Large-Scale Analyses of Angiosperm Nucleotide-Binding Site-Leucine-Rich Repeat Genes Reveal Three Anciently Diverged Classes with Distinct Evolutionary Patterns

Abstract

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 exon-intron structures and DNA motif sequences. A total of seven ancient TNL, 14 CNL, and two RNL lineages were discovered in the ancestral angiosperm, from which all current NBS-LRR gene repertoires were evolved. A pattern of gradual expansion 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.

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.

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.

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.

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.

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.

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.

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.