GENE-39634; No. of pages: 9; 4C: Gene xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Gene
1
Tracing the origin and evolution of plant TIR-encoding genes
2Q1
Xiaoqin Sun a, Hui Pang a, Mimi Li a, Jianqun Chen b,⁎, Yueyu Hang a,⁎⁎
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a
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a r t i c l e
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Article history: Received 10 January 2014 Received in revised form 17 April 2014 Accepted 24 April 2014 Available online xxxx
a b s t r a c t
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i n f o
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Institute of Botany, Jiangsu Province and Chinese Academy of Sciences, Jiangsu Province Key Laboratory for Plant Ex Situ Conservation, Nanjing 210014, China State Key Laboratory of Pharmaceutical Biotechnology, School of Life Sciences, Nanjing University, Nanjing 210093, China
P
Toll-interleukin-1 receptor (TIR)-encoding proteins represent one of the most important families of disease resistance genes in plants. Studies that have explored the functional details of these genes tended to focus on only a few limited groups; the origin and evolutionary history of these genes were therefore unclear. In this study, focusing on the four principal groups of TIR-encoding genes, we conducted an extensive genome-wide survey of 32 fully sequenced plant genomes and Expressed Sequence Tags (ESTs) from the gymnosperm Pinus taeda and explored the origins and evolution of these genes. Through the identification of the TIR-encoding genes, the analysis of chromosome positions, the identification and analysis of conserved motifs, and sequence alignment and phylogenetic reconstruction, our results showed that the genes of the TIR-X family (TXs) had an earlier origin and a wider distribution than the genes from the other three groups. TIR-encoding genes experienced largescale gene duplications during evolution. A skeleton motif pattern of the TIR domain was present in all spermatophytes, and the genes with this skeleton pattern exhibited a conserved and independent evolutionary history in all spermatophytes, including monocots, that followed their gymnosperm origin. This study used comparative genomics to explore the origin and evolutionary history of the four main groups of TIR-encoding genes. Additionally, we unraveled the mechanism behind the uneven distribution of TIR-encoding genes in dicots and monocots. © 2014 Published by Elsevier B.V.
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Keywords: TIR-encoding genes T genes Disease resistance genes Gene evolution Monocots Gene clusters
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Q1311 12 13 14 15 16 17
b
36 34 33
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1. Introduction
38 39
Disease resistance genes (R genes) in plants are important functional genes that prevent and respond to the invasion of pathogens. Most R genes encode members of an extremely polymorphic superfamily of nucleotide-binding leucine-rich repeat (NLR) receptors (Maekawa et al., 2011). Specific NLR proteins can be activated by specific pathogen effectors via direct interaction as receptor and ligand respectively (Dodds et al., 2006). Alternatively, a specific NLR can be activated by sensing effector-mediated alteration in a host virulence target or a decoy protein of that target (Dangl and Jones, 2001; van der Hoorn and Kamoun, 2008). In further support of the latter, experimentally established interaction networks of the Arabidopsis thaliana proteins and the bacterial and oomycete effectors have revealed that independently evolved effectors converge onto the hubs of the immune system network (Mukhtar et al., 2011). NLR activation coordinates effectortriggered immunity that limits pathogen proliferation.
48 49 50 51 52
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44 45
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42 43
18 19 20 21 22 23 24 25 26 27 28 29 30 31
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40 41
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journal homepage: www.elsevier.com/locate/gene
Abbreviations: CC, coiled-coil; HNL, α/β-hydrolase-NBS-LRR; LRR, leucine-rich repeat; NBS, nucleotide binding site; NLR, nucleotide-binding leucine-rich repeat; Pkinase, protein kinase; PNL, protein-kinase-NBS-LRR; TIR, Toll-interleukin-1 receptor. ⁎ Correspondence to: J. Chen, State Key Laboratory of Pharmaceutical Biotechnology, School of Life Sciences, Nanjing University, 22 Hankou Road, Nanjing 210093, China. ⁎⁎ Correspondence to: Y. Hang, Jiangsu Province Key Laboratory for Plant Ex Situ Conservation, Institute of Botany, Jiangsu Province and Chinese Academy of Sciences, 1 Qianhuhoucun Road, Nanjing 210014, China. E-mail addresses: [email protected] (J. Chen), [email protected] (Y. Hang).
The R genes encode a number of conserved domains, including the nucleotide binding site (NBS), leucine-rich repeat (LRR), protein kinase (Pkinase), toll/interleukin-1 receptor (TIR) and coiled-coil (CC) domains (Jacob et al., 2013). TIR-encoding genes represent one of the most important families of disease resistance genes in plants and act as signal mediators that respond to pathogens (Katagiri and Tsuda, 2010; Lewis et al., 2010; Römer et al., 2009; Zhang et al., 2012). These genes can be further categorized into several groups according to the variable C-terminal domains: TIR-NBS-LRR (TNL), TIR-NBS (TN), TIRLRR (TL) and TIR-X (TX, where X is any domain other than NBS or LRR) (Nandety et al., 2013). The function of TIR-encoding genes is believed to be involved in immunity, yet some studies on Arabidopsis TIR-encoding genes suggest their possible roles beyond. Arabidopsis CHS1 which encodes a TN protein confers cold resistance by limiting chloroplast damage and cell death at low temperature (Zbierzak et al., 2013). Overexpression analysis and phylogenetic highly conservation suggest that the TNX proteins may have other nondefense roles in monocots and other plants (Nandety et al., 2013). Studies of the TIR-encoding genes (mostly involving TNLs) have primarily focused on gene cloning, the functional verification of the genes and the molecular mechanisms of disease resistance (Anderson et al., 1985; Franchel et al., 2012; Gassmann et al., 1999; Lawrence et al., 1995; Meyers et al., 1999; Parker et al., 1997; Whitham et al., 1994). In contrast, the analysis of genetic polymorphisms and gene evolution has been limited to only a few genes or individual plant species (reviewed in Jacob et al., 2013). The functions of TXs and TNs (the
http://dx.doi.org/10.1016/j.gene.2014.04.060 0378-1119/© 2014 Published by Elsevier B.V.
Please cite this article as: Sun, X., et al., Tracing the origin and evolution of plant TIR-encoding genes, Gene (2014), http://dx.doi.org/10.1016/ j.gene.2014.04.060
53 54 55 56 57 58 Q2 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 Q3 74 Q4 75 76 77 78
90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107
t1:3
Phylum
Class
Species
a
R
t1:39
Dicotyledoneae
U
N
C
O
Bryophyta Pteridophyta Gymnospermae Angiospermae
Monocotyledoneae
Total
119
2.1. Data sampling
120
A total of 32 different whole-genome sequenced species were selected, including two species in Chlorophyta, one species in Bryophyta, one species in Pteridophyta and 28 species in Angiospermae. The TIRencoding genes in Pinus taeda L. (Meyers et al., 2002) were also incorporated into the dataset to represent the gymnosperms. These species were sampled because they represent the five major taxonomic phyla (Chlorophyta, Bryophyta, Pteridophyta, Gymnospermae and Angiospermae) in the plant kingdom. The complete genome sequences and corresponding annotation information were downloaded from online databases (Table S1). The complete set of TIR-encoding genes was identified in a reiterative manner. Three analytical steps were followed to compile the final set of sequences. First, for each species, protein entries matching the TIR domain (Pfam: PF01582) were identified as TIR-encoding genes using BLASTP with an E-value cutoff of 10−4. The presence of the TIR
Chlamydomonas reinhardtii Volvox carteri Physcomitrella patens Selaginella moellendorffii Hieron. P. taeda L. Aquilegia coerulea E. James Vitis vinifera L. Manihot esculenta L. Populus trichocarpa (Torr. & Gray) Ricinus communis L. Glycine max Merr. Medicago truncatula Gaertn. Lotus corniculatus L. var. japonicus Regel Prunus persica (L.) Batsch Cucumis sativis L. Eucalyptus grandis Hill ex Maiden Citrus clementina Hort. ex Tan. Citrus sinensis (L.) Osb. Carica papaya L. Arabidopsis thaliana L. Arabidopsis lyrata L. Thellungiella halophila (C. A. Mey.) O. E. Schulz Brassica rapa L. Mimulus guttatus DC. Solanum lycopersicum L. Solanum tuberosum L. Musa acuminata Colla Brachypodium distachyon (L.) Beauv Oryza sativa L. Sorghum bicolor (L.) Moench Setaria italica (L.) Beauv. Panicum virgatum L. Zea mays L. 33
R
Chlorophyta
E
t1:4 t1:5 t1:6 t1:7 t1:8 t1:9 t1:10 t1:11 t1:12 t1:13 t1:14 t1:15 t1:16 t1:17 t1:18 t1:19 t1:20 t1:21 t1:22 t1:23 t1:24 t1:25 t1:26 t1:27 t1:28 t1:29 t1:30 t1:31 t1:32 t1:33 t1:34 t1:35 t1:36 t1:37 t1:38
2. Materials and methods
F
88 89
O
86 87
C
85
R O
Table 1 Distribution of TIR-encoding genes in the sequenced plant genomes.
83 84
109 110
P
t1:1 t1:2
81 82
A. thaliana have suggested that TXs were derived from TNs or TNLs and co-evolved as functional units (Meyers et al., 2002). Data from poplar and grapevine also support this finding (Yang et al., 2008). Because few studies have investigated the origin and evolutionary history of the TIR-encoding genes and have instead mainly focused on TNLs, the evolution of the four major groups of TIR-encoding genes remains unclear. In this study, the origin, evolutionary history and relationships between the four groups of TIR-encoding genes in the plant kingdom were investigated using 32 whole-genome sequenced plants, including two members of Chlorophyta.
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other two groups of TIR-encoding genes) remain unclear; however, the diversification and conservation of these genes suggest that some of these proteins have important functions (Nandety et al., 2013). For example, Arabidopsis CHS1, which encodes a TN protein, confers cold resistance (Zbierzak et al., 2013). Overexpression analysis of Arabidopsis TXs and TNs in tobacco or Arabidopsis suggested that TXs and TNs might play roles in disease resistance (Nandety et al., 2013). Previous studies have shown that the TLs and TXs originated from eubacteria (Yue et al., 2012) and that the TNs and TNLs were first detected in the moss Physcomitrella patens (Akita and Valkonen, 2002; Xue et al., 2012; Yue et al., 2012). The frequency of the appearance of conserved motifs in the NBS domain of TNLs has changed during the evolutionary progression from mosses to tracheophytes and from gymnosperms to spermatophytes (Yue et al., 2012). Some studies have also proposed that TNLs evolved differently during the early differentiation of dicots and monocots. Indeed, TNLs are completely absent in monocots, while these genes differentiated and expanded to form an important family of resistance genes in dicots (Bai et al., 2002; Cannon et al., 2002; Pan et al., 2000). In support of this hypothesis, TNLs could not be identified using either degenerate PCR or database searching in nine cereal genera (Cannon et al., 2002; Meyers et al., 1999); furthermore, TNLs could not be detected in monocot species other than cereals, including Dracaena marginata Lam., Sansevieria trifasciata Hort ex D. Prain, Spathiphyllum sp., Carex blanda Dewey, Musa acuminata Colla and Elaeis guineensis Jacg (Tarr and Alexander, 2009). In contrast, dozens to hundreds of TNLs have been discovered in dicots (reviewed by Jacob et al., 2013), indicating that the TNLs have undergone massive expansion in these plants. Unfortunately, few studies have addressed the evolution of TNs, TLs or TXs. The few reports describing the phylogenetic relationships and chromosomal positions of TNs, TXs and TNLs in
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X. Sun et al. / Gene xxx (2014) xxx–xxx
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2
All
2 1 13 1 5 1 31 45 177 43 182 272 157 169 25 754 112 113 11 162 158 98 155 2 37 102 1 1 2 2 2 4 3 2843
TNL
0 0 7 0 2 0 15 26 84 24 53 26 25 128 12 281 57 33 6 102 90 52 91 0 18 44 0 0 0 0 0 0 0 1176
TN
0 0 3 1 2 0 6 3 28 7 92 164 47 15 8 204 29 33 1 19 23 7 23 0 5 15 0 0 0 0 0 0 0 735
TL
0 0 0 0 0 0 1 2 9 0 0 0 1 1 0 11 3 4 0 0 0 0 0 0 2 2 0 0 0 0 0 0 0 36
TX All
Ta
2 1 3 0 1 1 9 14 56 12 37 82 84 25 5 258 23 43 4 41 45 39 41 2 12 41 1 1 2 2 2 4 3 896
2 1 3 0 1 1 7 8 33 5 36 82 68 19 4 194 16 27 2 23 30 16 22 2 6 24 1 1 2 2 2 4 3 647
The column shows the distribution of T genes, a sub-category of TXs which contain only the TIR domain(s).
Please cite this article as: Sun, X., et al., Tracing the origin and evolution of plant TIR-encoding genes, Gene (2014), http://dx.doi.org/10.1016/ j.gene.2014.04.060
111 112 113 114 115 116 117 118
121 122 123 124 125 126 127 128 129 130 131 132 133 134 135
N
C
Table 2 Clusters and composition of TIR-encoding genes in the sequenced plant genomes. Species
Clusters
T
TN
TL
TNL
TTN
TTL
TTNL
TTX
t2:3 t2:4 t2:5 t2:6 t2:7 t2:8 t2:9 t2:10 t2:11 t2:12 t2:13 t2:14 t2:15 t2:16 t2:17 t2:18 t2:19 t2:20 t2:21
Vitis vinifera Manihot esculenta Populus trichocarpa Ricinus communis Glycine max Medicago truncatula Prunus persica Cucumis sativis Eucalyptus grandis Citrus clementina Citrus sinensis Carica papaya Arabidopsis thaliana Arabidopsis lyrata Thellungiella halophila Brassica rapa Solanum lycopersicum Total
O
TNTX
TLTNL
TLTX
1 0
0 0
0 0
TNTL
TNTNL
R
7 6
0 0
1 0
0 0
2 2
0 0
0 0
2 2
0 0
0 0
0 0
21
0
0
0
4
1
0
1
0
0
1
0
1
1
7
0
0
0
2
0
0
1
0
0
2
0
0
0
34 36
6 2
15 8
0 0
2 0
2 13
0 0
3 3
0 0
0 0
4 2
0 0
0 0
32 4 97
1 0 9
1 0 6
0 0 0
17 0 10
0 0 11
0 0 2
6 1 9
1 0 5
0 0 0
3 2 12
0 0 1
9
0
1
0
0
1
0
1
1
0
0
16 2 22
0 0 0
2 0 0
0 0 0
3 1 5
3 0 0
0 0 0
2 0 4
0 0 1
1 0 0
22
3
0
0
8
0
0
0
0
19
0
1
0
9
0
0
1
29 6
1 0
3 0
0 0
10 2
0 0
0 1
369
22
38
0
77
31
3
TNLTX
R
0 1
T-TNTL 0 0
E
T-TNTX
T-TLTNL
T-TLTX
T-TNLTX
TN-TLTNL
TNTL-TX
TNTNL-TX
TLTNLTX
T-TNTLTNL
T-TNTL-TX
1 0
0 0
0 0
0 1
0 0
0 0
0 0
0 0
0 0
0 0
0 0
T-TNTNL
T-TLTNLTX
TN-TLTNLTX
T-TN-TLTNL-TX
0 0
0 0
0 0
0 0
T-TNTNLTX
1
0
2
0
1
0
0
0
0
2
1
0
0
2
2
0
1
1
0
0
0
0
1
0
0
0
0
0
0
0
0
0
0
0 0
0 0
C
0
0 0
1 8
0 0
0 0
0 0
0 0
0 0
0 0
0 0
0 0
0 0
0 0
1 0
0 0
0 0
0 0
1 0 1
0 0 1
1 0 5
0 0 0
1 0 11
0 0 3
0 0 0
E
0 0 0
0 0 1
0 0 0
0 0 0
0 0 1
0 0 0
0 0 2
0 0 1
0 1 6
0 0 0
0 0 0
0 0 0
0
0
0
1
0
2
1
0
0
0
0
0
0
0
0
0
0
0
0
1
3 0 6
0 0 1
0 0 0
0 0 0
1 1 0
0 0 0
0 0 1
0 0 0
0 0 0
0 0 0
0 0 0
0 0 0
1 0 1
0 0 0
0 0 0
0 0 0
0 0 0
0 0 0
0 0 0
0 0 0
0
5
0
0
0
1
0
1
0
0
0
0
0
0
2
0
0
0
2
0
0
0
2
0
3
0
0
0
2
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
0
2 0
2 0
0 0
6 1
0 1
0 0
0 0
1 1
0 0
0 0
1 0
0 0
0 0
1 0
0 0
0 0
2 0
0 0
0 0
0 0
0 0
0 0
0 0
0 0
38
12
1
50
4
3
2
17
0
28
5
1
1
7
0
0
9
1
1
12
2
0
2
T
D
P 0 0 3
R O
O
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X. Sun et al. / Gene xxx (2014) xxx–xxx
Please cite this article as: Sun, X., et al., Tracing the origin and evolution of plant TIR-encoding genes, Gene (2014), http://dx.doi.org/10.1016/ j.gene.2014.04.060
t2:1 t2:2
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4 t3:1 t3:2
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Table 3 The twenty most significant motifs in the TIR regions of aligned sequences, as identified by the MEME algorithm. E value
1
NYASSSWCLDEL
3.5e−20825
t3:5
2
QIV[LI]P[VI]FY[KD]VDPS[DE]V
6.1e−21128
t3:6
3
FLSFRGED[TV]RK
4.2e−13537
t3:7
4
KAIEES[RK]I[SA][IV][VI][VI][FL]S[KE]
t3:8
5
[NG]FT[SD]HLYKALx
t3:9
6
DDEEL[ER][RK]G[ED]EISP[EA]L
t3:10
7
RKQ[TR]GS[FY]G[EK]A[FL]AKHE
t3:11
8
VKI[LM]EC[KR][KE]xKG
t3:12
9
[KG][KE][DN]SEKV[KQ][RK]W[KR]
t3:13
10
Q[KA]GIxTF[RIK]
t3:14
11
RKQTGS[FY]G[ED]AF
t3:15
12
t3:16
13
t3:17
14
t3:18
15
t3:19
16
t3:20
P
R O
O
F
Consensusa
2.2e−16438
7.0e−8784
3.2e−10318
5.3e−7746
D
6.6e−5767
E
t3:4
Pictogramb
T
Motifa
2.4e−2546
1.1e−2399
E
ALTE[AV][AG]N[LI]SGW
1.5e−1201
LRPFLD[SN]K[NS]M[KR]PGD[KR]LF[DE]KI[DN]
1.5e−675
[EA][NK]IN[VF]FID[EK]xEx[RK]G[RK]DLxNL[FL]
2.2e−635
KGIxTF
6.3e−342
YASS[PR]WCLDEL
1.7e−240
17
VK[IM]K[EK][FR]MD[EQK]G[KR]
4.8e−209
t3:21
18
TLL[NH]SH[KN][RK]LHG[VI]DDAIDKSKK[VI]L[TS]TMSRR[IM]TRNKWIV[AG]S[VI]IGALVFAI[VI]I
2.0e−170
t3:22
19
RKLITAF[RK]DNEIERS[HR]SL[WD]P[DE]
8.0e−140
U
N
C
O
R
1.1e−1711
R
C
t3:3
Please cite this article as: Sun, X., et al., Tracing the origin and evolution of plant TIR-encoding genes, Gene (2014), http://dx.doi.org/10.1016/ j.gene.2014.04.060
X. Sun et al. / Gene xxx (2014) xxx–xxx
5
Table 3 (continued) t3:23
Motifa
t3:24
20
t3:25 t3:26
a
Consensusa
E value
[TN][VI]A[GR]LL[YH]D[HR][LF]S
4.1e−104
Motif IDs and consensus sequences were generated by MEME analysis. The second column for each motif shows the amino acid frequency distribution graphed using WebLogo, where the sizes of the characters represent the frequencies of occurrence.
2.3. Identification and analysis of conserved motifs present within the TIR domain
161
169
The conserved motifs in the amino acid sequences of the TIR regions were identified and analyzed with MEME 4.6.1 (Bailey and Elkan, 1994). For MEME, minimum and maximum motif lengths of 10 and 50, respectively, were set, and 20 motifs were requested using the zero- or oneoccurrence-per-sequence model. Based on expectation maximization, conserved motifs were identified in a group of sequences without a priori assumptions about the alignments. The individual profile for each conserved motif was assessed, and after tiling, only the conserved motifs with P-values ≤10−4 and no overlap were reported.
170
2.4. Phylogenetic analysis of the T genes with the skeleton motif pattern
171
The evolutionary history of the T genes (genes containing only the TIR domain(s) were hereafter referred to as “T genes”) with the skeleton motif pattern was inferred using the neighbor-joining method (Saitou and Nei, 1987). In addition to the 58 identified T genes with the skeleton motif pattern, up to 101 matches were discovered from the Picea abies genome (http://congenie.org) during the conservation search using one dicot-typed T gene (Acoerulea v1.019315 in Aquilegia coerulea) and two monocot-typed T genes (Macuminata Achr9T24500_001 in M. acuminata and LOC_Os07g37950.1 in Oryza sativa). To compensate for the lack of fully sequenced genome data for gymnosperms, we added five T genes (the top five hits from the BLAST search) from P. abies to the profile. The maximum-likelihood phylogenetic tree shows the homology of the T genes across plants. The bootstrap consensus tree inferred from 1000 replicates is taken to represent the evolutionary
164 165 166 167 168
172 173 174 175 176 177 178 179 180 181 182 183 184
C
E
R
R
162 163
N C O
151 152 Q6
U
149 150
F
159 160
147 148
Whole-genome sequences from 32 different species (two species in Chlorophyta, one species in Bryophyta, one species in Pteridophyta and 28 species in Angiospermae) were selected to represent all evolutionary nodes in the plant kingdom. The sequence from P. taeda L. was also incorporated into the dataset to represent the gymnosperms. We discovered 2843 TIR-encoding genes in all 33 plant species (Table 1). We identified two and one TX genes in Chlamydomonas reinhardtii and Volvox carteri (Chlorophyta), respectively; 13 TNLs, TNs and TXs in P. patens (Bryophyta); one TN in Selaginella moellendorffii Hieron. (Pteridophyta); five TNLs, TNs and TXs in P. taeda (Gymnospermae) and 2821 TIR-encoding genes of all four types in Angiospermae. The TNLs and TNs accounted for 41.36% (1176/2, 843) and 25.85% (735/2843), respectively, of all TIR-encoding genes. TNLs and TNs were first observed in P. patens, and present in P. taeda and all dicots except A. coerulea E. James and Mimulus guttatus DC. These two groups are absent in monocots, while TN was found in S. moellendorffii. Fewer TLs, which accounted for 1.27% (36/2843) of all TIR-encoding genes, were found in ten species of dicots. The TXs comprised 31.52% (896/2843) of all the TIR-encoding genes; 72.21% (647/896) of the TXs were from a subgroup of genes containing only the TIR domain(s), hereafter referred to as “T genes”. The remaining 29.79% of the TXs had more than 100 domains or domain combinations for X. The TXs exhibited an ancient origin in early green algae and could be identified in all plant species, except S. moellendorffii. Moreover, the TXs found in the seven monocot species were all T genes.
189 190
O
157 158
TIR-encoding genes were aligned using the ClustalW software (Thompson et al., 1994); the genetic parameters were computed using the Geneious 6.0.3 software. Adjacent homologous genes separated by less than 200 kb were defined as clusters (Holub, 2001).
145 146
188
R O
155 156
143 144
187
3.1. Distribution of the TIR-encoding genes in 33 plant species
P
2.2. Sequence alignment and chromosomal position analysis
141 142
3. Results
D
154
139 140
history of the analyzed taxa. Evolutionary analyses were conducted in 185 MEGA5 (Tamura et al., 2011). 186
T
153
domain in the identified genes was confirmed by conducting a Pfam search with an E-value cutoff of 10− 4 (Finn et al., 2010). A total of 2744 proteins were identified in all plant species. In the second analytical step, the TIR domains of the selected protein sequences were aligned using ClustalW (Thompson et al., 1994). These alignments were used to build a plant-specific HMM profile using hmmbuild in HMMER version 3.0 (Mistry et al., 2013); this profile was then used to identify proteins encoded in each genome using hmmsearch (E b 10−4). The 110 newly discovered sequences that had a confirmed TIR domain, according to the results of the Pfam search and visual inspection, were incorporated into the HMM for the third analysis. Eight new sequences were discovered in six dicot species; however, none of these sequences contained a TIR domain and were therefore discarded. These three analytical steps identified 2843 annotated genes that encoded homologs of TIR proteins. A Pfam search and SMART were used to describe the domain architecture of each TIR-encoding gene (Finn et al., 2010; Letunic et al., 2009). The default threshold values for both databases were used (E-value b 10−4).
137 Q5 138
E
136
b
Pictogramb
191 192 193 194 195 196 197 198 199 200 201 202 203 204 205 206 207 208 209 210 211 212 213 214
3.2. Distribution and evolution of the TIR-encoding genes on the 215 chromosomes 216 We found that 1846 (64.93%) TIR-encoding genes formed 369 chromosomal clusters. However, this percentage could be slightly underestimated because of the presence of unanchored scaffold sequences. On average, five TIR-encoding genes were detected in a cluster. Few TIR-encoding genes were identified in C. reinhardtii, V. carteri, S. moellendorffii or in the seven monocots; 13 TIR-encoding genes present in P. patens could not be clustered because of incomplete annotation. The numbers of clusters varied significantly in all dicots, except A. coerulea and M. guttatus, which lacked clusters of TIR-encoding genes (Table 2). As many as 97 clusters were detected in Eucalyptus grandis Hill ex Maiden. Two types of gene clusters were identified in plants: homogeneous and heterogeneous clusters. Homogeneous clusters contained only one group of TIR-encoding genes; for example, the Bra001160– Bra001175 cluster in Brassica rapa L. is composed of four TNLs, and the POPTR0019s12840.1–POPTR0019s12910.1 cluster in Populus trichocarpa contains three T genes. Heterogeneous clusters consisted of more than one group of TIR-encoding genes; for example, the Medtr4g018120.1–Medtr4g018780.1 cluster in Medicago truncatula
Please cite this article as: Sun, X., et al., Tracing the origin and evolution of plant TIR-encoding genes, Gene (2014), http://dx.doi.org/10.1016/ j.gene.2014.04.060
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3.4. Phylogeny and conservation of the TIR-encoding genes with the skeleton 297 motif pattern 298 Interestingly, the TIR-encoding genes with the skeleton motif pat- 299 tern in the TIR domain were all T genes. This special subgroup consisted 300 of 58T genes that were distributed in all 29 spermatophyte species, 301
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To further explore the evolutionary relationship between the different groups of TIR-encoding genes in different species, the 20 most conserved motifs in the amino acid sequences of the TIR regions from the TIR domain datasets were identified using MEME (Table 3). Consistent with previous studies of the TIR domain, a number of motifs were found in our analysis; in particular, motifs 1, 2 and 3 corresponded to the previously described motifs 2, 3, and 1, respectively, of A. thaliana (Meyers et al., 2002). A total of 492 composite motif patterns for the TIR domains existed in the analyzed plants (Fig. 1). C. reinhardtii and V. carteri had only one pattern (independent motif 1). The TIR domains of P. patens had a total of 11 composite patterns composed of 4–7 motifs. The TIR domain of S. moellendorffii had only one composite pattern composed of six motifs. The TIR domains of P. taeda had a total of more than five composite
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patterns. The TIR domains of monocots had only one composite pattern of six motifs, while the TIR domains of angiosperm dicots had 475 composite patterns (Fig. 1). It's notable that the motif pattern 3-20-13-4-1-2 was present in all species of gymnosperms and angiosperms, suggesting a high conservation among seed plants. Unique composite patterns were detected in C. reinhardtii, V. carteri, P. patens and S. moellendorffii; these patterns were very different from the composite patterns found in gymnosperms and angiosperms. Five TIR-encoding genes in P. taeda had five composite patterns in the TIR domains, four of which were unique to P. taeda; the fifth composite pattern in this species (3-20-13-4-1-2) was also found in angiosperms. The TIRencoding genes in dicots had 474 unique patterns. Because the motif pattern 3-20-13-4-1-2 was present in all 27 species of gymnosperms and angiosperms, the gymnosperms might constitute an evolutionary node of the TIR-encoding genes. This composite pattern first observed in the gymnosperms and appears to represent a skeleton pattern of motifs in the TIR domain of TIR-encoding genes from all spermatophytes. By alignment and comparison of the skeleton pattern and multiple patterns of motifs in the TIR domain in spermatophytes, it can be inferred that two ways might be involved in evolution of the TIR domains from gymnosperms to angiosperms (Fig. 2). The skeleton pattern could evolve to a variety of patterns through motif insertion or superseding, which is prevalent in dicots; taken for example, the replacement of motif 20 and motif 13 with motif 5 and motif 6, the insertion of motif 10 between motif 5 and motif 6 and the insertion of motif 8 between motif 1 and motif 2 could have resulted in a shift from the skeleton pattern to motif 3-5-10-6-4-1-8-2. The other way by maintaining the skeleton pattern as the only composite pattern usually occurred in monocots.
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contains 13 TNs and three T genes, and the Glyma16g33590.1– Glyma16g34110.1 cluster in Glycine max Merr. contains 15 TNs, two TNLs and a single T gene. The number of heterogeneous clusters was greater than or equal to the number of homogeneous clusters in most species (Table 2); however, G. max, Prunus persica (L.) Batsch and Thellungiella halophila (C. A. Mey.) O. E. Schulz contained more homogeneous clusters than heterogeneous clusters. The gene clusters composed of TNLs, hereafter referred to as a TNL cluster, accounted for 21% of all gene clusters; those composed of TNLTN, TNL-T or TN genes accounted for 14%, 10% and 10% of all gene clusters, respectively (Table 2). The T, T-TN, TNL-TX and T-TN-TNL clusters accounted for 6%, 8%, 5% and 8%, respectively, of all clusters, while the remaining 22 types of clusters accounted for less than 3%. Because the number of detectable TL genes in plants is relatively low, the percentage of gene clusters with TLs was almost zero. However, 32 of all 34 TL genes (94.12%) reside in homogeneous or heterogeneous clusters. In most dicotyledon species, the TNL, TNL-TN, TNL-T and TN clusters account for more than 50% of all TIR-gene clusters in each species.
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Fig. 1. The composite patterns of motifs in the TIR domains in the plant kingdom. The numbers in the boxes indicate the motifs shown in Table 3. A full list of composite patterns in the TIR domains of dicotyledoneae is provided in Table S2.
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C. reinhardtii, which represents the earliest evolutionary node of plants, has only one TIR-encoding gene. The number of TIR-encoding genes increases to 13 in three groups of P. patens. The genome of P. patens underwent whole genome duplication, leading to a general increase in the gene family complexity and the acquisition of genes involved in tolerating environmental stresses (Rensing et al., 2008). The increase in the gene number and complexity of the TIR-encoding gene family suggests that the bryophytes were a key group on the evolutionary path to land-dwelling species; to tolerate stresses, e.g., certain pathogens encountered in the terrestrial environment, the bryophytes had to rapidly evolve under strong natural selection pressure (Ponce de León and Montesano, 2013). In further support of this conclusion, diverse novel classes of NBS-encoding genes, including α/β-hydrolaseNBS-LRR (HNL) and protein-kinase-NBS-LRR (PNL), were discovered in the moss P. patens and the liverwort Marchantia (Xue et al., 2012). Moreover, the fusion of NBS and LRR domains, which represents a critical step in the origin of the plant R genes, first occurred in bryophytes (Yue et al., 2011). Consequently, the striking expansion of the R gene family and their frequent recombination throughout all land plant lineages provided a powerful arsenal for land plants, allowing them to quickly and effectively evolve novel resistance specificities to fight diverse pathogens in the terrestrial environment (Leister, 2004). The evolution of the reproduction mode might also play a vital role in the expansion and diversification of the plant R genes (Dimijian, 2005). Sexual reproduction is far more prevalent than asexual reproduction in higher plants, which may lead to frequent genetic recombination and thus result in novel R genes against pathogens, parasites, predators or harsh environmental conditions (Morran et al., 2011).
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Previous studies (Cannon et al., 2002; Mchale et al., 2006; Yang et al., 2008) showed that TNLs and TNs that exhibit species-specific expansion are highly differentiated. Our results confirmed that TNLs, TNs and TLs are not homologous among species; however, the genes that belong to a special subgroup of T genes in all species of spermatophytes share the same composite motif pattern in the TIR domain, indicating that the TIR-encoding genes of spermatophytes may have originated from
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Of all the whole-genome sequenced plants, S. moellendorffii has the smallest genome (approximately 100 Mb) and only one TN gene, indicating that the TIR-encoding genes in Pteridophyta are less diverse and numerous than those in Bryophyta. Yue et al. (2012) proposed that a large-scale gene loss in S. moellendorffii decreased the number of TIR-encoding genes in this species. S. moellendorffii is the only species in Pteridophyta that has had its whole genome sequenced; in contrast, Gleichenites (also in Pteridophyta) is closely evolutionarily related to gymnosperms (Pryer et al., 2001). Therefore, S. moellendorffii may not represent most species in Pteridophyta; more information from other species of pteridophytes is needed. The gymnosperm P. taeda has five TIR-encoding genes; however, this number may be an underestimation as the statistics used in the calculation were based on the EST database and not on genome sequence. Given the distributions of the TIRencoding genes in Pteridophyta, Bryophyta and Gymnospermae, some species in Pteridophyta may maintain a considerable number of TIRencoding genes, while species in Bryophyta may maintain these genes in multiple groups. Furthermore, because gymnosperms have fewer species, stable growth environments and a stable pathogen spectrum, these plants may maintain fewer and less diverse TIR-encoding genes than angiosperms (Liu and Ekramoddoullah, 2003, 2007). According to the APG III system (Angiosperm Phylogeny Group, 2009), angiosperms can be classified as basal angiosperms, magnoliids, monocots or eudicots. The evolutionary history of the TIR-encoding genes in the basal angiosperms and magnoliid lineages cannot be assessed due to the lack of genome sequence data. The monocots, including the seven species with fewer T genes that were used in this study, constitute a relatively independent monophyletic group that evolved after the basal angiosperms and magnoliids but before the eudicots. The TIR-encoding genes in the eudicots are characterized by remarkably high levels of gene duplication and complex diverse architectures; additionally, 64.93% of the clustered TIR-encoding genes are present in all eudicots. This indicates that the eudicots have significantly expanded the spectrum of potential disease resistance through massive duplication and subsequent differentiation of the TIR-encoding genes; in contrast, monocots resist pathogens through other families of resistance genes, e.g., CC-NBS-LRR (CNL) (Bai et al., 2002; Pan et al., 2000; Tian et al., 2004). Dissimilar mechanisms of disease resistance that exist in both angiosperm lineages also provide evidence of the remarkably uneven distribution of TIR-encoding genes in monocots and dicots.
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including the seven monocots (Table S3; Fig. 3). These T genes had an identity of 48.5% (55.7% for the Dicotyledoneae clade and 58.3% for the Monocotyledoneae clade), suggesting that these special T genes were conserved across spermatophytes. The conservation of the T genes with the skeleton motif pattern was further examined using plant genomic sequences from GenBank. One dicot-type T gene (Acoerulea v1.019315 in A. coerulea) and two monocot-type T genes (Macuminata Achr9T24500_001 in M. acuminata and LOC_Os07g37950.1 in O. sativa) were used to identify homologs. All three T genes yielded homologs in a BLAST search against sequences from gymnosperms, basal angiosperms, monocots and magnoliids (Table S4). The identity of the matches ranged from 27% to 50%, indicating that these special T genes form a highly conserved group across all plants that contain these homologs. Although these special T genes are not found in lower plants, important motifs in the skeleton pattern of these T genes, in particular motif 13, have been detected in P. patens.
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Fig. 2. Examples of summarized and aligned motifs for TIR domain. The skeleton motif pattern 3-20-13-4-1-2 was represented by four TIR-encoding genes in P. taeda, A. thaliana and O. sativa, and the motif pattern 3-5-10-6-4-1-8-2 is represented by ten TIR-encoding genes in M. truncatula and G. max. The numbers above or below boxes indicate the motifs shown in Table 3.
Please cite this article as: Sun, X., et al., Tracing the origin and evolution of plant TIR-encoding genes, Gene (2014), http://dx.doi.org/10.1016/ j.gene.2014.04.060
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can be traced back to gymnosperms may have existed in ancestral angiosperms. Furthermore, the skeleton motif pattern present in the TIR domain of TIR-encoding genes first appeared in gymnosperms. This skeleton pattern is shared by every species of spermatophyte and subsequently evolved into a variety of composite motif patterns. These results provide strong evidence that the gymnosperms are a key evolutionary node for the TIR-encoding genes. T genes are indispensable for each species, and the evolutionary pathways for composite motif patterns in the TIR domain from gymnosperms to angiosperms are based on T genes. Thus, the T genes must be ancestors of the other TIR resistance genes; this finding could explain why T genes are present in all spermatophyte species, including in the earliest plant lineages. Furthermore, the diversification and conservation of the TX genes suggests that these genes may have important functions, including cold resistance (Arabidopsis CHS1, Zbierzak et al., 2013) and disease resistance (Arabidopsis TNs and TXs, Nandety et al., 2013). The distributed clusters of TIR-encoding genes were observed in species of Arabidopsis, rice, grapevine and poplar (Meyers et al., 2002; Yang et al., 2008; Zhou et al., 2004); in this study, the TIR-encoding genes were often distributed in clusters. In addition to being present in Arabidopsis, grapevine and poplar (Meyers et al., 2002; Yang et al., 2008), clusters of TNLs, TNs and T genes were widely observed in the majority of dicots. TNs and TXs are thought to be required by the TNLs for downstream signaling; as a result, TNLs, TNs and TXs may have coevolved during natural selection to favor the development of functional allele units (Meyers et al., 2002). Because TLs lack NBS domains, they have rarely been studied by other investigators. In this study, 94.12% of the TLs were located in clusters; this finding implies that TLs may maintain certain functions, thus creating a selective advantage for species with co-localized TIR-encoding genes.
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Although the origins of TNLs seems to date back to very early land plant lineages, TNLs are known to be absent from monocots (Yue et al., 2012). Two hypotheses have been proposed to explain the lack of distribution for the groups of TIR-encoding genes other than TNLs: either the few TIR-encoding genes in ancestral angiosperms were completely lost in cereals, or the TIR-encoding genes differentiated and expanded and were eventually lost in cereals. However, certain investigators have argued that none of the existing mechanisms could successfully explain the complete elimination of such a large and complicated family of genes (Bai et al., 2002; Cannon et al., 2002; Pan et al., 2000). To our knowledge, no appropriate hypotheses have been proposed for the loss of TNLs in cereals or monocots or for the loss of other TIR-encoding genes, e.g., the TNs and TLs. Our study revealed that T genes (in limited numbers) represent the only group of TIRencoding genes in monocots; in contrast, dozens to hundreds of TIRencoding genes can be found in most dicots. The mechanism behind this uneven distribution of TIR-encoding genes in dicots and monocots is unclear. T genes are present at each evolutionary node of plant phylogeny, including Chlorophyta, Bryophyta and Gymnospermae. As a result, it has been proposed that the presence of T genes in monocots is a conserved continuous evolutionary event. Because T genes and other groups of TIR-encoding genes are continuously present in gymnosperms, we inferred that the TIR-encoding genes differentiated during the evolution from gymnosperms to basal angiosperms. Certain species of basal angiosperms that only had T genes subsequently evolved into monocots; other species of basal angiosperms that had diverse TIR-encoding genes differentiated and expanded to form a large family of resistance genes in eudicots. Monocots may only have T genes because of pathogenic stresses or species-specific mechanisms of disease resistance. The presence of only one or two T gene(s) in the eudicot species A. coerulea and M. guttatus and the absence of TIR-type resistance genes in Beta vulgaris L. (Tian et al., 2004) suggest that additional factors
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Fig. 3. Phylogeny and conservation of the T genes with the skeleton motif pattern. Numbers at branches are bootstrap percentages (the values less than 50% are not shown).
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a common ancestor. Moreover, these conserved T genes might represent ancestral genes that are maintained in each plant during evolution. It has been proposed that a few TIR-encoding genes were present early in the evolution of angiosperms and that the abundant diversity of TIR-encoding genes was subsequently shaped by massive speciesspecific expansion in modern plant lineages, particularly in the dicots (Bai et al., 2002; Pan et al., 2000; Tarr and Alexander, 2009; Tian et al., 2004). Our study showed that a selection of TIR-encoding genes that
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This work was supported by the National Natural Science Foundation of China (30930008; 31200177).
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may repress gene expansion in some dicot species, resulting in a conserved evolution similar to that observed in monocots. 470 Besides the TXs observed in monocots, Nandety et al. (2013) identi471 fied 16 TNs (in Fig. 1, Nandety et al., 2013) in four monocot species in 472 contrast to no detection of TNs in this study. Based on a Pfam search 473 with an E-value cutoff of 10–4 and visual inspection, we found that the 474 16 TNs identified in monocot species lacked a typical TIR domain. 475 None of the TNs (except Os11g36760) got significant matches to the 476 TIR domain; these genes were therefore not considered as typical TIR477 encoding genes and were discarded in our analysis. The assumed TN 478 gene Os11g36760 in rice exhibited significant matches to TIR_2 and 479 AAA_16, and then was designated a TX gene instead here. 480 Supplementary data to this article can be found online at http://dx. 481 Q11 doi.org/10.1016/j.gene.2014.04.060.
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Contents lists available at ScienceDirect
Gene
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Tracing the origin and evolution of plant TIR-encoding genes
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Xiaoqin Sun a, Hui Pang a, Mimi Li a, Jianqun Chen b,⁎, Yueyu Hang a,⁎⁎
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Article history: Received 10 January 2014 Received in revised form 17 April 2014 Accepted 24 April 2014 Available online xxxx
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Institute of Botany, Jiangsu Province and Chinese Academy of Sciences, Jiangsu Province Key Laboratory for Plant Ex Situ Conservation, Nanjing 210014, China State Key Laboratory of Pharmaceutical Biotechnology, School of Life Sciences, Nanjing University, Nanjing 210093, China
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Toll-interleukin-1 receptor (TIR)-encoding proteins represent one of the most important families of disease resistance genes in plants. Studies that have explored the functional details of these genes tended to focus on only a few limited groups; the origin and evolutionary history of these genes were therefore unclear. In this study, focusing on the four principal groups of TIR-encoding genes, we conducted an extensive genome-wide survey of 32 fully sequenced plant genomes and Expressed Sequence Tags (ESTs) from the gymnosperm Pinus taeda and explored the origins and evolution of these genes. Through the identification of the TIR-encoding genes, the analysis of chromosome positions, the identification and analysis of conserved motifs, and sequence alignment and phylogenetic reconstruction, our results showed that the genes of the TIR-X family (TXs) had an earlier origin and a wider distribution than the genes from the other three groups. TIR-encoding genes experienced largescale gene duplications during evolution. A skeleton motif pattern of the TIR domain was present in all spermatophytes, and the genes with this skeleton pattern exhibited a conserved and independent evolutionary history in all spermatophytes, including monocots, that followed their gymnosperm origin. This study used comparative genomics to explore the origin and evolutionary history of the four main groups of TIR-encoding genes. Additionally, we unraveled the mechanism behind the uneven distribution of TIR-encoding genes in dicots and monocots. © 2014 Published by Elsevier B.V.
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Keywords: TIR-encoding genes T genes Disease resistance genes Gene evolution Monocots Gene clusters
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1. Introduction
38 39
Disease resistance genes (R genes) in plants are important functional genes that prevent and respond to the invasion of pathogens. Most R genes encode members of an extremely polymorphic superfamily of nucleotide-binding leucine-rich repeat (NLR) receptors (Maekawa et al., 2011). Specific NLR proteins can be activated by specific pathogen effectors via direct interaction as receptor and ligand respectively (Dodds et al., 2006). Alternatively, a specific NLR can be activated by sensing effector-mediated alteration in a host virulence target or a decoy protein of that target (Dangl and Jones, 2001; van der Hoorn and Kamoun, 2008). In further support of the latter, experimentally established interaction networks of the Arabidopsis thaliana proteins and the bacterial and oomycete effectors have revealed that independently evolved effectors converge onto the hubs of the immune system network (Mukhtar et al., 2011). NLR activation coordinates effectortriggered immunity that limits pathogen proliferation.
48 49 50 51 52
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18 19 20 21 22 23 24 25 26 27 28 29 30 31
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journal homepage: www.elsevier.com/locate/gene
Abbreviations: CC, coiled-coil; HNL, α/β-hydrolase-NBS-LRR; LRR, leucine-rich repeat; NBS, nucleotide binding site; NLR, nucleotide-binding leucine-rich repeat; Pkinase, protein kinase; PNL, protein-kinase-NBS-LRR; TIR, Toll-interleukin-1 receptor. ⁎ Correspondence to: J. Chen, State Key Laboratory of Pharmaceutical Biotechnology, School of Life Sciences, Nanjing University, 22 Hankou Road, Nanjing 210093, China. ⁎⁎ Correspondence to: Y. Hang, Jiangsu Province Key Laboratory for Plant Ex Situ Conservation, Institute of Botany, Jiangsu Province and Chinese Academy of Sciences, 1 Qianhuhoucun Road, Nanjing 210014, China. E-mail addresses: [email protected] (J. Chen), [email protected] (Y. Hang).
The R genes encode a number of conserved domains, including the nucleotide binding site (NBS), leucine-rich repeat (LRR), protein kinase (Pkinase), toll/interleukin-1 receptor (TIR) and coiled-coil (CC) domains (Jacob et al., 2013). TIR-encoding genes represent one of the most important families of disease resistance genes in plants and act as signal mediators that respond to pathogens (Katagiri and Tsuda, 2010; Lewis et al., 2010; Römer et al., 2009; Zhang et al., 2012). These genes can be further categorized into several groups according to the variable C-terminal domains: TIR-NBS-LRR (TNL), TIR-NBS (TN), TIRLRR (TL) and TIR-X (TX, where X is any domain other than NBS or LRR) (Nandety et al., 2013). The function of TIR-encoding genes is believed to be involved in immunity, yet some studies on Arabidopsis TIR-encoding genes suggest their possible roles beyond. Arabidopsis CHS1 which encodes a TN protein confers cold resistance by limiting chloroplast damage and cell death at low temperature (Zbierzak et al., 2013). Overexpression analysis and phylogenetic highly conservation suggest that the TNX proteins may have other nondefense roles in monocots and other plants (Nandety et al., 2013). Studies of the TIR-encoding genes (mostly involving TNLs) have primarily focused on gene cloning, the functional verification of the genes and the molecular mechanisms of disease resistance (Anderson et al., 1985; Franchel et al., 2012; Gassmann et al., 1999; Lawrence et al., 1995; Meyers et al., 1999; Parker et al., 1997; Whitham et al., 1994). In contrast, the analysis of genetic polymorphisms and gene evolution has been limited to only a few genes or individual plant species (reviewed in Jacob et al., 2013). The functions of TXs and TNs (the
http://dx.doi.org/10.1016/j.gene.2014.04.060 0378-1119/© 2014 Published by Elsevier B.V.
Please cite this article as: Sun, X., et al., Tracing the origin and evolution of plant TIR-encoding genes, Gene (2014), http://dx.doi.org/10.1016/ j.gene.2014.04.060
53 54 55 56 57 58 Q2 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 Q3 74 Q4 75 76 77 78
90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107
t1:3
Phylum
Class
Species
a
R
t1:39
Dicotyledoneae
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Bryophyta Pteridophyta Gymnospermae Angiospermae
Monocotyledoneae
Total
119
2.1. Data sampling
120
A total of 32 different whole-genome sequenced species were selected, including two species in Chlorophyta, one species in Bryophyta, one species in Pteridophyta and 28 species in Angiospermae. The TIRencoding genes in Pinus taeda L. (Meyers et al., 2002) were also incorporated into the dataset to represent the gymnosperms. These species were sampled because they represent the five major taxonomic phyla (Chlorophyta, Bryophyta, Pteridophyta, Gymnospermae and Angiospermae) in the plant kingdom. The complete genome sequences and corresponding annotation information were downloaded from online databases (Table S1). The complete set of TIR-encoding genes was identified in a reiterative manner. Three analytical steps were followed to compile the final set of sequences. First, for each species, protein entries matching the TIR domain (Pfam: PF01582) were identified as TIR-encoding genes using BLASTP with an E-value cutoff of 10−4. The presence of the TIR
Chlamydomonas reinhardtii Volvox carteri Physcomitrella patens Selaginella moellendorffii Hieron. P. taeda L. Aquilegia coerulea E. James Vitis vinifera L. Manihot esculenta L. Populus trichocarpa (Torr. & Gray) Ricinus communis L. Glycine max Merr. Medicago truncatula Gaertn. Lotus corniculatus L. var. japonicus Regel Prunus persica (L.) Batsch Cucumis sativis L. Eucalyptus grandis Hill ex Maiden Citrus clementina Hort. ex Tan. Citrus sinensis (L.) Osb. Carica papaya L. Arabidopsis thaliana L. Arabidopsis lyrata L. Thellungiella halophila (C. A. Mey.) O. E. Schulz Brassica rapa L. Mimulus guttatus DC. Solanum lycopersicum L. Solanum tuberosum L. Musa acuminata Colla Brachypodium distachyon (L.) Beauv Oryza sativa L. Sorghum bicolor (L.) Moench Setaria italica (L.) Beauv. Panicum virgatum L. Zea mays L. 33
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Chlorophyta
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t1:4 t1:5 t1:6 t1:7 t1:8 t1:9 t1:10 t1:11 t1:12 t1:13 t1:14 t1:15 t1:16 t1:17 t1:18 t1:19 t1:20 t1:21 t1:22 t1:23 t1:24 t1:25 t1:26 t1:27 t1:28 t1:29 t1:30 t1:31 t1:32 t1:33 t1:34 t1:35 t1:36 t1:37 t1:38
2. Materials and methods
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88 89
O
86 87
C
85
R O
Table 1 Distribution of TIR-encoding genes in the sequenced plant genomes.
83 84
109 110
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t1:1 t1:2
81 82
A. thaliana have suggested that TXs were derived from TNs or TNLs and co-evolved as functional units (Meyers et al., 2002). Data from poplar and grapevine also support this finding (Yang et al., 2008). Because few studies have investigated the origin and evolutionary history of the TIR-encoding genes and have instead mainly focused on TNLs, the evolution of the four major groups of TIR-encoding genes remains unclear. In this study, the origin, evolutionary history and relationships between the four groups of TIR-encoding genes in the plant kingdom were investigated using 32 whole-genome sequenced plants, including two members of Chlorophyta.
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other two groups of TIR-encoding genes) remain unclear; however, the diversification and conservation of these genes suggest that some of these proteins have important functions (Nandety et al., 2013). For example, Arabidopsis CHS1, which encodes a TN protein, confers cold resistance (Zbierzak et al., 2013). Overexpression analysis of Arabidopsis TXs and TNs in tobacco or Arabidopsis suggested that TXs and TNs might play roles in disease resistance (Nandety et al., 2013). Previous studies have shown that the TLs and TXs originated from eubacteria (Yue et al., 2012) and that the TNs and TNLs were first detected in the moss Physcomitrella patens (Akita and Valkonen, 2002; Xue et al., 2012; Yue et al., 2012). The frequency of the appearance of conserved motifs in the NBS domain of TNLs has changed during the evolutionary progression from mosses to tracheophytes and from gymnosperms to spermatophytes (Yue et al., 2012). Some studies have also proposed that TNLs evolved differently during the early differentiation of dicots and monocots. Indeed, TNLs are completely absent in monocots, while these genes differentiated and expanded to form an important family of resistance genes in dicots (Bai et al., 2002; Cannon et al., 2002; Pan et al., 2000). In support of this hypothesis, TNLs could not be identified using either degenerate PCR or database searching in nine cereal genera (Cannon et al., 2002; Meyers et al., 1999); furthermore, TNLs could not be detected in monocot species other than cereals, including Dracaena marginata Lam., Sansevieria trifasciata Hort ex D. Prain, Spathiphyllum sp., Carex blanda Dewey, Musa acuminata Colla and Elaeis guineensis Jacg (Tarr and Alexander, 2009). In contrast, dozens to hundreds of TNLs have been discovered in dicots (reviewed by Jacob et al., 2013), indicating that the TNLs have undergone massive expansion in these plants. Unfortunately, few studies have addressed the evolution of TNs, TLs or TXs. The few reports describing the phylogenetic relationships and chromosomal positions of TNs, TXs and TNLs in
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2
All
2 1 13 1 5 1 31 45 177 43 182 272 157 169 25 754 112 113 11 162 158 98 155 2 37 102 1 1 2 2 2 4 3 2843
TNL
0 0 7 0 2 0 15 26 84 24 53 26 25 128 12 281 57 33 6 102 90 52 91 0 18 44 0 0 0 0 0 0 0 1176
TN
0 0 3 1 2 0 6 3 28 7 92 164 47 15 8 204 29 33 1 19 23 7 23 0 5 15 0 0 0 0 0 0 0 735
TL
0 0 0 0 0 0 1 2 9 0 0 0 1 1 0 11 3 4 0 0 0 0 0 0 2 2 0 0 0 0 0 0 0 36
TX All
Ta
2 1 3 0 1 1 9 14 56 12 37 82 84 25 5 258 23 43 4 41 45 39 41 2 12 41 1 1 2 2 2 4 3 896
2 1 3 0 1 1 7 8 33 5 36 82 68 19 4 194 16 27 2 23 30 16 22 2 6 24 1 1 2 2 2 4 3 647
The column shows the distribution of T genes, a sub-category of TXs which contain only the TIR domain(s).
Please cite this article as: Sun, X., et al., Tracing the origin and evolution of plant TIR-encoding genes, Gene (2014), http://dx.doi.org/10.1016/ j.gene.2014.04.060
111 112 113 114 115 116 117 118
121 122 123 124 125 126 127 128 129 130 131 132 133 134 135
N
C
Table 2 Clusters and composition of TIR-encoding genes in the sequenced plant genomes. Species
Clusters
T
TN
TL
TNL
TTN
TTL
TTNL
TTX
t2:3 t2:4 t2:5 t2:6 t2:7 t2:8 t2:9 t2:10 t2:11 t2:12 t2:13 t2:14 t2:15 t2:16 t2:17 t2:18 t2:19 t2:20 t2:21
Vitis vinifera Manihot esculenta Populus trichocarpa Ricinus communis Glycine max Medicago truncatula Prunus persica Cucumis sativis Eucalyptus grandis Citrus clementina Citrus sinensis Carica papaya Arabidopsis thaliana Arabidopsis lyrata Thellungiella halophila Brassica rapa Solanum lycopersicum Total
O
TNTX
TLTNL
TLTX
1 0
0 0
0 0
TNTL
TNTNL
R
7 6
0 0
1 0
0 0
2 2
0 0
0 0
2 2
0 0
0 0
0 0
21
0
0
0
4
1
0
1
0
0
1
0
1
1
7
0
0
0
2
0
0
1
0
0
2
0
0
0
34 36
6 2
15 8
0 0
2 0
2 13
0 0
3 3
0 0
0 0
4 2
0 0
0 0
32 4 97
1 0 9
1 0 6
0 0 0
17 0 10
0 0 11
0 0 2
6 1 9
1 0 5
0 0 0
3 2 12
0 0 1
9
0
1
0
0
1
0
1
1
0
0
16 2 22
0 0 0
2 0 0
0 0 0
3 1 5
3 0 0
0 0 0
2 0 4
0 0 1
1 0 0
22
3
0
0
8
0
0
0
0
19
0
1
0
9
0
0
1
29 6
1 0
3 0
0 0
10 2
0 0
0 1
369
22
38
0
77
31
3
TNLTX
R
0 1
T-TNTL 0 0
E
T-TNTX
T-TLTNL
T-TLTX
T-TNLTX
TN-TLTNL
TNTL-TX
TNTNL-TX
TLTNLTX
T-TNTLTNL
T-TNTL-TX
1 0
0 0
0 0
0 1
0 0
0 0
0 0
0 0
0 0
0 0
0 0
T-TNTNL
T-TLTNLTX
TN-TLTNLTX
T-TN-TLTNL-TX
0 0
0 0
0 0
0 0
T-TNTNLTX
1
0
2
0
1
0
0
0
0
2
1
0
0
2
2
0
1
1
0
0
0
0
1
0
0
0
0
0
0
0
0
0
0
0 0
0 0
C
0
0 0
1 8
0 0
0 0
0 0
0 0
0 0
0 0
0 0
0 0
0 0
0 0
1 0
0 0
0 0
0 0
1 0 1
0 0 1
1 0 5
0 0 0
1 0 11
0 0 3
0 0 0
E
0 0 0
0 0 1
0 0 0
0 0 0
0 0 1
0 0 0
0 0 2
0 0 1
0 1 6
0 0 0
0 0 0
0 0 0
0
0
0
1
0
2
1
0
0
0
0
0
0
0
0
0
0
0
0
1
3 0 6
0 0 1
0 0 0
0 0 0
1 1 0
0 0 0
0 0 1
0 0 0
0 0 0
0 0 0
0 0 0
0 0 0
1 0 1
0 0 0
0 0 0
0 0 0
0 0 0
0 0 0
0 0 0
0 0 0
0
5
0
0
0
1
0
1
0
0
0
0
0
0
2
0
0
0
2
0
0
0
2
0
3
0
0
0
2
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
0
2 0
2 0
0 0
6 1
0 1
0 0
0 0
1 1
0 0
0 0
1 0
0 0
0 0
1 0
0 0
0 0
2 0
0 0
0 0
0 0
0 0
0 0
0 0
0 0
38
12
1
50
4
3
2
17
0
28
5
1
1
7
0
0
9
1
1
12
2
0
2
T
D
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Please cite this article as: Sun, X., et al., Tracing the origin and evolution of plant TIR-encoding genes, Gene (2014), http://dx.doi.org/10.1016/ j.gene.2014.04.060
t2:1 t2:2
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Table 3 The twenty most significant motifs in the TIR regions of aligned sequences, as identified by the MEME algorithm. E value
1
NYASSSWCLDEL
3.5e−20825
t3:5
2
QIV[LI]P[VI]FY[KD]VDPS[DE]V
6.1e−21128
t3:6
3
FLSFRGED[TV]RK
4.2e−13537
t3:7
4
KAIEES[RK]I[SA][IV][VI][VI][FL]S[KE]
t3:8
5
[NG]FT[SD]HLYKALx
t3:9
6
DDEEL[ER][RK]G[ED]EISP[EA]L
t3:10
7
RKQ[TR]GS[FY]G[EK]A[FL]AKHE
t3:11
8
VKI[LM]EC[KR][KE]xKG
t3:12
9
[KG][KE][DN]SEKV[KQ][RK]W[KR]
t3:13
10
Q[KA]GIxTF[RIK]
t3:14
11
RKQTGS[FY]G[ED]AF
t3:15
12
t3:16
13
t3:17
14
t3:18
15
t3:19
16
t3:20
P
R O
O
F
Consensusa
2.2e−16438
7.0e−8784
3.2e−10318
5.3e−7746
D
6.6e−5767
E
t3:4
Pictogramb
T
Motifa
2.4e−2546
1.1e−2399
E
ALTE[AV][AG]N[LI]SGW
1.5e−1201
LRPFLD[SN]K[NS]M[KR]PGD[KR]LF[DE]KI[DN]
1.5e−675
[EA][NK]IN[VF]FID[EK]xEx[RK]G[RK]DLxNL[FL]
2.2e−635
KGIxTF
6.3e−342
YASS[PR]WCLDEL
1.7e−240
17
VK[IM]K[EK][FR]MD[EQK]G[KR]
4.8e−209
t3:21
18
TLL[NH]SH[KN][RK]LHG[VI]DDAIDKSKK[VI]L[TS]TMSRR[IM]TRNKWIV[AG]S[VI]IGALVFAI[VI]I
2.0e−170
t3:22
19
RKLITAF[RK]DNEIERS[HR]SL[WD]P[DE]
8.0e−140
U
N
C
O
R
1.1e−1711
R
C
t3:3
Please cite this article as: Sun, X., et al., Tracing the origin and evolution of plant TIR-encoding genes, Gene (2014), http://dx.doi.org/10.1016/ j.gene.2014.04.060
X. Sun et al. / Gene xxx (2014) xxx–xxx
5
Table 3 (continued) t3:23
Motifa
t3:24
20
t3:25 t3:26
a
Consensusa
E value
[TN][VI]A[GR]LL[YH]D[HR][LF]S
4.1e−104
Motif IDs and consensus sequences were generated by MEME analysis. The second column for each motif shows the amino acid frequency distribution graphed using WebLogo, where the sizes of the characters represent the frequencies of occurrence.
2.3. Identification and analysis of conserved motifs present within the TIR domain
161
169
The conserved motifs in the amino acid sequences of the TIR regions were identified and analyzed with MEME 4.6.1 (Bailey and Elkan, 1994). For MEME, minimum and maximum motif lengths of 10 and 50, respectively, were set, and 20 motifs were requested using the zero- or oneoccurrence-per-sequence model. Based on expectation maximization, conserved motifs were identified in a group of sequences without a priori assumptions about the alignments. The individual profile for each conserved motif was assessed, and after tiling, only the conserved motifs with P-values ≤10−4 and no overlap were reported.
170
2.4. Phylogenetic analysis of the T genes with the skeleton motif pattern
171
The evolutionary history of the T genes (genes containing only the TIR domain(s) were hereafter referred to as “T genes”) with the skeleton motif pattern was inferred using the neighbor-joining method (Saitou and Nei, 1987). In addition to the 58 identified T genes with the skeleton motif pattern, up to 101 matches were discovered from the Picea abies genome (http://congenie.org) during the conservation search using one dicot-typed T gene (Acoerulea v1.019315 in Aquilegia coerulea) and two monocot-typed T genes (Macuminata Achr9T24500_001 in M. acuminata and LOC_Os07g37950.1 in Oryza sativa). To compensate for the lack of fully sequenced genome data for gymnosperms, we added five T genes (the top five hits from the BLAST search) from P. abies to the profile. The maximum-likelihood phylogenetic tree shows the homology of the T genes across plants. The bootstrap consensus tree inferred from 1000 replicates is taken to represent the evolutionary
164 165 166 167 168
172 173 174 175 176 177 178 179 180 181 182 183 184
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R
162 163
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151 152 Q6
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149 150
F
159 160
147 148
Whole-genome sequences from 32 different species (two species in Chlorophyta, one species in Bryophyta, one species in Pteridophyta and 28 species in Angiospermae) were selected to represent all evolutionary nodes in the plant kingdom. The sequence from P. taeda L. was also incorporated into the dataset to represent the gymnosperms. We discovered 2843 TIR-encoding genes in all 33 plant species (Table 1). We identified two and one TX genes in Chlamydomonas reinhardtii and Volvox carteri (Chlorophyta), respectively; 13 TNLs, TNs and TXs in P. patens (Bryophyta); one TN in Selaginella moellendorffii Hieron. (Pteridophyta); five TNLs, TNs and TXs in P. taeda (Gymnospermae) and 2821 TIR-encoding genes of all four types in Angiospermae. The TNLs and TNs accounted for 41.36% (1176/2, 843) and 25.85% (735/2843), respectively, of all TIR-encoding genes. TNLs and TNs were first observed in P. patens, and present in P. taeda and all dicots except A. coerulea E. James and Mimulus guttatus DC. These two groups are absent in monocots, while TN was found in S. moellendorffii. Fewer TLs, which accounted for 1.27% (36/2843) of all TIR-encoding genes, were found in ten species of dicots. The TXs comprised 31.52% (896/2843) of all the TIR-encoding genes; 72.21% (647/896) of the TXs were from a subgroup of genes containing only the TIR domain(s), hereafter referred to as “T genes”. The remaining 29.79% of the TXs had more than 100 domains or domain combinations for X. The TXs exhibited an ancient origin in early green algae and could be identified in all plant species, except S. moellendorffii. Moreover, the TXs found in the seven monocot species were all T genes.
189 190
O
157 158
TIR-encoding genes were aligned using the ClustalW software (Thompson et al., 1994); the genetic parameters were computed using the Geneious 6.0.3 software. Adjacent homologous genes separated by less than 200 kb were defined as clusters (Holub, 2001).
145 146
188
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155 156
143 144
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3.1. Distribution of the TIR-encoding genes in 33 plant species
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2.2. Sequence alignment and chromosomal position analysis
141 142
3. Results
D
154
139 140
history of the analyzed taxa. Evolutionary analyses were conducted in 185 MEGA5 (Tamura et al., 2011). 186
T
153
domain in the identified genes was confirmed by conducting a Pfam search with an E-value cutoff of 10− 4 (Finn et al., 2010). A total of 2744 proteins were identified in all plant species. In the second analytical step, the TIR domains of the selected protein sequences were aligned using ClustalW (Thompson et al., 1994). These alignments were used to build a plant-specific HMM profile using hmmbuild in HMMER version 3.0 (Mistry et al., 2013); this profile was then used to identify proteins encoded in each genome using hmmsearch (E b 10−4). The 110 newly discovered sequences that had a confirmed TIR domain, according to the results of the Pfam search and visual inspection, were incorporated into the HMM for the third analysis. Eight new sequences were discovered in six dicot species; however, none of these sequences contained a TIR domain and were therefore discarded. These three analytical steps identified 2843 annotated genes that encoded homologs of TIR proteins. A Pfam search and SMART were used to describe the domain architecture of each TIR-encoding gene (Finn et al., 2010; Letunic et al., 2009). The default threshold values for both databases were used (E-value b 10−4).
137 Q5 138
E
136
b
Pictogramb
191 192 193 194 195 196 197 198 199 200 201 202 203 204 205 206 207 208 209 210 211 212 213 214
3.2. Distribution and evolution of the TIR-encoding genes on the 215 chromosomes 216 We found that 1846 (64.93%) TIR-encoding genes formed 369 chromosomal clusters. However, this percentage could be slightly underestimated because of the presence of unanchored scaffold sequences. On average, five TIR-encoding genes were detected in a cluster. Few TIR-encoding genes were identified in C. reinhardtii, V. carteri, S. moellendorffii or in the seven monocots; 13 TIR-encoding genes present in P. patens could not be clustered because of incomplete annotation. The numbers of clusters varied significantly in all dicots, except A. coerulea and M. guttatus, which lacked clusters of TIR-encoding genes (Table 2). As many as 97 clusters were detected in Eucalyptus grandis Hill ex Maiden. Two types of gene clusters were identified in plants: homogeneous and heterogeneous clusters. Homogeneous clusters contained only one group of TIR-encoding genes; for example, the Bra001160– Bra001175 cluster in Brassica rapa L. is composed of four TNLs, and the POPTR0019s12840.1–POPTR0019s12910.1 cluster in Populus trichocarpa contains three T genes. Heterogeneous clusters consisted of more than one group of TIR-encoding genes; for example, the Medtr4g018120.1–Medtr4g018780.1 cluster in Medicago truncatula
Please cite this article as: Sun, X., et al., Tracing the origin and evolution of plant TIR-encoding genes, Gene (2014), http://dx.doi.org/10.1016/ j.gene.2014.04.060
217 218 219 220 221 222 223 224 225 226 227 228 229 230 231 232 233 234 235
264 265 266 267 268
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262 263
269 270 271 272 273 274 275 276 277 278 279 280 281 282 283 284 285 286 287 288 289 290 291 292 293 294 295 296
3.4. Phylogeny and conservation of the TIR-encoding genes with the skeleton 297 motif pattern 298 Interestingly, the TIR-encoding genes with the skeleton motif pat- 299 tern in the TIR domain were all T genes. This special subgroup consisted 300 of 58T genes that were distributed in all 29 spermatophyte species, 301
E
260 261
R
258 259
R
256 257
O
251 252
C
249 250
N
247 248
U
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To further explore the evolutionary relationship between the different groups of TIR-encoding genes in different species, the 20 most conserved motifs in the amino acid sequences of the TIR regions from the TIR domain datasets were identified using MEME (Table 3). Consistent with previous studies of the TIR domain, a number of motifs were found in our analysis; in particular, motifs 1, 2 and 3 corresponded to the previously described motifs 2, 3, and 1, respectively, of A. thaliana (Meyers et al., 2002). A total of 492 composite motif patterns for the TIR domains existed in the analyzed plants (Fig. 1). C. reinhardtii and V. carteri had only one pattern (independent motif 1). The TIR domains of P. patens had a total of 11 composite patterns composed of 4–7 motifs. The TIR domain of S. moellendorffii had only one composite pattern composed of six motifs. The TIR domains of P. taeda had a total of more than five composite
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patterns. The TIR domains of monocots had only one composite pattern of six motifs, while the TIR domains of angiosperm dicots had 475 composite patterns (Fig. 1). It's notable that the motif pattern 3-20-13-4-1-2 was present in all species of gymnosperms and angiosperms, suggesting a high conservation among seed plants. Unique composite patterns were detected in C. reinhardtii, V. carteri, P. patens and S. moellendorffii; these patterns were very different from the composite patterns found in gymnosperms and angiosperms. Five TIR-encoding genes in P. taeda had five composite patterns in the TIR domains, four of which were unique to P. taeda; the fifth composite pattern in this species (3-20-13-4-1-2) was also found in angiosperms. The TIRencoding genes in dicots had 474 unique patterns. Because the motif pattern 3-20-13-4-1-2 was present in all 27 species of gymnosperms and angiosperms, the gymnosperms might constitute an evolutionary node of the TIR-encoding genes. This composite pattern first observed in the gymnosperms and appears to represent a skeleton pattern of motifs in the TIR domain of TIR-encoding genes from all spermatophytes. By alignment and comparison of the skeleton pattern and multiple patterns of motifs in the TIR domain in spermatophytes, it can be inferred that two ways might be involved in evolution of the TIR domains from gymnosperms to angiosperms (Fig. 2). The skeleton pattern could evolve to a variety of patterns through motif insertion or superseding, which is prevalent in dicots; taken for example, the replacement of motif 20 and motif 13 with motif 5 and motif 6, the insertion of motif 10 between motif 5 and motif 6 and the insertion of motif 8 between motif 1 and motif 2 could have resulted in a shift from the skeleton pattern to motif 3-5-10-6-4-1-8-2. The other way by maintaining the skeleton pattern as the only composite pattern usually occurred in monocots.
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contains 13 TNs and three T genes, and the Glyma16g33590.1– Glyma16g34110.1 cluster in Glycine max Merr. contains 15 TNs, two TNLs and a single T gene. The number of heterogeneous clusters was greater than or equal to the number of homogeneous clusters in most species (Table 2); however, G. max, Prunus persica (L.) Batsch and Thellungiella halophila (C. A. Mey.) O. E. Schulz contained more homogeneous clusters than heterogeneous clusters. The gene clusters composed of TNLs, hereafter referred to as a TNL cluster, accounted for 21% of all gene clusters; those composed of TNLTN, TNL-T or TN genes accounted for 14%, 10% and 10% of all gene clusters, respectively (Table 2). The T, T-TN, TNL-TX and T-TN-TNL clusters accounted for 6%, 8%, 5% and 8%, respectively, of all clusters, while the remaining 22 types of clusters accounted for less than 3%. Because the number of detectable TL genes in plants is relatively low, the percentage of gene clusters with TLs was almost zero. However, 32 of all 34 TL genes (94.12%) reside in homogeneous or heterogeneous clusters. In most dicotyledon species, the TNL, TNL-TN, TNL-T and TN clusters account for more than 50% of all TIR-gene clusters in each species.
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Fig. 1. The composite patterns of motifs in the TIR domains in the plant kingdom. The numbers in the boxes indicate the motifs shown in Table 3. A full list of composite patterns in the TIR domains of dicotyledoneae is provided in Table S2.
Please cite this article as: Sun, X., et al., Tracing the origin and evolution of plant TIR-encoding genes, Gene (2014), http://dx.doi.org/10.1016/ j.gene.2014.04.060
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4.1. Evolution of TIR-encoding genes in the plant kingdom
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C. reinhardtii, which represents the earliest evolutionary node of plants, has only one TIR-encoding gene. The number of TIR-encoding genes increases to 13 in three groups of P. patens. The genome of P. patens underwent whole genome duplication, leading to a general increase in the gene family complexity and the acquisition of genes involved in tolerating environmental stresses (Rensing et al., 2008). The increase in the gene number and complexity of the TIR-encoding gene family suggests that the bryophytes were a key group on the evolutionary path to land-dwelling species; to tolerate stresses, e.g., certain pathogens encountered in the terrestrial environment, the bryophytes had to rapidly evolve under strong natural selection pressure (Ponce de León and Montesano, 2013). In further support of this conclusion, diverse novel classes of NBS-encoding genes, including α/β-hydrolaseNBS-LRR (HNL) and protein-kinase-NBS-LRR (PNL), were discovered in the moss P. patens and the liverwort Marchantia (Xue et al., 2012). Moreover, the fusion of NBS and LRR domains, which represents a critical step in the origin of the plant R genes, first occurred in bryophytes (Yue et al., 2011). Consequently, the striking expansion of the R gene family and their frequent recombination throughout all land plant lineages provided a powerful arsenal for land plants, allowing them to quickly and effectively evolve novel resistance specificities to fight diverse pathogens in the terrestrial environment (Leister, 2004). The evolution of the reproduction mode might also play a vital role in the expansion and diversification of the plant R genes (Dimijian, 2005). Sexual reproduction is far more prevalent than asexual reproduction in higher plants, which may lead to frequent genetic recombination and thus result in novel R genes against pathogens, parasites, predators or harsh environmental conditions (Morran et al., 2011).
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Previous studies (Cannon et al., 2002; Mchale et al., 2006; Yang et al., 2008) showed that TNLs and TNs that exhibit species-specific expansion are highly differentiated. Our results confirmed that TNLs, TNs and TLs are not homologous among species; however, the genes that belong to a special subgroup of T genes in all species of spermatophytes share the same composite motif pattern in the TIR domain, indicating that the TIR-encoding genes of spermatophytes may have originated from
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Of all the whole-genome sequenced plants, S. moellendorffii has the smallest genome (approximately 100 Mb) and only one TN gene, indicating that the TIR-encoding genes in Pteridophyta are less diverse and numerous than those in Bryophyta. Yue et al. (2012) proposed that a large-scale gene loss in S. moellendorffii decreased the number of TIR-encoding genes in this species. S. moellendorffii is the only species in Pteridophyta that has had its whole genome sequenced; in contrast, Gleichenites (also in Pteridophyta) is closely evolutionarily related to gymnosperms (Pryer et al., 2001). Therefore, S. moellendorffii may not represent most species in Pteridophyta; more information from other species of pteridophytes is needed. The gymnosperm P. taeda has five TIR-encoding genes; however, this number may be an underestimation as the statistics used in the calculation were based on the EST database and not on genome sequence. Given the distributions of the TIRencoding genes in Pteridophyta, Bryophyta and Gymnospermae, some species in Pteridophyta may maintain a considerable number of TIRencoding genes, while species in Bryophyta may maintain these genes in multiple groups. Furthermore, because gymnosperms have fewer species, stable growth environments and a stable pathogen spectrum, these plants may maintain fewer and less diverse TIR-encoding genes than angiosperms (Liu and Ekramoddoullah, 2003, 2007). According to the APG III system (Angiosperm Phylogeny Group, 2009), angiosperms can be classified as basal angiosperms, magnoliids, monocots or eudicots. The evolutionary history of the TIR-encoding genes in the basal angiosperms and magnoliid lineages cannot be assessed due to the lack of genome sequence data. The monocots, including the seven species with fewer T genes that were used in this study, constitute a relatively independent monophyletic group that evolved after the basal angiosperms and magnoliids but before the eudicots. The TIR-encoding genes in the eudicots are characterized by remarkably high levels of gene duplication and complex diverse architectures; additionally, 64.93% of the clustered TIR-encoding genes are present in all eudicots. This indicates that the eudicots have significantly expanded the spectrum of potential disease resistance through massive duplication and subsequent differentiation of the TIR-encoding genes; in contrast, monocots resist pathogens through other families of resistance genes, e.g., CC-NBS-LRR (CNL) (Bai et al., 2002; Pan et al., 2000; Tian et al., 2004). Dissimilar mechanisms of disease resistance that exist in both angiosperm lineages also provide evidence of the remarkably uneven distribution of TIR-encoding genes in monocots and dicots.
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including the seven monocots (Table S3; Fig. 3). These T genes had an identity of 48.5% (55.7% for the Dicotyledoneae clade and 58.3% for the Monocotyledoneae clade), suggesting that these special T genes were conserved across spermatophytes. The conservation of the T genes with the skeleton motif pattern was further examined using plant genomic sequences from GenBank. One dicot-type T gene (Acoerulea v1.019315 in A. coerulea) and two monocot-type T genes (Macuminata Achr9T24500_001 in M. acuminata and LOC_Os07g37950.1 in O. sativa) were used to identify homologs. All three T genes yielded homologs in a BLAST search against sequences from gymnosperms, basal angiosperms, monocots and magnoliids (Table S4). The identity of the matches ranged from 27% to 50%, indicating that these special T genes form a highly conserved group across all plants that contain these homologs. Although these special T genes are not found in lower plants, important motifs in the skeleton pattern of these T genes, in particular motif 13, have been detected in P. patens.
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Fig. 2. Examples of summarized and aligned motifs for TIR domain. The skeleton motif pattern 3-20-13-4-1-2 was represented by four TIR-encoding genes in P. taeda, A. thaliana and O. sativa, and the motif pattern 3-5-10-6-4-1-8-2 is represented by ten TIR-encoding genes in M. truncatula and G. max. The numbers above or below boxes indicate the motifs shown in Table 3.
Please cite this article as: Sun, X., et al., Tracing the origin and evolution of plant TIR-encoding genes, Gene (2014), http://dx.doi.org/10.1016/ j.gene.2014.04.060
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can be traced back to gymnosperms may have existed in ancestral angiosperms. Furthermore, the skeleton motif pattern present in the TIR domain of TIR-encoding genes first appeared in gymnosperms. This skeleton pattern is shared by every species of spermatophyte and subsequently evolved into a variety of composite motif patterns. These results provide strong evidence that the gymnosperms are a key evolutionary node for the TIR-encoding genes. T genes are indispensable for each species, and the evolutionary pathways for composite motif patterns in the TIR domain from gymnosperms to angiosperms are based on T genes. Thus, the T genes must be ancestors of the other TIR resistance genes; this finding could explain why T genes are present in all spermatophyte species, including in the earliest plant lineages. Furthermore, the diversification and conservation of the TX genes suggests that these genes may have important functions, including cold resistance (Arabidopsis CHS1, Zbierzak et al., 2013) and disease resistance (Arabidopsis TNs and TXs, Nandety et al., 2013). The distributed clusters of TIR-encoding genes were observed in species of Arabidopsis, rice, grapevine and poplar (Meyers et al., 2002; Yang et al., 2008; Zhou et al., 2004); in this study, the TIR-encoding genes were often distributed in clusters. In addition to being present in Arabidopsis, grapevine and poplar (Meyers et al., 2002; Yang et al., 2008), clusters of TNLs, TNs and T genes were widely observed in the majority of dicots. TNs and TXs are thought to be required by the TNLs for downstream signaling; as a result, TNLs, TNs and TXs may have coevolved during natural selection to favor the development of functional allele units (Meyers et al., 2002). Because TLs lack NBS domains, they have rarely been studied by other investigators. In this study, 94.12% of the TLs were located in clusters; this finding implies that TLs may maintain certain functions, thus creating a selective advantage for species with co-localized TIR-encoding genes.
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Although the origins of TNLs seems to date back to very early land plant lineages, TNLs are known to be absent from monocots (Yue et al., 2012). Two hypotheses have been proposed to explain the lack of distribution for the groups of TIR-encoding genes other than TNLs: either the few TIR-encoding genes in ancestral angiosperms were completely lost in cereals, or the TIR-encoding genes differentiated and expanded and were eventually lost in cereals. However, certain investigators have argued that none of the existing mechanisms could successfully explain the complete elimination of such a large and complicated family of genes (Bai et al., 2002; Cannon et al., 2002; Pan et al., 2000). To our knowledge, no appropriate hypotheses have been proposed for the loss of TNLs in cereals or monocots or for the loss of other TIR-encoding genes, e.g., the TNs and TLs. Our study revealed that T genes (in limited numbers) represent the only group of TIRencoding genes in monocots; in contrast, dozens to hundreds of TIRencoding genes can be found in most dicots. The mechanism behind this uneven distribution of TIR-encoding genes in dicots and monocots is unclear. T genes are present at each evolutionary node of plant phylogeny, including Chlorophyta, Bryophyta and Gymnospermae. As a result, it has been proposed that the presence of T genes in monocots is a conserved continuous evolutionary event. Because T genes and other groups of TIR-encoding genes are continuously present in gymnosperms, we inferred that the TIR-encoding genes differentiated during the evolution from gymnosperms to basal angiosperms. Certain species of basal angiosperms that only had T genes subsequently evolved into monocots; other species of basal angiosperms that had diverse TIR-encoding genes differentiated and expanded to form a large family of resistance genes in eudicots. Monocots may only have T genes because of pathogenic stresses or species-specific mechanisms of disease resistance. The presence of only one or two T gene(s) in the eudicot species A. coerulea and M. guttatus and the absence of TIR-type resistance genes in Beta vulgaris L. (Tian et al., 2004) suggest that additional factors
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Fig. 3. Phylogeny and conservation of the T genes with the skeleton motif pattern. Numbers at branches are bootstrap percentages (the values less than 50% are not shown).
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a common ancestor. Moreover, these conserved T genes might represent ancestral genes that are maintained in each plant during evolution. It has been proposed that a few TIR-encoding genes were present early in the evolution of angiosperms and that the abundant diversity of TIR-encoding genes was subsequently shaped by massive speciesspecific expansion in modern plant lineages, particularly in the dicots (Bai et al., 2002; Pan et al., 2000; Tarr and Alexander, 2009; Tian et al., 2004). Our study showed that a selection of TIR-encoding genes that
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This work was supported by the National Natural Science Foundation of China (30930008; 31200177).
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may repress gene expansion in some dicot species, resulting in a conserved evolution similar to that observed in monocots. 470 Besides the TXs observed in monocots, Nandety et al. (2013) identi471 fied 16 TNs (in Fig. 1, Nandety et al., 2013) in four monocot species in 472 contrast to no detection of TNs in this study. Based on a Pfam search 473 with an E-value cutoff of 10–4 and visual inspection, we found that the 474 16 TNs identified in monocot species lacked a typical TIR domain. 475 None of the TNs (except Os11g36760) got significant matches to the 476 TIR domain; these genes were therefore not considered as typical TIR477 encoding genes and were discarded in our analysis. The assumed TN 478 gene Os11g36760 in rice exhibited significant matches to TIR_2 and 479 AAA_16, and then was designated a TX gene instead here. 480 Supplementary data to this article can be found online at http://dx. 481 Q11 doi.org/10.1016/j.gene.2014.04.060.
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