Genome-Wide Analysis of Nucleotide-Binding Site (NBS) Disease Resistance (R) Genes in Sacred Lotus
Genome-Wide Analysis of Nucleotide-Binding Site (NBS)Disease Resistance (R)Genes in Sacred Lotus (Nelumbo
nucifera Gaertn.)Reveals Their Transition Role During Early Evolution of Land Plants
Rui Zong Jia &Ray Ming &Yun J.Zhu
Received:29March 2013/Accepted:6June 2013/Published online:6July 2013#Springer Science+Business Media New York 2013
Abstract Nucleotide-binding site (NBS)containing genes comprise the largest class in identified plant resistance genes.A total of 137NBS class resistance genes were identified from the newly sequenced sacred lotus genome (Nelumbo nucifera Gaertn.)through a reiterative computational sequence analysis.Three distinct groups of NBS-encoding genes were classified:5with Toll/interleukin-1receptor homology (TIR)domain at N-terminal (TIR-NBS [-LRR (leucine-rich repeat)]),37with CC (coiled coil)domain (CC-NBS [-LRR]),and 95with neither TIR nor CC at N-terminal (NBS [-LRR]).Sequence analysis revealed high divergence of NBS-LRR genes in sacred lotus.The result of cluster and syntenic analysis of NBS genes suggested a duplication and recombination event,which is consistent with the correspondent result of whole genome analysis.In addition,we also identified 52NBS genes which have a putative NACHT domain embedded in the NBS domains.This characteristic has only been reported in animals,fungi and bacteria,suggesting that NACHT and NBS domains shared a similar ancient origin;and
sacred lotus NBS (NACHT)genes may represent a transition role during the early evolution of disease resistance in land plants.
Keywords Disease resistance gene .Divergence .NBS-encoding gene .Sacred lotus .Synteny Abbreviations NBS Nucleotide-binding site NB-ARC NB,ARC1,and ARC2
NACHT NAIP,CIITA,HET-E,and TP1TIR Toll/interleukin-1receptor (TIR)
homology domain
CC Coiled coil domain LRR Leucine-rich repeat R Resistance
Introduction
Plant disease resistance (R)genes confer resistance to patho-gens,and play critical roles in plant immune responses.Triggered by pathogen invasion,R gene cognates pathogen avirulence genes (Avr)in a GENE-for-GENE interaction lead-ing to hypersensitive response,termed as programmed cell death (Flor 1971;Chisholm et al.2006).There are two major classes of R genes:the NBS containing genes (McHale et al.2006)and the cell surface pattern recognition receptors (PRR)(Song et al.1995).NBS containing genes comprise the largest class in identified plant R genes.R proteins recognize a con-served pathogen-associated or microbe-associated molecular pattern (PAMP or MAMP),to trigger a series of nonspecific innate immune system responses (Chisholm et al.2006).
Communicated by Paul Arruda
Electronic supplementary material The online version of this article (doi:10.1007/s12042-013-9122-4)contains supplementary material,which is available to authorized users.
R.Z.Jia :Y .J.Zhu (*)
Hawaii Agriculture Research Center,P.O.Box 100,Kunia,HI 96759,USA
e-mail:JZhu@https://www.sodocs.net/doc/198932842.html,
R.Ming
Department of Plant Biology,University of Illinois,Urbana,IL 61801,USA
Y .J.Zhu
Institute of Tropical Bioscience and Biotechnology,China Academy of Tropical Agricultural Sciences,Haikou,Hainan 571101,People ’s Republic of China
Tropical Plant Biol.(2013)6:98–116DOI 10.1007/s12042-013-9122-4
Most of the characterized NBS genes encode an N-terminal variable domain,a central NBS domain,and a C-terminal leucine-rich repeat(LRR)domain.The NBS do-mains contain the three-layered alpha-beta fold and subse-quent short alpha-helical region characteristic of the AAA+ ATPase domain superfamily(van der Biezen and Jones 1998).It is proposed that NBS domains are involved in signaling pathways by binding and hydrolysis of ATP,in-ducing conformational changes of the overall protein,lead-ing to the formation of the apoptosome,and subsequently cell defense(Yan et al.2005).The LRR motif is typically involved in pathogen recognition specificity in protein–pro-tein interactions and ligand binding with pathogen-derived molecules.The well characterized modeling for NBS do-main for plants is a NB-ARC domain(Pfam:PF00931, current version17)(Punta et al.2012).
The NBS family of proteins can be subdivided further based on the presence or absence of an N-terminal Toll/Interleukin-1 Receptor(TIR)motif(Cannon et al.2002;Meyers et al.2003) and most of those proteins lacking a TIR may have a coiled-coil(CC)motif(Pan et al.2000).TIR and its associated signaling pathways may represent the most ancient host de-fense system found in mammals,insects and plants(Kopp and Medzhitov1999).The alpha-helical CC motif is the principal subunit of oligomerization motifs in proteins;a particular coiled-coil domain determines its oligomerization state,rigid-ity and ability to function as a molecular recognition system (Burkhard et al.2001).NACHT domain(named after NAIP, CIITA,HET-E and TP1)as a central domain,like NBS in plant,was reported to be mainly distributed in animals,fungi and bacteria(Koonin and Aravind2000).They work as recep-tors to perceive non-self and modified-self molecules inside host cells and mediate innate immune responses(Ausubel 2005;Ronald and Beutler2010).
Proteins that have homology with the plant NB-ARC-LRR protein function in mammalian defense response which is dom-inated by NACHT-LRR proteins,together called NLR (Maekawa et al.2011).Sharing similar biological function and protein architecture,the NLR family of receptors in plants and animals perceive signals and mediate innate immune responses to microbial pathogens.While in plants NLR typically detects strain-specific pathogen effectors,in animals NLR normally recognizes conserved microbe/damage associated molecular patterns(Maekawa et al.2011).The NB-ARC domain consists of three subdomains:NB,ARC1and ARC2,and highly con-served methionine–histidine–aspartate(MHD)motif at C-terminus of ARC2(van Ooijen et al.2008).The NB-ARC domain is a functional A TPase domain that hydrolyzes A TP in vitro(Tameling et al.2006).An example is the immunity in tomato(Solanum lycopersicum)to Pseudomonas syringae bac-teria by expression of Pto kinase activity and N-myristoylation for signaling via Pto interacting with a unique Prf(NB-ARC-LRR domain protein for recognition)(Mucyn et al.2006).The NACHT domain was used to reconstruct the evolutionary his-tory of vertebrate NACHT-containing proteins(Hughes2006).
There are two different theories for how the NACHT-related and NBS-related resistance proteins are thought to be involved in the immune system,namely convergent evolution (Ausubel2005;Ronald and Beutler2010;Maekawa et al. 2011)and independent-origin evolution(Yue et al.2011). The convergent evolution theory explains the evolution of the innate immune systems of plants and animals as a conse-quence of the biochemical constrains of building sensors for non-self and signaling cascades from a limited number of eukaryotic protein modules in both phyla(Maekawa et al. 2011).Also,it is thought as a consequence of convergent evolution reflecting inherent constraints on how an innate immune system can be constructed(Ausubel2005). Meanwhile,the theory of independent origin and evolution considers that,by counting the usage frequency patterns among all major kingdoms of organisms on the planet,the NACHT domain remains quite conserved from prokaryotes to eukaryotes,while the NBS domains shows considerable flex-ibility in different evolutionary lineages(Y ue et al.2011).
Sacred lotus(Nelumbo nucifera Gaertn.)is known for its agricultural,medicinal and cultural(religious)importance. Sacred lotus is cultivated for its edible rhizomes,seeds and leaves,which have been used as food for over4,000years in Asia.Its seeds exhibit exceptional longevity,as long as1,300-years,and its rhizomes remain healthy for more than50years. The genome-wide survey of defense/disease resistance asso-ciated NBS-encoding genes in sacred lotus was briefly de-scribed in the recently published draft genome sequence (238Mb containing26,685genes)(Ming et al.2013).In this study,a detailed analysis of the predicted NBS-containing resistance genes was carried out to identify a resource for comparative genomics and to characterize the disease resis-tance involved in innate immune system evolution. Results
Identification and Classification of NBS Genes
One hundred and thirty-seven(137)NBS-encoding resistance genes were identified from the draft genome of sacred lotus. Comparing to other sequenced plants,sacred lotus has less NBS genes except papaya(54),spike mosses(38),and moss (61)(Table1).Arabidopsis thaliana has a similar number of predicted total genes as that in sacred lotus while it has almost twice the number of NBS genes(208)than sacred lotus. Grapevine and rice both have535NBS genes,nearly four-times the number in sacred lotus.Poplar has416NBS genes, three-times more than sacred lotus.The variation of the total number of NBS genes in different plants is high,perhaps due to the difference in genome sizes(Richter and Ronald2000)and
the selection pressure of diseases and stresses during their evolution(Michelmore and Meyers1998).
NBS genes have been subdivided into the functionally distinct TIR-domain-containing(TNL)and CC-domain-containing(CNL)subfamilies(McHale et al.2006).Out of 137genes,5genes(4%)were classified as belonging to the TNL subfamily,and37genes(27%)were classified as belong-ing to the CNL subfamily.However,a large number(95also 69%)of NBS genes were without either CC domain or TIR domains and were assigned to“other”non-regular subfamily (Table1).In A.thaliana,all the NBS genes are classified as either TNL or CNL subfamilies,while NBS genes in other plants,including sacred lotus,are classified into TNL or CNL families,and also another subfamily with neither TIR nor CC domains.All monocots have less than0.1%of NBS genes belonging to the TNL subfamily.There were no TNL genes identified from sorghum,maize,or purple false brome,and only3TNL genes(0.06%of total NBS genes)were found in rice.On the other hand,all dicots have more than10%of NBS genes belonging to the TNL subfamily:The percentages of NBS genes belonging to the TNL subfamily are71%,20%, 21%,and13%in Arabidopsis,grapevine,poplar and papaya, respectively.The vascular spikemoss has3%of total NBS genes as TNL,and a non-vascular moss,has16%of total NBS genes as TNL.TNL were reported to be common in dicots and gymnosperms but rare in monocots and magnoliid orders(Tarr and Alexander2009).It is not clear whether the TNL were lost independently or prior to plant divergence.
The remarkable difference in percentages of TNL genes in total NBS genes among dicot and monocot plants led us to further analyze the distribution of genes in the CNL subfamily. The percentage of CNL genes in total NBS genes among dicots ranged from11%to43%and those in monocots ranged from 31%to86%,suggesting that the number of CNL varies among plants.The absence of the CNL subfamily in spike mosses and moss suggested that the CNL subfamily may have diversified during the evolution of vascular plants.The num-bers of non-regular NBS subfamily were also compared.The highest numbers of non-regular NBS were found in spike mosses(92%)and moss(82%),followed by papaya(76%), sacred lotus(69%),rice(68%),and maize(64%).In A. thaliana,there is no non-regular NBS found.The non-regular NBS encoding genes are considered to be highly divergent or simply truncated structures or pseudogenes with an incomplete coding sequence(Zhou et al.2004;Cheng et al.2010). Motif Architectures of Predicted NBS Encoding Genes
A highly conserved NBS motif with the consensus sequence VIxIVGMGGLGKTTLAxxVYN(Fig.1)was identified from NBS encoding genes using MEME analysis(Bailey et al. 2009).This conserved motif was found in119out of the137 NBS genes in sacred lotus,with cut off P-value10-9,and in some genes it appeared multiple times.Genome comparison of all published NBS genes(PFAM,https://www.sodocs.net/doc/198932842.html,, PF00391,v17,12,258sequences)showed the amino acid
Table1The total number of predicted NBS-encoding(R)genes identified in the whole genome of sequenced Angiosperms
Species Groups b Sacred
lotus(Nn)Mouse-ear
cress(At)
Grape
(Vv)
Poplar
(Pt)
Papaya
(Cp)
Rice
(Os)
Sorghum
(Sb)
Maize
(Zm)
Purple false
brome(Bd)
Spike
mosses(Sm)
Moss
(Pp)
Genome(Mb)2381254874853723897302,500272212.5480 No.of genes26,68525,49830,43445,55524,74637,54434,49632,00025,53222,27338,354 NBS-encoding(R)genes
TIR TN12314101300014 TNL49597786000006
Sub-total51471118873000110 CC CN106261427104404821 CNL27552031204160391915700
Sub-total376122913461671435920521 Other N300366218451047016293 NL65015913223320273618647
Sub-total95019519441365131106343550 Total R137208535416545352741652393861
The total number of predicted protein-encoding genes and the genome size of each species are also provided.See references for details
Bold number is highlighted for the number from Sacred lotus(Nn)
a References for each plant genome and NBS-encoding gene annotation was listed as footnote.Nn:Nelumbo nucifera(Ming et al.2013);At: Arabidopsis thaliana(Meyers et al.2003);Vv:Vitis vinifera(Yang et al.2008);Pt:Populus trichocarpa(Yang et al.2008);Cp:Carica papaya (Porter et al.2009);Os:Oryza sativa(Zhou et al.2004);Sb:Sorghum bicolor(Cheng et al.2010);Zm:Zea mays;Bd:Brachypodium distachyon(Tan and Wu2012);Sm:Selaginella moellendorffii;Pp:Physcomitrella patens
b TN:TIR_NB-ARC,TNL:TIR_NB-ARC_LRR,CN:CC_NB_ARC,CNL:CC_NB-ARC_LRR,N:NB-ARC,NL:NB-ARC_LRR
residues to be highly conserved.Glycine (G)residue in relative entropy position 6,9,and 11,and lysine (L)residue at position 12,were highly conserved,consistent with PFAM database.Furthermore,most of the residues in sacred lotus showed a similar preference with other NBS genes in the database.Exceptions to this were relative entropy 2where isoleucine residue (I)had a higher distribution than valine (V),relative entropy 10where lysine (L)was higher than isoleucine (I)and at relative entropy 21,asparagine (A)was preferred in sacred lotus instead of lysine (L).
To verify and classify other motif architectures in NBS-encoding genes,each candidate was evaluated using PFAM database including the CC motif,TIR motif as well as LRR,and other possible disease resistance associated motifs such as RPW8domain (Arabidopsis powdery mildew resistance gene),Pkinase domain,and WKRY domain (Table 2).We identified 10genes with CC-NBS (CN)and 28genes with CC-NBS-LRR (CNL).CNL is the most common architecture among sacred lotus NBS genes.In addition,we were able to find one gene with TIR-NBS (TN),4genes with TIR-NBS-LRR architectures (TNL)retaining 2~5LRR motifs.The TNL with diverged putative LRR motifs were considered to play different roles in plant defense (Wan et al.2012).
In addition,5NBS genes have the RPW8domain;NNU_16329has one copy of RPW8(CC)-NBS-LRR,NNU_006591has two copies of RPW8-NBS-LRR domains,NNU_003698has RPW-NBS domain,and NNU6589and NNU_011785have RPW8-NBS-LRR domain.RPW8is known as Arabidopsis broad-spectrum mildew resistance protein which confers resistance to powdery mildew by inducing localized,salicylic acid-dependent defenses (Xiao et al.2001).Two genes,NNU_016985and NNU_014596,were found to contain a Pkinase domain at the C-terminal as Pkinase-NBS-LRR-LRR.Such a Pkinase-NBS-LRR-LRR architecture has been found in only 12sequences in the PFAM database,all from the wheat family (2from common wheat,8from durum wheat,and 2from goatgrass)(Punta et al.2012).The Pkinase domain was reported to be involved in the NBS gene catalytic function of protein kinases.Tsn1(D7PDA0_9POAL),a wheat disease resistance-like
gene,
C o n t r i b u t i o n
C o n t r i b u t i o
n
1
123451718192021
678910111213
141516A
B
Relative Entropy
Relative Entropy
Partial NBS motif in sacred lotus
4
NB-ARC motif from Pfam database
Relative Entropy
C o n t r i b u t i o n
C
NACHT motif from Pfam database
4
Fig.1Conserved NBS motif from predicted sacred lotus NBS genes.a NBS motif logo revealed via MEME analysis.Contribution was calcu-lated by surveying the percentage of amino acid appearance.Relative entropy is reflected for each position of corresponding sequence.b Partially HMM logo of NB-ARC (PF00931)domain (truncated from original generated by https://www.sodocs.net/doc/198932842.html, ).Up to date,logo of PF00931(version 17)was generated from 12,258sequences.c Partially HMM logo of NACHT (PF05729)domain (truncated from original logo in https://www.sodocs.net/doc/198932842.html, ).Up to date,logo of PF05729(version 7)was generated from 6,525sequences
which has the typical architecture of Pkinase-NBS-LRRs,governs the effector-triggered susceptibility to necrotrophic pathogens (Faris et al.2010).Another domain called FMN-red domain (NADPH-dependent FMN reductase domain)was also found in the C-terminal of one NBS gene (NNU_024486).This novel domain combination was never reported before in the PFAM database.A Blast-P search (NCBI)identified one protein (XP_002281054,disease re-sistance protein RPP13-like from Vitis vinifera )that was highly matched to NNU_024486gene.Further analysis of this NBS gene with NBS-FMN-red architecture is needed to predict its function.One WRKY domain was also discovered
Table 2Predicted motif architecture of sacred lotus NBS-encoding genes and their most similar Genbank entries by
BLAST-P
to be in the T-terminal of NBS gene(NNU_007278).There are a total of9sequences containing the WRKY domain in PFAM database and all of them are from Arabidopsis(6 genes from A.thaliana:WRK52,WRK16,Q0WWA0, C4B7M5,C4B7M6,and E1B328;and3from A.lyrata: D7MKX1,D7MKY8,and D7MUR9).A typical model of NBS-LRR-WRKY architecture was found in the WRK52, also known as a novel structure combining domains in the Arabidopsis disease resistance protein RRS1.In A thaliana, RRS1-R-mediated resistance is partially salicylic acid-and partially NDR1-dependent conferring specific resistance to Ralstonia solanacearum(Deslandes et al.2002).
a Motif architectures of each predicted R gene were distinguished with multi-domains
b E:for E-value of the most similar Genbank entries via BLAST-P
c S:indicate species,Vv:Vitis vinifera,Pt:Populus trichocarpa,Rc:Ricinus communis,Os:Oryza sativa,Mt:Medicago truncatula,Gm:Glycine max,Md:Malus x domestica,Pa:Platanus x acerifolia,Sb:Solanum bulbocastanum,At:Arabidopsis thaliana,Pd:Pinus taeda
Comparison of NBS and NACTH Domains
Fifty-two(52)out of137sacred lotus NBS-encoding genes were found to have the NACTH domain embedded in the NBS domain(Supplementary Table1).Comparison of HMM logo of NACHT and NBS domains(Fig.1b and c) showed a similarity of amino acid patterns in both domains.
A phylogenetic analysis of NBS and NACHT domains among animals and plants revealed independent recruitment in evolution(Yue et al.2011).Since the NBS domain and NACHT sequences from eubacteria and archaebacteria show fundamental differences,Yue et al.(2011)proposed that those two domains either diverged a long time ago or actu-ally originated independently.
In addition to differences in domain structures,NBS-LRR genes from the subclasses also differ considerably in their phy-letic distribution and downstream signaling pathways, suggesting possible functional divergence.Analogous to the case for plants,animal innate immune systems also rely on two groups of receptors,the transmembrane Toll-like receptors (TLRs)and cytosolic Nod-like receptors(NLRs)(Ausubel 2005).TLRs are characterized by an extracellular LRR domain and an intracellular TIR domain,whereas NLRs often dem-onstrate a tripartite domain architecture consisting of a vari-able N-terminal domain,a central NACHT domain and also a C-terminal LRR domain,like NBS-LRR proteins in plants. However,the NACHT’s have been placed in a separate phy-logeny,suggesting different evolutionary histories of these two domains.
In this study,we found52out of the137NBS genes from the sacred lotus genome to have some similarity to the NACHT domain which is completely or partially embedded in the NBS domain(Supplementary Table1).Evidence has suggested that fundamental differences between the NBS and NACHT do-mains existed before the split of eubacteria,archaebacteria and eukaryotes(Y ue et al.2011).While NBS genes are found in most land plants,NACHT are mostly found in animals,fungi and bacteria.Therefore,these two apparently similar domains must have either diverged a long time ago or actually originated independently.It is interesting to explore the distinct role of sacred lotus in the largely unknown evolutionary history for the NBS and NACHT domains.Phylogenetic analysis of NBS domains from plant,and NACHT domains from animals re-vealed that the NBS domains of the spikemoss and sacred lotus were hyper-distributed among the animal and plant kingdoms (Fig.2and Supplementary Figure1).Based on our current finding,we developed a putative hypothesis that the evolution of the NBS genes(also retaining NACHT domain)in spikemoss and sacred lotus may play a transitionary role during the evolu-tion of land plants.By surveying the number of NBS and NACHT among plants,fungi,bacteria and animals,we were able to present the distinct distribution of the NBS(retaining NACHT)genes of sacred lotus(Supplementary Figure1).It was found that they were clustered with domains found not only in plant but also in the animal kingdom.However,current knowl-edge cannot explain the evolutionary role of NBS embedded with NACHT.Our data may suggest that there is a common origin of NBS and NACHT domains in early evolution and those NBS domains retaining a NACHT domain in sacred lotus may indicate a transitionary role or incomplete divergence before separation of the plant and animal kingdoms.
Divergence of the NBS Encoding Genes
The amino acid sequence of NBS-encoding genes in three sub-families;TNL,CNL and NL,were aligned separately to inves-tigate their sequence diversity and phylogenetic relations (Fig.3).We adopted Arabidopsis disease resistance cluster/groups(Meyers et al.2003).Four TNL genes of sacred lotus were clustered with Arabidopsis RPS4disease resistance TNL_B/C group,and one of them NNU_005300was clustered with At4G23440,a TN group(Fig.3a).RPS4is a resistance gene against P.syringae in Arabidopsis corresponding to the avirulence gene avrRps4(Hinsch and Staskawicz1996),and also confers resistance to oomycetes by EDS1-dependent path-way(Aarts et al.1998).All37CNL genes of sacred lotus were grouped into five subgroups according to the Arabidopsis sys-tem;CNL_A,CNL_B/B3(RPS2),CNL_C(RPP13),CNL_D (RPP8),and CNL others(Fig.3b).The role of RPS2in Arabidopsis is specifically to the recognize Pseudomonas syringae strains expressing the avirulence gene avrRpt2 (Kunkel et al.1993).Mutation studies on rps2,ndr1,and Atrar1revealed that RPS2initiates the signaling based upon perception of RIN4disappearance rather than the direct recog-nition of AvrRpt2(Axtell and Staskawicz2003).Three RPS2-like NBS genes in sacred lotus;NNU_007430,NNU003655 and NNU_007362,were identified.In addition,three RPP13-like NBS genes(CNL_C)were found in the sacred lotus ge-nome.RPP13in A.thaliana specifies resistance to downy mildew(Peronospora parasitica)(Bittner-Eddy et al.2000). The RPP13locus contains either a single gene capable of recognizing multiple isolates or a group of tightly linked genes such as pbs2,pad4-1,npr1-1,and rps5-1genes(Bittner-Eddy and Beynon2001).We also identified five CNL_D(RPP8-like) resistance genes in sacred lotus.RPP8in Arabidopsis confers resistance to turnip crinkle virus and oomycete pathogens (Cooley et al.2000).RPP8is induced by pathogens and salicylic acid treatment and WRKY transcription factors regulate the expression of surveillance genes upstream of the defense-signaling cascade(Cooley et al.2000).The remaining13CNL NBS genes were not clustered into any Arabidopsis subgroups. An additional95of N-or NL-type NBS genes were highly diversified,and were grouped into20subgroups(Fig.3c). Sequences in this group were hyper-variable suggesting high divergence of their functions.
Distribution of NBS Genes Across the Sacred Lotus Chromosome
The majority of sacred lotus NBS-encoding genes(80out of 137)were distributed among the nine linkage groups except LG7and LG8.An additional55NBS-encoding genes were scattered in28scaffolds which could not be assigned to linkage groups(Fig.4).The majority(109out of137, 80%)of NBS-encoding genes are members of complex loci with more than2NBS genes located in a single scaffold.Nine (9)NBS genes were clustered in S82on LG9;8NBS genes were clustered in S16on LG1and S70on LG2;5NBS genes were clustered in S196on LG1;4NBS genes were clustered in S39 on LG5;3NBS genes were clustered respectively in S85,S8on LG2,S32on LG9and S13on LG4;finally,2NBS genes were clustered respectively in S121,S119,S15on LG1,in S20,S23, S41on LG3,in S235on LG4,in S4,S21,S60on LG6,in S126 on LG9.A total of80NBS genes were clustered in scaffolds on these linkage groups.In scaffolds which were not mapped to any linkage groups,7NBS genes were clustered in S59and S67,5 NBS genes were cluster in S104,4NBS genes were clustered in S6,S100,3NBS genes were clustered in S138,S322and finally 2NBS genes were found to be clustered in S51,S76,S64,S131. The total NBS genes clustered in this group was49. Superclusters have been reported in poplar(Kohler et al. 2008),with31NBS genes super-clustered on Linkage group XIX,18NBS genes super-clustered on Linkage group II and13 NBS genes super-clustered on Linkage group XI.Clustered NBS genes are also present in B.distachyon(Tan and Wu 2012),rice(Monosi et al.2004;Zhou et al.2004),and papaya (Porter et al.2009).Thus highly tandem repeats with clusters of NBS genes found in sacred lotus(Fig.4)and other crops suggest that these NBS genes arose from whole genome duplication (WGD)events or have been maintained without substantial divergence(Porter et al.2009).
Further analysis revealed18syntenic pairs among the NBS-encoding genes in sacred lotus,supporting the WGD event(Ming et al.2013).Interestingly,among them,two pairs, NNU_022164/NNU_007845and NNU_000216/NNU_01 6337,were not all NBS-coding genes(Supplementary Table2).Sequence analysis showed that NNU_007845and NNU_016337were missing the entire NBS domains and only LRR domains remained,suggesting potential divergence of NBS genes by losing domains after WGD event.
The sacred lotus that holds the world record for seed longevity(200~1,300years),is a primitive angiosperm and one of the oldest plants surviving from the last ice age(Shen-Miller et al.1995,2002).The unique environmental stresses during its growth might engage and curtail the specialization of disease resistance genes in sacred https://www.sodocs.net/doc/198932842.html,parative ge-nome analyses with other genome-sequenced plants indicated that plant genomes usually encode a large number of NBS
(208, 0)
(38, 24)
(61, 32)
Fig.2Overview of the distribution of NBS encoding genes and NACHT encoding genes.a The number of genes containing the NBS and NACHT domains in bacteria,archaea,fungi,animals,and plants, shown as branches of a phylogenetic tree.Red area with number indicates the number of species or genus containing the NB-ARC domain,Green area with number indicates the number of species containing NACHT domain.b The distribution of NBS and NACHT domains in green plants.The phylogenetic tree was re-regenerated from the Taxonomy Browser(https://www.sodocs.net/doc/198932842.html,/taxonomy).The first number in brackets is the total number of NBS encoding genes and the second number is the number of NBS encoding genes that possessed NACHT domains.The numbers are referred to in Table1,except that data from castor bean indicated with“asterisk”,was directly adopted from PFAM database
genes,with a great diversity in the number of NBS-LRR genes as well as in the distribution of subfamilies of NBS-LRR genes.An interesting example is provided by TNL genes, which are absent from grass genomes surveyed to date(Bai et al.2002).Typically,dicot plants contain more TNL(more than13%of NBS genes,Table1),for example Arabidopsis (147or70.7%),Vitis vinifera(111or20.7%),populus(88or 21.2%)and Carica papaya(7or13%)(Meyers et al.2003; Yang et al.2008;Porter et al.2009).With the availability of the sacred lotus genome,analysis of NBS-encoding genes revealed the number of TNL genes(3.5%of total NBS genes) in sacred lotus,falling between dicot plants and grass species. Therefore,it is of particular interest to try to elucidate the origin and evolutionary history of this important gene family and also the evolutionary relationship between TIR-and non-TIR-NBS-LRR genes.Meanwhile,the CC domain was iden-tified among the NBS genes in both dicot and monocot plants with ranges from11.1%in C.papaya to55.8%in Sorghum bicolor.This comparison supports the evolution theory in-volving ancestral polyploidy events with substantial gene expansion and diversification of this gene family in plants (Yue et al.2011).Such substantial gene expansion of this gene family in the land plants was possibly driven by their func-tional specialization in terms of plant immunity(Tan et al. 2007).
Discussion
The draft genome of sacred lotus appears to contain distinct NBS encoding genes(Ming et al.2013).A total of137genes were identified as NBS encoding https://www.sodocs.net/doc/198932842.html,paring the sacred lotus with other plants which have had their genome se-quenced,the number of subfamilies of TIR-NBS-LRR genes was higher than in monocots,but lower than in dicots and most similar to spikemoss,suggesting that the sacred lotus is an ancient plant in terms of https://www.sodocs.net/doc/198932842.html,parison of the sub-families of CC-NBS-LRR genes in sacred lotus and Arabidopsis showed that sacred lotus has the most types of CC-NBS-LRR genes and shows great divergence in the NBS-LRR subgroup.Eighteen(18)syntenic pairs of were found among137NBS encoding genes which points to a WGD event.Additionally,52out of137NBS-encoding genes contained a NACTH domain anchored/embedded in the NBS domain.A unique character of NACHT is that it is only found in disease resistance genes in animals and fungi.Thus our analysis indicates that the NACHT or NBS domains were largely curtailed by environmental stresses and may play a role in the evolution of land plants.Our data may suggest that there is a common origin of NBS and NACHT domains in early evolution and those NBS domains retaining a NACHT domain in sacred lotus may indicate a transitionary role or incomplete divergence before the separation of plant and animal kingdoms.Sacred Lotus Genome Contains NBS Encoding Genes
in Distinct Subgroups
We have characterized the complete set of137NBS encoding genes in the current version of the sacred lotus genome.This number represented~0.5%of all predicted ORFs,the same percentage as that in Arabidopsis(Meyers et al.2003).Based on the gene sequence,protein motif and domain architecture,we classified them into2TNL sub-groups and5CNL subgroups,compared to8and4corre-sponding subgroups in Arabidopsis.Meanwhile,we also classified20subgroups in NL groups showing great diver-gence.Genome-wide analysis previously resulted in416 poplar NBS genes being classified into10subgroups based on the motif architectures,and535grapevine NBS genes being classified into14subgroups(Yang et al.2008).Due to
NNU_015526_(CNL)
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Fig.3Phylogeny of subfamilies of TNL(a),CNL(b),and N or NL(c). Reference genes of each subgroup of TNL and CNL proteins were adopted from A.thaliana(Meyers et al.2003).Bootstrapped(1,000 replicates)neighbor-joining trees from distance matrices were constructed using the Clustal-W aligned NBS sequences.Numbers less than50%(clustering50out of100times)were omitted due to possible collapse of the branch.Branch lengths proportional to genetic distance are indicated in the scale bar
NNU 024486
XP 002274414 (RPP8-like)NNU 001119NNU 003698NNU 023855
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XP 003634282 (RGA2-like)NNU 015616NNU 002459NNU 025043NNU 025044NNU 021135NNU 021136NNU 019222NNU 026022NNU 019855NNU 019860NNU 025365
XP 002271203 (RGA4-like)NNU 021753NNU 008143NNU 015503NNU 015544NNU 015580NNU 015581NNU 015582
XP 002297751 (R gene)NNU 004711NNU 025149NNU 007361
XP 003633547 (RPP13-like)NNU 021757NNU 016519NNU 019401NNU 018021NNU 018024NNU 018042NNU 018043NNU 002767NNU 020088NNU 015617NNU 024027NNU 019403NNU 016275NNU 016495NNU 016496NNU 016500NNU 016497NNU 019909NNU 021817NNU 016506NNU 022698NNU 007431NNU 024034NNU 012695NNU 026649NNU 012693NNU 016498NNU 018018NNU 009351NNU 018364NNU 021232NNU 013084NNU 015525NNU 018067NNU 004589
XP 002264704 (hypothetical R protein)NNU 011393NNU 006589NNU 011785NNU 015009NNU 008651NNU 020790NNU 007278NNU 008652NNU 020792NNU 024928NNU 014763NNU 004084NNU 025600NNU 014596NNU 014605NNU 017556NNU 026508NNU 020623NNU 007355NNU 018366
XP 002326566 (R protein)AFP82245(R protein)NNU 016976NNU 016978NNU 016979NNU 016984NNU 016985NNU 008736NNU 019089
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Fig.3(continued)
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Fig.4Distribution of identified NBS encoding genes across linkage groups (LG).a Each linkage group is shown in Centimorgans (cM);b Individual scaffolds were scaled in Mbp.The corresponding position of each predicted NBS gene was indicated with ascaffold number (Ming et al.2013)
the missing TNL group,519rice NBS genes were classified into7subgroups(Zhou et al.2004),and274sorghum NBS genes,also missing a TNL group,were classified into13 subgroups(Cheng et al.2010).The observed diversity of these NBS-LRR genes indicates the variety of recognition molecules available in detecting disease pathogens.
All dicot plants which have been sequenced so far have more than10%NBS with a TIR domain,and all genome sequenced monocots have less than1%.Arabidopsis is an exception among dicots with a ratio of approximately2:1of TNL and CNL(Meyers et al.2003).Sacred lotus has~4% which is in between the dicot and monocot plants and is similar to moss or spikemoss.Apparently,during the evolu-tion of land plants,TNL subgroups may have less preferen-tially amplified and diversified in cereal plant lineages(Bai et al.2002).Before work on the sacred lotus,researchers attempted to deduce the evolution of TNL and CNL genes and suggested that additional data as to the R gene compo-sition of other plant families was required to infer evolution events(Bai et al.2002;Cannon et al.2002;Meyers et al. 2003).Sacred lotus,an ancient plant species,with its specific feature of R gene composition could help to reveal how TNL and CNL were involved in land plant evolution.A recent study tracing both origin and evolutionary history concluded that TNL probably had an earlier origin than CNL(Yue et al. 2011).However,the molecular roles and evolution status of TNL and CNL remain to be confirmed in future studies. NB-ARC and NACHT Shared a Similar Ancient Origin, and Diverged During Evolution
Sharing a similar biological function and protein architec-ture,the NLR family of receptors in plants and animals,NBS and NACHT,perceive signals and mediate innate immune responses to microbial pathogens.While plant NLR typical-ly detect strain-specific pathogen effectors,animal NLR normally recognize conserved microbe/damage associated molecular patterns(Maekawa et al.2011).The similarity between NACHT and NB-ARC,as well as the R gene architectures suggest that resistance genes in plant and ani-mal systems may have evolved by the convergent evolution theory(Ausubel2005;Maekawa et al.2011).A trace of NACHT motif in sacred lotus NBS domain could lead us to conclude that NACHT and NB-ARC domains shared a similar ancient origin,and this transition status in sacred lotus confirms it as an early evolving land plant.
Genetic Events Shaped the Composition of the NBS Encoding Genes
Genome duplication plays a critical role in complex genetic systems like disease resistance,as a means of creating new loci,altering genes by recombination,or generating repeat sequences(Ronald and Beutler2010).Many NBS encoding genes were reported as clustered gene groups in all plants sequenced to date(Meyers et al.2003;Monosi et al.2004; Zhou et al.2004;Kohler et al.2008;Porter et al.2009;Tan and Wu2012)Including sacred lotus,NBS-LRR loci in plants are found both as isolated genes(singletons)and tightly linked arrays of related genes(Leister2004).Those gene clusters containing NBS-LRR genes from different phylogenetic clades may be driven by selection pressures on allelic variation created by mutations and re-assorted by recombination between alleles and sometimes between dif-ferent gene family members(Hulbert et al.2001).A sug-gested model for gene clusters in the genome is by chance or by certain mechanism to select NBS-LRR genes into clusters (Leister2004).
The duplication and recombination of NBS-LRR genes in sacred lotus also resulted in divergence.It has been sug-gested that the diversification of NBS-LRR genes in Arabidopsis resulted from intra-locus crossovers,sequence exchange,and selection mechanisms(Leister2004).To un-derstand the evolution of NBS-LRR in sacred lotus,the synteny was screened among the NBS-encoding genes,as well as non-NBS-encoding genes.Arabidopsis has the less synteny may resulting from heterogeneous originated dis-tance by chance association rather than by ectopic recombi-nation(Baumgarten et al.2003).Sacred lotus was also found less synteny among the NBS-encoding gene,may indicates those NBS gene different origin.Less selection mechanisms for clusters available in current knowledge,one of them was that different NBS-LRR genes mediated resistance against the same or different pathogen,and such a positive selection to promote the co-segregation to increase the total fitness to the plant(Hulbert et al.2001;Leister2004).
Function of NBS Encoding Genes
A fair amount of biochemical data has been reported to describe the function of the NBS genes in plants(Gassmann et al.1999;Zhang and Gassmann2003,2007;Wirthmueller et al.2007).The observed number of NBS proteins in sacred lotus represents a major part of the spectrum of recognition molecules to detect diverse pathogens.Thirty-one(31)CNL and TNL genes in sacred lotus have uninterrupted full-length ORFs.All full-length TNL genes were subgrouped with Arabidopsis RPS4gene which confers specific resistance to strains of Pseudomonas syringae pv.tomato expressing avrRps4(Gassmann et al.1999).The full length of RPS4is required to mediate disease resistance,indicating regulation at the protein level rather than as regulatory RNAs(Zhang and Gassmann2003).The PRS4accumulation could trigger the EDS1-dependent defense(Wirthmueller et al.2007),as well
as other signaling components,e.g.SGT1and HSP90,in activation of cell death with HR(hypersensitive response) (Zhang et al.2004).Negative regulators such as srfr(suppres-sor of rps4)mediate RPS4-dependent gene-for-gene disease resistance(Kwon et al.2004).Expression of truncated RPS4 proteins in Nicotiana benthamiana induced HR-like cell death in the absence of AvrRps4,suggesting that RPS4is regulated at multiple levels,including gene expression,alternative splic-ing,and protein stability(Zhang and Gassmann2007).RRS1 and RPS4are required for a dual resistance gene system that prevents infection of Colletotrichum higginsianum,R. solanacearum and Pst-avrRps4.The CNL genes in sacred lotus were mostly grouped together with Arabidopsis RPS2,RPP8, and RPP13.RPS2is also an Arabidopsis disease resistance locus specifying the recognition of specific Pseudomonas syringae strains expressing the avirulence gene avrRpt2(Kunkel et al. 1993).RPP8and RPP13confer resistance to downy-mildew via different avirulence determinants in Peronospora parasitica (Bittner-Eddy et al.2000;Mohr et al.2010).
Compared to Arabidopsis,sacred lotus apparently has fewer full-length NBS encoding genes.However,few sacred lotus pathogens/insect pests are known so far. Cercospora nymplaeacea(lotus leaf brown spot disease), Alternaria nelumbii(lotus leaf brown spot disease), Fusarium bulbigenum(lotus rot disease),Colletotrichum gloeosporioides(wilting),Cylindrocladium hawksworthii (leaf spot and rhizome rot),Prodenia litura,and waterlily aphid are common pathogens/insect pests of sacred lotus found in China(Xu1990;Chen et al.2005).Further molec-ular and biological analysis of the genes involved in the interaction of sacred lotus with its pathogens and insect pests would provide some insight as to the functions of the NBS-encoding genes.
Materials and Methods
Sacred Lotus Genome Sequence
The drafted genome of sacred lotus‘China Antique’became available in GenBank under genome ID14095:http:// https://www.sodocs.net/doc/198932842.html,/genome/14095(Ming et al.2013). MAKER(v2.22),SNAP and Augustus were used for gene prediction and annotation of NBS-encoding genes.A total of 26,685protein coding genes were predicted from the sacred lotus genome sequence.
Identification of NBS-Encoding Genes
Methods used in this study to identify the sacred lotus NBS type R genes and analyze motif architecture have been de-scribed by Porter et al.(2009).Hidden Markov Models method(HMMER,v3.0)was used to search for NBS(NB-ARC,Pfam family PF00931domain,up-to-date version17, https://www.sodocs.net/doc/198932842.html,)(Punta et al.2012).Preliminary search with lower stringency(expectation-value cut off of 1.0)to make sure covering all putative NBS proteins.The retrieved candidate proteins were further validated by MEME(Multiple Expectation Maximization for Motif Elicitation,version4.8.1,https://www.sodocs.net/doc/198932842.html,)(Bailey et al.2009),and NBCI Conserved Domain search tool to search against CDD(conserved domain database,v3.05, https://www.sodocs.net/doc/198932842.html,)(Marchler-Bauer et al.2011). Thus the critical validation yields a more stringent threshold (E-value10-6)to filter out false hits.Furthermore,BLASTP (https://www.sodocs.net/doc/198932842.html,)was conducted to confirm the candidate proteins(Altschul et al.1997).
Identification of TIR,CC,LRR and Other Motif Architectures
All candidate NBS genes were further searched manually and automatically against the HMM database(Pfam-A.hmm,v26.0) (Punta et al.2012)to find any potential architecture,such as TIR (Pfam domain module:PF01582,PF13676),LRR(Pfam do-main module:PF00560,PF12799,PF13306,PF13504, PF13516,PF13855,PF07723and PF07725),NACHT(Pfam domain module,PF05729).Additional CC domain analysis was carried out using Paircoil2program,with0.025as cut off value for P-score(McDonnell et al.2006).
Phylogenetic Analysis of NBS Resistance Gene
Full NBS-encoding amino acid sequences,truncated NBS-domains and truncated NACHT domain sequences were aligned separately using CLUSTALW program(Thompson et al.1994).Pairwise distance measurement was taken using PAUP(v4.0)to construct Neighbor-Joining method(NJ) (Saitou and Nei1987)with multiple alignment resampling set bootstrap at1,000(Felsenstein1996).The MEGA(v5.0) was used to view the phylogenetic tree(Tamura et al.2011).
Linkage Group Assignment
Based on the restriction associated DNA sequencing (RADseq)markers and simple sequence repeat(SSR) markers,the sacred lotus genome was mapped into9 linkage groups to anchor the assembled scaffolds(Ming et al.2013).Scaffold and Megascaffold(multi-scaffold) position were identified for each supercontig containing predicted NBS-encoding genes.In accordance with previ-ous genome-wide syntenic analysis(Ming et al.2013),the synteny among the predicted NBS-coding genes was also investigated.
Acknowledgments We thank Dr.Ming-Li Wang at the Hawai‘i Ag-riculture Research Center(HARC)for providing assistance with bioin-formatics and Dr.Heather McCafferty at HARC for critical reviewing of the manuscript.This work was supported partially by a cooperative agreement(No.CA58-5320-3-460)between the U.S.Department of Agriculture-Agricultural Research Service and HARC. References
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