Long-term evolution of nucleotide-binding site-leucine-rich repeat genes: understanding gained from and beyond the legume family

Abstract

Proper utilization of plant disease resistance genes requires a good understanding of their short- and long-term evolution. Here we present a comprehensive study of the long-term evolutionary history of nucleotide-binding site (NBS)-leucine-rich repeat (LRR) genes within and beyond the legume family. The small group of NBS-LRR genes with an amino-terminal RESISTANCE TO POWDERY MILDEW8 (RPW8)-like domain (referred to as RNL) was first revealed as a basal clade sister to both coiled-coil-NBS-LRR (CNL) and Toll/Interleukin1 receptor-NBS-LRR (TNL) clades. Using Arabidopsis (Arabidopsis thaliana) as an outgroup, this study explicitly recovered 31 ancestral NBS lineages (two RNL, 21 CNL, and eight TNL) that had existed in the rosid common ancestor and 119 ancestral lineages (nine RNL, 55 CNL, and 55 TNL) that had diverged in the legume common ancestor. It was shown that, during their evolution in the past 54 million years, approximately 94% (112 of 119) of the ancestral legume NBS lineages experienced deletions or significant expansions, while seven original lineages were maintained in a conservative manner. The NBS gene duplication pattern was further examined. The local tandem duplications dominated NBS gene gains in the total number of genes (more than 75%), which was not surprising. However, it was interesting from our study that ectopic duplications had created many novel NBS gene loci in individual legume genomes, which occurred at a significant frequency of 8% to 20% in different legume lineages. Finally, by surveying the legume microRNAs that can potentially regulate NBS genes, we found that the microRNA-NBS gene interaction also exhibited a gain-and-loss pattern during the legume evolution.

Figure 1.

The phylogenetic tree of four investigated legume species (M. truncatula, pigeon pea, common bean, and soybean). Two WGD events are indicated with triangles: one occurred approximately 59 MYA in the common ancestor of the four investigated legumes, and the other occurred approximately 13 MYA in the Glycine spp. lineage alone (Schmutz et al., 2010). The numbers at the branch nodes indicate divergence times (Lavin et al., 2005; Stefanovic et al., 2009). [See online article for color version of this figure.]

Figure 2.

Phylogenetic relationships of NBS-encoding genes in each of the four legume genomes. The TNL clade (blue), CNL clade (red), and RNL clade (black) are shown. The scale bars represent numbers of nucleotide substitutions per site. Support values (SH-aLRT values) for basal nodes are indicated.

Figure 3.

Phylogenetic tree of nTNL subclass NBS genes based on conserved NBS domain sequences. A total of 842 nTNL subclass NBS sequences were used: 202 sequences from M. truncatula (shown in red), 131 from pigeon pea (blue), 216 from common bean (orange), 234 from soybean (green), and 59 from Arabidopsis (At; black). Two TNL sequences from Arabidopsis (purple) were used as an outgroup. The reconstructed nTNL phylogeny is divided into 23 nTNL-rosid NBS gene families and 64 nTNL-legume NBS gene families (see “Materials and Methods”). The presence of Arabidopsis or legume sequences in nTNL-rosid families is indicated with check marks. The scale bar represents the number of nucleotide substitutions per site. The support values (SH-aLRT values) for basal nodes are indicated.

Figure 4.

Phylogenetic tree of TNL subclass NBS genes based on conserved NBS domain sequences. A total of 623 nTNL subclass NBS sequences were used: 197 sequences from M. truncatula (shown in red), 95 from pigeon pea (blue), 88 from common bean (orange), 144 from soybean (green), and 99 from Arabidopsis (At; black). Two nTNL sequences from Arabidopsis (purple) were used as an outgroup. The reconstructed TNL phylogeny is divided into eight TNL-rosid NBS gene families and 55 TNL-legume NBS gene families (see “Materials and Methods”). The presence of Arabidopsis or legume sequences in nTNL-rosid families is indicated with check marks. The scale bar represents the number of nucleotide substitutions per site. The support values (SH-aLRT values) for basal nodes are indicated.

Figure 5.

Number variations of nTNL and TNL subclass NBS genes at different stages of legume evolution. Differential gene losses and gains are indicated by numbers with − or + on each branch. WGD events are indicated with triangles as in Figure 1. [See online article for color version of this figure.]

Figure 6.

A typical example of Medicago spp. NBS gene evolution showing three different duplication types: local tandem duplication, ectopic duplication, and segmental duplication. A, Collinearity shared by Medicago spp. NBS gene loci 68 and 69 on chromosome 5 and locus 134 on chromosome 8. NBS genes are indicated by red boxes, non-NBS genes are indicated by white boxes, with syntenic genes linked by lines. B, Phylogenetic analysis revealed three monophyletic NBS gene groups (A, B, and B′). Two relevant Arabidopsis sequences were used as an outgroup, and all major nodes were supported with high confidence. C, A postulated evolutionary history of these NBS genes: steps 1 and 2 occurred before the Medicago spp. lineage separated from the other legumes, and step 3 occurred mainly in the Medicago spp. lineage.

Figure 7.

An integrated map of NBS gene loci showing synteny relationships among four legume genomes. The eight chromosomes of M. truncatula are used as background to map NBS loci from different legume genomes to their syntenic positions. The NBS loci are indicated by squares in different colors: red, M. truncatula; blue, pigeon pea; yellow, common bean; and green, soybean. If two or more loci are mapped to one syntenic position, then a slash is added to the square.

Figure 8.

An evolutionary history of the legume nTNL family containing known functional genes Rpg1-b and Rsv1. A, The reconstructed phylogeny of NBS genes belonging to nTNL-legume family 45 with the soybean Rsv1 gene incorporated. A total of 15 subfamilies that had evolved in the common ancestor of pigeon pea, common bean, and soybean are labeled. Soybean Rpg1-b and Rsv1 are indicated by red triangles. B, NBS gene subfamilies were differentially lost among later-evolving legume lineages. Common ancestors (α, β, and γ) are indicated by pink dots. The presence/absence of 15 subfamilies in pigeon pea, common bean, soybean, and common ancestor β and γ are indicated with purple/gray bars. C, Graph showing the recombination event that occurred on soybean Rpg1-b. Major parental sequence (PI96983 Rsv1) and minor parental sequence (13g26310) are shown in light blue and purple, respectively. Also, the sequence recombination boundary before the NBS domain is shown with a 111-bp-long alignment.

Figure 9.

Predicted regulation of microRNAs on NBS genes in M. truncatula and soybean. A, Summary of microRNA families predicted to target NBS genes in M. truncatula and soybean. The prediction process was performed by using the psRNATarget server with default settings, in which both the mismatch penalty (expectation value) and target accessibility (allowed maximum energy to unpair the target site) were taken into consideration. B, Numbers of TNL and nTNL subclass NBS genes that were predicted to be targeted by microRNAs in M. truncatula and soybean. C, Localization of microRNA target sites at different domains of NBS genes. D, An example showing the predicted NBS gene regulation pattern within nTNL-legume family 45 by three major microRNAs (miR5376, miR1510, and miR159). Certain NBS gene members were predicted to be regulated by the three microRNAs (shown with red triangles), and they all meet two conditions: the calculated mismatch penalty (expectation value) is no more than 3, and the estimated target accessibility value does not exceed 25 kcal mol⁻¹. Nucleotide mutations, insertions, and deletions at the potential targeting sites often could be detected on genes that were not predicted to be targeted by a given microRNA. All missing nucleotides are indicated with dashes.