Large-Scale Analyses of Angiosperm Nucleotide-Binding
Site-Leucine-Rich Repeat Genes Reveal Three Anciently
Diverged Classes with Distinct Evolutionary Patterns1
Zhu-Qing Shao 2, Jia-Yu Xue 2, Ping Wu, Yan-Mei Zhang, Yue Wu, Yue-Yu Hang, Bin Wang*, and
Jian-Qun Chen*
State Key Laboratory of Pharmaceutical Biotechnology, School of Life Sciences, Nanjing University, Nanjing,
210023, China (Z.-Q.S., P.W., Y.-M.Z., Y.W., B.W., J.-Q.C.); and Institute of Botany, Jiangsu Province and
Chinese Academy of Sciences, Nanjing, 210014, China (J.-Y.X., Y.-M.Z., Y.-Y.H.)
Nucleotide-binding site-leucine-rich repeat (NBS-LRR) genes make up the largest plant disease resistance gene family (R genes),
with hundreds of copies occurring in individual angiosperm genomes. However, the expansion history of NBS-LRR genes
during angiosperm evolution is largely unknown. By identifying more than 6,000 NBS-LRR genes in 22 representative
angiosperms and reconstructing their phylogenies, we present a potential framework of NBS-LRR gene evolution in the
angiosperm. Three anciently diverged NBS-LRR classes (TNLs, CNLs, and RNLs) were distinguished with unique exonintron structures and DNA motif sequences. A total of seven ancient TNL, 14 CNL, and two RNL lineages were discovered
in the ancestral angiosperm, from which all current NBS-LRR gene repertoires were evolved. A pattern of gradual expansion
during the first 100 million years of evolution of the angiosperm clade was observed for CNLs. TNL numbers remained stable
during this period but were eventually deleted in three divergent angiosperm lineages. We inferred that an intense expansion of
both TNL and CNL genes started from the Cretaceous-Paleogene boundary. Because dramatic environmental changes and an
explosion in fungal diversity occurred during this period, the observed expansions of R genes probably reflect convergent
adaptive responses of various angiosperm families. An ancient whole-genome duplication event that occurred in an
angiosperm ancestor resulted in two RNL lineages, which were conservatively evolved and acted as scaffold proteins for
defense signal transduction. Overall, the reconstructed framework of angiosperm NBS-LRR gene evolution in this study may
serve as a fundamental reference for better understanding angiosperm NBS-LRR genes.
Plant disease resistance (R) genes are a set of genes that
confer resistance to various pathogens. Among all known
types of R genes, nucleotide-binding site-leucine-rich repeat (NBS-LRR, NBS for short) genes represent the largest
class, encompassing more than 80% of the characterized
R genes (Meyers et al., 1999; Meyers et al., 2005; McHale
1
This work was supported by the National Natural Science Foundation of China (30930008, 31170210, 91231102, 31300190, 31400201,
31470327, 31500191, and 31570217), the Natural Science Foundation
of Jiangsu Province (BK20130565), the Fundamental Research Funds
for the Central Universities (2062140215, 20620140546, and
20620140558), and the National Postdoctoral Science Foundation of
China (2013M540435 and 2014T70503).
2
These authors contributed equally to the article.
* Address correspondence to [email protected] or binwang@nju.
edu.cn.
The author responsible for distribution of materials integral to the
findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is:
Jian-Qun Chen ([email protected]).
J.-Q.C., B.W., J.-Y.X., and Z.-Q.S. conceived and designed the project; J.-Y.X., Z.-Q.S., and B.W. obtained and analyzed the data; P.W.,
Y.-M.Z., Y.W., and Y.-Y.H. participated in the data analysis and discussion; Z.-Q.S. and J.-Y.X. drafted the initial manuscript; B.W. complemented the writing; J.-Q.C. supervised the analysis and writing;
all authors contributed to discussion of the results, reviewed the manuscript, and approved the final article.
www.plantphysiol.org/cgi/doi/10.1104/pp.15.01487
et al., 2006; Friedman and Baker, 2007). Since the first
comprehensive study on NBS genes in Arabidopsis
(Arabidopsis thaliana; Meyers et al., 2003), NBS genes have
been surveyed across various plant genomes, most of
which belong to the rosid lineage of eudicots and the
Poaceae of monocots (Monosi et al., 2004; Zhou et al.,
2004; Yang et al., 2006; Ameline-Torregrosa et al., 2008;
Yang et al., 2008; Mun et al., 2009; Porter et al., 2009; Li
et al., 2010; Zhang et al., 2011; Lozano et al., 2012; Luo
et al., 2012; Andolfo et al., 2013; Shao et al., 2014).
NBS genes are historically divided into two classes,
namely, TIR-NBS-LRR (TNL) and the non-TIR-NBSLRR (nTNL), which are differentiated by the presence
of a Toll/IL-1 Receptor-like (TIR) domain in the protein
amino terminus (Meyers et al., 1999; Bai et al., 2002;
Zhou et al., 2004). Because most nTNL genes encode a
coiled-coil (CC) domain at the N terminus, the nTNL
genes often are called CC-NBS-LRR (CNL) genes
(Meyers et al., 2003; Ameline-Torregrosa et al., 2008).
However, recent studies have revealed that apart from
CNL genes, a small group of nTNL genes that possess a
special N-terminal domain, RPW8 (resistance to powdery mildew8) domain, likely represent a distinct class
of NBS genes (RPW8-NBS-LRR [RNL]; Xiao et al., 2001;
Cannon et al., 2004; Bonardi et al., 2011; Collier et al.,
2011; Shao et al., 2014; Zhang et al., 2016). Investigations of NBS genes in the Fabaceae (rosid I lineage) and
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Shao et al.
Brassicaceae (rosid II lineage) have shown that RNLs
and CNLs diverged prior to the divergence of these two
families (Shao et al., 2014; Zhang et al., 2016). Interestingly, although both CNL and RNL genes are present in
monocots and dicots, TNL genes only are present in
dicots (Meyers et al., 2003; Tarr and Alexander, 2009;
Andolfo et al., 2013; Shao et al., 2014). Considering the
abundance of TNLs in various dicot species, their absence in monocots is perplexing.
For several years, our knowledge of NBS gene evolution has been based on investigations of individual
angiosperm genomes (Meyers et al., 2003; AmelineTorregrosa et al., 2008; Kohler et al., 2008). The number of NBS genes varied dramatically among different
species, even among different accessions of the same
species (Zhang et al., 2010). However, the relative
contributions of recent and ancient expansions to the
abundance of NBS genes in modern plant genomes
remain elusive. Recent studies report the evolutionary
analyses of NBS genes across multiple species in a plant
family (Cannon et al., 2002; Guo et al., 2011; Luo et al.,
2012; Lin et al., 2013; Shao et al., 2014; Zhang et al.,
2016). While studies in Brassicaceae and Poaceae both
revealed abundant and recent losses of NBS genes in
various species after the divergence of these two families, a study involving Fabaceae has suggested the opposite scenario in which all surveyed species recently
underwent a multitude of NBS expansions (Luo et al.,
2012; Shao et al., 2014; Zhang et al., 2016). Although
distinct evolutionary patterns of NBS genes have been
identified at the family level, hundreds of NBS gene
lineages arose in the ancestors of Fabaceae, Brassicaceae,
and Poaceae, indicating that NBS genes may have
probably undergone ancient expansions prior to familial divergence. Therefore, a phylogenetic analysis
at a larger scale is necessary to better understand the
expansion history of NBS genes in angiosperms.
Several functional NBS genes have been identified in
angiosperm species (Liu et al., 2007); however, their time
of emergence and relationship with other NBS genes
have not been established. For example, several CNL
genes have been identified in both soybean (Glycine max;
Rpg1b and Rpg1r) and Arabidopsis (RPM1 and RPS2)
against the bacterial pathogen Pseudomonas syringae by
guarding the host protein RIN4 (Bent et al., 1994; Mackey
et al., 2002; Mackey et al., 2003; Ashfield et al., 2014).
While the Rpg1b and Rpg1r genes were deduced to
emerge after the divergence of Fabaceae species, the
evolutionary time point of the origin of RPM1 and RPS2
genes in Arabidopsis is largely unknown (Ashfield et al.,
2014; Shao et al., 2014). Because all these R genes have
likely adopted a similar strategy to defend against the
same pathogen, investigating their evolution may elucidate the mechanism underlying the establishment and
maintenance of resistance in various plant species.
To obtain a more comprehensive understanding of
NBS gene evolution, it is essential to analyze more
species/lineages to determine how the current NBS
genes have evolved and diverged from ancestral NBS
lineages in the ancestral angiosperm. This also will
provide when and how resistance loci emerged or disappeared in certain lineages. In this study, a total of 22
plant species (Fig. 1) from key systematic clades in
eudicots, monocots, and the early diverging basal angiosperm were chosen to depict the ancient evolutionary history of NBS genes in angiosperms. More than
6,000 NBS genes were identified; family level phylogenetic trees were constructed first and then expanded
to higher systematic levels to eventually analyze all of
the angiosperms. Ancient taxonomies of NBS genes
were traced to the origin of angiosperms, and a total of
23 ancestral NBS genes were recovered in the last
common ancestor of angiosperms. This angiosperm
NBS frame provides a fundamental reference for understanding the evolutionary history of NBS genes as
well as insight into the finer details of particular NBS
gene classes.
RESULTS
Identification and Classification of NBS Genes from
22 Angiosperm Species
A total of 6,135 NBS genes were identified in 22 angiosperm species using previously described procedures (Shao et al., 2014; Zhang et al., 2016). Five of the
22 genomes were surveyed in this study, Amborella
trichopoda, Musa acuminata (banana), Phyllostachys heterocycla (bamboo), Capsicum annuum (hot pepper), and
Sesamum indicum (sesame), whereas the others have
been previously reported (Meyers et al., 2003; Luo et al.,
2012; Malacarne et al., 2012; Andolfo et al., 2013; Shao
et al., 2014; Zhang et al., 2016). A. trichopoda, a basal
angiosperm, had a total of 105 NBS genes, including 15
TNLs, one RNL, and 89 CNLs (Fig. 1; Supplemental
Table S1). The NBS genes varied among the 22 surveyed
species (Fig. 1). Of all species surveyed, Medicago truncatula possessed the highest number of NBS genes (571),
more than 6-fold of that observed in Thellungiella salsuginea (88). RNL genes were detected in all of the
surveyed species, although at lower numbers (1–18).
A large proportion of NBS genes were either TNLs or
CNLs, although TNLs were not detected in eight of 22
species, including seven monocots and S. indicum. Because the basal angiosperm A. trichopoda harbored TNL
genes, these genes were most likely also present in the
ancestor of angiosperms but independently lost in certain lineages. In species with both TNL and CNL genes,
CNLs often outnumbered TNLs, except for the four
Brassicaceae genomes analyzed (Zhang et al., 2016).
The asterid genomes showed a lower ratio of TNL to
CNL genes, with the C. annuum exhibiting the highest
level of disparity (TNL:CNL = 1:18).
Phylogenetic Analysis of NBS Genes in Major
Angiosperm Clades
The 22 angiosperm species surveyed in this study
occupy important phylogenetic positions (Fig. 1). To
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Evolution of NBS-LRR Genes in Angiosperms
Figure 1. Identification and classification
of NBS genes in 22 angiosperm genomes.
The phylogenetic relationship was constructed according to the APG III system
(Bremer et al., 2009). The divergence times
at different nodes of angiosperms were
combined from previous studies (Lavin
et al., 2005; Stefanovic et al., 2009;
D’Hont et al., 2012; Zhang et al., 2012;
Peng et al., 2013; Yang et al., 2013; Kim
et al., 2014; Wang et al., 2014; Zeng et al.,
2014). The total number of NBS genes and
their classification in each species are
shown. The number of ancestral NBS
genes recovered by phylogenetic analysis
at each node is shown in a green circle,
and an asterisk after the number indicates
that it is not directly unraveled by phylogenetic analysis.
trace the evolutionary history of NBS genes, we examined multiple angiosperm clades, including rosids (Ro),
asterids (As), eudicots (Ed), monocots (Mo), mesangiospermae (Ma), and all angiosperms (An; Fig. 1). It is
widely recognized that the NBS domain effectively
distinguishes NBS genes into TNL and nTNL classes
(Bai et al., 2002; Meyers et al., 2003). Although a sister
relationship between CNLs and RNLs has been established in rosids, it has not been examined at angiosperm
level. Therefore, phylogenetic analyses of TNLs and
nTNLs (including both CNLs and RNLs) were separately performed in this study. We first conducted
phylogenetic analysis of three major angiosperm lineages, namely, monocots, asterids, and rosids, which
cover seven, four, and 10 genomes, respectively.
The M. acuminata genome, together with six Poaceae
genomes, including Oryza sativa, Brachypodium distachyon,
P. heterocycla, Zea mays, Sorghum bicolor, and Setaria italica,
were used to elucidate the evolution of NBS genes in
monocots. Because these species did not harbor TNL
genes, phylogenetic analysis of only nTNL genes was
performed. The reconstructed monocot nTNL phylogeny
(Supplemental Fig. S1) revealed that nTNL genes from the
six Poaceae species formed 456 monophyletic lineages,
indicating the presence of at least 456 ancestral nTNL
genes in the Poaceae common ancestor (;62 million years
ago [MYA]; Peng et al., 2013, Zhang et al., 2012). These
Poaceae nTNL genes formed 57 monophyletic lineages
when grouped with M. acuminata nTNL genes (one RNL
lineage designated as Mo-R1, and 56 CNL lineages
identified as Mo-C1 to Mo-C56), suggesting that nTNL
genes from the seven monocot genomes were actually
derived from 57 ancestral Mo-NBS genes ;110 MYA (Fig.
2; Supplemental Fig. S1; D’Hont et al., 2012). From their
common ancestor, the Poaceae and M. acuminata inherited
51 and 32 ancestral Mo-NBS genes, respectively. Among
these, 26 were shared by both Poaceae and M. acuminata.
Several ancestral genes in the Poaceae underwent extensive duplications (from 51 to 456) during the time interval
of 48 million years. For example, the NBS gene Mo-C56
duplicated into 197 genes during this time period
(Supplemental Fig. S1). As multiple species radiated from
the Poaceae common ancestor, in some species NBS gene
numbers contracted drastically (Z. mays, 139 from 456)
and in others only slightly (S. italica and P. heterocycla, 424
and 344 from 456, respectively), whereas expansion was
only found in the O. sativa genome (from 456 to 498).
Within the asterids four species were analyzed, including three Solanaceae species, namely, Solanum
lycopersicum, Solanum tuberosum, and C. annuum, as well
as S. indicum from Pedaliaceae. NBS genes from the
three Solanaceae genomes formed 19 TNL monophyletic lineages (Supplemental Fig. S2) and 147 nTNL
monophyletic lineages (Supplemental Fig. S3), suggesting that at least 166 ancestral NBS genes were present in their last common ancestor. The Solanaceae
nTNL genes formed 55 monophyletic lineages with
nTNL genes from S. indicum (two RNL lineages
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Figure 2. Cladograms depicting reconciled phylogenetic relationships of ancestral NBS genes in the divergence nodes of
monocots, rosids, and asterids. The presence of a NBS gene lineage in different species and families are indicated with species or
lineage-specific icons. The maximum likelihood trees of each cladogram are presented in Supplemental Figures S1 to S5.
designated as As-R1 and As-R2, and 53 CNL lineages
identified as As-C1 to As-C53), indicating a minimum of
55 ancestral genes in the asterid common ancestor (Fig. 2;
Supplemental Fig. S2). Solanales inherited more As-nTNL
lineages than S. indicum, which represents Lamiales (47
versus 30). No TNL genes were detected in the S. indicum
genome; therefore, the accurate number of ancestral TNL
genes at this asterid node was not estimated.
Rosids are a major lineage of eudicots that consists of
three sublineages, rosid I (fabids), rosid II (malvids),
and the basal rosids. Previous work analyzed NBS
genes in Fabaceae and Brassicaceae (Shao et al., 2014;
Zhang et al., 2016), which belong to the rosid I sublineage and the rosid II sublineage, respectively. A total
of 64 nTNL and 55 TNL ancestral lineages were discovered in the last common ancestor of the four
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Evolution of NBS-LRR Genes in Angiosperms
Fabaceae species, and 80 nTNL and 148 TNL ancestral
lineages were identified in the last common ancestor of
the five surveyed Brassicaceae species. In this study,
one representative sequence from each of the 119
Fabaceae ancestral NBS lineages and the 228 Brassicaceae ancestral NBS lineages were used to represent the
diversity of ancient NBS genes in these two families,
and then combined with NBS genes identified in Vitis
vinifera (basal rosid) to construct NBS phylogenies for
the whole rosid lineage. A total of 61 ancestral Ro-NBS
genes were identified (four RNLs, 49 CNLs, and eight
TNLs) in the common ancestor of the rosids (;108 MYA;
Fig. 2; Supplemental Figs. S4 and S5), among which the
Fabaceae of rosid I lineage inherited 35 Ro-NBS genes
(two RNLs, 27 CNLs, and six TNLs), Brassicaceae of
rosid II lineage inherited 19 (two RNLs, 12 CNLs, and
five TNLs), and basal rosids inherited 33 (two RNLs,
29 CNLs, and two TNLs). Less than half of the Ro-NBS
genes (26/61) were detected in two of the lineages, and
35/61 were observed in one descending lineage (Fig. 2),
thereby reflecting a pattern of frequent NBS gene loss
after their split from the common rosid ancestor. The
35 and 19 Ro-NBS genes inherited by Fabaceae and
Brassicaceae lineages expanded to 119 (55 TNLs and 64
nTNLs) and 228 (148 TNLs and 80 nTNLs) NBS genes in
the Fabaceae ancestor (;54 MYA) and the Brassicaceae
ancestor (;42 MYA), respectively (Shao et al., 2014;
Zhang et al., 2016). The NBS genes continued to expand
in Fabaceae (Shao et al., 2014), whereas these genes
contracted as the Brassicaceae diverged into multiple
species (Zhang et al., 2016). Although the Brassicaceae
inherited fewer ancestral TNLs than CNLs (five versus
12) from the rosid ancestor, this lineage vigorously expanded its TNLs during the time interval of ;108 MYA
to ;42 MYA. A 30-fold expansion was observed in the
Brassicaceae TNLs (from five to 148), far beyond that of
legume TNLs (;9-fold, from six to 55; Shao et al., 2014;
Zhang et al., 2016).
Reconstruction of NBS Gene Phylogenies at the
Whole-Angiosperm Scale
Representative NBS sequences from ancestral lineages
of asterids, rosids, and monocots were used to construct
ancestral NBS gene lineages at the divergence nodes of
eudicots and mesangiospermae. A total of eight Ed-TNL
and 49 Ed-nTNL genes (47 Ed-CNLs and two Ed-RNLs)
were recovered at the eudicot node (Supplemental Figs.
S6 and S7), whereas 37 Ma-nTNL genes (35 Ma-CNLs and
two Ma-RNLs) were detected at the mesangiospermae
node (Supplemental Fig. S8). Independent loss and differential inheritance of ancestral NBS genes were also
observed at these two nodes. Representative genes from
each ancestral Ma-nTNL lineage were compiled with
A. trichopoda nTNL genes to reconstruct angiosperm
nTNL phylogeny (Supplemental Fig. S9). Because a
mesangiospermae TNL phylogeny could not be constructed due to the absence of TNL genes in monocots, a
TNL phylogeny was generated at the angiosperm level
using eudicot representative TNLs and A. trichopoda
TNLs (Supplemental Fig. S10). Figure 3 shows that all
TNL genes clustered into seven ancestral angiosperm
lineages (designated as An-T1 to An-T7), and all nTNL
genes clustered into 16 ancestral lineages (two RNL lineages designated as An-R1 and An-R2, and 14 CNL lineages identified as An-C1 to An-C14), indicating at least
23 NBS genes were present in the last angiosperm ancestor (;225 MYA). The basal angiosperm A. trichopoda
inherited only eight of the 23 An-NBS genes, whereas the
mesangiospermae inherited 19 (Fig. 3). The angiosperm
nTNL tree was composed of two monophyletic groups,
namely, RNL and CNL (Supplemental Fig. S9). Therefore, despite the extensive discrepancy in gene numbers
between the two groups, this study revealed that the
RNL is another ancient class of NBS genes that has a sister
relationship with the CNL class in angiosperms.
The angiosperm NBS gene phylogenies have
revealed the evolutionary state of NBS genes at critical
angiosperm nodes, which indicated that differential
inheritance of ancestral NBS genes and lineage-specific
expansions are major themes of NBS gene evolution.
Characterization of Class-Specific Signatures among Three
Ancient NBS Gene Classes
Because the results of phylogenetic analysis suggest
that RNLs represent an ancient NBS gene class independent of the CNL and TNL classes, characterization
of distinctive features of each class was performed.
Distinctive Signatures in Conserved NBS Motifs Differentiate
the Three Classes
The NBS domain is composed of a number of characteristic motifs that are conserved and shared by all
NBS genes, whereas other motifs are more variable
(DeYoung and Innes, 2006). All three classes of NBS
genes share five functionally important motifs: P-loop,
Kinase-2, RNBS-B, GLPL, and RNBS-D. The amino acid
sequence conservation of these five motifs was estimated using sequence logos using MEME software
(Fig. 4; Bailey and Elkan, 1995). All motifs were highly
conserved at most sites among all classes, except for the
RNBS-D motif, which was poorly conserved in the TNL
and CNL classes. The class-specific features within
these motifs also were identified. The Kinase-2 motif
had a conserved “DDVW” sequence in the RNL and
CNL genes, but frequently appeared as “DDVD” in the
TNL genes. A “TSR” sequence in the RNBS-B motif of
RNLs was conserved, but frequently appeared as
“TTR” and “TTRD” in the CNL and TNL genes, respectively. CNL did not have specific highly conserved
motifs, but could be distinguished by a RNL like
Kinase-2 motif and a TNL like RNBS-B motif.
Intron-Exon Structures Also Distinguish NBS Classes
The absence/presence, position, and phase of introns
often are regarded as important parameters in phylogeny (Qiu et al., 1998; Sánchez et al., 2003; Qian et al.,
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Figure 3. Cladograms depicting reconciled phylogenetic relationships of ancestral NBS genes in the common ancestor of angiosperms. The maximum likelihood trees of each cladogram are provided in Supplemental Figures S9 and S10.
2015). In a previous study, the intron positions and
phases were determined to be distinctive among the
classes of NBS genes in bryophytes (Xue et al., 2012). In
this study, three classes of NBS genes in angiosperms
also differed in intron characteristics (Fig. 4). Three introns with conserved positions and phases were shared
by most TNL genes, with the first intron separating the
TIR domain and the NBS domain (phase 2), the second
intron separating the NBS domain and the whole LRR
domain (phase 0), and the third intron separating the
first and the remaining LRR domains (phase 0). Angiosperm RNLs share four class-specific introns, with
the exception of RNLs in monocots, which did not
possess the second intron (Fig. 4). Because the second
intron is present in both A. trichopoda and dicot RNL
genes, intron loss likely occurred in the monocot lineage. For the CNL class, conserved introns were not
detected across all CNL lineages, especially in the basal
lineages. In fact, a number of CNL genes were intronless, and were not concentrated in a specific lineage but
distributed across all ancestral angiosperm nTNL lineages. Although some CNL genes had introns, these
were more likely to have been gained at later evolutionary stages. This finding is in agreement with the
intronless structure reported in bryophytes (Xue et al.,
2012).
The Three NBS Classes Show Distinct Evolutionary
Patterns in Angiosperms
Our step-by-step phylogenetic analysis not only
supports the presence of three distinct NBS classes
in the common ancestor of angiosperms, but also
unraveled the ancient NBS gene lineages at multiple
angiosperm evolutionary nodes (Fig. 5). Due to the loss
of TNL genes in S. indicum and the monocot lineage, the
TNL ancestral states at the asterid and mesangiospermae
nodes could not be directly obtained from phylogenetic
analyses. However, based on the recovered ancestral
NBS lineages at the divergence nodes of Solanaceae
(Supplemental Fig. S2), dicots (Supplemental Fig. S6),
and all angiosperms (Supplemental Fig. S10), both
asterid and mesangiospermae nodes were deduced to
have at least five TNL lineages.
Above the family divergence nodes (An, Ma, Mo, Ed,
As, Ro, and Er), CNL genes were abundant but only
slightly more than those of earlier ancestral nodes (Fig.
5). This finding indicates that CNL genes adopted a
gradual expansion strategy in the early stage of angiosperm evolution (from ;225 MYA as angiosperms diverged to around 100 MYA as asterids, rosids, and
monocots diverged; Fig. 1; D’Hont et al., 2012; Wang
et al., 2014; Zeng et al., 2014). Intensive expansion of
CNL genes seems to have occurred in the familial ancestors of Solanaceae and Poaceae, reaching hundreds
of copies. TNL genes apparently followed a different
evolutionary path, as these were completely lost in
monocots and in S. indicum. Although inherited by dicots, TNL numbers remained relatively constant for a
long evolutionary period, with similar numbers of ancestral TNL genes identified at various ancestral nodes
(An, Ma, Ed, As, Ro, and Er; Fig. 5). The expansion of
TNLs mainly occurred prior to the divergence of families, such as in the Fabaceae ancestor, 55 TNLs were
expanded from six of eight Ro-TNLs. A similar pattern
was observed in the Brassicaceae ancestor, in which
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Evolution of NBS-LRR Genes in Angiosperms
Figure 4. Motif and exon-intron structure differences among the three NBS classes. Top, The conservation of amino acid sequences at five motifs in the NBS domain is shown. Bottom, Proposed ancient exon-intron structure of TNL, CNL, and RNL
classes.
four Ro-TNL lineages expanding to 148 genes. The TNL
genes were probably maintained at a lower number for
more than 100 million years (from ;225 MYA as angiosperm diverged to around 100 MYA as rosids and
asterids diverged) and only began to expand thereafter.
This may explain why TNL genes were easily lost in
multiple independent angiosperm lineages.
RNL genes are differentiated from the other two
classes by their relatively conserved evolutionary pattern. Nearly all species from basal angiosperm, monocots, and asterids have one or two RNL genes, whereas
a slight expansion of RNL genes was observed in certain rosid genomes (Fig. 1). Tracing the evolutionary
history of RNL genes revealed two anciently diverged
lineages in the common ancestor of angiosperms (Fig.
6). One RNL lineage contained the Arabidopsis ADR1
gene, whereas the other included the tobacco (Nicotiana
benthamiana) NRG1 orthologs. Both lineages were inherited by dicot species, except for S. indicum, which
lost the NRG1 lineage, similar to all monocot genomes.
Several whole-genome duplications (WGDs) occurred
throughout angiosperm evolution, causing gene duplications and functional divergence (Jiao et al., 2011;
Vanneste et al., 2014). The separation of the ADR1 and
NRG1 lineages prior to the divergence of angiosperms
prompted us to investigate the involvement of an ancient WGD event. The syntenic relationships between
chromosome fragments containing ADR1 or NRG1
orthologs were conserved in angiosperm genomes. Figure 6 (detailed in Supplemental Table S2) shows the
synteny between ADR1- and NRG1-containing chromosomal regions between the dicot genomes of Phaseolus
vulgaris (rosid lineage) and S. lycopersicum (asterid lineage). The syntenic blocks were also detected in the
monocot genome of S. bicolor, in the absence of the NRG1
gene. Moreover, most RNL flanking gene pairs conserved in the duplicated blocks within the three species
have high Ks values (.1; Supplemental Table S2), indicating that these genes may have resulted from one ancient WGD event. Because the most recent WGD event
shared by monocots and dicots occurred in the common
ancestor of all angiosperms (Jiao et al., 2011; Vanneste
et al., 2014), the syntenic data and phylogenetic results
support that RNL genes separated into two lineages
sometime before this time point.
Deciphering the Evolution of Arabidopsis NBS Genes
By performing phylogenetic analyses at different
evolutionary nodes, we have established a framework
for the evolution of angiosperm NBS genes, which
could help decipher the evolution of NBS genes in
specific species as well as gain a much deeper view of
the evolution of functionally characterized NBS genes.
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Shao et al.
Figure 5. Comparison of the evolutionary
patterns of CNL and TNL classes in angiosperms. A, Reconstruction of the NBS gene
loss and gain events during angiosperm
evolution. The number of ancestral NBS
genes at each node is depicted in a solid
circle. The thickness of each terminal
branch is roughly proportional to the
number of NBS genes in the genome or the
ancestral genes in the family. Gene loss and
gain events at different evolutionary states
are indicated by minus and plus symbols,
respectively. An asterisk after the number
indicates that the number is not directly
recovered from phylogenetic analysis. B,
Extensive expansion of CNL and TNL
classes at the K-P boundary. The numbers
of CNL (red circles) and TNL (blue circles)
genes at different divergence nodes above/
at family level, including all angiosperms
(An), mesangiospermae (Ma), monocots
(Mo), eudicots (Ed), rosids (Ro), asterids
(As), Poaceae (P), Solanaceae (S), Fabaceae
(F), and Brassicaceae (B), were plotted to
reveal their expansion history.
In total, five out of 14 An-CNL genes were inherited
by the Arabidopsis genome (Fig. 7). Gradual duplications occurred in four of these An-CNLs (An-C2, -C5,
-C6, and C8), with An-C5 and An-C6 expanding to a
few copies. Two An-CNLs (An-C2 and An-C8) produced more than 80% of Arabidopsis CNL genes,
mainly occurring prior to the divergence of the Brassicaceae family. One CNL gene (At3g07040) represented
an ancient gene (An-C14) that was conserved from the
angiosperm ancestor to Arabidopsis without duplication. Together, the evolutionary paths of the five ancestral CNL genes resulted in the current CNL profile of
Arabidopsis genome (Fig. 7).
The structural changes were also traced using the
evolutionary framework of angiosperm NBS genes. For
example, nearly all Arabidopsis CNL genes showed an
intronless structure regardless of their origin (Meyers
et al., 2003), which is in accordance with our proposed
intronless structure for the ancestral angiosperm CNL
gene. However, a group of Arabidopsis CNL genes
were determined to possess two shared introns. All
these genes originated from a lineage that diverged
from An-C8 (Fig. 7) in the common ancestor of
mesangiospermae, with the sister lineage remaining
intronless. Based on the phylogenies of CNLs at different taxonomic levels, the two introns were frequently
detected in orthologs of Brassicaceae species. The first
intron also was found in orthologous genes of the legume family, grape, and the asterid species, but they
were absent in monocots and A. trichopoda. Therefore,
the exon-intron structure detected in these Arabidopsis
CNL genes gradually formed from two independent
intron gain events, one in the common ancestor of
eudicots and the other acquired in the common ancestor of Brassicaceae (Fig. 7).
Furthermore, the angiosperm evolutionary framework of NBS genes enabled us to determine when a
functional NBS gene likely arose during angiosperm
evolution. Five functional CNL genes against different
pathogens have been identified in Arabidopsis (Liu
et al., 2007). According to the recovered evolutionary
history of Arabidopsis CNL genes in Figure 7, RPM1
was the most ancient one among the five genes, possibly originating from the common ancestor of angiosperms, whereas the RPP8/HRT was most recent,
possibly originating after Arabidopsis speciation. The
origin of the other three genes could be traced to the
divergence nodes of rosids, Brassicaceae, and Arabidopsis. Among the five genes, RPS2, RPS5, and RPM1
were R genes against P. syringae, whereas RPP8/HRT
and RPP13 were both against Peronospora parasitica,
indicating functional R genes continuously arose
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Evolution of NBS-LRR Genes in Angiosperms
Figure 6. Evolutionary history of RNL genes inferred from phylogenetic and syntenic analyses. Top, The inheritance of the two
RNL lineages in different angiosperm lineages. The inferred duplication is marked with a green circle, whereas inferred losses are
indicated by black circles. Bottom, Intra- and intergenome syntenic blocks containing RNL genes detected within and among
P. vulgaris, S. lycopersicum, and S. bicolor genomes.
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Shao et al.
Figure 7. A few ancestral angiosperm NBS genes that are responsible in generating the current Arabidopsis CNL repertoire via
gradual expansion. The ancient states of Arabidopsis at different evolutionary nodes were retrieved from the constructed NBS
phylogenies. Offspring generated from the same ancestral gene at different divergence nodes are indicated by boxes of the same
color. Black triangles indicate the birth of known functional genes. Gray arrows indicate intron gain events. The exon-intron
structure of each ancestral lineage is shown.
against the same pathogen during plant evolution.
Actually, apart from these genes, many other functional
genes may have appeared in the history and then been
lost during plant evolution. However, such cases often
could not be traced due to their absence in model plant
genomes.
The adoption of the angiosperm NBS evolutionary
framework in Arabidopsis provides new insights into
the evolutionary history of the NBS gene in this species.
To facilitate the use of this framework in the analysis of
NBS genes in other plants that were not included in this
study, we generated hidden Markov model (HMM)
profiles (Supplemental Dataset S1) for the 23 ancestral
NBS lineages of angiosperms. Taken together, the angiosperm NBS evolutionary framework established in
this study provides a fundamental reference for the
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Evolution of NBS-LRR Genes in Angiosperms
evolutionary paths, duplication patterns, and possible
functionalization time frames for a deeper understanding of NBS genes in various plant species.
DISCUSSION
Three Ancient NBS Gene Classes in the Ancestral
Angiosperm Genome
In this study, we performed genome-wide analyses
of NBS genes in 22 species that belonged to major angiosperm clades. We identified 105 NBS genes from the
A. trichopoda genome, and detected all three NBS classes
(TNL, CNL, and RNL) in this basal angiosperm species.
Unlike the well-separated TNL and CNL classes, the
phylogenetic place of RNL genes has not been fully
established. For a long time, RNL genes were treated as
a special lineage of CNLs, but with their specific function and distinct domain composition, these genes have
only been recently differentiated (Meyers et al., 2003;
Bonardi et al., 2011; Collier et al., 2011). Recent studies
involving legumes and the Brassicaceae have revealed a
sister relationship between RNLs and CNLs (Shao et al.,
2014; Zhang et al., 2016). However, whether this NBS
class was derived from an ancestral CNL gene before
the divergence of the rosid lineage or if these genes
were of a more ancient origin remained elusive. If the
former case were true, then we would expect RNLs
nested as a sublineage of angiosperm CNLs; otherwise,
a sister relationship would be consistently detected
with different datasets. To clarify this issue, CNLs and
RNLs were combined in phylogenetic analysis in this
study that supports the sister relationship stemming
from an ancient origin, prior to the divergence of the
ancestral angiosperm. Class-specific signatures were
further detected among the CNL, RNL, and TNL classes at several motifs in the conserved NBS domain (Fig.
3), which was suggestive of an ancient separation of the
three NBS classes. Furthermore, distinct intron-exon
structures among the three NBS classes provide additional evidence that supports the presence of three ancient classes of NBS genes in the ancestral angiosperm.
Differential Expansion during Angiosperm Evolution
Shaped TNLs and CNLs
Although both TNL and CNL genes could be dated
back to at least the arrival of land plants (Akita and
Valkonen, 2002; Xue et al., 2012) and were inherited by
the common ancestor of angiosperms, the TNL genes
are apparently outnumbered by CNLs in most angiosperms, except for the Brassicaceae (Zhang et al., 2016).
The phylogeny of TNLs also showed a lower level of
topological complexity and a relatively shorter branch
length compared to that in CNLs. All these discrepancies reflect different evolutionary patterns for TNLs
and CNLs, and the results of this study suggest that the
long-term contraction of TNLs and gradual expansion
of CNLs in the early stages of angiosperm evolution
(from ;225 MYA as angiosperms diverged to around
100 MYA as asterids, rosids, and monocots diverged)
may have been responsible for these differences. While
,10 TNL genes were detected in rosids, eudicots, and
probably mesangiospermae and asterid nodes, the
number of CNL genes rapidly expanded to 35 early in
the common ancestor of mesangiospermae and increased to 47 at the emergence of dicots (Fig. 5).
The long-term steady number of TNL genes also explains their independent loss in several angiosperm lineages. The absence of TNLs was first reported in cereals
and major monocot lineages (Bai et al., 2002; Zhou et al.,
2004; Yang et al., 2006; Li et al., 2010), then in two eudicot
species, including an asterid, Mimulus guttatus, and a
basal eudicot, Aquilegia coerulea (Collier et al., 2011). Because both the basal angiosperm A. trichopoda and eudicots harbor TNL genes, it is highly likely that the monocot
lineage lost its TNLs near the dicot/monocot split.
M. guttatus and S. indicum are from two different families
of Lamiales, indicating that the Lamiales lineage might
have lost its TNL genes soon after its separation with its
sister lineage, Solanales. The presence of TNL genes in
basal angiosperms and in both the rosid and asterid lineages of eudicots suggests that the loss of TNLs in the
basal eudicot A. coerulea also stems from an independent
loss event. Therefore, at least three independent gene loss
events occurred during angiosperm evolution. Furthermore, the near singleton status of TNL genes at the early
stages of angiosperm evolution suggests that these events
may have possibly occurred. Otherwise, deletion of an
entire gene family consisting of dozens of members in
three independent lineages would be unlikely, especially
when these were dispersed in the genome.
Another interesting finding is that both CNLs and
TNLs exhibit a recent expansion prior to the divergence
of angiosperm families, with more than 100 NBS genes
independently arising prior to the separation of each of
the four families investigated in this study. Because the
divergence of these angiosperm families occurred in the
Paleogene and the divergence of their higher level
taxonomies all occurred in the Cretaceous (Fig. 1; Lavin
et al., 2005; Stefanovic et al., 2009; D’Hont et al., 2012;
Zhang et al., 2012; Peng et al., 2013; Yang et al., 2013;
Kim et al., 2014; Wang et al., 2014; Zeng et al., 2014), the
observed intensive expansion of NBS genes in angiosperms more likely coincided with the CretaceousPaleogene (K-P) boundary (;66 MYA). This finding
suggests that different lineages of angiosperms either
underwent a process to reinforce resistance or experienced increased selective pressure from environmental
pathogens during this period. The K-P boundary is an
important period when mass extinction greatly impacted the evolution of various species. Angiosperms
underwent drastic genomic and phenotypic changes to
survive the climatic and environmental changes in this
period, including prevalently detected WGDs across
different angiosperm lineages and increased leaf vein
density (Blonder et al., 2014; Vanneste et al., 2014).
A hypothesis has been proposed that the documented
bloom in fungal diversity during this period triggered
the decline of reptiles, as well as promoted the expansion
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Shao et al.
of mammalian species, which have a more evolved
immune system and body temperature (Vajda and
McLoughlin, 2004; Casadevall, 2012). Actually, most
fungal families diverged during the Cretaceous, and
most fungal genera diverged at the K-P boundary, such
as Botryosphaeriacea and Fusarium, which are two
fungal groups with many species that cause severe
pathogenicity (Ma et al., 2013; Slippers et al., 2013;
Hedges et al., 2015). Therefore, the fungal bloom during
the K-P boundary may have enhanced the selection
pressure on plant R genes, which consequently accelerated NBS gene expansions at this period, prior to the
divergence of angiosperm families.
An Ancient WGD Gave Rise to Two Functionally
Divergent RNL Lineages
WGDs frequently occur during plant evolution (Jiao
et al., 2011; Li et al., 2015; Qian et al., 2015). However,
evidence from various studies has revealed that most
NBS duplicons that originate from polyploidy are
preferentially lost during evolution, possibly to avoid
fitness costs (Bergelson et al., 2001; Cannon et al., 2004;
Nobuta et al., 2005; Ashfield et al., 2012; Shao et al.,
2014). Therefore, NBS genes resulting from ancient
WGD are rarely detected. Our phylogenetic analysis
has revealed that RNL genes were derived from two
ancient lineages, namely, ADR1 and NRG1, which
separated prior to the divergence of angiosperms. By
surveying the syntenic regions in P. vulgaris and
S. lycopersicum genomes, we determined that ADR1 and
NRG1 orthologous genes were located on syntenic
blocks in both genomes, suggesting that the two genes
resulted from an ancient WGD. Despite having lost the
NRG1 orthologs, the two syntenic blocks were also
detected in the monocot genome of S. bicolor, suggesting that the WGD event was shared by dicots and
monocots. Recent studies involving angiosperms have
indicated that the most recent WGD shared by monocots and dicots occurred in the common ancestor of
angiosperms (Jiao et al., 2011; Vanneste et al., 2014).
Therefore, although syntenic blocks that contain ADR1
and NRG1 orthologs were not detected in A. trichopoda,
our data suggest that the WGD contributing to the
generation of two RNL lineages occurred in the ancestral angiosperm. This finding also is validated by
the results of phylogenetic analysis, which places the
one RNL gene in A. trichopoda in the NRG1 lineage with
high statistical support but not basal to all RNL
genes (Supplemental Fig. S9). The absence of an ADR1
orthologous gene in A. trichopoda might be attributable
to either the incomplete coverage of the genome sequence or a species-specific gene loss. By searching the
transcriptome data of the 1,000 plants project (1KP; www.
onekp.com), we determined that both ADR1 and NRG1
orthologous genes are prevalently distributed in various
basal angiosperms (data not shown). Taken together, both
the phylogenetic analysis and the synteny results indicate
that an ancient WGD involving the ancestral angiosperm
gave rise to the two RNL lineages.
Functional RNL genes have been identified in both
RNL lineages: ADR1 in Arabidopsis and NRG1 in tobacco. However, interestingly, neither ADR1 nor NRG1
functions as a canonical R gene that detects specific
pathogens (Collier et al., 2011). ADR1 and its paralogs
are considered helper genes that transduce signals
downstream from other R genes (Bonardi et al., 2011).
Similarly, NRG1 assists the N gene in fighting against
the Tobacco mosaic virus (Peart et al., 2005). Although
neither ADR1 nor NRG1 acts as a pathogen detector,
these are indispensable in resistance reactions. The
distinct function, extremely conserved sequence, and
gene structure suggest a downstream and crucial role
for RNLs in disease resistance pathways. The simultaneous loss of TNL and NRG1 orthologs in M. guttatus,
A. coerulea, and the Poaceae family has been speculated
to be due to their close functional interdependence
(Collier et al., 2011). It has been hypothesized that TNL
genes only transfer their signal to NRG1 genes, whereas
CNL genes could transfer their signal through either the
ADR1 or NRG1 gene. The observed functional differentiation of ADR1 and NRG1 lineages suggests that
divergence occurred after the two RNL copies emerged
in the ancestral angiosperm WGD. As signal transduction protein-encoding genes, RNL genes are not
involved in recognizing specific pathogens and thus are
not under the selection of pathogens. Our results show
that RNL lineages only kept a few copies in their genomes, except in several rosid species. The different
evolutionary patterns among the three NBS classes reflect their distinct functions as either pathogen detectors
or signal transducers, which further suggests that the
NBS family diverged early to contribute to the plant
immune system from multiple aspects.
An Angiosperm Framework for Understanding NBS
Gene Evolution
Most previous studies of NBS genes were mainly
performed on individual species, which hindered in
better understanding NBS gene evolution at the wholeangiosperm scale. The results reported in this study
(covering .6,000 NBS genes from 22 genomes) provide
a fundamental phylogenetic framework of NBS genes
in angiosperms. Because the whole dataset is too large
for phylogenetic analysis and subsequent gene loss/
gain reconcile, we used a step-by-step method for
phylogenetic analysis as described earlier. In such
procedure, the phylogenetic relationships among all
angiosperm NBS genes could not be fully displayed in a
single tree, and supporting values for some internal
nodes may decrease because only a few representative
sequences were used in the construction of high taxonomic level NBS phylogenies. However, our data suggest that the results from this approach are generally
robust, and the taxonomy of NBS genes often were not
affected by different datasets used. For example, one of
our major findings, the sister relationship of CNL and
RNL, is well supported in nTNL phylogenies constructed by different datasets (Supplemental Figs. S1,
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Evolution of NBS-LRR Genes in Angiosperms
S3, S4, S6, and S9). Furthermore, during the procedure,
with more data incorporated, the shared introns in
a CNL lineage (An-C8; Fig. 7) also were in perfect
agreement with its phylogenetic relationships. Therefore, to a large extent, the phylogenetic framework of
angiosperm NBS provided in this study is reliable for
the estimation of the evolutionary history of this large
gene family. Using this framework as reference, NBS
gene profiles in a particular plant genome could be fully
classified to better understand the evolutionary dynamics. For example, the 52 CNL genes present in the
Arabidopsis genome were traced to five ancestral CNL
genes in the common ancestor of angiosperms (Fig. 7).
Gradual changes in gene content in these ancestral
lineages were observed at five intermediate divergence
nodes and the node to Arabidopsis speciation, which
not only revealed different fates for the five ancestral
genes during their evolution but also uncovered the
extent of gene expansion at different nodes.
Furthermore, the five known Arabidopsis functional
CNL genes were traced to four ancient NBS genes and
arose from different angiosperm nodes (Fig. 7). Notably, two of the five R genes, RPS2 and RPM1, defend
against P. syringae by guarding the Arabidopsis RIN4
protein. The state change of RIN4 that is monitored by
RPS2 and RPM1 also are modified by two different effectors of P. syringae (Mackey et al., 2002; Mackey et al.,
2003), suggesting a long-term coevolutionary arms race
among Arabidopsis R genes and P. syringae (Fig. 7).
Angiosperms may have first evolved the RPM1 gene as
a mechanism against P. syringae infection by detecting
effector (AvrRpm1)-induced phosphorylation of RIN4.
On the other hand, P. syringae evolved another effector
(AvrRpt2) to cleave the RIN4 protein and evade the
activation of RPM1, and plants further evolved another
CNL gene (RPS2, probably prior to the node of rosid
divergence) to monitor the cleavage of RIN4 and activate the immune system. The long history of R genes
guarding RIN4 also suggests that this protein is an
ancient target of pathogens, which in turn unravels its
important function in plant immune signal transduction (Liu et al., 2009; Hou et al., 2011). It should be noticed that although we traced the emergence time of
these genes in angiosperm lineages, we were not able to
accurately determine when they obtained specific
functions. The resistance specificity may have existed
prior to the duplication event but subsequently been
lost from some duplicated copy; alternatively, the
specificity may have arisen after the duplication event.
In conclusion, by identifying more than 6,000 NBS
genes from 22 angiosperm species, this study has successfully constructed the phylogeny of angiosperm
NBS genes. A total of 23 ancestral NBS genes were
identified in angiosperm ancestors, and three distinct
NBS classes were revealed. Although exhibiting different evolutionary patterns, both TNL and CNL genes
underwent extensive expansion at the K-P boundary. In
contrast, RNL genes conservatively evolved during
angiosperm evolution, which mirrors their distinct
pathogen detection or signal transduction roles in plant
disease resistance. The angiosperm level NBS phylogenetic framework constructed in this study provides
a fundamental reference for further studies on NBS
genes.
MATERIALS AND METHODS
Database
The genomic sequences, annotations, and gene models for 13 out of 22 genomes (except four Fabaceae and five Brassicaceae genomes) were used in this
study. Among these, data on Vitis vinifera, Solanum lycopersicum, Solanum
tuberosum, Brachypodium distachyon, Setaria italica, Sorghum bicolor, Oryza sativa,
and Zea mays were downloaded from the Phytozome database (http://www.
phytozome.net/; Goodstein et al., 2012). Files of Amborella trichopoda, Capsicum
annuum, Sesamum indicum, Phyllostachys heterocycla, and Musa acuminata were
downloaded from the Pepper Genome Database (http://peppersequence.
genomics.cn/page/species/index.jsp; Qin et al., 2014), Sinbase (http://ocrigenomics.org/Sinbase/manual.htm; Wang et al., 2014), BambooGDB (http://
www.bamboogdb.org/page/download.jsp; Peng et al., 2013), Banana Genome
Hub (http://banana-genome.cirad.fr; D’Hont et al., 2012), and the Amborella
genome database (http://www.amborella.org; Amborella-Genome-Project,
2013), respectively.
Identification of NBS-Encoding Genes
The NBS genes in the 13 genomes were identified as described in previous
studies (Shao et al., 2014; Shao et al., 2015; Zhang et al., 2016). Briefly, a HMM
search was first performed for protein sequences in each genome using
hmmer3.0 (http://hmmer.org) using default parameter settings, with the
NB-ARC domain (Pfam: PF00931) as query. The amino acid sequence of the
NB-ARC domain was then used to run a BLASTp search against all protein
sequences in each genome, with the threshold expectation value set to 1.0. All
hits obtained using HMM or BLAST searches were then merged together, and
the redundant hits were removed. The remaining sequences were further
subjected to Pfam analysis (http://pfam.sanger.ac.uk/) to further exclude sequences that do not have a detectable NBS domain at an E value of 1024. When
two or more transcripts were annotated for a gene from alternative splicing, the
longest form with an NBS domain was selected. Finally, the identified NBS
domain-encoding genes were examined to see whether these encode the TIR,
RPW8, CC, or LRR domains using Pfam and COILS analyses (Lupas et al.,
1991). The exon position and intron phase of each gene were transformed from
the gff3 file of the reference genome.
Sequence Alignment and Phylogenetic Analysis
Sequence alignment and phylogenetic analysis were performed as described
previously (Shao et al., 2014). Amino acid sequences of the NBS domain were
aligned using ClustalW (Edgar, 2004) with default options (Thompson et al.,
1994) and then manually corrected in MEGA 5.0 (Tamura et al., 2011). The
resulting amino acid sequence alignments were used to guide the alignments of
the nucleotide coding sequences. Genes with very short NBS domains were
eliminated from the matrix because these interfered in fine alignment and
subsequent construction of phylogenetic trees. All alignments used for phylogenetic analysis in the presented study are provided in Supplemental Dataset
S2. Phylogenetic analysis was conducted using a maximum likelihood method
as provided in the Phyml program (Guindon and Gascuel, 2003), which was
integrated in the Seaview package (Gouy et al., 2010). Branch support was
assessed with the aLRT statistic (Guindon et al., 2010). Gene losses and gains at
each divergence node were reconciled using Notung software (Stolzer et al.,
2012). The number of ancestral NBS genes at different divergence nodes was
determined using monophyletic NBS lineages as described elsewhere (Shao
et al., 2014).
Because merging all data generates large datasets for both nTNL and TNL
classes, we adopted a step-by-step strategy to recover the full picture of NBS gene
evolution in angiosperms. Generally, phylogenetic analysis was first performed
at a low taxonomy level, mainly containing NBS genes from a few genomes.
Then, representative NBS sequences from these angiosperm lineages were
compiled with NBS genes in more plant genomes to construct the NBS gene
phylogenies at higher taxonomy levels, finally reaching the whole-angiosperm
level.
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The conserved motifs within the NBS domain of three NBS classes were
identified from the amino acid sequence using Multiple Expectation Maximization for Motif Elicitation (Bailey and Elkan, 1995), and the generated sequence
logos were manually modified to compare their differences among different
classes.
Synteny Analyses
Genome synteny between duplicate gene pairs was examined by searching the
Plant Genome Duplication Database (http://chibba.agtec.uga.edu/duplication/;
Tang et al., 2008).
Supplemental Data
The following supplemental materials are available.
Supplemental Figure S1. Phylogenetic tree of monocot nTNL genes.
Supplemental Figure S2. Phylogenetic tree of asterid nTNL genes.
Supplemental Figure S3. Phylogenetic tree of asterid TNL genes.
Supplemental Figure S4. Phylogenetic tree of rosid nTNL genes.
Supplemental Figure S5. Phylogenetic tree of rosid TNL genes.
Supplemental Figure S6. Phylogenetic tree of dicot nTNL genes.
Supplemental Figure S7. Phylogenetic tree of dicot TNL genes.
Supplemental Figure S8. Phylogenetic tree of mesangiospermae nTNL
genes.
Supplemental Figure S9. Phylogenetic tree of angiosperm nTNL genes.
Supplemental Figure S10. Phylogenetic tree of angiosperm TNL genes.
Supplemental Table S1. NBS-encoding genes detected in angiosperm genomes examined in this study.
Supplemental Table S2. Inter- or intraspecies RNL-containing syntenic
blocks.
Supplemental Dataset S1. HMM profiles for the 23 ancestral NBS lineages.
Supplemental Dataset S2. Alignments used for phylogenetic analysis in
this study.
Received September 18, 2015; accepted February 1, 2016; published February 2,
2016.
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