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Large-Scale Analyses of Angiosperm Nucleotide-Binding Site-Leucine-Rich Repeat Genes Reveal PDF

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Large-Scale Analyses of Angiosperm Nucleotide-Binding Site-Leucine-Rich Repeat Genes Reveal Three Anciently 1 Diverged Classes with Distinct Evolutionary Patterns Zhu-Qing Shao2, Jia-Yu Xue2, Ping Wu, Yan-Mei Zhang, Yue Wu, Yue-Yu Hang, Bin Wang*, and Jian-Qun Chen* State Key Laboratory of Pharmaceutical Biotechnology, School of Life Sciences, Nanjing University, Nanjing, 210023, China (Z.-Q.S., P.W., Y.-M.Z., Y.W., B.W., J.-Q.C.); and Institute of Botany, Jiangsu Province and Chinese Academy of Sciences, Nanjing, 210014, China (J.-Y.X., Y.-M.Z., Y.-Y.H.) Nucleotide-bindingsite-leucine-richrepeat(NBS-LRR)genesmakeupthelargestplantdiseaseresistancegenefamily(Rgenes), with hundreds of copies occurring in individual angiosperm genomes. However, the expansion history of NBS-LRR genes during angiosperm evolution is largely unknown. By identifying more than 6,000 NBS-LRR genes in 22 representative angiosperms and reconstructing their phylogenies, we present a potential framework of NBS-LRR gene evolution in the angiosperm. Three anciently diverged NBS-LRR classes (TNLs, CNLs, and RNLs) were distinguished with unique exon- intron structures and DNA motif sequences. A total of seven ancient TNL, 14 CNL, and two RNL lineages were discovered in the ancestral angiosperm, from which all current NBS-LRR gene repertoires were evolved. A pattern of gradual expansion duringthefirst100millionyearsofevolutionoftheangiospermcladewasobservedforCNLs.TNLnumbersremainedstable duringthisperiodbutwereeventuallydeletedinthreedivergentangiospermlineages.Weinferredthatanintenseexpansionof bothTNLandCNLgenes startedfrom theCretaceous-Paleogene boundary.Becausedramaticenvironmental changesandan explosion in fungal diversity occurred during this period, the observed expansions of R genes probably reflect convergent adaptive responses of various angiosperm families. An ancient whole-genome duplication event that occurred in an angiosperm ancestor resulted in two RNL lineages, which were conservatively evolved and acted as scaffold proteins for defense signal transduction. Overall, the reconstructed framework of angiosperm NBS-LRR gene evolution in this study may serveasa fundamentalreferenceforbetterunderstandingangiosperm NBS-LRRgenes. Plantdiseaseresistance(R)genesareasetofgenesthat et al., 2006; Friedman and Baker, 2007). Since the first conferresistancetovariouspathogens.Amongallknown comprehensive study on NBS genes in Arabidopsis types of R genes, nucleotide-binding site-leucine-rich re- (Arabidopsisthaliana;Meyersetal.,2003),NBSgeneshave peat(NBS-LRR,NBSforshort)genesrepresentthelargest been surveyed across various plant genomes, most of class,encompassingmorethan80%ofthecharacterized which belong to the rosid lineage of eudicots and the Rgenes(Meyersetal.,1999;Meyersetal.,2005;McHale Poaceae of monocots (Monosi et al., 2004; Zhou et al., 2004; Yang et al., 2006; Ameline-Torregrosa et al., 2008; 1ThisworkwassupportedbytheNationalNaturalScienceFoun- Yang et al., 2008; Mun et al., 2009; Porter et al., 2009; Li dationofChina(30930008,31170210,91231102,31300190,31400201, et al., 2010; Zhang et al., 2011; Lozano et al., 2012; Luo 31470327,31500191,and31570217),theNaturalScienceFoundation etal.,2012;Andolfoetal.,2013;Shaoetal.,2014). ofJiangsuProvince(BK20130565),theFundamentalResearchFunds NBS genes are historically divided into two classes, for the Central Universities (2062140215, 20620140546, and namely, TIR-NBS-LRR (TNL) and the non-TIR-NBS- 20620140558),andtheNationalPostdoctoralScienceFoundationof LRR (nTNL), which are differentiated by the presence China(2013M540435and2014T70503). 2Theseauthorscontributedequallytothearticle. ofaToll/IL-1Receptor-like(TIR)domainintheprotein amino terminus (Meyers et al., 1999; Bai et al., 2002; *[email protected]@nju. edu.cn. Zhouetal.,2004).BecausemostnTNLgenesencode a Theauthorresponsiblefordistributionofmaterialsintegraltothe coiled-coil (CC) domain at the N terminus, the nTNL findingspresented in this article in accordancewith the policy de- genes often are called CC-NBS-LRR (CNL) genes scribed in the Instructions for Authors (www.plantphysiol.org) is: (Meyers et al., 2003; Ameline-Torregrosa et al., 2008). Jian-QunChen([email protected]). However,recentstudieshaverevealedthatapartfrom J.-Q.C.,B.W.,J.-Y.X.,andZ.-Q.S.conceivedanddesignedthepro- CNLgenes,asmallgroupofnTNLgenesthatpossessa ject;J.-Y.X.,Z.-Q.S.,andB.W.obtainedandanalyzedthedata;P.W., special N-terminal domain, RPW8 (resistance to pow- Y.-M.Z.,Y.W.,andY.-Y.H.participatedinthedataanalysisanddis- derymildew8)domain,likelyrepresentadistinctclass cussion;Z.-Q.S.andJ.-Y.X.draftedtheinitialmanuscript;B.W.com- ofNBSgenes(RPW8-NBS-LRR[RNL];Xiaoetal.,2001; plementedthewriting;J.-Q.C.supervisedtheanalysisandwriting; Cannon et al., 2004; Bonardi et al., 2011; Collier et al., allauthorscontributedtodiscussionoftheresults,reviewedtheman- uscript,andapprovedthefinalarticle. 2011; Shao et al., 2014; Zhang et al., 2016). Investiga- www.plantphysiol.org/cgi/doi/10.1104/pp.15.01487 tionsofNBSgenesintheFabaceae(rosidIlineage)and Plant Physiology(cid:1), April 2016, Vol. 170, pp. 2095–2109, www.plantphysiol.org (cid:3)2016AmericanSociety ofPlant Biologists. All RightsReserved. 2095 Downloaded from on April 1, 2019 - Published by www.plantphysiol.org Copyright © 2016 American Society of Plant Biologists. All rights reserved. Shao et al. Brassicaceae (rosid II lineage) have shown that RNLs providewhenandhowresistancelociemergedordis- andCNLsdivergedpriortothedivergenceofthesetwo appearedincertainlineages.Inthisstudy,atotalof22 families(Shaoetal.,2014;Zhang etal.,2016).Interest- plant species (Fig. 1) from key systematic clades in ingly,althoughbothCNLandRNLgenesarepresentin eudicots, monocots, and the early diverging basal an- monocots and dicots, TNL genes only are present in giosperm were chosen to depict the ancientevolution- dicots (Meyers et al., 2003; Tarr and Alexander, 2009; ary history of NBS genes in angiosperms. More than Andolfoetal.,2013;Shaoetal.,2014).Consideringthe 6,000 NBS genes were identified; family level phylo- abundance of TNLs in various dicot species, their ab- genetictreeswereconstructedfirstandthenexpanded senceinmonocotsisperplexing. to higher systematic levels to eventually analyze all of For several years, our knowledge of NBS gene evo- the angiosperms. Ancient taxonomies of NBS genes lution has been based on investigations of individual weretracedtotheoriginofangiosperms,andatotalof angiosperm genomes (Meyers et al., 2003; Ameline- 23 ancestral NBS genes were recovered in the last Torregrosa et al., 2008; Kohler et al., 2008). The num- common ancestor of angiosperms. This angiosperm ber of NBS genes varied dramatically among different NBS frame provides a fundamental reference for un- species, even among different accessions of the same derstanding the evolutionary history of NBS genes as species (Zhang et al., 2010). However, the relative well as insight into the finer details of particular NBS contributions of recent and ancient expansions to the geneclasses. abundance of NBS genes in modern plant genomes remain elusive. Recent studies report the evolutionary analysesofNBSgenesacrossmultiplespeciesinaplant RESULTS family(Cannonetal.,2002;Guoetal.,2011;Luoetal., 2012; Lin et al., 2013; Shao et al., 2014; Zhang et al., Identification andClassificationofNBS Genesfrom 2016). While studies in Brassicaceae and Poaceae both 22AngiospermSpecies revealed abundant and recent losses of NBS genes in A total of 6,135 NBS genes were identified in 22 an- variousspeciesafterthedivergenceofthesetwofami- giosperm species using previously described proce- lies, a study involvingFabaceae hassuggested theop- dures(Shaoetal.,2014;Zhangetal.,2016).Fiveofthe posite scenario in which all surveyed species recently 22 genomes were surveyed in this study, Amborella underwent a multitude of NBS expansions (Luo et al., trichopoda, Musa acuminata (banana), Phyllostachys het- 2012; Shao et al., 2014; Zhang et al., 2016). Although erocycla(bamboo),Capsicumannuum(hotpepper),and distinct evolutionarypatternsof NBS geneshave been Sesamum indicum (sesame), whereas the others have identified at the family level, hundreds of NBS gene beenpreviouslyreported(Meyersetal.,2003;Luoetal., lineagesaroseintheancestorsofFabaceae,Brassicaceae, 2012; Malacarne et al.,2012; Andolfo et al., 2013; Shao and Poaceae, indicating that NBS genes may have et al., 2014; Zhang et al., 2016). A. trichopoda, a basal probably undergone ancient expansions prior to fa- angiosperm,hadatotalof105NBSgenes,including15 milial divergence. Therefore, a phylogenetic analysis TNLs, one RNL, and 89 CNLs (Fig. 1; Supplemental at a larger scale is necessary to better understand the TableS1).TheNBSgenesvariedamongthe22surveyed expansionhistoryofNBSgenesinangiosperms. species(Fig.1).Ofallspeciessurveyed,Medicagotrun- Several functional NBS genes have been identified in catulapossessedthehighestnumberofNBSgenes(571), angiospermspecies(Liuetal.,2007);however,theirtime more than 6-fold of that observed in Thellungiella sal- of emergence and relationship with other NBS genes suginea (88). RNL genes were detected in all of the have not been established. For example, several CNL surveyed species, although at lower numbers (1–18). geneshavebeenidentifiedinbothsoybean(Glycinemax; A large proportion of NBS genes were either TNLs or Rpg1b and Rpg1r) and Arabidopsis (RPM1 and RPS2) CNLs,althoughTNLswerenotdetectedineightof22 against the bacterial pathogen Pseudomonas syringae by species, including seven monocots and S. indicum. Be- guardingthehostproteinRIN4(Bentetal.,1994;Mackey causethebasalangiospermA.trichopodaharboredTNL et al., 2002; Mackey et al., 2003; Ashfield et al., 2014). genes,thesegenesweremostlikelyalsopresentinthe While the Rpg1b and Rpg1r genes were deduced to ancestorofangiospermsbutindependentlylostincer- emerge after the divergence of Fabaceae species, the tainlineages.InspecieswithbothTNLandCNLgenes, evolutionarytimepointoftheoriginofRPM1andRPS2 CNLs often outnumbered TNLs, except for the four genesinArabidopsisislargelyunknown(Ashfieldetal., Brassicaceae genomes analyzed (Zhang et al., 2016). 2014; Shao et al., 2014). Because all these R genes have The asterid genomes showed a lower ratio of TNL to likely adopted a similar strategy to defend against the CNL genes, with the C. annuum exhibiting the highest samepathogen,investigatingtheirevolutionmayeluci- levelofdisparity(TNL:CNL=1:18). date the mechanism underlying the establishment and maintenanceofresistanceinvariousplantspecies. To obtain a more comprehensive understanding of Phylogenetic AnalysisofNBSGenesinMajor NBS gene evolution, it is essential to analyze more AngiospermClades species/lineages to determine how the current NBS genes have evolved and diverged from ancestral NBS The 22 angiosperm species surveyed in this study lineages in the ancestral angiosperm. This also will occupy important phylogenetic positions (Fig. 1). To 2096 Plant Physiol. Vol. 170, 2016 Downloaded from on April 1, 2019 - Published by www.plantphysiol.org Copyright © 2016 American Society of Plant Biologists. All rights reserved. Evolution of NBS-LRR Genes in Angiosperms Figure1. Identification andclassification ofNBSgenesin22angiospermgenomes. The phylogenetic relationship was con- structed according to the APG III system (Bremeretal.,2009).Thedivergencetimes at different nodes of angiosperms were combined from previous studies (Lavin et al., 2005; Stefanovic et al., 2009; D’Hont et al., 2012; Zhang et al., 2012; Pengetal.,2013;Yangetal.,2013;Kim etal.,2014;Wangetal.,2014;Zengetal., 2014).ThetotalnumberofNBSgenesand their classification in each species are shown. The number of ancestral NBS genesrecoveredbyphylogeneticanalysis at each node is shown in a green circle, andanasteriskafterthenumberindicates thatitisnotdirectlyunraveledbyphylo- geneticanalysis. tracetheevolutionaryhistoryofNBSgenes,weexam- lineage designated as Mo-R1, and 56 CNL lineages inedmultipleangiospermclades,includingrosids(Ro), identified as Mo-C1 to Mo-C56), suggesting that nTNL asterids (As), eudicots (Ed), monocots (Mo), mesan- genes from the seven monocot genomes were actually giospermae(Ma),andallangiosperms(An;Fig.1).Itis derivedfrom57ancestralMo-NBSgenes;110MYA(Fig. widely recognized that the NBS domain effectively 2;SupplementalFig.S1;D’Hontetal.,2012).Fromtheir distinguishes NBS genes into TNL and nTNL classes commonancestor,thePoaceaeandM.acuminatainherited (Bai et al., 2002; Meyers et al., 2003). Although a sister 51and32ancestralMo-NBSgenes,respectively.Among relationship between CNLs and RNLs has been estab- these,26weresharedbybothPoaceaeandM.acuminata. lishedinrosids,ithasnotbeenexaminedatangiosperm SeveralancestralgenesinthePoaceaeunderwentexten- level. Therefore, phylogenetic analyses of TNLs and siveduplications(from51to456)duringthetimeinterval nTNLs (including both CNLs and RNLs) were sepa- of 48 million years. For example, the NBS gene Mo-C56 rately performed in this study. We first conducted duplicated into 197 genes during this time period phylogenetic analysis of three major angiosperm line- (SupplementalFig.S1).Asmultiplespeciesradiatedfrom ages, namely, monocots, asterids, and rosids, which thePoaceaecommonancestor,insomespeciesNBSgene coverseven,four,and10genomes,respectively. numbers contracted drastically (Z. mays, 139 from 456) The M. acuminata genome, together with six Poaceae andinothersonlyslightly(S.italicaandP.heterocycla,424 genomes,includingOryzasativa,Brachypodiumdistachyon, and344from456,respectively),whereasexpansionwas P.heterocycla,Zeamays,Sorghumbicolor,andSetariaitalica, onlyfoundintheO.sativagenome(from456to498). were used to elucidate the evolution of NBS genes in Within the asterids four species were analyzed, in- monocots. Because these species did not harbor TNL cluding three Solanaceae species, namely, Solanum genes, phylogenetic analysis of only nTNL genes was lycopersicum,Solanumtuberosum,andC.annuum,aswell performed.ThereconstructedmonocotnTNLphylogeny as S. indicum from Pedaliaceae. NBS genes from the (SupplementalFig.S1)revealedthatnTNLgenesfromthe three Solanaceae genomes formed 19 TNL monophy- six Poaceae species formed 456 monophyletic lineages, letic lineages (Supplemental Fig. S2) and 147 nTNL indicating the presence of at least 456 ancestral nTNL monophyletic lineages (Supplemental Fig. S3), sug- genesinthePoaceaecommonancestor(;62millionyears gestingthatatleast166ancestralNBSgeneswerepre- ago [MYA]; Peng et al., 2013, Zhang et al., 2012). These sent in their last common ancestor. The Solanaceae Poaceae nTNL genes formed 57 monophyletic lineages nTNL genes formed 55 monophyletic lineages with whengroupedwithM.acuminatanTNLgenes(oneRNL nTNL genes from S. indicum (two RNL lineages Plant Physiol. Vol. 170, 2016 2097 Downloaded from on April 1, 2019 - Published by www.plantphysiol.org Copyright © 2016 American Society of Plant Biologists. All rights reserved. Shao et al. Figure 2. Cladograms depicting reconciled phylogenetic relationships of ancestral NBS genes in the divergence nodes of monocots,rosids,andasterids.ThepresenceofaNBSgenelineageindifferentspeciesandfamiliesareindicatedwithspeciesor lineage-specificicons.ThemaximumlikelihoodtreesofeachcladogramarepresentedinSupplementalFiguresS1toS5. designated as As-R1 and As-R2, and 53 CNL lineages Rosidsareamajorlineageofeudicotsthatconsistsof identifiedasAs-C1toAs-C53),indicatingaminimumof three sublineages, rosid I (fabids), rosid II (malvids), 55ancestralgenesintheasteridcommonancestor(Fig.2; and the basal rosids. Previous work analyzed NBS SupplementalFig.S2).SolanalesinheritedmoreAs-nTNL genes in Fabaceae and Brassicaceae (Shao et al., 2014; lineages than S. indicum, which represents Lamiales (47 Zhang et al., 2016), which belong to the rosid I sub- versus30).NoTNLgenesweredetectedintheS.indicum lineageandtherosidIIsublineage,respectively.Atotal genome;therefore,theaccuratenumberofancestralTNL of 64 nTNL and 55 TNL ancestral lineages were dis- genesatthisasteridnodewasnotestimated. covered in the last common ancestor of the four 2098 Plant Physiol. Vol. 170, 2016 Downloaded from on April 1, 2019 - Published by www.plantphysiol.org Copyright © 2016 American Society of Plant Biologists. All rights reserved. Evolution of NBS-LRR Genes in Angiosperms Fabaceaespecies,and80nTNLand148TNLancestral TNLs (Supplemental Fig. S10). Figure 3 shows that all lineageswereidentifiedinthelastcommonancestorof TNL genes clustered into seven ancestral angiosperm the five surveyed Brassicaceae species. In this study, lineages (designated as An-T1 to An-T7), and all nTNL one representative sequence from each of the 119 genesclusteredinto16ancestrallineages(twoRNLlin- Fabaceae ancestral NBS lineages and the 228 Brassica- eagesdesignatedasAn-R1andAn-R2,and14CNLlin- ceaeancestralNBSlineageswereusedtorepresentthe eagesidentifiedasAn-C1toAn-C14),indicatingatleast diversity of ancient NBS genes in these two families, 23 NBS genes were present in the last angiosperm an- and then combined with NBS genes identified in Vitis cestor(;225 MYA). ThebasalangiospermA. trichopoda vinifera (basal rosid) to construct NBS phylogenies for inheritedonlyeightofthe23An-NBSgenes,whereasthe thewholerosidlineage.Atotalof61ancestralRo-NBS mesangiospermae inherited 19 (Fig. 3).The angiosperm genes were identified (four RNLs, 49 CNLs, and eight nTNLtreewascomposedoftwo monophyleticgroups, TNLs)inthecommonancestoroftherosids(;108MYA; namely, RNL and CNL (Supplemental Fig. S9). There- Fig.2;SupplementalFigs.S4andS5),amongwhichthe fore,despitetheextensivediscrepancyingenenumbers Fabaceae of rosid I lineage inherited 35 Ro-NBS genes between the two groups, this study revealed that the (two RNLs, 27 CNLs, and six TNLs), Brassicaceae of RNLisanotherancientclassofNBSgenesthathasasister rosid II lineage inherited 19 (two RNLs, 12 CNLs, and relationshipwiththeCNLclassinangiosperms. five TNLs), and basal rosids inherited 33 (two RNLs, The angiosperm NBS gene phylogenies have 29CNLs,andtwoTNLs).LessthanhalfoftheRo-NBS revealedtheevolutionarystateofNBSgenesatcritical genes(26/61)weredetectedintwoofthelineages,and angiosperm nodes, which indicated that differential 35/61wereobservedinonedescendinglineage(Fig.2), inheritanceofancestralNBSgenesandlineage-specific thereby reflecting a pattern of frequent NBS gene loss expansionsaremajorthemesofNBSgeneevolution. after their split from the common rosid ancestor. The 35 and 19 Ro-NBS genes inherited by Fabaceae and CharacterizationofClass-SpecificSignaturesamongThree Brassicaceaelineagesexpandedto119(55TNLsand64 AncientNBS GeneClasses nTNLs)and228(148TNLsand80nTNLs)NBSgenesin theFabaceaeancestor(;54MYA)andtheBrassicaceae Because the results of phylogenetic analysis suggest ancestor (;42 MYA), respectively (Shao et al., 2014; that RNLs represent an ancient NBS gene class inde- Zhangetal.,2016).TheNBSgenescontinuedtoexpand pendentofthe CNL and TNLclasses,characterization in Fabaceae (Shao et al., 2014), whereas these genes ofdistinctivefeaturesofeachclasswasperformed. contracted as the Brassicaceae diverged into multiple species (Zhang etal.,2016).AlthoughtheBrassicaceae Distinctive Signatures in Conserved NBS Motifs Differentiate inheritedfewerancestralTNLsthanCNLs(fiveversus theThreeClasses 12)fromtherosidancestor,thislineagevigorouslyex- pandeditsTNLsduringthetimeintervalof;108MYA The NBS domain is composed of a number of char- to;42MYA.A30-foldexpansionwasobservedinthe acteristic motifs that are conserved and shared by all BrassicaceaeTNLs(fromfiveto148),farbeyondthatof NBS genes, whereas other motifs are more variable legumeTNLs(;9-fold,fromsixto55;Shaoetal.,2014; (DeYoung and Innes, 2006). All three classes of NBS Zhangetal.,2016). genessharefivefunctionallyimportantmotifs:P-loop, Kinase-2,RNBS-B,GLPL,andRNBS-D.Theaminoacid sequence conservation of these five motifs was esti- ReconstructionofNBSGenePhylogeniesatthe mated using sequence logos using MEME software Whole-AngiospermScale (Fig.4;BaileyandElkan,1995).Allmotifswerehighly RepresentativeNBSsequencesfromancestrallineages conservedatmostsitesamongallclasses,exceptforthe ofasterids,rosids,andmonocotswereusedtoconstruct RNBS-Dmotif,whichwaspoorlyconservedintheTNL ancestral NBS gene lineages at the divergence nodes of and CNL classes. The class-specific features within eudicotsandmesangiospermae.AtotalofeightEd-TNL these motifs also were identified. The Kinase-2 motif and49Ed-nTNLgenes(47Ed-CNLsandtwoEd-RNLs) had a conserved “DDVW” sequence in the RNL and were recovered at the eudicot node (Supplemental Figs. CNLgenes,butfrequentlyappearedas“DDVD”inthe S6andS7),whereas37Ma-nTNLgenes(35Ma-CNLsand TNL genes. A“TSR”sequence in the RNBS-B motif of two Ma-RNLs) were detected at the mesangiospermae RNLs was conserved, but frequently appeared as node (Supplemental Fig. S8). Independent loss and dif- “TTR” and “TTRD” in the CNL and TNL genes, re- ferential inheritance of ancestral NBS genes were also spectively.CNLdidnothavespecifichighlyconserved observedatthesetwonodes.Representativegenesfrom motifs, but could be distinguished by a RNL like each ancestral Ma-nTNL lineage were compiled with Kinase-2motifandaTNLlikeRNBS-Bmotif. A. trichopoda nTNL genes to reconstruct angiosperm nTNL phylogeny (Supplemental Fig. S9). Because a Intron-ExonStructuresAlso DistinguishNBSClasses mesangiospermae TNL phylogeny could not be con- structedduetotheabsenceofTNLgenesinmonocots,a Theabsence/presence,position,andphaseofintrons TNL phylogeny wasgeneratedattheangiospermlevel often are regarded as important parameters in phy- using eudicot representative TNLs and A. trichopoda logeny(Qiuetal.,1998;Sánchezetal.,2003;Qianetal., Plant Physiol. Vol. 170, 2016 2099 Downloaded from on April 1, 2019 - Published by www.plantphysiol.org Copyright © 2016 American Society of Plant Biologists. All rights reserved. Shao et al. Figure3. CladogramsdepictingreconciledphylogeneticrelationshipsofancestralNBSgenesinthecommonancestorofan- giosperms.ThemaximumlikelihoodtreesofeachcladogramareprovidedinSupplementalFiguresS9andS10. 2015). In a previous study, the intron positions and unraveled the ancient NBS gene lineages at multiple phases were determined to be distinctive among the angiospermevolutionarynodes(Fig.5).Duetotheloss classesofNBSgenesinbryophytes(Xueetal.,2012).In ofTNLgenesinS.indicumandthemonocotlineage,the this study, three classes of NBS genes in angiosperms TNLancestralstatesattheasteridandmesangiospermae alsodifferedinintron characteristics (Fig.4).Threein- nodescouldnotbedirectlyobtainedfromphylogenetic tronswithconservedpositionsandphaseswereshared analyses. However, based on the recovered ancestral bymostTNLgenes,withthefirstintronseparatingthe NBS lineages at the divergence nodes of Solanaceae TIRdomainandtheNBSdomain(phase2),thesecond (Supplemental Fig. S2), dicots (Supplemental Fig. S6), intronseparatingtheNBSdomainandthewholeLRR and all angiosperms (Supplemental Fig. S10), both domain (phase 0), and the third intron separating the asterid and mesangiospermae nodes were deduced to first and the remaining LRR domains (phase 0). An- haveatleastfiveTNLlineages. giosperm RNLs share four class-specific introns, with Abovethefamilydivergencenodes(An,Ma,Mo,Ed, the exception of RNLs in monocots, which did not As, Ro, and Er), CNL genes were abundant but only possess the second intron (Fig. 4). Because the second slightlymorethanthoseofearlierancestralnodes(Fig. intron is present in both A. trichopoda and dicot RNL 5). This finding indicates that CNL genes adopted a genes, intron loss likely occurred in the monocot line- gradualexpansionstrategyintheearlystageofangio- age. For the CNL class, conserved introns were not sperm evolution (from ;225 MYA as angiosperms di- detectedacrossallCNLlineages,especiallyinthebasal verged to around 100 MYA as asterids, rosids, and lineages. In fact, a number of CNL genes were intron- monocots diverged; Fig. 1; D’Hont et al., 2012; Wang less,andwerenotconcentratedinaspecificlineagebut et al., 2014; Zeng et al., 2014). Intensive expansion of distributedacrossallancestralangiospermnTNLline- CNL genes seems to have occurred in the familial an- ages. Although some CNL genes had introns, these cestors of Solanaceae and Poaceae, reaching hundreds were more likely to have been gained at later evolu- of copies. TNL genes apparently followed a different tionary stages. This finding is in agreement with the evolutionary path, as these were completely lost in intronlessstructurereportedinbryophytes (Xue etal., monocotsandinS.indicum.Althoughinheritedbydi- 2012). cots, TNL numbers remained relatively constant for a long evolutionary period, with similar numbers of an- cestralTNLgenesidentifiedatvariousancestralnodes TheThree NBSClassesShowDistinct Evolutionary (An, Ma, Ed, As, Ro, and Er; Fig. 5). The expansion of Patterns inAngiosperms TNLs mainly occurred prior to the divergence of fam- Our step-by-step phylogenetic analysis not only ilies, such as in the Fabaceae ancestor, 55 TNLs were supports the presence of three distinct NBS classes expandedfromsixofeightRo-TNLs.Asimilarpattern in the common ancestor of angiosperms, but also was observed in the Brassicaceae ancestor, in which 2100 Plant Physiol. Vol. 170, 2016 Downloaded from on April 1, 2019 - Published by www.plantphysiol.org Copyright © 2016 American Society of Plant Biologists. All rights reserved. Evolution of NBS-LRR Genes in Angiosperms Figure4. Motifandexon-intronstructuredifferencesamongthethreeNBSclasses.Top,Theconservationofaminoacidse- quencesatfivemotifsintheNBSdomainisshown.Bottom,Proposedancientexon-intronstructureofTNL,CNL,andRNL classes. fourRo-TNLlineagesexpandingto148genes.TheTNL chromosome fragments containing ADR1 or NRG1 geneswereprobablymaintainedatalowernumberfor orthologs were conserved in angiosperm genomes. Fig- more than 100 million years (from ;225 MYA as an- ure 6 (detailed in Supplemental Table S2) shows the giosperm diverged to around 100 MYA as rosids and syntenybetweenADR1-andNRG1-containingchromo- asteridsdiverged)andonlybegantoexpandthereafter. somal regions between the dicot genomes of Phaseolus This may explain why TNL genes were easily lost in vulgaris (rosid lineage) and S. lycopersicum (asterid line- multipleindependentangiospermlineages. age). The syntenic blocks were also detected in the RNL genes are differentiated from the other two monocotgenomeofS.bicolor,intheabsenceoftheNRG1 classes by their relatively conserved evolutionary pat- gene. Moreover, most RNL flanking gene pairs con- tern. Nearly all species from basal angiosperm, mono- servedin theduplicatedblocks within the threespecies cots,andasteridshaveoneortwoRNLgenes,whereas havehigh Ksvalues(.1; Supplemental Table S2), indi- a slight expansion of RNL genes was observed in cer- cating thatthesegenesmay haveresultedfrom onean- tain rosid genomes (Fig. 1). Tracing the evolutionary cient WGD event. Because the most recent WGD event history of RNL genes revealed two anciently diverged sharedbymonocotsanddicotsoccurredinthecommon lineages in the common ancestor of angiosperms (Fig. ancestor of all angiosperms (Jiao et al., 2011; Vanneste 6). One RNL lineage contained the Arabidopsis ADR1 et al., 2014), the syntenic data and phylogenetic results gene,whereastheotherincludedthetobacco(Nicotiana support that RNL genes separated into two lineages benthamiana) NRG1 orthologs. Both lineages were in- sometimebeforethistimepoint. herited by dicot species, except for S. indicum, which losttheNRG1lineage,similartoallmonocotgenomes. DecipheringtheEvolutionofArabidopsisNBSGenes Several whole-genome duplications (WGDs) occurred throughout angiosperm evolution, causing gene du- By performing phylogenetic analyses at different plications and functional divergence (Jiao et al., 2011; evolutionary nodes, we have established a framework Vannesteetal.,2014).TheseparationoftheADR1and for the evolution of angiosperm NBS genes, which NRG1lineagesprior tothedivergence ofangiosperms could help decipher the evolution of NBS genes in prompted us to investigate the involvement of an an- specific species as well as gain a much deeper view of cient WGD event. The syntenic relationships between theevolutionoffunctionallycharacterizedNBSgenes. Plant Physiol. Vol. 170, 2016 2101 Downloaded from on April 1, 2019 - Published by www.plantphysiol.org Copyright © 2016 American Society of Plant Biologists. All rights reserved. Shao et al. Figure5. Comparisonoftheevolutionary patternsofCNLandTNLclassesinangio- sperms.A,ReconstructionoftheNBSgene loss and gain events during angiosperm evolution. The number of ancestral NBS genesateachnodeisdepictedinasolid circle. The thickness of each terminal branch is roughly proportional to the numberofNBSgenesinthegenomeorthe ancestralgenesinthefamily.Genelossand gaineventsatdifferentevolutionarystates areindicatedbyminusandplussymbols, respectively.Anasteriskafterthenumber indicates that the number is not directly recovered from phylogenetic analysis. B, Extensive expansion of CNL and TNL classesattheK-Pboundary.Thenumbers ofCNL(redcircles)andTNL(bluecircles) genesatdifferentdivergencenodesabove/ atfamilylevel,includingallangiosperms (An), mesangiospermae (Ma), monocots (Mo), eudicots (Ed), rosids (Ro), asterids (As),Poaceae(P),Solanaceae(S),Fabaceae (F), andBrassicaceae(B), wereplottedto revealtheirexpansionhistory. Intotal,fiveoutof14An-CNLgeneswereinherited detected in orthologs of Brassicaceae species. The first by the Arabidopsis genome (Fig. 7). Gradual duplica- intron also was found in orthologous genes of the le- tions occurred in four of these An-CNLs (An-C2, -C5, gume family, grape, and the asterid species, but they -C6, and C8), with An-C5 and An-C6 expanding to a were absent in monocots and A. trichopoda. Therefore, few copies. Two An-CNLs (An-C2 and An-C8) pro- theexon-intronstructuredetectedintheseArabidopsis duced more than 80% of Arabidopsis CNL genes, CNL genes gradually formed from two independent mainlyoccurringpriortothedivergenceoftheBrassi- intron gain events, one in the common ancestor of caceaefamily.OneCNLgene(At3g07040)represented eudicots andtheother acquiredinthecommonances- anancientgene(An-C14)thatwasconservedfromthe torofBrassicaceae(Fig.7). angiosperm ancestor to Arabidopsis without duplica- Furthermore, the angiosperm evolutionary frame- tion. Together, the evolutionary paths of the five an- work of NBS genes enabled us to determine when a cestralCNLgenesresultedinthecurrentCNLprofileof functional NBS gene likely arose during angiosperm Arabidopsisgenome(Fig.7). evolution.FivefunctionalCNLgenesagainstdifferent The structural changes were also traced using the pathogens have been identified in Arabidopsis (Liu evolutionaryframeworkofangiospermNBSgenes.For et al., 2007). According to the recovered evolutionary example,nearlyallArabidopsisCNLgenesshowedan history of Arabidopsis CNL genes in Figure 7, RPM1 intronless structure regardless of their origin (Meyers wasthemostancientoneamongthefivegenes,possi- etal.,2003),whichisinaccordancewithourproposed bly originating from the common ancestor of angio- intronless structure for the ancestral angiosperm CNL sperms, whereas the RPP8/HRT was most recent, gene. However, a group of Arabidopsis CNL genes possibly originating after Arabidopsis speciation. The were determined to possess two shared introns. All origin of the other three genes could be traced to the these genes originated from a lineage that diverged divergence nodes of rosids, Brassicaceae, and Arabi- from An-C8 (Fig. 7) in the common ancestor of dopsis.Amongthefivegenes,RPS2,RPS5,andRPM1 mesangiospermae, with the sister lineage remaining were R genes against P. syringae, whereas RPP8/HRT intronless. Based on the phylogenies of CNLs at dif- and RPP13 were both against Peronospora parasitica, ferenttaxonomiclevels,thetwointronswerefrequently indicating functional R genes continuously arose 2102 Plant Physiol. Vol. 170, 2016 Downloaded from on April 1, 2019 - Published by www.plantphysiol.org Copyright © 2016 American Society of Plant Biologists. All rights reserved. Evolution of NBS-LRR Genes in Angiosperms Figure6. EvolutionaryhistoryofRNLgenesinferredfromphylogeneticandsyntenicanalyses.Top,Theinheritanceofthetwo RNLlineagesindifferentangiospermlineages.Theinferredduplicationismarkedwithagreencircle,whereasinferredlossesare indicatedbyblackcircles.Bottom,Intra-andintergenomesyntenicblockscontainingRNLgenesdetectedwithinandamong P.vulgaris,S.lycopersicum,andS.bicolorgenomes. Plant Physiol. Vol. 170, 2016 2103 Downloaded from on April 1, 2019 - Published by www.plantphysiol.org Copyright © 2016 American Society of Plant Biologists. All rights reserved. Shao et al. Figure7. AfewancestralangiospermNBSgenesthatareresponsibleingeneratingthecurrentArabidopsisCNLrepertoirevia gradualexpansion.TheancientstatesofArabidopsisatdifferentevolutionarynodeswereretrievedfromtheconstructedNBS phylogenies.Offspringgeneratedfromthesameancestralgeneatdifferentdivergencenodesareindicatedbyboxesofthesame color.Blacktrianglesindicatethebirthofknownfunctionalgenes.Grayarrowsindicateintrongainevents.Theexon-intron structureofeachancestrallineageisshown. against the same pathogen during plant evolution. theevolutionaryhistoryoftheNBSgeneinthisspecies. Actually,apartfromthesegenes,manyotherfunctional Tofacilitatetheuseofthisframeworkintheanalysisof genesmayhaveappearedinthehistoryandthenbeen NBSgenesinotherplantsthatwerenotincludedinthis lostduringplantevolution.However,suchcasesoften study, we generated hidden Markov model (HMM) couldnotbetracedduetotheirabsenceinmodelplant profiles (Supplemental Dataset S1) for the 23 ancestral genomes. NBS lineages of angiosperms. Taken together, the an- The adoption of the angiosperm NBS evolutionary giosperm NBS evolutionary framework established in framework in Arabidopsis provides new insights into this study provides a fundamental reference for the 2104 Plant Physiol. Vol. 170, 2016 Downloaded from on April 1, 2019 - Published by www.plantphysiol.org Copyright © 2016 American Society of Plant Biologists. All rights reserved.

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in the ancestral angiosperm, from which all current NBS-LRR gene repertoires were activation of RPM1, and plants further evolved another.
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