates inG andH,n = 3 replicates in M]. **P < 0.01; two-tailed, two-sample t test.

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The use of CRISPR-Cas for miRNA studies is hampered by thesmall size of the mature miRNA sequences (∼21 nt); the re-quirement of a protospacer adjacent motif sequence (50); and theexistence of large gene families encoding some miRNAs, such asmiR166 with 14 loci, in rice (51). Most importantly, the observedphenotypes were stable across generations (SI Appendix, Fig. S1).This transgenerational stability is essential if STTM lines are to beused for research purposes or as breeding resources.

ConclusionsSTTM lines are a valuable resource for functional miRNAstudies as well as potential breeding materials for rice improve-ment. We have discovered the strong agronomic potential ofsome miRNAs and revealed previously unknown functions oflong-studied miRNAs. We also provide evidence for evolution-ary divergences in the functions of some miRNAs betweenmonocots and eudicots. Our work suggests that targetingmiRNAs can influence entire gene networks and improve complexagronomic traits. The transgenic lines generated in this study canbe screened for responses to biotic and abiotic stresses, nutrientuse efficiency, and photosynthetic efficiency, for example, toreveal new and exciting targets for agronomic improvement inthe future.

MethodsPlant Materials and Growth Conditions. Rice (Oryza sativa L.) variety Nip-ponbare (ssp. japonica) was used in this study. Most of the WT and trans-genic plants were grown under natural field conditions in the Shanghairegion of China (30°N, 121°E) during the normal rice-growing season frommid-May to mid-October and in Lingshui, China (18°N, 110°E) during thenormal rice-growing season from mid-December to mid-April. SomeSTTM156 plants were also grown in a greenhouse with a 30/24 ± 1 °C day/night temperature, 50–70% relative humidity, and a light/dark period of14 h/10 h. For hydroponic culture experiments, plants were grown in solu-tion as described by Yoshida et al. (52) in a greenhouse.

Trait Measurements. Plant height, tiller number, grain length, grain width,1,000-grainweight, panicle length, grain number per panicle, internode length,and diameter of the third internode were measured at full maturity. Plantheight was measured in the paddy fields. Grain length and width were mea-sured using an SC-A grain analysis system (Wseen Company). The 1,000-grainweightwasweighted using an SC-A grain analysis system after fully filled grainswere dried at 42 °C in an oven for 2 wk.

Phenotyping and Histological Experiments. All of the materials, exceptSTTM156and correspondingWTseedling-stageplants,weregrownundernaturalfield conditions. The plants were moved to pots for photographing to display thedifferences more clearly. Most plant materials were photographed with a NikonD3000 digital camera and an Olympus BX53 microscope. Grain length and widthphotographs were generated using an SC-A grain analysis system.

For the iodine-potassium iodide (I2-KI) staining of pollen grains, the spikeletwas collected near anthesis and fixed in FAA (50% ethanol, 10% formalin, and5% acetic acid) solution. The pollen grains were squeezed out to a microscopeslide, stained with1.0% I2-KI, and photographed using the Olympus BX53microscope.

For root transverse sectionobservations, thematerialswere collectedand fixedovernight at 4 °C in FAA solution and dehydrated in a graded ethanol series. Thesamples were then embedded in Technovit 7100 resin (Hereaus Kulzer), and2-μm sections were made using a Leica RM 2265 programmable rotary micro-tome (Leica Microsystems). After staining with 0.05% Toluidine Blue, transversesections were photographed using an Olympus BX53 microscope.

Vector Construction and Transformation of Rice. STTM vectors were con-structed as described by Tang et al. (19). For the OX-miR398 vector, a PCRfragment amplified from genomic DNA using primers OE-miR398a-F andOX-miR398a-R was cloned into the BamHI/SpeI sites of the binary vectorpTCK303 (53) between the maize Ubi 1 promoter and the Nos terminator. Toconstruct the mOs07g46990 vector, a 4,181-bp genomic fragment, includingthe 1,406-bp promoter and coding sequence for Os07g46990, was amplifiedand cloned into the BamHI/HindIII sites of the binary vector pCAMBIA 1300.To introduce mismatches into Os07g46990, a fragment amplified by PCR usingmOs07g46990-5-F and mOs07g46990-5-R was first cloned into the BamHI/SalIsites of pCAMBIA 1300. A second fragment was then amplified usingmOs07g46990-3-F and mOs07g46990-3-R and cloned into the HindIII/SalI sitesof the vector described above. All constructs were introduced into the

Fig. 4. Examples of developmental alterations observed in STTM159, STTM160, STTM166, STTM171, STTM441, and STTM1428 rice lines. (A) Gross morphologiesof WT and STTM159 plants at maturity. (Scale bar, 10 cm.) (B) Leaf morphologies of WT and STTM159 plants at maturity. (Scale bar, 5 cm.) (C) Grain length of WTand STTM159 plants. (Scale bar, 1 cm.) (D) Grain width of WT and STTM159 plants. (Scale bar, 1 cm.) (E) Gross morphologies of WT and STTM166 plants. (Scale bar,10 cm.) (F) Leaf morphologies of WT and STTM166 plants at maturity. (G) Gross morphologies of WT and STTM160 plants. (Scale bar, 10 cm.) (H) Spikelets of WTand STTM160 after removing the palea and lemma. (Scale bar, 1 mm.) (I) Gross morphologies of WT and STTM171 plants at maturity. (Scale bar, 10 cm.)(J) Comparison of flag leaf angle between WT and STTM171 plants. (K) Gross morphologies of WT and STTM441 plants at maturity. (Scale bar, 10 cm.) I2-KIstaining of pollen grains fromWT (L) and STTM1428 plants (M) is shown. (Scale bars, 100 μm.) (N) Expression levels of miR159 inWT and STTM159 plants detectedusing Northern blotting. U6 was used as a loading control. (O) Expression levels of miR160 and miR156 in WT and STTM160 plants detected using Northernblotting. U6 was used as a loading control. (P) Expression levels of miR171 and miR398 in WT and STTM171 plants detected using Northern blotting. U6 was usedas a loading control. (Q) Expression levels of miR166 in WT and STTM166 plants determined by stem-loop qRT-PCR. U6 was used to normalize samples. (R) Ex-pression levels of predicted miR159 targets in WT and STTM159 plants determined by qRT-PCR. (S) Expression levels of predicted miR160 target in WT andSTTM160 plants determined by qRT-PCR. (T) Expression levels of predicted miR166 targets in WT and STTM166 plants determined by qRT-PCR. Data are presentedas mean ± SD (n = 3 replicates in Q–T). **P < 0.01; two-tailed, two-sample t test.

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Agrobacterium tumefaciens strain EHA105 and subsequently transferred intojaponica variety Nipponbare by A. tumefaciens-mediated rice transformationas previously described (54). Sequences of the primers used are listed in SIAppendix, Table S3.

Small RNA Northern Blotting Analysis. Total RNA was isolated from roots,shoots, leaves, and panicles at different stages using a TRIzol Reagent kit(Invitrogen) according to themanufacturer’s protocol. About 40 μg of total RNAwas electrophoresed on a denaturing 19% polyacrylamide gel, transferred toNytran Super Charge Nylon Membranes (Schleicher & Schuell BioScience), andcross-linked using a Stratagene UV Crosslinker. DNA oligonucleotides comple-mentary to the different miRNA sequences were synthesized and labeled with[32P]-γ-ATP (PerkinElmer) using T4 polynucleotide kinase (Takara). The mem-branes were prehybridized with PerfectHyb (Sigma–Aldrich) hybridizing withthe labeled probes. After washing, the membranes were autoradiographedusing film (X-Omat BT Film; Carestream). U6 was used as a loading control. Theprobe sequences are listed in SI Appendix, Table S3.

RNA Extraction and qRT-PCR. Total RNA was isolated from roots, shoots,leaves, and panicles at different stages using the TRIzol Reagent kit accordingto the manufacturer’s protocol. After treatment with RNase-free DNase I(Promega), total RNA (1 μg) was reverse-transcribed using the TransScript IIOne-Step gDNA Removal and cDNA Synthesis SuperMix kits (TransGen Bio-tech). For stem-loop qRT-PCR, SuperScript III Reverse Transcriptase (Invitrogen)was used for reverse transcription. The reverse transcription products wereused as templates for qRT-PCR performed on a CFX96 real-time PCR system(Bio-Rad) using SYBR Premix EX Taq (Takara) according to the manufacturer’sprotocol. ACTIN1 and U6 snRNA were used to normalize samples in normalqRT-PCR and stem-loop qRT-PCR, respectively; relative expression levels weremeasured using the 2−ΔΔCt analysis method. The sequences used in qRT-PCRare listed in SI Appendix, Table S3.

ACKNOWLEDGMENTS. This work was supported by the Chinese Academy ofSciences and by US NIH Grants R01GM070795 and R01GM059138 (to J.-K.Z.).H.Z. acknowledges the support of the International Postdoctoral ExchangeFellowship Program of China under Grant 20140029.

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