《园艺作物育种学》课程教学资源（学术研究）Genome-wide analysis and identification of TIR-NBS-LRR genes in Chinese cabbage（Brassica rapa ssp. pekinensis）reveal expression patterns to TuMV infection

Physiological and Molecular Plant Pathology 90(2015)89-97 Contents lists available at ScienceDirect PMPP Physiological and Molecular Plant Pathology ELSEVIER journal homepage:www.elsevier.com/locate/pmpp Genome-wide analysis and identification of TIR-NBS-LRR genes in CrossMark Chinese cabbage (Brassica rapa ssp.pekinensis)reveal expression patterns to TuMV infection Shanwu Lv.1,Changwei Z..Jun Tang,Yanxiao Li,Zhen Wang,Dahua Jiang Xilin Hou a.◆ State Key Laboratory of Crop Genetics and Germplasm Enhancement.Key Laboratory of Biology and Germplasm Enhancement of Horticultural Crops in East China,Ministry of Agriculture,College of Horticulture,Nanjing Agricultural University.Nanjing PR China Institute of Botany.Jiangsu Province and Chinese Sciences of Academy.Nanjing.PR China ARTICLE INFO ABSTRACT Article history: TIR-NBS-LRR (TNL)genes greatly affect plant growth and development.Ninety TNL-type genes were Accepted 1 April 2015 identified and characterized in Chinese cabbage (Brassica rapa ssp.pekinensis).Tissue-expression Available online 10 April 2015 profiling revealed different expression levels in different tissues.gRT-PCR analysis revealed the expression patterns of 69 genes challenged by Turnip mosaic virus (TuMV):42 genes were up-regulated Keywords: and 11 genes down-regulated:genes were grouped according to their different expression patterns. Genome-wide analysis TIR-NBS-LRR genes Sixteen candidate genes were identified as responding to TuMV infection.This study supplies infor- Gene expression patterns mation on resistance genes involved in Chinese cabbage's response against TuMV,and furthers the Turnip mosaic virus understanding of resistance mechanisms in B.rapa crops. Chinese cabbage 2015 Elsevier Ltd.All rights reserved. Introduction and avr genes appear in hosts and pathogens simultaneously then plants display disease resistance,but if not,disease occurs [2]. Plants are subject to attack from pathogens and herbivores Currently,over 100 reference resistance genes and 104,310 pu- during their growth and development.As sessile organisms,they tative genes make up the super R gene family(Plant Resistance have developed a series of sophisticated defense mechanisms, Genes database:PRGdb;http://prgdb.org)[3].Based on putative regulated by gene-for-gene disease resistance(R)genes,to resist protein domains,R genes are divided into five classes:CNL (CC- infringement from diverse biotic challenges [1.In many cases, NBS-LRR).TNL (TIR-NBS-LRR).RLK (Receptor like kinases).RLP plant-pathogen interactions are triggered by pathogen avirulence (Receptor like proteins).and Pot (Ser/Thr kinase protein)[4.5].The (avr)genes and corresponding plant disease R genes.If relevant R 'nucleotide-binding site plus leucine-rich repeat'(NBS-LRR)is an R gene superfamily,which is involved in encoding putative inter- cellular responders [2.6].This superfamily is abundant in plants. containing 149 genes in Arabidopsis [7.535 related sequences in rice(Oryza sativa)[8].and approximate 400 genes in Populus tri- Abbreviations:avr gene,avirulence gene:CC.coiled-coil:CNL CC-NBS-LRR:CP coat protein:dpi,days post-inoculation;LF,least fractionated blocks:MF1,medium chocarpa (9].The domains of NBS-LRR genes are characterized by fractionated blocks:MF2,most fractionated blocks:ML,maximum-likelihood:MW two kinds of N-terminal ends:either a coiled-coil(CC)or a Toll molecular weight:MYA,million years ago:pl.proteins'isoelectric point:PRGdb. Interleukin-1 receptor(TIR)domain,referred to as TIR-NBS-LRRs plant resistance genes database:qRT-PCR,quantitative reverse transcription PCR: (TNLs)or CC-NBS-LRRs (CNLs),respectively [5.10].The NBS RFLP.restricted fragment length polymorphisms:R gene,resistance gene:RL domain is essential for ATP/GTP binding activity,and consists of resistant line:RLK,receptor like kinases:RLP.receptor like proteins:RPKM,reads per kilobase per million:SL susceptible line:SSR,simple sequence repeat:TNL TIR- kinase-2,kinase-3,P-loop,and GLPL motifs [11,12].The LRR NBS-LRR:TIR,toll/interleukin-1 receptor:TuMV,turnip mosaic virus:WGT,whole domain can mediate interactions directly or indirectly with path- genome triplication. ogen molecules [13].The CC domain may be related to protein-- Corresponding author.Tel./fax:+86(0)25 84395917. protein interaction and signaling [5].The function of the TIR E-mail address:hxi@njau.edu.cn (X.Hou). 1 These authors contributed equally to this work domain is mainly related to resistance specificity and signaling: htp:/dx.doi.org/10.1016j.pmpp.2015.04.001 0885-5765/2015 Elsevier Ltd.All rights reserved

Genome-wide analysis and identification of TIR-NBS-LRR genes in Chinese cabbage (Brassica rapa ssp. pekinensis) reveal expression patterns to TuMV infection Shanwu Lv a, 1 , Changwei Z. a, 1 , Jun Tang b , Yanxiao Li a , Zhen Wang a , Dahua Jiang a , Xilin Hou a, * a State Key Laboratory of Crop Genetics and Germplasm Enhancement, Key Laboratory of Biology and Germplasm Enhancement of Horticultural Crops in East China, Ministry of Agriculture, College of Horticulture, Nanjing Agricultural University, Nanjing, PR China b Institute of Botany, Jiangsu Province and Chinese Sciences of Academy, Nanjing, PR China article info Article history: Accepted 1 April 2015 Available online 10 April 2015 Keywords: Genome-wide analysis TIR-NBS-LRR genes Gene expression patterns Turnip mosaic virus Chinese cabbage abstract TIR-NBS-LRR (TNL) genes greatly affect plant growth and development. Ninety TNL-type genes were identified and characterized in Chinese cabbage (Brassica rapa ssp. pekinensis). Tissue-expression profiling revealed different expression levels in different tissues. qRT-PCR analysis revealed the expression patterns of 69 genes challenged by Turnip mosaic virus (TuMV): 42 genes were up-regulated, and 11 genes down-regulated; genes were grouped according to their different expression patterns. Sixteen candidate genes were identified as responding to TuMV infection. This study supplies infor￾mation on resistance genes involved in Chinese cabbage's response against TuMV, and furthers the understanding of resistance mechanisms in B. rapa crops. © 2015 Elsevier Ltd. All rights reserved. Introduction Plants are subject to attack from pathogens and herbivores during their growth and development. As sessile organisms, they have developed a series of sophisticated defense mechanisms, regulated by gene-for-gene disease resistance (R) genes, to resist infringement from diverse biotic challenges [1]. In many cases, plantepathogen interactions are triggered by pathogen avirulence (avr) genes and corresponding plant disease R genes. If relevant R and avr genes appear in hosts and pathogens simultaneously then plants display disease resistance, but if not, disease occurs [2]. Currently, over 100 reference resistance genes and 104,310 pu￾tative genes make up the super R gene family (Plant Resistance Genes database; PRGdb; http://prgdb.org) [3]. Based on putative protein domains, R genes are divided into five classes: CNL (CC￾NBS-LRR), TNL (TIR-NBS-LRR), RLK (Receptor like kinases), RLP (Receptor like proteins), and Pot (Ser/Thr kinase protein) [4,5]. The ‘nucleotide-binding site plus leucine-rich repeat’ (NBS-LRR) is an R gene superfamily, which is involved in encoding putative inter￾cellular responders [2,6]. This superfamily is abundant in plants, containing 149 genes in Arabidopsis [7], 535 related sequences in rice (Oryza sativa) [8], and approximate 400 genes in Populus tri￾chocarpa [9]. The domains of NBS-LRR genes are characterized by two kinds of N-terminal ends: either a coiled-coil (CC) or a Toll/ Interleukin-1 receptor (TIR) domain, referred to as TIR-NBS-LRRs (TNLs) or CC-NBS-LRRs (CNLs), respectively [5,10]. The NBS domain is essential for ATP/GTP binding activity, and consists of kinase-2a, kinase-3a, P-loop, and GLPL motifs [11,12]. The LRR domain can mediate interactions directly or indirectly with path￾ogen molecules [13]. The CC domain may be related to proteine￾protein interaction and signaling [5]. The function of the TIR domain is mainly related to resistance specificity and signaling; Abbreviations: avr gene, avirulence gene; CC, coiled-coil; CNL, CC-NBS-LRR; CP, coat protein; dpi, days post-inoculation; LF, least fractionated blocks; MF1, medium fractionated blocks; MF2, most fractionated blocks; ML, maximum-likelihood; MW, molecular weight; MYA, million years ago; pI, proteins' isoelectric point; PRGdb, plant resistance genes database; qRT-PCR, quantitative reverse transcription PCR; RFLP, restricted fragment length polymorphisms; R gene, resistance gene; RL, resistant line; RLK, receptor like kinases; RLP, receptor like proteins; RPKM, reads per kilobase per million; SL, susceptible line; SSR, simple sequence repeat; TNL, TIR￾NBS-LRR; TIR, toll/interleukin-1 receptor; TuMV, turnip mosaic virus; WGT, whole genome triplication. * Corresponding author. Tel./fax: þ86 (0) 25 84395917. E-mail address: hxl@njau.edu.cn (X. Hou). 1 These authors contributed equally to this work. Contents lists available at ScienceDirect Physiological and Molecular Plant Pathology journal homepage: www.elsevier.com/locate/pmpp http://dx.doi.org/10.1016/j.pmpp.2015.04.001 0885-5765/© 2015 Elsevier Ltd. All rights reserved. Physiological and Molecular Plant Pathology 90 (2015) 89e97

S.Lv et al Physiological and Molecular Plant Pathology 90 (2015)89-97 plant resistance proteins use their TIR domain in the detection of ancestral genome of mesohexaploid B.rapa 28 were used to map pathogens [14].CNLs are more common in dicotyledon and cereal TNL encoding gene loci on the ten chromosomes and three sub- crops,and their relevant functions are similar to TNLs domains [15. genomes,using the method described previously [29]. A feature of TNLs and CNLs is their species-specific distribution. TNL genes are numerous in dicots,yet few exist in cereals [8. Conserved motif and phylogenetic analysis Furthermore,TNL gene sequences are more easily amplified from dicot genomes than cereal genomes.The truncation of TIR-NBS(TN) Pepstats (http://www.ebi.ac.uk/Tools/seqstats/emboss_ or TIR-X(TX)type protein domains in domesticated cereal plants pepstats)was used to identify putative TNL characteristics, may have led to loss of TNL genes in monocot plants such as rice, including isoelectric point(pl)and molecular weight(MW).MEME wheat(Triticum spp.),and maize(Zea mays)[16.17].TNL genes are Suite 30 was used to identify conserved motifs in TNL proteins. mainly involved in recognition of species-specific pathogens.Non- The conserved motifs were used for phylogenetic analysis using the TIR-NBS genes (including CNLs)are derived from at least two maximum-likelihood (ML)method,with the bootstrap value set as species,demonstrating their ancestors originated before the 1000 by MEGA v5.0 [311. divergence between species (before monocot and dicot):non-TIR- NBS genes function in basic defense,it may be due to the adapta- Expression analysis of TIR-NBS-LRR genes in different tissues tion to biotic habitats [18]. Brassica crops provide food,oilseed,fodder,and condiments. Illumina RNA-Seq data obtained from BRAD [32]provided the Numerous materials have been developed for the investigation of groundwork for analyses of TNL expression patterns in Chinese genome evolution;these are largely based on Arabidopsis thaliana and cabbage.Gene expression patterns were obtained as previously Brassica rapa [19.Chinese cabbage (B.rapa ssp.pekinensis)is an described 29].Heat map and hierarchical clustering were important member of the Brassicaceae family.Completion of the employed to display the different expression levels using the RPKM Chinese cabbage Chiifu-401-42 draft genome sequence allows (reads per kilobase per million)value in Cluster v3.0(http://bonsai genome-wide identification analyses 20,21.Chinese cabbage quality hgc.jp/-mdehoon/software/cluster/software.htm). and yield are susceptible to constant barrages from biotic stresses. Among these,the Turnip mosaic virus (TuMV)belongs to the Potyvirus Plant materials,growth conditions,and virus treatments genus,it affects numerous plants in Asia,North America,and Europe, and does serious harm to Brassica crops and numerous other types of Seeds of the Chinese cabbage (B.rapa.ssp.pekinensis)cultivar vegetables[22.The host range of TuMV is wide,with the virus known "Baotoulian",which is less susceptible to TuMV,were used for this to infect 318 kinds of dicotyledoneae:harm to Brassica plants is study.They were planted in pots,and grown in an artificial climate particularly acute,causing severe systemic vein clearing,necrosis, chamber under the following cultural conditions:25C/18C day/ stunting.and plant death[23,24].It is difficult to avoid TuMV infection night temperature;photoperiod,16 h light/8 h dark;relative hu- because of the wide range of host species and its transmission by midity,70%.Four-week-old plants,at the four-leaf stage,were aphids,furthermore,the efficiency of pesticides used against it is inoculated with TuMV.The TuMV(pathotype C4)came from viru- declining 25].Thus,natural plant resistance may prove an effective liferous Chinese cabbages.Leaves showing obvious disease symp- and environmentally friendly method to control TuMV. toms were ground in a porcelain mortar in phosphate buffer There have been numerous studies on the NBS-LRR domain (0.02 mol/L,pH 7.0).The virus was mechanically transmitted to 6.7.15,261.However,none of these analyzed the TNL family in three leaves of each plant using a friction inoculation method,and Chinese cabbage,an important model plant for the genomic study mock leaves simultaneously inoculated by phosphate buffer alone. of Brassica crops.Nor has the challenge by TuMV been specifically Inoculated plants were exposed in the same growth conditions as investigated.In this study.genome-wide analysis identified 90 described above,as were the control group.Inoculated leaf samples putative TNL encoding genes using the Chinese cabbage genome were collected under a continuous time process(0,1,3,7.14,and 21 database.Deeper analysis was performed on these genes,including days post inoculation(dpi))using three biological repetitions for identifying gene locations on 10 chromosomes,subgenome distri- each time point,and stored at-80C for further analysis. bution,phylogenetic analysis,and transcript profiling of different organs.Quantitative Real-Time PCR(gRT-PCR)analysis was used to RNA isolation and fluorescence quantitative real-time PCR reveal gene expression patterns.Sixteen genes were identified as significantly responding to TuMV infection.These results provide a Total RNA was extracted from samples using an RNA extraction foundation for further identification and more efficient screening of kit (TaKaRa RNAiso Reagent,Takara,Dalian,China)in accordance resistance genes,and will prove crucial for further investigations of with manufacturer's instructions,and 2 ug total RNA were reverse relevant underlying mechanisms. transcribed using PrimeScriptTM RT reagent Kit with gDNA Eraser (Perfect Real Time;Takara,Dalian,China).Products used as tem- Materials and methods plates for qRT-PCR were diluted 10 times with ddH2O.SYBR Premix Ex TagTM(Takara.Dalian.China)was employed to identify target Identification and chromosome locations of TNL genes gene expression,using a Fluorescent Quantity PCR Detecting Sys- tem(Bio-Rad ICycler iQ5 Hercules,CA,USA),in accordance with To analyze the genome-wide TNL encoding genes,we retrieved manufacturer's protocols.Specific primers were designed based on the genome-wide sequences of Chinese cabbage from the Brassica the TNL encoding gene sequences using Beacon Designer v7.9 database(Version 1.5)(BRAD;http://brassicadb.org/brad/)[20.211. (Supplementary Table 1).Actin (Bra028615.1)was used as the Then,hmmbuild in HMMER v3.0(http://hmmer.janelia.org/)was reference gene [33].PCR conditions and the calculation method of used to screen the whole TNL encoding gene family according to gene expression were as described previously [34].The relative the specific hidden Markov model(HMM)profile.SMART v7.0[27] expression levels of TNL encoding genes in response to TuMV were and Pfam database (http://pfam.sanger.ac.uk/)analyses identified analyzed using Cluster v3.0 (http://bonsai.hgc.jp/-mdehoon/ 90 putative genes.Chromosome size and centromere position from software/cluster/software.htm);gene and array clusters were BRAD,and least fractionated (LF),medium fractionated(MF)and created using the Euclidean Distance method,and visualized using most fractionated(MF2)blokes subgenome data from the diploid JavaTree View v3.0 software (http://jtreeview.sourceforge.net/)

plant resistance proteins use their TIR domain in the detection of pathogens [14]. CNLs are more common in dicotyledon and cereal crops, and their relevant functions are similar to TNLs domains [15]. A feature of TNLs and CNLs is their species-specific distribution. TNL genes are numerous in dicots, yet few exist in cereals [8]. Furthermore, TNL gene sequences are more easily amplified from dicot genomes than cereal genomes. The truncation of TIR-NBS (TN) or TIR-X (TX) type protein domains in domesticated cereal plants may have led to loss of TNL genes in monocot plants such as rice, wheat (Triticum spp.), and maize (Zea mays) [16,17]. TNL genes are mainly involved in recognition of species-specific pathogens. Non￾TIR-NBS genes (including CNLs) are derived from at least two species, demonstrating their ancestors originated before the divergence between species (before monocot and dicot); non-TIR￾NBS genes function in basic defense, it may be due to the adapta￾tion to biotic habitats [18]. Brassica crops provide food, oilseed, fodder, and condiments. Numerous materials have been developed for the investigation of genome evolution; these are largely based on Arabidopsis thaliana and Brassica rapa [19]. Chinese cabbage (B. rapa ssp. pekinensis) is an important member of the Brassicaceae family. Completion of the Chinese cabbage Chiifu-401-42 draft genome sequence allows genome-wide identification analyses [20,21]. Chinese cabbage quality and yield are susceptible to constant barrages from biotic stresses. Among these, the Turnip mosaic virus (TuMV) belongs to the Potyvirus genus, it affects numerous plants in Asia, North America, and Europe, and does serious harm to Brassica crops and numerous other types of vegetables [22]. The host range of TuMV is wide, with the virus known to infect 318 kinds of dicotyledoneae; harm to Brassica plants is particularly acute, causing severe systemic vein clearing, necrosis, stunting, and plant death [23,24]. It is difficult to avoid TuMV infection because of the wide range of host species and its transmission by aphids, furthermore, the efficiency of pesticides used against it is declining [25]. Thus, natural plant resistance may prove an effective and environmentally friendly method to control TuMV. There have been numerous studies on the NBS-LRR domain [6,7,15,26]. However, none of these analyzed the TNL family in Chinese cabbage, an important model plant for the genomic study of Brassica crops. Nor has the challenge by TuMV been specifically investigated. In this study, genome-wide analysis identified 90 putative TNL encoding genes using the Chinese cabbage genome database. Deeper analysis was performed on these genes, including identifying gene locations on 10 chromosomes, subgenome distri￾bution, phylogenetic analysis, and transcript profiling of different organs. Quantitative Real-Time PCR (qRT-PCR) analysis was used to reveal gene expression patterns. Sixteen genes were identified as significantly responding to TuMV infection. These results provide a foundation for further identification and more efficient screening of resistance genes, and will prove crucial for further investigations of relevant underlying mechanisms. Materials and methods Identification and chromosome locations of TNL genes To analyze the genome-wide TNL encoding genes, we retrieved the genome-wide sequences of Chinese cabbage from the Brassica database (Version 1.5) (BRAD; http://brassicadb.org/brad/) [20,21]. Then, hmmbuild in HMMER v3.0 (http://hmmer.janelia.org/) was used to screen the whole TNL encoding gene family according to the specific hidden Markov model (HMM) profile. SMART v7.0 [27] and Pfam database (http://pfam.sanger.ac.uk/) analyses identified 90 putative genes. Chromosome size and centromere position from BRAD, and least fractionated (LF), medium fractionated (MF) and most fractionated (MF2) blokes subgenome data from the diploid ancestral genome of mesohexaploid B. rapa [28] were used to map TNL encoding gene loci on the ten chromosomes and three sub￾genomes, using the method described previously [29]. Conserved motif and phylogenetic analysis Pepstats (http://www.ebi.ac.uk/Tools/seqstats/emboss_ pepstats) was used to identify putative TNL characteristics, including isoelectric point (pI) and molecular weight (MW). MEME Suite [30] was used to identify conserved motifs in TNL proteins. The conserved motifs were used for phylogenetic analysis using the maximum-likelihood (ML) method, with the bootstrap value set as 1000 by MEGA v5.0 [31]. Expression analysis of TIR-NBS-LRR genes in different tissues Illumina RNA-Seq data obtained from BRAD [32] provided the groundwork for analyses of TNL expression patterns in Chinese cabbage. Gene expression patterns were obtained as previously described [29]. Heat map and hierarchical clustering were employed to display the different expression levels using the RPKM (reads per kilobase per million) value in Cluster v3.0 (http://bonsai. hgc.jp/~mdehoon/software/cluster/software.htm). Plant materials, growth conditions, and virus treatments Seeds of the Chinese cabbage (B. rapa. ssp. pekinensis) cultivar “Baotoulian”, which is less susceptible to TuMV, were used for this study. They were planted in pots, and grown in an artificial climate chamber under the following cultural conditions: 25C/18 C day/ night temperature; photoperiod, 16 h light/8 h dark; relative hu￾midity, 70%. Four-week-old plants, at the four-leaf stage, were inoculated with TuMV. The TuMV (pathotype C4) came from viru￾liferous Chinese cabbages. Leaves showing obvious disease symp￾toms were ground in a porcelain mortar in phosphate buffer (0.02 mol/L, pH 7.0). The virus was mechanically transmitted to three leaves of each plant using a friction inoculation method, and mock leaves simultaneously inoculated by phosphate buffer alone. Inoculated plants were exposed in the same growth conditions as described above, as were the control group. Inoculated leaf samples were collected under a continuous time process (0, 1, 3, 7, 14, and 21 days post inoculation (dpi)) using three biological repetitions for each time point, and stored at 80 C for further analysis. RNA isolation and fluorescence quantitative real-time PCR Total RNA was extracted from samples using an RNA extraction kit (TaKaRa RNAiso Reagent, Takara, Dalian, China) in accordance with manufacturer's instructions, and 2 mg total RNA were reverse transcribed using PrimeScript™ RT reagent Kit with gDNA Eraser (Perfect Real Time; Takara, Dalian, China). Products used as tem￾plates for qRT-PCR were diluted 10 times with ddH2O. SYBR® Premix Ex Taq™ (Takara, Dalian, China) was employed to identify target gene expression, using a Fluorescent Quantity PCR Detecting Sys￾tem (Bio-Rad ICycler iQ5 Hercules, CA, USA), in accordance with manufacturer's protocols. Specific primers were designed based on the TNL encoding gene sequences using Beacon Designer v7.9 (Supplementary Table 1). Actin (Bra028615.1) was used as the reference gene [33]. PCR conditions and the calculation method of gene expression were as described previously [34]. The relative expression levels of TNL encoding genes in response to TuMV were analyzed using Cluster v3.0 (http://bonsai.hgc.jp/~mdehoon/ software/cluster/software.htm); gene and array clusters were created using the Euclidean Distance method, and visualized using JavaTree View v3.0 software (http://jtreeview.sourceforge.net/). 90 S. Lv et al. / Physiological and Molecular Plant Pathology 90 (2015) 89e97

S.Lv et aL Physiological and Molecular Plant Pathology 90(2015)89-97 91 A01 A02 A03 A04 A05 Bra023350(-） Bra006094(-) Bra006146(+) QBra011666(+ a006452-) Bra039798(-) 3r8008556(+) ra013400(+ 0a023670(- Br023710(+) Br022850(+ Bra025493(+) Bra033939(+】 OBra025467(+) OBra008053(-) Bra008055t) Bra032045(+) QBra000759(+ Bra0011800 Bra001161) Bra021754(-） Bra038872 + Bra021952(-) QBa021953(+) 6ra001162( 3ra02195B(-) a001175( 8e0220371+ Bra022039(+) 51555> Brad2071(-) Ba01288-】 9Bra012540(+) Bra034079(+)】 Bra036413(-) Bra01910(-j Bra036417(+ Brag1940g(-） 8r8019273.- Bra017807(+) A06 A07 A08 A09 A10 Bra027791(+ Bra027x90(+】 Bra0277801+】 Bra01395 Bra027779 Bra027778(- Bra027775(-） Bra018037-) Bra027772-) ○Bra002154(+) Bra020861(-) Bra002153(+) Bra010496(+) Ba027510(+ Bra002117(+) Ba028500(-' DBra010552(+) OBra027599(+) CBra028499(-) Bra010551-) Bra027595(+) Bra017544+) Bra009141(-) Bra010590(+) Bra010589r+1 Bra010663(+ ○Ba003867(+ Bra016311(-) OBra029431(-) Bra003997 Bra06314(+) Bra029454(-)） Bra004192(-) QBra016534 0Bra024652(+) Bra035126(+) Bra024651(+) Bra036799(-) 日ra0367911) Bra036790(+) Bra030998(-) 8ra030984(+】 0 Mb LF■MF1■MF2 Centromere 10Mb Fig.1.Distribution of 88 TNL encoding genes and duplication events on 10 Chinese cabbage chromosomes as well as 3 subgenomes.There was no gene mapped on chromosome A05 (with dotted line).The centromeres were shown based on the analysis result of Chinese cabbage genome sequencing and two genes(Bra011666,Bra000759)on the scaffold cannot be located onto certain chromosomes.The size of each chromosome was indicated by the scale in megabases(Mb)on the bottom.Different colors bars represented different subgenomes(LF.MF1.and MF2).16 pairs of duplicated TNL encoding genes were connected by orange straight lines on duplicated chromosomal segments.(For interpretation of the references to colour in this figure legend,the reader is referred to the web version of this article.)

Fig. 1. Distribution of 88 TNL encoding genes and duplication events on 10 Chinese cabbage chromosomes as well as 3 subgenomes. There was no gene mapped on chromosome A05 (with dotted line). The centromeres were shown based on the analysis result of Chinese cabbage genome sequencing and two genes (Bra011666, Bra000759) on the scaffold cannot be located onto certain chromosomes. The size of each chromosome was indicated by the scale in megabases (Mb) on the bottom. Different colors bars represented different subgenomes (LF, MF1, and MF2). 16 pairs of duplicated TNL encoding genes were connected by orange straight lines on duplicated chromosomal segments. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) S. Lv et al. / Physiological and Molecular Plant Pathology 90 (2015) 89e97 91

92 S.Lv et al Physiological and Molecular Plant Pathology 90(2015)89-97 Results useful genes were retained in the lineage following the whole genome triplication(WGT)event [37,38]. Identification,chromosome distribution,and duplication events of TNL family genes in Chinese cabbage Conserved motifs and phylogenetic analysis of TNL encoding genes in Chinese cabbage Ninety genes,comprising all TNL domains in the whole Chinese cabbage genome,were identified.Characteristics,including chro- The 90 Chinese cabbage putative TNL encoding proteins were mosomal position,gene molecular weight,isoelectric point,do- classified into four different groups(I-IV)based on conserved mains and corresponding positions,and homologous genes in domain and protein motif structures(Fig.2)using MEME suite and A.thaliana were analyzed(Supplementary Table 2).Of the 90 pre- MEGAv5.0.The resultant phylogenetic tree harbored 32,14,29,and dicted TNL encoding genes,88 annotated genes physically mapped 15 members in groups I,Il,Ill,and IV,respectively.The logos and to nine of the ten chromosomes.The remaining two genes, regular expression sequence of protein motifs are given in Table 2. Bra036019 and Bra040210,were located on two unanchored scaf- Every gene contained at least eight motifs,indicating that motifs folds,Scaffold000111 and Scaffold000191,respectively(Fig.1).The are pivotal for gene function.Of the 15 motifs,motifs 1 and 3 were chromosomal distribution of Chinese cabbage TNL encoding genes found in the TIR domain of every gene.Motif 1 was located in front was non-random;this is similar to A.thaliana and rice [7,35].In of motif 3 in groups I and ll but was reversed in groups Ill and IV, numerous plants,NBS-LRR (containing TNL encoding genes)loci with the exception of Bra027772.The two genes adjacent to have been characterized with isolated genes and gene clusters. Bra027772 in the phylogenetic tree were Bra027779 (upper)and NBS-LRR gene clusters mainly result from different heterogeneous Bra019409 (lower)(Fig.2).Bra019409 is located on chromosome 3. clusters.A large number of genes (66/88,76%)were located on while both Bra027772 and Bra027779 are located on chromosome chromosomes A02.A03.A08,and A09(Fig.1).with the remaining 9.Thus,the Bra027772 and Bra027779 duplication might have 24%on the other chromosomes.Chromosome A09 had the most resulted from tandem duplication,whereas Bra027772 and TNL encoding genes(22/88.25%),while no TNL encoding genes Bra019409 likely resulted from whole genome duplication after were present on chromosome A05(Fig.1).This is thought to be a truncation. result of R-gene evolution,as clusters facilitated for trending to mispair and produce frequent haplotypic diversity[36]. Expression patterns of TNL family genes in Chinese cabbage in In addition,having been reported to be relative to A.thaliana response to TuMV the almost complete triplication of the B.rapa genome was divided into triplicate segments,and orthologous divergence traced back to To select genes for expression pattern analysis and screening in between 5 and 9 million years ago(MYA).The triplicate segments response to TuMV infection,TNL encoding genes were analyzed in contained LF,MF and MF2 [20].In this study,88 genes could be different tissues using Illumina mRNA-seq data available from mapped to the three fractionated blocks:MF1 contained 36 genes BRAD (http://brassicadb.org/brad/genomeDominanceData.php) (42%);MF2,34 genes(39%);and LF.18 genes(20%;Fig.1). Among these,12 genes (Bra002117,Bra017542,Bra017544 Furthermore,16 pairs (involving 23 genes)of duplicated TNL Bra010663.Bra028499.Bra022037. Bra036799.Bra038872 encoding genes (Fig.1)were identified on the chromosomal seg- Bra016594.Bra039798.Bra027595,and Bra030998)were deemed ments (Table 1).Most of these genes (20/23,87%)were located on not integral as a result of non-expression,or from their spatial and six chromosomes (A01,A02,A03,A08,A09,and A10).However, temporal expression patterns.Several TNL encoding genes showed there was no significant difference among the three subgenomes up-regulation of expression in a tissue specific manner,e.g.. (26%.35%.and 39%,respectively).Some duplicated genes only Bra008055 was highly expressed in leaves (RPKM =55.6),while contained two members,e.g.,Bra011666 and Bra010552;Bra023647 expression levels of Bra006146 and Bra027772,were high in stems and Bra002117:and Bra025467 and Bra028499,while others (RPKM=64.2)and roots(RPKM=32.1),respectively.Furthermore included up to four genes,e.g.,Bra008053,Bra003867,Bra016594. many genes displayed spatial up-regulated expression patterns. and.Bra030998.These results demonstrated TNL encoding gene Roots contained most up-regulated TNL encoding genes(50,60%). retention and loss in Chinese cabbage at the chromosomal and followed by stems(48.53%),while leaves had the lowest number subgenome level,thus genes of no use must have been lost,while (42,47%).Some TNL encoding genes expressed highly in the same Table 1 Colinearity of duplicated TNL family genes in Chinese cabbage. Gene pairs Gene names Chromosome number Subgenome Gene names Chromosome number Subgenome 1 Bra010552 A08 MF2 Bra011666 A01 LF 2 Bra012541 A03 MF1 Bra013400 A01 3 Bra013400 A01 F Bra027595 A09 MF2 4 Bra008053 A02 MF1 Bra016594 A0S MF1 5 Bra008053 A02 MFI Bra030998 A09 MF2 6 Bra021953 A02 MF2 Bra027599 A09 MF2 7 Bra002117 A10 F Bra023647 A02 Ba006452 A03 MF1 Bra023670 A02 9 Bra002154 A10 LF Bra023670 A02 MF2 1 Bra025467 A04 MF2 Bra028499 A07 MF1 Bra000759 A03 MF1 Bra029431 A09 Bra002154 A10 F Bra006452 A03 Bra012540 A03 MF1 Bra027595 A09 14 Bra003867 A07 MF2 Bra016594 A08 1 Bra016311 A08 MF1 Br2024652 A09 LF 16 Bra016594 A08 MF1 Bra030998 A09 MF2 LF:frac ionat ed bl ocks,MF1: the fractionated ocks,and MF2 the most fractionatec blocks

Results Identification, chromosome distribution, and duplication events of TNL family genes in Chinese cabbage Ninety genes, comprising all TNL domains in the whole Chinese cabbage genome, were identified. Characteristics, including chro￾mosomal position, gene molecular weight, isoelectric point, do￾mains and corresponding positions, and homologous genes in A. thaliana were analyzed (Supplementary Table 2). Of the 90 pre￾dicted TNL encoding genes, 88 annotated genes physically mapped to nine of the ten chromosomes. The remaining two genes, Bra036019 and Bra040210, were located on two unanchored scaf￾folds, Scaffold000111 and Scaffold000191, respectively (Fig. 1). The chromosomal distribution of Chinese cabbage TNL encoding genes was non-random; this is similar to A. thaliana and rice [7,35]. In numerous plants, NBS-LRR (containing TNL encoding genes) loci have been characterized with isolated genes and gene clusters. NBS-LRR gene clusters mainly result from different heterogeneous clusters. A large number of genes (66/88, 76%) were located on chromosomes A02, A03, A08, and A09 (Fig. 1), with the remaining 24% on the other chromosomes. Chromosome A09 had the most TNL encoding genes (22/88, 25%), while no TNL encoding genes were present on chromosome A05 (Fig. 1). This is thought to be a result of R-gene evolution, as clusters facilitated for trending to mispair and produce frequent haplotypic diversity [36]. In addition, having been reported to be relative to A. thaliana, the almost complete triplication of the B. rapa genome was divided into triplicate segments, and orthologous divergence traced back to between 5 and 9 million years ago (MYA). The triplicate segments contained LF, MF and MF2 [20]. In this study, 88 genes could be mapped to the three fractionated blocks: MF1 contained 36 genes (42%); MF2, 34 genes (39%); and LF, 18 genes (20%; Fig. 1). Furthermore, 16 pairs (involving 23 genes) of duplicated TNL encoding genes (Fig. 1) were identified on the chromosomal seg￾ments (Table 1). Most of these genes (20/23, 87%) were located on six chromosomes (A01, A02, A03, A08, A09, and A10). However, there was no significant difference among the three subgenomes (26%, 35%, and 39%, respectively). Some duplicated genes only contained two members, e.g., Bra011666 and Bra010552; Bra023647 and Bra002117; and Bra025467 and Bra028499, while others included up to four genes, e.g., Bra008053, Bra003867, Bra016594, and. Bra030998. These results demonstrated TNL encoding gene retention and loss in Chinese cabbage at the chromosomal and subgenome level, thus genes of no use must have been lost, while useful genes were retained in the lineage following the whole genome triplication (WGT) event [37,38]. Conserved motifs and phylogenetic analysis of TNL encoding genes in Chinese cabbage The 90 Chinese cabbage putative TNL encoding proteins were classified into four different groups (IeIV) based on conserved domain and protein motif structures (Fig. 2) using MEME suite and MEGA v5.0. The resultant phylogenetic tree harbored 32, 14, 29, and 15 members in groups I, II, III, and IV, respectively. The logos and regular expression sequence of protein motifs are given in Table 2. Every gene contained at least eight motifs, indicating that motifs are pivotal for gene function. Of the 15 motifs, motifs 1 and 3 were found in the TIR domain of every gene. Motif 1 was located in front of motif 3 in groups I and II but was reversed in groups III and IV, with the exception of Bra027772. The two genes adjacent to Bra027772 in the phylogenetic tree were Bra027779 (upper) and Bra019409 (lower) (Fig. 2). Bra019409 is located on chromosome 3, while both Bra027772 and Bra027779 are located on chromosome 9. Thus, the Bra027772 and Bra027779 duplication might have resulted from tandem duplication, whereas Bra027772 and Bra019409 likely resulted from whole genome duplication after truncation. Expression patterns of TNL family genes in Chinese cabbage in response to TuMV To select genes for expression pattern analysis and screening in response to TuMV infection, TNL encoding genes were analyzed in different tissues using Illumina mRNA-seq data available from BRAD (http://brassicadb.org/brad/genomeDominanceData.php). Among these, 12 genes (Bra002117, Bra017542, Bra017544, Bra010663, Bra028499, Bra022037, Bra036799, Bra038872, Bra016594, Bra039798, Bra027595, and Bra030998) were deemed not integral as a result of non-expression, or from their spatial and temporal expression patterns. Several TNL encoding genes showed up-regulation of expression in a tissue specific manner, e.g., Bra008055 was highly expressed in leaves (RPKM ¼ 55.6), while expression levels of Bra006146 and Bra027772, were high in stems (RPKM ¼ 64.2) and roots (RPKM ¼ 32.1), respectively. Furthermore, many genes displayed spatial up-regulated expression patterns. Roots contained most up-regulated TNL encoding genes (50, 60%), followed by stems (48, 53%), while leaves had the lowest number (42, 47%). Some TNL encoding genes expressed highly in the same Table 1 Colinearity of duplicated TNL family genes in Chinese cabbage. Gene pairs Gene names Chromosome number Subgenomea Gene names Chromosome number Subgenomea 1 Bra010552 A08 MF2 Bra011666 A01 LF 2 Bra012541 A03 MF1 Bra013400 A01 LF 3 Bra013400 A01 LF Bra027595 A09 MF2 4 Bra008053 A02 MF1 Bra016594 A08 MF1 5 Bra008053 A02 MF1 Bra030998 A09 MF2 6 Bra021953 A02 MF2 Bra027599 A09 MF2 7 Bra002117 A10 LF Bra023647 A02 MF2 8 Bra006452 A03 MF1 Bra023670 A02 MF2 9 Bra002154 A10 LF Bra023670 A02 MF2 10 Bra025467 A04 MF2 Bra028499 A07 MF1 11 Bra000759 A03 MF1 Bra029431 A09 LF 12 Bra002154 A10 LF Bra006452 A03 MF1 13 Bra012540 A03 MF1 Bra027595 A09 MF2 14 Bra003867 A07 MF2 Bra016594 A08 MF1 15 Bra016311 A08 MF1 Bra024652 A09 LF 16 Bra016594 A08 MF1 Bra030998 A09 MF2 a LF: fractionated blocks, MF1: the medium fractionated blocks, and MF2: the most fractionated blocks. 92 S. Lv et al. / Physiological and Molecular Plant Pathology 90 (2015) 89e97

S.Lv et aL Physiological and Molecular Plant Pathology 90(2015)89-97 93 Name uced Motif Location 024852 01631 m口口-DD000口a口上 1r0t含314 0■-00口口口口a口▣ 1口口00口口0-口一 aa口口D口 口■■口 圆▣■口00 回 明■ ■●T一门 O■D ■T 2 ▣ 4224 802546 ■▣ 032045 8a02175 ■ a02791 8a027775 9a02777g 口回■中口口 a02777 1口口 9ra01940 Ba01941d ■回回四 日r00140 国 2 ▣ 童■■■ ra07510 02417 02641 ■■■ ra036799 08R a02 Bra0339 a02207 Ba001175 □w4☐□ Fig.2.Phylogenetic tree and conserved motifs distribution of TNL proteins.The left part exhibited the multiple alignments of 90 full-length TNL proteins generated by MEGA and the combined P-values were shown on the leaf side.The tree was divided into four groups named as I-IV.The right side was schematic conserved motifs and the sizes of motifs were indicated by the scale.Different motifs were indicated by different colors LOGOs from 1 to15 at the bottom and the detailed information of them was shown in Table 2

Fig. 2. Phylogenetic tree and conserved motifs distribution of TNL proteins. The left part exhibited the multiple alignments of 90 full-length TNL proteins generated by MEGA and the combined P-values were shown on the leaf side. The tree was divided into four groups named as IeIV. The right side was schematic conserved motifs and the sizes of motifs were indicated by the scale. Different motifs were indicated by different colors LOGOs from 1 to15 at the bottom and the detailed information of them was shown in Table 2. S. Lv et al. / Physiological and Molecular Plant Pathology 90 (2015) 89e97 93

S.Lv et al Physiological and Molecular Plant Pathology 90 (2015)89-97 tissue,demonstrating functional homoousia,while expression of contained three genes,Bra011666,Bra023350,and Bra023647,genes some TNL encoding genes was diversified in different tissues, from this group behaved similar to the first,but also showed a steep showing functional dissimilarity (Supplementary Fig. 1, rise at 7 dpi,excepting 1 dpi day(Fig.4,i right).Thus,these genes Supplementary Table.3). can respond to a virus not only at the early stage,but also at later Among the 90 genes,69 genes specifically expressed in leaves, stages.As plant samples after 7 dpi were from uninoculated leaves, and were subjected to expression profile analysis via gRT-PCR. they are expected to be involved in virus propagation and trans- The -AACT data was analyzed using cluster software and visual- portation.Group ii,which included Bra012689,Bra016594,and ized via a heat map that depicted overall variation tendency(Fig.3. Bra002154 contained one correlation;expression descended at Supplementary Table.4).In accordance with the gene cluster and 1 dpi,and showed peak values at 3 dpi(Fig.4,ii).They exhibited expression level results at different sampling times (especially. obvious hysteresis in expression levels compared with the virus. early stages).genes were divided into three categories:i,up- They therefore may have participated in viral defense as their regulated genes:ii,mediumly expressed genes;iii,down- expression ascended when virus content was high,and dropped regulated genes (Supplementary Table.5,Fig.3).In group i,59.4% when virus content was lower,and were close to normal levels at (42/69)of genes were up-regulated at most stages.Eleven genes the end.The last group (iii)belonged to the correlation which belonged to group ii(15.9%);these were mainly down-regulated at exhibited peak values at 1 dpi,the correlations were similar to early stages(1 dpi,3 dpi,and 7 dpi).Group iii contained 16 genes group i,but their expression values were much lower at 1 dpi. (23.2%,16/69);these were downregulated at most stages. The expression level of coat protein(CP)gene was analyzed at Discussion different sampling times to measure the in vitro virus content change trend (Fig.4).Virus content fluctuated significantly from 0 dpi to 21 dpi,with peak(2C955.4)and valley values As one of most important vegetables cultivated worldwide and a model plant,Chinese cabbage is widely used in genetic research (2-4CT =1.9)occurring at 1 dpi and 21 dpi,respectively.The Due to its importance,breeding targets of Chinese cabbage include reason for the rapid drop after 1 dpi was that the cultivar "Bao- numerous traits,such as high quality and yield,high nutrient toulian"was less susceptible toTuMV.As a ground Chinese cabbage content,tolerance to extreme temperatures,and resistance to organ solution was used to infect plants,the initial value(0 dpi)was not zero(2-AACT=4.3).Thus,small amounts of the virus were pathogens.Considerable work has been carried out on TNL family genes involved in resistant to pathogens [7.16,39.There have been derived from the inoculation solution itself.Virus content at 14 dpi several bioinformatical analysis studies on TNL family genes in B. and 21 dpi was very similar,it was at a low level and tended to be rapa [17,38,40].The release of the B.rapa genome has greatly stable(2-AACT =2.0;Fig.4).When comparing these groups with enhanced this research.Since A.thaliana was first sequenced in the expression tendency closely related to virus content variation, 2000,numerous other species,including potato (Solanum tuber- each group had a different correlation;representative genes are osum L),tomato (Solanum lycopersicum L).and rice have been shown in Fig.4.Group i contained two kinds of correlations:the sequenced.The overall bioinformatical information provides an first,including the four genes Bra022039,Bra028499,Bra019273 efficient means for analyzing a huge amount of sequence data to and Bra021754,was initially lowly expressed,then expression reveal genomic and underlying genetic mechanisms raised,with a steep rise correlating to virus content.Expression The distribution of Chinese cabbage TNL encoding genes on levels returned to their initial low and stable levels at the final chromosomes was uneven,with many genes clustered:25%(22/88) stages(Fig.4.i left).Therefore,these genes responded to the virus of genes were located on chromosome 9,while no gene located to at early stages of infection,and furthermore they showed unifor- chromosome 5.A.thaliana has undergone three duplication events mity with the virus,showing the same change tendency at almost [41]:a whole genome triplication event(WGT:y)shared with most every stage.This indicated that these genes were likely to be eudicots,and two more recent whole genome duplications(WGD: involved in virus propagation.The second correlation in group i. 8 then a)shared with species with Brassicales order lineages. Table 2 Information of motif LOGOs shown in Fig.2. Motif ID e value Width Sites Regular expression sequence Description Motif 1 3.5e-1891 50 50 IKRJNYASSSWCLIDNJELVEI[ML]KCIRKJEELGQITIJVIMIJIPT][IV]FYIEGJVDPSIDHJVIRK]KOTGIDEJFGKIAV]F TIR domain Motif 2 3.9e-1329 44 50 VDDLIVEJQLEDJAILMJAIKGJETSWFG[PS]GSRII[VITTT[EQKJD[KQR]KILIJLKRJAHGI[NDJH[IVJYIEKHJV Motif 3 12e-2148 70 50 SS[LS][SPJRNWR[YH][DHJVFPSF[HRJG[EPJDVRKITG]FLSH[LVFIJ[LRJKE[FL]K[RS]KGITAF[IKJD[NQJEI[EKJR[SG] TIR domain IEQK]SI[GAJPELIVIKJIORJAIRIGEJSRIIV]ASJIVIVILVILS Motif 4 9.3e-1551 50 50 PSIRKNJDF[DE]IGD][FLIVG[MIJEAH[IMLI[ERAJIKE]IMIL]IKSISLLCL[ED]SIDENJEVIRK]M[IV]GIWGPIASP] GIGKTTIARJAV]LIFY] Motif 5 1.3e-1229 6 50 FE[EKJLAREVTKLAGNLPLGLRVIMLJGS[YS][LF]RG[MKRJSK[DEQJEW[EIJINDEJALPRLRITS]SLD Motif 6 1.3e-1887 70 50 LRLLHWDX[YFCJP[MLJ[RTK]CLPSKFINSR]PEFLVTEK]LIM[RP]NSKLEKLWEG[IVT][QK]PLT[NCSLK[WK] MDLSGSXNLKE[ILJPDLS[TKNJATINS]LE Motif7 3.8e-1046 36 50 WIRKJQALITAJIDEJVAINTJIAG[YE][HDJSSNWDNEAIDK][MLJIEK[IV]ATDVSNK Motif 8 1.0e-1205 50 50 RA[CG]ILS]D[DEJY[GS]LKL[RHJLQJEKJQ[FLJLS[KQE][IL]ILF]NQKD[IM]KIIVIxHLG[VAJIAVI][KQJERLKD [KQJ[KRJVLIV][VIJLD[DE] Motif9 3.3e-1091 50 50 S[VITJURKJIFVI[SGJY[DEJALSDIIDE][EKNJIDE]IKQISAJILIJFLHIACFFN[GYJEK[VIJIDEJXVIKEJE[MCJLA Motif 10 7.3e-1118 50 50 PSSI[GQR]NLHN]KLKKLE[MLXGCIST]IKNJLE[VT]LPTINGJINLKEJSLED]IERJL[NDJLS[GD]CSIRSJL [RKISTIFPIDEQJI Motif 11 15e_7q9 36 50 LDN]LSGCSSLVIEKJLPSSIGN[ALJITIJNL[EKJ[KEJLNLSGCSSLVEL Motif 12 5.2e-540 28 50 KGC[RK][KNJLVSLPIQEP]LPDSLLFLDA[HE]IGD]CESL[EK] Motif 13 5.5e-532 19 50 EALIQEJIIMIIFL]CIRQI[YSJAFIGRJQIKN]ISYIP[PK]DG Motif 14 7.6e-441 19 50 EPGIKOJROFLVDAKIDEJICIDEJVLIAE] Motif 15 4.7e-505 26 50 xKYA[VCIILFJPGEEVPIAS]IYE]FTTHYJIRQJATIGSIISGNJSLTI a Alternative amino acids

tissue, demonstrating functional homoousia, while expression of some TNL encoding genes was diversified in different tissues, showing functional dissimilarity (Supplementary Fig. 1, Supplementary Table. 3). Among the 90 genes, 69 genes specifically expressed in leaves, and were subjected to expression profile analysis via qRT-PCR. The DDCT data was analyzed using cluster software and visual￾ized via a heat map that depicted overall variation tendency (Fig. 3, Supplementary Table. 4). In accordance with the gene cluster and expression level results at different sampling times (especially, early stages), genes were divided into three categories: i, up￾regulated genes; ii, mediumly expressed genes; iii, down￾regulated genes (Supplementary Table. 5, Fig. 3). In group i, 59.4% (42/69) of genes were up-regulated at most stages. Eleven genes belonged to group ii (15.9%); these were mainly down-regulated at early stages (1 dpi, 3 dpi, and 7 dpi). Group iii contained 16 genes (23.2%, 16/69); these were downregulated at most stages. The expression level of coat protein (CP) gene was analyzed at different sampling times to measure the in vitro virus content change trend (Fig. 4). Virus content fluctuated significantly from 0 dpi to 21 dpi, with peak (2DDCT ¼ 955.4) and valley values (2DDCT ¼ 1.9) occurring at 1 dpi and 21 dpi, respectively. The reason for the rapid drop after 1 dpi was that the cultivar “Bao￾toulian” was less susceptible to TuMV. As a ground Chinese cabbage organ solution was used to infect plants, the initial value (0 dpi) was not zero (2DDCT ¼ 4.3). Thus, small amounts of the virus were derived from the inoculation solution itself. Virus content at 14 dpi and 21 dpi was very similar, it was at a low level and tended to be stable (2DDCT ¼ 2.0; Fig. 4). When comparing these groups with the expression tendency closely related to virus content variation, each group had a different correlation; representative genes are shown in Fig. 4. Group i contained two kinds of correlations: the first, including the four genes Bra022039, Bra028499, Bra019273, and Bra021754, was initially lowly expressed, then expression raised, with a steep rise correlating to virus content. Expression levels returned to their initial low and stable levels at the final stages (Fig. 4, i left). Therefore, these genes responded to the virus at early stages of infection, and furthermore they showed unifor￾mity with the virus, showing the same change tendency at almost every stage. This indicated that these genes were likely to be involved in virus propagation. The second correlation in group i, contained three genes, Bra011666, Bra023350, and Bra023647, genes from this group behaved similar to the first, but also showed a steep rise at 7 dpi, excepting 1 dpi day (Fig. 4, i right). Thus, these genes can respond to a virus not only at the early stage, but also at later stages. As plant samples after 7 dpi were from uninoculated leaves, they are expected to be involved in virus propagation and trans￾portation. Group ii, which included Bra012689, Bra016594, and Bra002154 contained one correlation; expression descended at 1 dpi, and showed peak values at 3 dpi (Fig. 4, ii). They exhibited obvious hysteresis in expression levels compared with the virus. They therefore may have participated in viral defense as their expression ascended when virus content was high, and dropped when virus content was lower, and were close to normal levels at the end. The last group (iii) belonged to the correlation which exhibited peak values at 1 dpi, the correlations were similar to group i, but their expression values were much lower at 1 dpi. Discussion As one of most important vegetables cultivated worldwide and a model plant, Chinese cabbage is widely used in genetic research. Due to its importance, breeding targets of Chinese cabbage include numerous traits, such as high quality and yield, high nutrient content, tolerance to extreme temperatures, and resistance to pathogens. Considerable work has been carried out on TNL family genes involved in resistant to pathogens [7,16,39]. There have been several bioinformatical analysis studies on TNL family genes in B. rapa [17,38,40]. The release of the B. rapa genome has greatly enhanced this research. Since A. thaliana was first sequenced in 2000, numerous other species, including potato (Solanum tuber￾osum L.), tomato (Solanum lycopersicum L.), and rice have been sequenced. The overall bioinformatical information provides an efficient means for analyzing a huge amount of sequence data to reveal genomic and underlying genetic mechanisms. The distribution of Chinese cabbage TNL encoding genes on chromosomes was uneven, with many genes clustered: 25% (22/88) of genes were located on chromosome 9, while no gene located to chromosome 5. A. thaliana has undergone three duplication events [41]: a whole genome triplication event (WGT: g) shared with most eudicots, and two more recent whole genome duplications (WGD: b then a) shared with species with Brassicales order lineages. Table 2 Information of motif LOGOs shown in Fig. 2. Motif ID e value Width Sites Regular expression sequencea Description Motif 1 3.5e1891 50 50 [KR]NYASSSWCL[DN]ELVEI[ML]KC[RK]EELGQ[TI]V[MI][PT][IV]FY[EG]VDPS[DH]V[RK]KQTG[DE]FGK[AV]F TIR domain Motif 2 3.9e1329 44 50 VDDL[VE]QL[ED]A[LM]A[KG]ETSWFG[PS]GSRII[VI]TT[EQK]D[KQR]K[LI]L[KR]AHGI[ND]H[IV]Y[EKH]V Motif 3 1.2e2148 70 50 SS[LS][SP]RNWR[YH][DH]VFPSF[HR]G[EP]DVRK[TG]FLSH[LVFI][LR]KE[FL]K[RS]KGITAF[IK]D[NQ]EI[EK]R[SG] [EQK]SI[GA]PEL[VIK][QR]AIR[GE]SR[IV][AS][IV]V[LV]LS TIR domain Motif 4 9.3e1551 50 50 PS[RKN]DF[DE][GD][FL]VG[MI]EAH[IML][ERA][KE][MIL][KS]SLLCL[ED]S[DEN]EV[RK]M[IV]GIWGP[ASP] GIGKTTIAR[AV]L[FY] Motif 5 1.3e1229 46 50 FE[EK]LAREVTKLAGNLPLGLRV[ML]GS[YS][LF]RG[MKR]SK[DEQ]EW[EI][NDE]ALPRLR[TS]SLD Motif 6 1.3e1887 70 50 LRLLHWDx[YFC]P[ML][RTK]CLPSKF[NSR]PEFLV[EK]LIM[RP]NSKLEKLWEG[IVT][QK]PLT[NCS]LK[WK] MDLSGSxNLKE[IL]PDLS[TKN]AT[NS]LE Motif 7 3.8e1046 36 50 W[RK]QAL[TA][DE]VA[NT]IAG[YE][HD]SSNWDNEA[DK][ML]IEK[IV]ATDVSNK Motif 8 1.0e1205 50 50 RA[CG][LS]D[DE]Y[GS]LKL[RH]LQ[EK]Q[FL]LS[KQE][IL][LF]NQKD[IM]K[IV]xHLG[VA][AVI][KQ]ERLKD [KQ][KR]VL[IV][VI]LD[DE] Motif 9 3.3e1091 50 50 S[VIT]L[RK][FV][SG]Y[DE]AL[SD][DE][EKN][DE][KQ][SA][LI]FLHIACFFN[GY]EK[VI][DE]xV[KE]E[MC]LA [KD]SFLDVR[HQ]GLK[VT] Motif 10 7.3e1118 50 50 PSSI[GQR]NL[HN]KLKKLE[ML]xGC[ST][KN]LE[VT]LPT[NG]INL[KE]SL[ED][ER]L[ND]LS[GD]CS[RS]L [RK][ST]FP[DEQ]I Motif 11 1.5e799 36 50 L[DN]LSGCSSLV[EK]LPSSIGN[AL][TI]NL[EK][KE]LNLSGCSSLVEL Motif 12 5.2e540 28 50 KGC[RK][KN]LVSLP[QEP]LPDSLLFLDA[HE][GD]CESL[EK] Motif 13 5.5e532 19 50 EAL[QE][IM][FL]C[RQ][YS]AF[GR]Q[KN][SY]P[PK]DG Motif 14 7.6e441 19 50 EPG[KQ]RQFLVDAK[DE]IC[DE]VL[AE] Motif 15 4.7e505 26 50 xKYA[VC][LF]PGEEVP[AS][YE]FT[HY][RQ]AT[GS][SGN]SLTI a Alternative amino acids. 94 S. Lv et al. / Physiological and Molecular Plant Pathology 90 (2015) 89e97

S.Lv et al Physiological and Molecular Plant Pathology 90(2015)89-97 95 B.rapa shares the same evolution process,including a WGT event In recent years,many studies have made progress in the genetic between 13 and 17 MYA [20.37].According to "two-step"genome breeding and molecular cloning of R genes,revealing that TNL triplication and differential subgenome methylation,Brassicaceae encoding genes are involved in defence mechanisms;most genes genomes formed from chromosomal rearrangements,and breaking mentioned in this work have been reported in Chinese cabbage or and fusion of the polyploidy ancestor:this comprised 24 genomic other plants.Two probable R genes (Bol037156 and Bol037157)to blocks,which can be divided into three subgenomes and displayed Fusarium wilt were homologous to Bra022039 and Bra012689. along the five chromosomes of A.thaliana [20,32].Only a short respectively [42].Kover and Cheverud [43]identified a QTL asso- segment (MF2:Bra039484-Bra025437)comes from mid- ciated with the bacterium Pseudomonas syringae,this QTL con- chromosome 5 of A.thaliana.According to previous A.thaliana tained At5g41540,which is orthologous with Bra028499.The studies,approximately 37%of TNL encoding genes are located on deduced amino acid sequence of RPS6,containing At5g46450 At-chromosome 5,and these did not map to the middle part of the orthologous to Bra022071,showed the highest similarity to the TNL chromosome [7.17].Wang et al.[20]showed there was almost no resistance protein RAC1,which determines resistance to the segmental collinearity of B.rapa and A.thaliana genomes in the oomycete pathogen Albugo candida [441.Bra013400 was ortholo- mid-regions of Arabidopsis chromosome Chr5 and Chinese cabbage gous to At4g19500.which was more transiently expressed in chromosome A05.Thus,few chromosome 5 genes from B.rapa are response to silverleaf whitefly feeding:although At4g19500 derived from A.thaliana chromosome 5[20]. showed decreased expression in A.thaliana [45].Bra013400 0 dpi 1 dpi 3 dpi 7 dpi 14 dpi 21 dpi 209 0 -2.00 3.00 Bra01055 39 55 Bra03 409 L 314 0 Bra Bra ii 030998 Bra009141 Fig.3.Expression profiles of 69 TNL encoding genes under the challenge of TuMV.Four-week-old Chinese cabbage samples were selected in different times(0 dpi.1 dpi.3 dpi,7 dpi. and 21 dpi).The qRT-PCR data was normalized by Actin gene(Bra028615.1).The -AACT was used in drawing heat map and hierarchical clustering.Euclidean distance was used in gene cluster and the color bar was on the top left

B. rapa shares the same evolution process, including a WGT event between 13 and 17 MYA [20,37]. According to “two-step” genome triplication and differential subgenome methylation, Brassicaceae genomes formed from chromosomal rearrangements, and breaking and fusion of the polyploidy ancestor; this comprised 24 genomic blocks, which can be divided into three subgenomes and displayed along the five chromosomes of A. thaliana [20,32]. Only a short segment (MF2: Bra039484eBra025437) comes from mid￾chromosome 5 of A. thaliana. According to previous A. thaliana studies, approximately 37% of TNL encoding genes are located on At-chromosome 5, and these did not map to the middle part of the chromosome [7,17]. Wang et al. [20] showed there was almost no segmental collinearity of B. rapa and A. thaliana genomes in the mid-regions of Arabidopsis chromosome Chr5 and Chinese cabbage chromosome A05. Thus, few chromosome 5 genes from B. rapa are derived from A. thaliana chromosome 5 [20]. In recent years, many studies have made progress in the genetic breeding and molecular cloning of R genes, revealing that TNL encoding genes are involved in defence mechanisms; most genes mentioned in this work have been reported in Chinese cabbage or other plants. Two probable R genes (Bol037156 and Bol037157) to Fusarium wilt were homologous to Bra022039 and Bra012689, respectively [42]. Kover and Cheverud [43] identified a QTL asso￾ciated with the bacterium Pseudomonas syringae, this QTL con￾tained At5g41540, which is orthologous with Bra028499. The deduced amino acid sequence of RPS6, containing At5g46450 orthologous to Bra022071, showed the highest similarity to the TNL resistance protein RAC1, which determines resistance to the oomycete pathogen Albugo candida [44]. Bra013400 was ortholo￾gous to At4g19500, which was more transiently expressed in response to silverleaf whitefly feeding; although At4g19500 showed decreased expression in A. thaliana [45], Bra013400 Fig. 3. Expression profiles of 69 TNL encoding genes under the challenge of TuMV. Four-week-old Chinese cabbage samples were selected in different times (0 dpi, 1 dpi, 3 dpi, 7 dpi, and 21 dpi). The qRT-PCR data was normalized by Actin gene (Bra028615.1). The DDCT was used in drawing heat map and hierarchical clustering. Euclidean distance was used in gene cluster and the color bar was on the top left. S. Lv et al. / Physiological and Molecular Plant Pathology 90 (2015) 89e97 95

S.Lv et al Physiological and Molecular Plant Pathology 90 (2015)89-97 1200 1200 18 000 16 1000 800。—Bra022039 800 i 12 童一B02849g 量一Bra01166 80D 8 600士-Bra02335 8 6 400米一Ba021754 400 一4一ra023547 6 ==◆▣Cp 200 200 0 Odpi 1dpi 3dpi 7dpi 14dpi 21dpi Odpl 1dpi 3dpi 7dpl 14dpi 21dpi 1200 1200 1000 1000 800一m012689 55 ★=0ga00g053 i 600±一Ba016594 600 一00805 X一Bna002154 一0一k022071 400.-◆-C A0W一a265配 200 +一a0275]0 200 +-CP 0 0dpl 1dpi 3dpi 7dpl 14dpi 21dpi Odpi Idpi 3dpi 7dpi 14dpi 21dpi Fig.4.The correlations between 16 genes expression levels and virus'content variation.i,ii.iii showed the expression correlations.The vertical axis represented the relative expression level and the 0 dpi.1 dpi.3 dpi,7 dpi.14 dpi.21 dpi(x-axis)indicate the treatment times.Different colour lines represented different genes and CP gene was shown in black dotted line in all subgraphs.Error bars represent the standard error of the mean based on three replicates. expression was found to be increased in this research.At1g63730 patterns were analyzed under normal and infectious conditions was orthologous with Bra021754,its expression was up-regulated Sixteen candidate genes were identified as responding to TuMV 6.59 fold in A.thaliana colonized by Pseudomonas putida infection.These genes are expected to be very important in the MTCC5279 [46].Shimizu et al.found that Bra022071 and Bra008053 response to pathogens,and their cloning and verification should be showed resistant line susceptible line expression when the focus of subsequent work.The expression pattern data,under responding to Fusarium [47].AtTN15(AT4G09420)was orthologous normal and infectious conditions,can be applied to construction of to Bra008055.this is known to interact with effectors from Pseu- a pathogen-defense regulation network model in Chinese cabbage domonas syringae,Ralstonia.solanacearum,Bremia.lactucae,and This work provides genetic resources for the genetic modification Hyaloperonospora.arabidopsidis,and with plant NBS-LRR proteins of plants and is conducive to the breeding of pathogen resistant 48.TAOl is the Mt-0 allele of the Col-0 gene At5g44510.this gene varieties. is orthologous with Bra027510 and contributes to disease resistance induced by the P.syringae effecter AvrB [491.All the above Chinese cabbage genes and orthologous/homologous genes belonged to the Authors'contributions sixteen genes that significantly responded to TuMV infection. Therefore,part of sixteen genes may be related to resistance or SL,CZ.YL ZW,and XH designed the research.SL and YL similar functions with viruses in Chinese cabbage. completed the experiments.SL and JT performed data analysis and Much genetic research into resistance against TuMV has been manuscript preparation.CZ,JT,DJ and XH participated in revising performed in Chinese cabbage,and recently some resistance genes the manuscript.All authors had read and approved the final version have been mapped on the Chinese cabbage genome.Two resistance of the manuscript. genes,recessive TuMV resistance 01 (retr01)and Conditional TuMV resistance 01 (ConTRO1).were mapped on the upper portion of chromosomes A04 and A08,respectively using RFLP probes [50]. The recessive Turnip mosaic virus resistance 02 (retr02)gene was Acknowledgements mapped to scaffold000060 or scaffold000104 on chromosome A04 using microsatellite and indel marker assays [51].In addition This work was supported by the grants from the National Nat- resistance and necrosis to tumv 1-2(rt1-2).TuRB07(TuMV RESIS. ural Science Foundation of China (31272172),the Fundamental TANCE in Brassica 07),and a novel TuMV resistance locus(TuMV-R) Research Funds for the Central Universities (KYTZ201401),the were mapped to chromosome A06 [52-541.Among these 13 Nature Science Foundation of Jiangsu Province(BK20141364).Ph.D. candidate genes above,six (Brao18862,Bra018863,Bra018835. Programs Foundation of Ministry of Education of China Bra018834,Bra018810,and Bra018810)belonged to the CNL family. (20110097120010).and China Postdoctoral Science Foundation Moreover,a novel microRNA induced by TuMV infection was re- 2012M521093): ported to target TNL on chromosome A0355.In this study,the sixteen predicted genes mainly mapped to chromosomes A02,A03. andA09(61.5%). Appendix A.Supplementary material Ninety TNL encoding genes were identified in the whole genome of Chinese cabbage,and these were anchored on 9 of the 10 Supplementary data related to this article can be found at http:/ chromosomes.Their characteristics,classification,and expression dk.doi.org/10.1016j-pmpp.2015.04.001

expression was found to be increased in this research. At1g63730 was orthologous with Bra021754, its expression was up-regulated 6.59 fold in A. thaliana colonized by Pseudomonas putida MTCC5279 [46]. Shimizu et al. found that Bra022071 and Bra008053 showed resistant line > susceptible line expression when responding to Fusarium [47]. AtTN15 (AT4G09420) was orthologous to Bra008055, this is known to interact with effectors from Pseu￾domonas syringae, Ralstonia. solanacearum, Bremia. lactucae, and Hyaloperonospora. arabidopsidis, and with plant NBS-LRR proteins [48]. TAO1 is the Mt-0 allele of the Col-0 gene At5g44510, this gene is orthologous with Bra027510 and contributes to disease resistance induced by the P. syringae effecter AvrB [49]. All the above Chinese cabbage genes and orthologous/homologous genes belonged to the sixteen genes that significantly responded to TuMV infection. Therefore, part of sixteen genes may be related to resistance or similar functions with viruses in Chinese cabbage. Much genetic research into resistance against TuMV has been performed in Chinese cabbage, and recently some resistance genes have been mapped on the Chinese cabbage genome. Two resistance genes, recessive TuMV resistance 01 (retr01) and Conditional TuMV resistance 01 (ConTR01), were mapped on the upper portion of chromosomes A04 and A08, respectively using RFLP probes [50]. The recessive Turnip mosaic virus resistance 02 (retr02) gene was mapped to scaffold000060 or scaffold000104 on chromosome A04 using microsatellite and indel marker assays [51]. In addition, resistance and necrosis to tumv 1-2 (rnt1-2), TuRB07 (TuMV RESIS￾TANCE in Brassica 07), and a novel TuMV resistance locus (TuMV-R) were mapped to chromosome A06 [52e54]. Among these 13 candidate genes above, six (Brao18862, Bra018863, Bra018835, Bra018834, Bra018810, and Bra018810) belonged to the CNL family. Moreover, a novel microRNA induced by TuMV infection was re￾ported to target TNL on chromosome A03 [55]. In this study, the sixteen predicted genes mainly mapped to chromosomes A02, A03, and A09 (61.5%). Ninety TNL encoding genes were identified in the whole genome of Chinese cabbage, and these were anchored on 9 of the 10 chromosomes. Their characteristics, classification, and expression patterns were analyzed under normal and infectious conditions. Sixteen candidate genes were identified as responding to TuMV infection. These genes are expected to be very important in the response to pathogens, and their cloning and verification should be the focus of subsequent work. The expression pattern data, under normal and infectious conditions, can be applied to construction of a pathogen-defense regulation network model in Chinese cabbage. This work provides genetic resources for the genetic modification of plants and is conducive to the breeding of pathogen resistant varieties. Authors' contributions SL, CZ, YL, ZW, and XH designed the research. SL and YL completed the experiments. SL and JT performed data analysis and manuscript preparation. CZ, JT, DJ and XH participated in revising the manuscript. All authors had read and approved the final version of the manuscript. Acknowledgements This work was supported by the grants from the National Nat￾ural Science Foundation of China (31272172), the Fundamental Research Funds for the Central Universities (KYTZ201401), the Nature Science Foundation of Jiangsu Province (BK20141364), Ph.D. Programs Foundation of Ministry of Education of China (20110097120010), and China Postdoctoral Science Foundation (2012M521093). Appendix A. Supplementary material Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.pmpp.2015.04.001. Fig. 4. The correlations between 16 genes expression levels and virus' content variation. i, ii, iii showed the expression correlations. The vertical axis represented the relative expression level and the 0 dpi, 1 dpi, 3 dpi, 7 dpi, 14 dpi, 21 dpi (x-axis) indicate the treatment times. Different colour lines represented different genes and CP gene was shown in black dotted line in all subgraphs. Error bars represent the standard error of the mean based on three replicates. 96 S. Lv et al. / Physiological and Molecular Plant Pathology 90 (2015) 89e97

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