RNA-seq approach to discriminate gene expression profiles in RIXI-overexpressing transgenic rice

PENG Yaoyao,HOU Chunxiao,ZHAN Yihua,HUANG Yingying,SUN Xiangyu,WENG Xiaoyan*

(1.State Key Laboratory of Plant Physiology and Biochemistry,College of Life Sciences,Zhejiang University,Hangzhou 310058, China;2.Zhejiang Academy of Agricultural Sciences,Hangzhou 310021,China;3.School of Medicine,Zhejiang University,Hangzhou 310058,China)

RNA-seq approach to discriminate gene expression profiles in RIXI-overexpressing transgenic rice

PENG Yaoyao1,HOU Chunxiao2,ZHAN Yihua1,HUANG Yingying3,SUN Xiangyu1,WENG Xiaoyan1*

(1.State Key Laboratory of Plant Physiology and Biochemistry,College of Life Sciences,Zhejiang University,Hangzhou 310058, China;2.Zhejiang Academy of Agricultural Sciences,Hangzhou 310021,China;3.School of Medicine,Zhejiang University,Hangzhou 310058,China)

Summary To investigate whether RIXI-overexpressing transgenic plants influence the expression of other genes,we analyzed transcriptomic changes between R7(RIXI-overexpression transgenic line 7)plants and WT (wild-type)plants using deep RNA sequencing combined with digital gene expression profile analysis.The differentially expressed genes between the WT and R7 libraries were identified by DEG-Seq(differentially expressed genes from RNA-seq)software.The profiling analysis revealed that the overexpression of RIXI in rice resulted in numerous changes in gene expressions,including upregulation of 391 genes and downregulation of 905 genes.These differentially expressed genes were categorized into 30 groups with broad functions using gene ontology(GO)assignments.Among the 30 groups,five groups(single-organism metabolic process,biological regulation,anion binding,small molecule binding and nucleotide binding)had more differentially expressed genes.Biological pathways affected by RIXI overexpression were mapped using the detected genes to reference canonical pathways in the Kyoto Encyclopedia of Genes and Genomes(KEGG).The differentially expressed genes were assigned to 98 KEGG pathways,and four enriched pathways were identified:metabolic,biosynthesis of secondary metabolites,plant-pathogen interaction and plant hormone signal transduction.The measurement of agronomic traits of R7 showed that the overexpression of RIXI did not influence the growth and development of rice.Thus,we conclude that the xylanase inhibitor gene RIXI may play a role in activation of a complex signal transduction network in response to various biotic and abiotic stresses,but does not have a negative influence on growth and development of rice plants.

Key words rice xylanase inhibitor;transgenic rice plant;RNA sequencing;plant defense

Plant endogenous xylanase inhibitors(XIs), discovered in 1997 by DEBYSER et al.[1]in wheat, are plant-produced proteinaceous inhibitors that inhibit xylanases.To date,three structurally different classes of XIs have been identified in plants,Triticum aestivum xylanase inhibitor(TAXI)-type[2],xylanaseinhibiting protein(XIP)-type[3]and thaumatin-like xylanase inhibitor(TLXI)-type[4].TAXI,XIP and TLXI genes have already been characterized in wheat, rice,maize,barley,sorghum and rye[5].

XIs are only active against xylanases of microbial origin and cannot inhibit the plant endogenous xylanases;therefore,it has been hypothesized that XIs function only in plant defense rather than in plant growth and development[6].Additional features that support possibly involvement of XIs in plant defense include the findings that XIs share sequence and structure homology with some pathogenesis-related (PR)proteins,and their expression is induced under stress conditions,and they inhibit the growth and germination of fungi.For example,XIPand TLXI can be classified as PR proteins of classes PR-5 and PR-8, respectively,based on their homology to thaumatinlike proteins and chitinases.Some members of TAXI and XIP gene families are significantly induced by wounding,pathogen infection and treatment with jasmonic acid(JA)and methyl jasmonate[78].A chitinase-like XIP from coffee,Coffea arabica, inhibited the germination of spores of soybean Asian rust Phakopsora pachyrhizi[9];and XIP and TAXI inhibited the growth of Rhizoctonia solani and Fusarium graminearum,respectively[10].

Rice was reported to contain only the XIP type XIs,including RIXI,OsXIP,riceXIP and OsHIXIP[8,1113].The RIXI gene(LOC_Os11g47580)was first cloned from rice in 2005,and it inhibited fungal GH11 xylanases,but did not inhibit GH11 xylanases from bacteria and GH10 xylanases from both fungi and bacteria[11];and RIXI was upregulated in rice seedlings infected by Magnaporthe grisea[14].

Although most studies agree that XIs are involved in plant defense,little is known about additional functions of XIs in rice growth,XI-related genes and their expression pattern.

The RNA-seq method generates absolute information,and is more sensitive for transcripts with low expression.In this study,to gain more insight into the function of XIs in plant development,deep RNA sequencing was employed in combination with digital gene expression(DGE)profiles to analyze the difference between the transcriptomes of R7(RIXI-overexpression transgenic line 7)and WT(wild-type). Additionally,the biological functions of RIXI,XI,and the effects of overexpressing RIXI on expressions of other rice genes as well as the mechanism of its role in plant defense were illustrated.

1　Materials and methods

1.1Plant materials and growth conditions

Rice(Oryza sativa L.ssp.japonica cv. Nipponbare)was used in this study.R7 plants were RIXI overexpressing transgenic rice plants with single copy integration[15],which were generated by placing RIXI under control of the strong cauliflower mosaic virus(CaMV)35S promoter in our laboratory.WTand R7 were grown in normal culture solution at p H 5.0- 5.5 in a glasshouse at 28°C with 16 h of light and 8 h of darkness.The leaves of 2-week-old plants were collected for sequencing.The experiments were repeated three times.The experimental materials for sequencing were obtained by mixing 50 leaves together in each group.

1.2Library construction and sequencing

At least 3μg of total RNA per sample was sent to the Beijing Genomics Institute for Solexa sequencing.Sequencing libraries were generated using Illumina TruSeq RNA sample preparation kit (Illumina,San Diego,CA,USA)following the manufacturer's recommendations.The clustering of the index-coded samples was performed on a cBot Cluster Generation System using TruSeq PE cluster kit v3-cBot-HS(Illumina,San Diego,CA, USA)according to the manufacturer's instructions.After cluster generation,the library preparations were sequenced on an Illumina Hiseq 2000 platform and 100 bp single-end reads were generated.

1.3Data analysis

Raw data(raw reads)of FASTQ format were first processed through in-house Perl scripts.In this step,clean data(clean reads)were obtained by removing reads containing adapters,those containing poly-N and low-quality reads from raw data.At the same time,Q20,Q30,GC content and the clean data were calculated.All downstream analyses were based on the clean highquality data.

Reference genome and gene model annotation files were directly downloaded from the genome website(ftp://ftp.ensemblgenomes.Org/pub/release-17/plants/fasta/oryza_sativa),and index of the reference genome was built using Bowtie v2.0.6, and single-end clean reads were aligned to the reference genome using Top Hat v2.0.9.HTSeq v0.5.4p3 was used to count the read numbers mapped to each gene.Then RPKM(reads per kilobase of exon model per million mapped reads) of each gene was calculated based on the length of the gene and read counts mapped to this gene[16]. To obtain statistical confirmation of the differences in gene expression between the WT and R7 plants, we used the RPKM to normalize the expression level of genes.All the uniquely mapped reads were used for calculating RPKM values.The differentially expressed genes between the WT and R7 libraries were identified using DEG-Seq software[17],and P-values were corrected using the method of BENJAMINI et al.[18].Genes with an adjusted P-value＜0.05 found by DEG-Seq were assigned as differentially expressed.

1.4GO and KEGG enrichment analysis of differentially expressed genes

Gene ontology(GO)enrichment analysis of differentially expressed genes was implemented by the GOseq R package,in which gene length bias was corrected.GO terms with corrected P-value＜0.05 were considered significantly enriched by differentially expressed genes.

Kyoto Encyclopedia of Genes and Genomes (KEGG)is a database resource for understanding high-level functions and utilities of the biological system,such as the cell,organism and ecosystem from molecular-level information,especially largescale molecular datasets generated by genome sequencing and other high-throughput experimental technologies(http://www.genome.jp/kegg/).We used KOBAS software to test the statistical enrichment of differentially expressed genes in KEGG pathways.

1.5Quantitative real-time PCR analysis

For quantitative real-time polymerase chain reaction(qPCR),total RNAs from seedlings of different genotypes were extracted using RNA prep pure plant kit(TianGen Biotech Co.Ltd.,Qiagen, http://www.qiagen.com),following the manufacturer's protocols.Total RNAs were converted into cDNAs using PrimeScript RT reagent kit(TaKaRa,now Clontech,http://www.clontech.com/takara).Realtime PCR analysis used SYBR Premix Ex Taq TMⅡ(TaKaRa)on a Roche LightCycler480 real-time PCR system,following the manufacturer's instructions (Roche,http://www.roche.com).Transcript levels of each mRNA were determined and normalized withthe level of actin mRNAs using theΔCtmethod[19]. Gene-specific primers are listed in Table 1.

1.6Measurement of agronomic traits

Ten plants from each genotype were randomly selected and evaluated for five growth and development related agronomic traits[20].Plant height and panicle length were measured as the average of the ten plants,and number of tiller per plant and panicles per plant were the average which evaluated for all tillers and panicles on the ten plants(panicles with less than ten seeds were not counted)[2122].Thousand-grain mass(TGM)was the average of a sample of 1 000 filled grains per plant.All data including at least three measurements were analyzed using SPSS 16.0(SPSS Inc.,Chicago, IL,USA)for analysis of variance(ANOVA)and Duncan's test.P＜0.05 was considered statistically significant.

Table 1 Genes selected for quantitative real-time PCR analysis

2　Results

2.1Comparison of gene expression level between the two libraries

To investigate the expression patterns of genes in WT and R7 plants,the RNA extracted from leaves was sequenced by Illumina Hiseq 2000 platform.Comparing the data between WT and R7 plants,each of two replicate experiments showed highly correlated expression values(R2＞0.92), indicating that the results from these experiments were reliable.

Compared with WT,a total of 1 296 significantly changed genes were detected in R7 plants,in which 391 genes were upregulated and 905 genes were downregulated.The results also revealed that a total of 96 genes were upregulated by at least threefolds,and 431 genes were downregulated by at least threefolds in R7 plants. Overexpression of RIXI gene may have an inhibitory effect on expression of some genes, because RIXI itself is an inhibitory protein,and overexpressing one gene in rice can influence the expression levels of some other genes.

2.2Functional categorization of differentially expressed genes

Gene expression was compared between R7 and WT rice plants,and GO(http://geneontology.org) assignments were used to classify the functions of the differentially expressed genes.The GO annotation of these genes was presented in Fig.1.These genes were mainly classified into the following categories: biological process and molecular function with 19 and 11 functional groups,respectively(Fig.1A,B). Among the 30 groups,five groups had more differentially expressed genes,such as single-organism metabolic process(GO:0044710)with 260 genes(62 upregulated genes and 198 downregulated genes)in the main category of biological process.Anion binding(GO:0043168)with 214 genes,small molecule binding(GO:0036094)with 212 genes in the main categories of molecular function.These results showed that RIXI may be involved in the molecular function and the regulation of biological process,and plays a vital role.

Fig.1 Histogram of gene ontology classification

2.3Pathway enrichment analysis of the differentially expressed genes

The biological pathways affected by RIXI expression were evaluated by mapping the detected genes to reference canonical pathways in the KEGG.Based on the expression pattern in WT and R7 plants,the differentially expressed genes were assigned to 98 KEGG pathways.The pathways with the greatest representation by unique genes were the metabolic pathways(KO: osa01100)with 87 members(6.1%of background genes in metabolic pathway),biosynthesis of secondary metabolites(KO:osa01110)with 62 members(8.5%),plant-pathogen interaction (KO:osa04626)with 25 members(26.0%)and plant hormone signal transduction(KO:osa04075) with 20 members(13.7%).These annotations provide a valuable resource for investigating the function of RIXI in rice growth and pathogen defense.

2.4Overexpression of RIXI modulates plant hormone signaling

The plant hormone signal transduction pathway contained a total of 146 genes,with 20 significantly upregulated or downregulated genes in R7 plants compared to WT.It should be pointedthat seven jasmonate-signaling genes in R7 plants were downregulated(Table 2).These genes(log-ratio range of-6.302 8 to-1.281 5)included rice jasmonate ZIM-domain(JAZ)proteins OsJAZ6,OsJAZ7, OsJAZ8,OsJAZ9,OsJAZ10,OsJAZ12 and OsJAZ13.

Compared with WT plants,two regulatory protein NPR1 genes(LOC_Os01g09800 and LOC_Os03g46440,log-ratio range of-0.995 1 and -1.661 6)in transgenic plants were downregulated (Table 2).Ethylene(ET)signaling-related genes, such as ET receptor-like protein and EIN3-binding F-box protein 1,were upregulated in R7 plants. Overexpression of RIXI also influenced abscisic acid(ABA)signaling,and enhanced the expression of one gibberellin(GA)20 oxidase gene (LOC_Os01g66100);while a chitin-inducible gibberellin-responsive protein 1 gene(CIGR1, LOC_Os07g36170)was downregulated,and two indole acetic acid(IAA)biosynthesis genes, indole-3-acetic acid-amido synthetases OsGH3.8 and OsGH3.2,were highly downregulated (Table 2).

Table 2 Selected genes of plant hormone signal transduction pathway with altered expression in the two samples

2.5RIXI expression and transcription factors(TFs)

Overexpression of RIXI induced changes in the expression of 95 TFs belonging to 23 TF families(Table 3),including AP2/EREBP,AUX/ IAA,BES1,b HLH,bZIP,C3 H,HSF,MYB, NAC,TCP,Trihelix,WRKY and TIFY,all of which play important roles in plant development and immunity.Of the 95 identified TF genes,30 were upregulated,including two ethylene-responsive factor transcriptional regulator genes(LOC_Os09g11460 and LOC_Os04g46220)belonging to the AP2/ EREBP family,two Aux/IAA TFs,seven b HLH genes,three C3H genes and one WRKY gene (LOC_Os08g38990).Meantime,65 TF genes were downregulated,including six MYB TFs, seven TIFY TFs and 13 WRKY TFs(Table 3).

Table 3 Number of transcription factor genes with altered expression in the two samples

2.6RIXI was involved in plant defense responses

The KEGG pathway enrichment analysis also revealed that the“plant-pathogen interaction”pathway was also significantly changed in R7 plants,indicating that RIXI overexpression influenced the expression of many genes involved in plant defense.This included such signal transduction components as Ca2+and G-protein signaling,protein phosphatase,protein kinases,and defense response proteins including cyclic nucleotidegated ion channel 2,calcium-binding protein and defense proteins against fungi or bacteria(Table 4).

Overexpression of RIXI also influenced the expression of genes involved in reactive oxygen species(ROS)production and antioxidants(Table 4).Of the responsive ROS-production genes (including amine oxidase,germin-like protein, respiratory burst oxidase homolog protein F and NADH dehydrogenase),three were upregulated and six were downregulated.Of the responsive genes involved in antioxidants,four were upregulated and 13 were downregulated(Table 4).

Table 4 Number of genes with altered expression involved in plant-pathogen interaction pathway

2.7Confirmation of tag-mapped genes by qPCR analyses

To evaluate the reproducibility of the profile, 10 genes were selected for qPCR assays,including PR genes,TF genes,JA and salicylic acid(SA) biosynthesis and signaling genes.Expression levels of these genes(Cht,OsPR1a,OsPR1b,PR4, AOS,LOX,PAL,N H1,Pid2 and Myb)had similar patterns with qPCR to those of Tag-seq analysis(Fig.2),indicating the validity of the profile.

Fig.2 Quantitative real-time PCR analysis for 10 transcripts in WT and R7 plants

2.8Characteristics of transgenic rice plants

The transgenic lines R7 appeared to grow and develop normally.Phenotype parameters were measured at the stage of plant maturity.There were no significant differences in plant height, panicle length,number of tillers and panicles compared with non-transgenic plants(WT)(Table 5).The transgenic lines R7 had slight higher values for TGM.These results indicate that the overexpression of RIXI has little influence during the development stages of transgenic rice plants and don't reduce the production of rice in total; moreover,it might have a positive effect on the yield of rice to a certain extent.

Table 5 Phenotype and thousand-grain mass(TGM)of R7 and WT plants

3　Discussion

This study is the first to study transcriptomic changes of transgenic rice(R7)and WT plants using deep RNA sequencing combined with DEG profile analysis.Compared with WT,the transcript levels of 1 296 genes in overexpressing transgenic R7 plants were differentially expressed. The annotated differentially expressed genes could be classified into two categories:biological process and molecular function with 19 and 11 functional groups,respectively,among which singleorganism metabolic process,anion binding,small molecule binding,small molecule binding and nucleotide binding have more different expression genes.These results showed that RIXI may be involved in the molecular function and regulation of biological process by controlling the small molecular binding which involved in the metabolic process.According to KEGG pathway enrichment analysis,most differentially expressed genes were involved in metabolism,biosynthesis of secondary metabolites,plant-pathogen interaction and plant hormone signal transduction.By altering metabolism,plants could make more energy available for synthesis of defense materials and elicitation of the defense response[23].Changes in metabolism might be a general phenomenon in transcriptional repressors of JA responses in Arabidopsis and rice[2425].This confirmed observations that XIs respond to stress via a JA-mediated signaling pathway[8].One of the major roles of ABA is to mediate adaptive responses to various environmental stresses during the plant growth.It has a critical role in the plant adaptation or acclimation to abiotic stresses,such as high salinity,drought,low temperature and mechanical wounding[26].The overexpression of RIXI also influenced ABA signaling,resulting in upregulation of two genes(LOC_Os02g15640 and LOC_Os01g59760)and downregulation of three genes(LOC_Os07g42940,LOC_Os03g16170 and plant responses to stresses.

As it has been hypothesized that RIXI plays an important role in plant defense[14],we focused on defense response genes,such as plant hormone signaling,TFs,signal transduction components and defense response protein genes.

Phytohormones are chemical messengers that coordinate cellular activities.The phytohormones of plants such as Auxin/IAAs,CK,GA,ABA, ET,BR and JA are chemical messengers that play distinct but overlapping roles in the regulation of defense response,growth and development of rice plants.In the present study,compared with WT, plants overexpressing RIXI downregulated JAZ proteins.JAZ transcriptional proteins acted asLOC_Os02g52780).The LOC_Os01g59760 and LOC_Os02g52780 encode OsbZIP09 and OsbZIP23, respectively,which are members of the basic leucine zipper(bZIP)TF family in rice and regulate the expression of a wide spectrum of stress-related genes in response to abiotic stresses through an ABA-dependent regulation pathway[2627]. In addition,we found that the overexpression of RIXI elevated the m RNA levels of many genes involved in ET biosynthesis and signal transduction.ET plays a highly pleiotropic role in plant growth and development,and participates in responses to various biotic and abiotic stresses. Most genes involved in JA,SA,ABA,and ET biosynthesis or signaling pathways were altered, indicating that RIXI-induced plant defense responses are mainly dependent on the JA,SA, ABA and ET pathways.

All major processes of life depend on differential gene expression,which is largely controlled by TFs'activity.Many TFs,such as MYB,NAC,WRKY,and TIFY family TFs, were downregulated or upregulated in R7 plants. Few genes of TFs,for example WRKY gene (OsWRKY30,LOC_Os08g38990)was highly increased in R7 plants.These downregulated TFs serve as negative regulators of defense signaling, for instance,OsWRKY71 has features characteristic of a transcriptional repressor of GA signaling in aleurone cells[28],and OsWRKY45-1 negatively regulates ABA signaling and,in addition,OsWRKY45-2 negatively regulates rice response to salt stress[29].TIFY 10C/OsJAZ8 negatively regulated the JA-induced resistance to pathogens in rice and acts as a repressor of JA signaling in rice[30].

Many genes involved in the biosynthesis pathways of secondary signaling compounds were regulated,including Ca2+signaling,ROS,G-protein,phosphatidylinositol signaling,and protein kinase.Compared with transcript levels in WT plants,the expression levels of many genes encoding protein kinases,such as receptor-like protein kinase,mitogen-activated protein kinase (MAPK)and calmodulin dependent protein kinase,were changed in RIXI-overexpressing rice plants.Protein kinases represent an important mechanism in rice defense signal transduction,and MAPK signaling cascades are conserved in signaling pathways and have been implicated in a wide variety of plant biotic and abiotic stress responses[31].This indicates that the constitutive expression of RIXI in rice plants activated a complex signal transduction network and enhanced rice resistance to stress.

In conclusion,RIXI,a rice XI,involved in biological process and molecular function, especially activated a complex signal transduction network in response to various biotic and abiotic stresses.Meanwhile,the overexpression of RIXI didn't have a negative influence on the growth and development of rice plants.More work should be done to understand the deep biological functions and regulatory mechanisms of this transgenic plant in future.

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彭耀耀1,侯春曉2,詹儀花1,黃瑩瑩3,孫翔宇1,翁曉燕1*(1.浙江大學(xué)生命科學(xué)學(xué)院,植物生理學(xué)與生物化學(xué)國(guó)家重點(diǎn)實(shí)驗(yàn)室,杭州310058;2.浙江省農(nóng)業(yè)科學(xué)院,杭州310021;3.浙江大學(xué)醫(yī)學(xué)院,杭州310058)

摘要為研究水稻植株中木聚糖酶抑制劑基因RIXI過(guò)量表達(dá)是否會(huì)引起其他基因的差異表達(dá),利用轉(zhuǎn)錄組測(cè)序(RNA-Seq)技術(shù)結(jié)合數(shù)字基因表達(dá)譜分析對(duì)RIXI過(guò)量表達(dá)單拷貝純合株系R7進(jìn)行基因差異表達(dá)分析.水稻全基因表達(dá)譜顯示,RIXI過(guò)表達(dá)引起水稻中大量基因的表達(dá)差異,包括391個(gè)上調(diào)基因,905個(gè)下調(diào)基因.GO分析將差異基因分成30個(gè)功能聚類,其中的5組聚類中含有較多的差異基因,分別為單個(gè)有機(jī)體代謝過(guò)程、生物調(diào)控、陰離子結(jié)合、小分子結(jié)合和核苷酸結(jié)合.利用KEGG數(shù)據(jù)庫(kù),通過(guò)Pathway顯著性富集確定差異表達(dá)基因參與主要生化代謝途徑和信號(hào)轉(zhuǎn)導(dǎo)途徑.與整個(gè)水稻基因組背景WT相比,R7的差異表達(dá)基因包含在98個(gè)KEGG通路中,包含差異基因數(shù)量最多的4個(gè)KEGG通路分別為代謝通路、次級(jí)代謝的生物合成、植物與病原菌互作和激素信號(hào)傳導(dǎo).農(nóng)藝性狀測(cè)量顯示,RIXI過(guò)表達(dá)對(duì)水稻生長(zhǎng)發(fā)育幾乎沒(méi)有影響.以上結(jié)果說(shuō)明,木聚糖酶抑制劑基因RIXI可能在各種生物和非生物脅迫中激活復(fù)雜的信號(hào)傳導(dǎo),但對(duì)水稻的生長(zhǎng)和發(fā)育沒(méi)有負(fù)面影響.

關(guān)鍵詞水稻木聚糖酶抑制劑;轉(zhuǎn)基因水稻;轉(zhuǎn)錄組測(cè)序;植物防御

RIXI過(guò)量表達(dá)轉(zhuǎn)基因水稻的全基因組表達(dá)譜分析(英文).浙江大學(xué)學(xué)報(bào)(農(nóng)業(yè)與生命科學(xué)版),2016, 42(6):643- 653

DOI:10.3785/j.issn.1008-9209.2016.01.142

CLC numberQ 945.78

Document codeA

Foundation item:Supported by the National Natural Science Foundation of China(Nos.30971702 and 31271632),and Science and Technology Department of Zhejiang Province,China(Nos.2013C32018 and 2016C32086).

*Corresponding author:WENG Xiaoyan(http://orcid.org/0000-0001-6559-3386),E-mail:xyweng@zju.edu.cn

Received:2016 01 14;Accepted:2016 04 06;Published online:2016 11 19

Biography:PENG Yaoyao(http://orcid.org/0000-0003-0168-1234),E-mail:pengyaoyao09@foxmail.com

URL:http://www.zjujournals.com/agr/CN/article/download ArticleFile.do?attach Type=PDF&id=10424