NRL3 Interacts with OsK4 to Regulate Heading Date in Rice

CHEN Wei,CAI Yicong,Shakeel AHMADWANG YakunAN RuihuTANG ShengjiaGUO NaihuiWEI XiangjinTANG ShaoqingSHAO GaonengJIAO GuiaiXIE LihongHU ShikaiSHENG ZhonghuaHU Peisong

(1Key Laboratory of Crop Physiology,Ecology and Genetic Breeding,Ministry of Education/ Collaboration Center for Double-Season Rice Modernization Production,Jiangxi Agricultural University/ Research Center of Super Rice Engineering and Technology,Jiangxi Province,Nanchang 330045,China;2State Key Laboratory of Rice Biology/ Key Laboratory of Rice Biology and Breeding,Ministry of Agriculture and Rural Affairs/ China National Rice Improvement Center/ China National Rice Research Institute,Hangzhou 310006,China;#These authors contributed equally to this work)

Abstract:NRL3 is essential for the growth and development of rice leaves.In this study,we found that the loss function of NRL3 also delayed heading date under natural long daylight and short daylight conditions.The yeast two-hybrid and the bimolecular fluorescence complementation proved that NRL3 interacts with OsK4,a Snf1-related kinase.OsK4 localized to the nucleus and expressed in various rice tissues.The rhythmic expression pattern of OsK4 was similar to NRL3 under long daylight and short daylight conditions.Knock-out mutants of OsK4 exhibited early heading under long daylight conditions,indicating that it acts as a negative regulator of heading date in rice.Interestingly,the OsK4 mutant under the nrl3 mutant background rescued the late heading phenotype of nrl3 under long daylight conditions,suggesting that OsK4 functions downstream of NRL3.Moreover,both NRL3 and OsK4 controlled heading date through regulating the expression of Hd3a and RFT1 genes.These findings shed light on the heading date regulation in rice and provide a sound theoretical base to improve regional adaptability of rice.

Key words:Oryza sativa;heading date;NRL3 gene;OsK4 gene;negative regulator;loss function

Rice (Oryza sativaL.) is one of the most important food crops globally,and is the primary source of energy for over half of the world’s population.Recently,the Corona Virus Disease 2019 (COVID-19) has severely affected the world’s food supply chain and food security.Therefore,improvement of rice grain yield is urgent to solve the global food crisis issue.Rice yield can be improved in several ways,such as enhancing grain size,perfecting plant architecture and increasing the number of panicles.However,heading date is one of the crucial agronomic traits that determine regional adaptability and high grain production of rice (Izawa,2007;Jung and Muller,2009).

The molecular mechanisms that regulate heading date have been extensively studied using molecular genomic methods inArabidopsis,a long daylight plant.TheGI-CO-FT(GIGANTEA-CONSTANS-FLOWERING LOCUS T) regulation pathway has been identified inArabidopsis.GI,a circadian oscillator protein,integrates signals from photoreceptors and circadian clock,up-regulatingCOmRNA levels in order to regulate the heading date (Simpson,2003).CO,a transcriptional activator,plays a key role as a major regulator of photoperiodic heading.It directly activates the expression of FT,a RAF kinase inhibitor protein that acts as a florigen promoting floral initiation under long-day (LD) conditions (Onouchi et al,2000;Samach et al,2000;Lifschitzet al,2006;Corbesier et al,2007).

Rice is a typical short daylight plant,and two distinct pathways have been reported to control its photoperiodic flowering (Turck et al,2008).TheOsGI-Hd1-Hd3a(Hd1) pathway is evolutionarily conserved in rice andArabidopsisbut is functionally different.Hd1is orthologous toCOinArabidopsis.UnlikeCO,which merely promotes flowering inArabidopsis,Hd1has a dual function in rice,promoting heading by activating the expressionofHd3aunder short-day (SD) conditions and delaying heading by suppressing the expression ofHd3aunder LD conditions (Yano et al,2000;Hayama et al,2003).Recently,two important repressors of flowering,DTH8 (days to heading on chromosome 8) and Ghd7 (grain number,plant height and heading date 7) have been identified in rice,which can form a heterotrimer complex with Hd1 for regulating flowering (Zhang et al,2017;Cai et al,2019).HAF1 encodes a C3HC4 RING domain-containing E3 ubiquitin ligase which interacts with Hd1 and degrades it via the 26S proteasome-dependent pathway (Yang et al,2015).OsK4is a repressor of heading date,and encodes a protein kinase.Theosk4mutants show early heading under LD.OsK4 interacts with HDR1,a Snf1-related kinase interactor,and depends on HDR1 to phosphorylate HD1 (Sun et al,2016).Additionally,theEhd1-Hd3a/ RFT1(Ehd1) is another photoperiodic flowering pathway,unique in rice (Doi et al,2004;Tsuji et al,2011).Ehd1encodes a B-type response regulator that activates the expression of two florigen genesHd3aandRFT1that promote flowering under LD and SD conditions (Doi et al,2004).Many genes that mediate flowering throughEhd1have been reported.Ehd3is a PHD-finger domain transcriptional regulator,which up-regulates the expression ofEhd1,thus promoting flowering (Matsubara et al,2011).Likewise,OsMADS51encodes a MAD-box transcription factor that acts on upstream ofEhd1and transmits signals fromOsGItoEhd1,thereby,activatingEhd1expression and promotes flowering under SD (Kimet al,2007).Similarly,Ehd4,a unique gene in rice,encodes a CCCH-type zinc finger transcription factor and activates the expression ofEhd1(Gao et al,2013).Conversely,several repressors ofEhd1have also been identified.For example,OsCOL10(Tan et al,2016),OsCOL4(Lee et al,2010),OsCOL13(Sheng et al,2016),DTH8(Wei et al,2010),HBF1(Brambilla et al,2017),Hd5(Fujino et al,2013),OsHAPL1(Zhu et al,2017),OsLFL1(Peng et al,2008),OsMFT1(Song et al,2018) andSIP1(Jiang et al,2018) genes delay flowering by repressing the expression ofEhd1.Among them,SIP1 directly binds the specific core motif within the promoter region ofEhd1,thus repressing its expression (Jianget al,2018).Additionally,theEhd1pathway is not independent of theHd1pathway.For instance,Ghd7,a strong repressor in theEhd1pathway,directly binds to the promoter region ofEhd1,down-regulating its expression and interacting with Hd1 (Nemoto et al,2016).Hd1 directly binds to the CCAAT-motif of theGhd7promoter region to activate its expression.However,DTH8 and OsHAP5Bcan form a trimeric complex with Hd1,enhancing its binding with the promoter region ofGhd7(Wang et al,2019).

Previous studies demonstrated thatNRL3(LOC_ Os03g19520) plays a vital role in the growth and development of rice,including leaf morphology,fertility,seed size and grain yield.The mutants exhibit rolling leaves,abnormal tapetum degeneration and microspore development,and slender seeds (Liu et al,2016;Zhao et al,2016;Ma et al,2017;Chen et al,2019).This study identified annrl3mutant,which showed delayed heading phenotype under LD and SD.We observed thatNRL3down-regulated the expression ofHd3aandRFT1in order to delay the heading.Furthermore,we found that NRL3 interacted with OsK4,andOsK4was genetically epistatic toNRL3.Thus,our findings revealed the link betweenNRL3andOsK4and provided insightful information on their involvement in regulating the heading date in rice.

RESULTS

NRL3 positively regulates heading date

In our previous study,we showed thatNRL3encodes an unknown function protein and plays a crucial role in the development of rice leaf morphology (Chen et al,2019).In this study,we found that thenrl3mutant plants headed approximately 7 d later than the wild type (WT),Zhongjiazao 17 (YK17),under natural long-day (NLD) (Hangzhou,China) and natural short-day (NSD) (Hainan,China) conditions (Fig.1-A and -B).Furthermore,to examine whether the increased vegetative period delayed the heading innrl3,we measured the leaf emergence rates in WT andnrl3under LD (14 h light/10 h dark) and SD (10 h light/14 h dark) conditions.The leaf emergence rates ofnrl3were similar to those of WT under LD and SD,implying that the delayed heading was not caused by the vegetative period,which might be due to the prolonged floral transition innrl3(Fig.1-C and -D).To further evaluate the function ofNRL3on heading date,an overexpression vector was constructed and transformed into thenrl3mutant plants.Compared to the WT andnrl3,the expression levels ofNRL3dramatically increased in the OE (overexpression) plants (Fig.1-F).The OE plants exhibited a normal heading date phenotype similar to WT under NLD and NSD (Fig.1-E,-G and -H).These data suggested thatNRL3is a positive regulator of heading date in rice.

NRL3 shows rhythmic expression pattern and regulates heading date through down-regulation of Hd3a, RFT1 and Ehd1

The WT plants were grown under LD and SD in a greenhouse for 20 d to identify the rhythmic expression ofNRL3.The results showed thatNRL3exhibited the similar diurnal rhythmic expression patterns under LD and SD.The expression levels began to rise during dawn,peaked before dusk,and then rapidly decreased (Fig.2-A and -B).

To reveal the molecular function ofNRL3in regulation of the heading date,we examined the transcript levels of heading date genes (Hd3a,RFT1,Ehd1andHd1) under LD and SD using qRT-PCR.The results showed that the transcript levels ofHd3a,RFT1andEhd1were dramatically decreased under both LD and SD innrl3compared to WT (Fig.2-C to -H).Furthermore,theHd1andOsGIgenes expression levels dramatically fluctuated in WT andnrl3,especially forHd1(Figs.2-I,2-J and S1).As a core regulator of heading date,Hd1is regulated by some other genes related to heading date,which might be the reason for its dramatical fluctuation.It suggested thatnrl3delays heading in rice by repressing the expression levels ofHd3a,RFT1andEhd1.

NRL3 interacts with OsK4

To elucidate the molecular mechanism ofNRL3in regulation of the heading date in rice,we performed a yeast two-hybrid (Y2H) assay to screen the interacting factors of NRL3.The Y2H assay identified many independent fragments.Among these,four independent clones were confirmed to interact with NRL3.Sequencing and BLAST analyses showed that four matching clones can potentially be OsK4 (LOC_Os08g37800),a Snf1-related kinase.Furthermore,to examine whether NRL3 interacts with OsK4,a Y2H assay of these two proteins was performed.It showed that NRL3 physically interacted with OsK4 in yeast cells (Fig.3-A).Additionally,a bimolecular fluorescence complementation (BiFC) assay was also performed to confirm the interaction between NRL3 and OsK4 in the epidermal cells ofNicotiana benthamiana.As expected,strong fluorescence signal was observed within the nucleus and cytoplasm when NRL3 and OsK4 were co-expressed.The negative control showed no fluorescence signal (Fig.3-B).These data combined suggested that NRL3 directly interacts with OsK4 in yeast and tobacco.

Expression pattern of OsK4

qRT-PCR assay was performed in different tissues (root,stem,leaf,leaf sheath,panicle,seed at 5 d and seed at 10 d) to examine the spatio-temporal expression pattern ofOsK4.The results showed thatOsK4was expressed in all tissues,and the highest expression levels were developed in the endosperm at 10 d after fertilization (Fig.4-A).Transient expression analysis in rice protoplasts was performed to investigate the subcellular localization of the OsK4 protein.It was observed that the green fluorescent protein (GFP) signal of the OsK4-GFP fusion protein was localized in the nucleus and merged with the known nucleus-localized signals of D53-mCherry (Fig.4-B).In addition,we found thatOsK4also had a rhythmic expression pattern,which was quite similar toNRL3(Fig.4-C and -D).

OsK4 negatively regulates heading date

To investigate the function ofOsK4,OsK4knockout transgenic plantsko-osk4were generated using the CRISPR/Cas9 system under the Nipponbarebackground.As a result,20 independent lines were obtained,and 2 homozygous lines were selected for further study (Fig.S2).Theko-osk4lines showed approximately 13 d heading earlier than its WT (Nipponbare) under NLD,but no difference was observed under NSD (Fig.5-A to -C).In addition,we examined the expression levels ofNRL3,Hd3a,RFT1andEhd1under NLD in theko-osk4plants.The expression levels ofHd3a,RFT1andEhd1were significantly increased in theko-osk4transgenic plants compared to WT,but the expression level ofNRL3was not significantly changed (Fig.5-D to -G).These results suggested thatOsK4controls heading date by regulatingthe expression ofHd3a,RFT1andEhd1.

OsK4 is genetically epistatic to NRL3

Considering the interaction between NRL3 and OsK4,and to further investigate the genetic relationship betweenNRL3andOsK4,we generatedosk4/nrl3double mutants in thenrl3mutant background via the CRISPR/Cas9 system.Ten independent positive CRISPR-based transgenic lines were obtained.Out of which,two homozygous lines with a threonine (T) inserted in the targeted region were selected for subsequent studies.Interestingly,thenrl3/osk4double mutants rescued the late heading phenotype ofnrl3under LD (Fig.6-A and -B).We also examined the mRNA levels and protein abundance ofOsK4in thenrl3andnrl3/osk4mutants,and found that the mRNA levels and protein abundance ofOsK4were significantly reduced in thenrl3/osk4double mutants compared with thenrl3mutant (Fig.6-C,-E and -F).Thus,it indicated thatOsK4is genetically epistatic toNRL3.

Moreover,to investigate whether the early heading ofnrl3/osk4under LDcauses the up-regulation of theHd3a,RFT1andEhd1genes,we examined their transcript levels in thenrl3andnrl3/osk4mutants grown under NLD.The results showed that the transcript levelsofHd3a,RFT1andEhd1were dramatically up-regulated innrl3/osk4,compared withnrl3(Fig.S3).

Furthermore,we detected the transcript levels and protein levels of OsK4 in the WT (Zhongjiazao 17),nrl3,OE-1andOE-2plants.The transcript levels ofOsK4were not markedly changed in the WT,nrl3,OE-1andOE-2plants (Fig.6-D).Interestingly,the OsK4 protein was highly accumulated in thenrl3mutant,whereas the protein levels in theOE-1andOE-2plants remained similar to WT (Fig.6-G and -H).Furthermore,we tested the protein stability of OsK4 in WT andnrl3,and compared to the total protein ofnrl3,the GST-OsK4 fusion proteins were much more sensitive to WT protein,indicating GST-OsK4 fusion proteins degraded much more quickly in the WT protein (Fig.6-I and -J).Together,these data suggested that NRL3 reduces the stability of the OsK4 protein,therefore,affecting its accumulation.

DISCUSSION

NRL3 positively regulates heading date in rice

Previously,we have reported thatNRL3encodes a protein that plays an important role in leaf morphology development (Chen et al,2019).However,its biological function in rice development was yet to be elucidated.Thus,in the present study,we studied its biological function and molecular mechanism for heading date in rice.We observed thatnrl3exhibited the late heading phenotype under NLD and NSD,compared to WT (Fig.1-A and -B).So far,at least two independent flowering pathways,Hd1pathway andEhd1pathway,have been revealed to regulate the floral transition in rice (Komiya et al,2009;Tsuji et al,2011).TheHd1pathway is conserved between rice andArabidopsis,whereas theEhd1pathway is unique to rice.In these pathways,the two florigen genes,Hd3aandRFT1,are the switches of rice floral transition (Sun et al,2014).Therefore,the expression ofHd1andEhd1was studied in bothnrl3and WT (Fig.2-G to -J).The expression ofEhd1was reduced under LD and SD innrl3compared with WT.Ehd1up-regulates the expression ofHd3aandRFT1to promote heading in rice (Doi et al,2004).Furthermore,almost all mutants related to heading date ultimately control the heading date by directly or indirectly regulating the expression levels ofHd3aandRFT1.OsRE1,a nucleus-localized bZIP transcription factor,which directly binds to the promoter region ofEhd1and represses its expression,thereby regulating the expression ofHd3aandRFT1(Chai et al,2021).SIP1,a positive regulator of heading date,whichup-regulates the expression ofHd3aandRFT1through interacting with OsTrx1 as well as altering H3K4me3 levels ofEhd1(Jiang et al,2018).SDG701encodes a methyl-transferase that specifically catalyzes H3K4 methylation,thus promoting the heading in rice by binding to chromatin and promoting H3K4me3 and enhancing the expression ofHd3aandRFT1(Liu et al,2017).To confirm whether the delayed heading phenotype innrl3is responsible for the down-regulation ofHd3aandRFT1,we examined the expression ofHd3aandRFT1innrl3and WT.As compared to WT,the expression ofHd3aandRFT1was significantly decreased innrl3under LD and SD conditions,which also corresponded to the late heading date ofnrl3(Fig.2-C to -F).These results suggested thatNRL3likely mediates the heading date primarily through the regulation of the expression ofHd3aandRFT1under LD and SD conditions.

NRL3 forms a complex with OsK4 to regulate heading date under LD conditions

NRL3is involved in the floral transition in rice and disrupts a delay in the heading date under LD and SD conditions.To investigate the molecular mechanism of NRL3 in the regulation of heading date,we identified an interaction protein OsK4 via the Y2H assay.NRL3 interacted with OsK4 in the nucleus and cytoplasm,which was strongly supported by the Y2H and BiFC assays (Fig.3).Moreover,the results showed that OsK4 is a negative regulator of heading date in rice under LD conditions,and theosk4mutant showed an early heading date phenotype (Fig.5-A and -B).Subsequently,we analyzed the expression patterns ofNRL3andOsK4under LD and SD conditions,and found that they had similar rhythmic expression patterns,with the expression escalating after dawn,peaking before dusk,and then decreasing rapidly (Figs.2-A,2-B,4-C and 4-D).The expression patterns implicated thatNRL3andOsK4might function together to mediate the heading date within the same pathway.In the current study,NRL3was found to be a positive regulator of heading,whereasOsK4repressed heading in rice (Fig.5-A to -C).However,theosk4mutant rescued the delayed heading phenotype ofnrl3(Fig.6-A and -B).Collectively,these results proved thatOsK4is a functional down-stream effector ofNRL3,andNRL3is dependent onOsK4to control the heading date under LD conditions in rice.In addition,we analyzed the levels of the transcript and protein ofOsK4innrl3.Remarkably,the OsK4protein levels were dramatically increased innrl3,whereas no significant differences were observed at the transcript level (Fig.6-D and -G),and the disruptionofNRL3affected the accumulation of OsK4.Similarly,NRL3 interacts with NAL9,a Clp protease homologue,which is a part of an evolutionarily conserved protein degradation pathway (Dong et al,2013;Chen et al,2019).Thus,it was suggested that NRL3 might play a critical role in protein degradation.Likewise,we observed that the protein abundance of OsK4 accumulated dramatically innrl3,however,its transcript level remained unchanged (Fig.6-D and -G).Consequently,we speculated that NRL3 may affect the accumulation of OsK4 by affecting the stability of OsK4.These results collectively indicated that the increased OsK4 protein level might regulate the late heading date phenotype in thenrl3plants.Moreover,OsK4 can phosphorylate Hd1,increase its activity,and repress the expression ofHd3aandEhd1(Sun et al,2016).Therefore,we hypothesized that NRL3 affects the protein accumulation of OsK4,thereby affecting the phosphorylated levels of the Hd1 protein,and further regulating the expression ofHd3a,RFT1andEhd1to control heading date in rice.

METHODS

Rice materials and growth conditions

Thenrl3mutants were identified from the YK17 (O.sativasubsp.indica) mutant library generated by ethyl methyl sulfone (EMS).All plants were grown under NLD (Hangzhou,Zhejiang,China) and NSD conditions (Lingshui,Hainan,China) to observe the phenotypes.The plants were also grown in chambers under LD (14 h light/10 h dark) and SD (10 h light/ 14 h dark) conditions to analyze the rhythmic expression.

Vector construction and rice transformation

The coding sequence ofNRL3was fused into the binary vector pCUBI1390 and transformed into thenrl3plants to creat overexpression transgenic plants.Theosk4mutants and thenrl3/osk4double mutants were generated by editingOsK4via the CRISPR/Cas9 system in Nipponbare andnrl3mutant backgrounds,respectively.At least two independent lines of T2homozygous plants were used for analysis.The primers are listed in Table S1.

Yeast two-hybrid assay

For Y2H analysis,the full-length cDNA sequences ofNRL3andOsK4were amplified and ligated into pGBKT7 and pGADT7 to make the bait plasmid and the prey plasmid,respectively.The pGBKT7-53 and pGADT7-T were co-transformed as the positive controls,and the pGBKT7 and pGADT7 were used as the negative controls.All plasmids were transformed into the Y2H Gold yeast strain,and grown on SD/-Leu-Trp medium for 3 d at 30 °C.Then,the transformants were tested on SD/ -His-Ade-Trp-Leu medium with X-α-gal at 30 °C for 3 d to detect the protein interactions.

BiFC analysis

The coding sequences ofNRL3andOsK4were cloned into the binary vectors pSPYNE and pSPYCE,respectively.Then,the recombinant plasmid was co-transformed into theAgrobacteriumstrain GV3101,cultivated in fresh LB (luria-bertani) broth medium at 28 °C,200 r/min until OD600reached 0.6.The bacterial solution was then centrifuged at 8 000 r/min and resuspended in MES solution (10 mmol/L MES,pH 5.6,10 mmol/L MgCl2and 0.2 mmol/L acetosyringone).Then,the resuspension was injected into tobacco leaves and the yellow fluorescent signal was observed using a laser scanning confocal microscope (LSM710,Karl Zeiss,Germany).

RNA extracted and qRT-PCR analysis

RNA was extracted from the root,stem,leaf,panicle,leaf sheath and seed (5 and 10 d after fertilization) for expression pattern analysis.Total RNA was extracted from the leaves of 60-day-old plants using a TRIZOL reagent (Invitrogen,Carlsbad,USA),and then reverse-transcribed into cDNA from 1 μg of total RNA following the manufacturer’s instructions (ReverTra Ace qPCR RT Kit;Toyobo,Osaka,Japan).qRT-PCR analysis was performed using a LightCycler 480 system (Roche,Basel,Switzerland) and SYBR Green Real-time PCR Master Mix (Toyobo,Japan).The relative expression levels of each gene were normalized to the riceActin(Os03g0718150) gene,which was used as an internal control.All qRT-PCR were measured by three individual replicates.The transcript levels were calculated by the 2-ΔΔCTmethod.The primer sequences are listed in Table S1.

Subcellular localization

The coding sequence ofOsK4excluding the stop codon was cloned into pAN580 to generate the construct 35S:OsK4-GFP.TheD53(Os11g0104300) cDNA was cloned into 163-mCherry vector to create the construct 35S:D53-mCherry,which expressed the fusion protein D53-mCherry and was used as a nucleus localization marker.All vectors,including the empty vector,were transiently expressed into rice protoplasts following previously described protocols (Zhang et al,2011).The GFP signal was detected using a laser scanning confocal microscopy (LSM710,Karl Zeiss,Germany).

Western blot assay

Total protein was extracted from the leaves of the WT,nrl3,nrl3/osk4andNRL3overexpression plants (OE-1andOE-2) at the heading stage using an extraction medium [25 mmol/L Tris-HCl,pH 7.4,150 mmol/L NaCl,1 mmol/L EDTA,1% Nonidet P-40,5% glycerol,1 mmol/L PMSF (phenyl methane sulfonyl fluoride) and Roche protease inhibitor].The extracts were centrifuged at 14 000 r/min for 5 min at 4 °C,and then the supernatant was collected (repeated three times).Proteins were separated by SDS-PAGE,and transferred to polyvinylidene fluoride (PVDF) membranes.The anti-NRL3 and anti-OsK4 antibodies (Synthesis by GenScript Biological Technology,China) were used in immunoblot assay,and β-ACTIN (CW0264M,CWBIO,China) was used as a control.

In vitro stability assay for OsK4 protein

The protein stability assay was performed following the protocol described by Wang et al (2009).The total protein was extracted from 9-day-old seedlings of YK17 andnrl3plants.The GST-OsK4 protein was purified using the GST Recombinant Protein Purification Kit (BBI,C600327-0001,Sangon Biotech,China) according to the manufacturer’s instructions.Equal amounts of YK17 andnrl3total protein were incubated with the GST-OsK4 fusion protein at 25 °C.The protease inhibitor MG132 (Sigma-Aldrich,Germany) with a final concentration of 50 μmol/L was used to suppress the degradation of GST-OsK4.

ACKNOWLEDGEMENTS

This study was supported by the China National Natural Science Foundation (Grant No.31871597),the Key Research and Development Program of Zhejiang Province,China (Grant No.2021C02063-2),the Key Research and Development Program of China National Rice Research Institute (Grant No.CNRRI-2020-02),the Science and Technology Project of Jiangxi Provincial Department of Education,China (Grant No.GJJ180217),and the Project supported by Jiangxi Youth Science Foundation,China (Grant No.20202BAB215001).

SUPPLEMENTAL DATA

The following materials are available in the online version of this article at http://www.sciencedirect.com/journal/rice-science;http://www.ricescience.org.

Fig.S1.Expression analysis ofOsGI.

Fig.S2.Twoko-osk4mutations obtained by CRISPR/Cas9 system.

Fig.S3.Expression analyses ofHd3a,RFT1andEhd1innrl3andosk4/nrl3mutants.

Table S1.Primers used in this study.