Large-scale, lineage-specific expansion of a bric-a-brac/tramtrack/broad complex ubiquitin-ligase gene family in rice

Abstract

Selective ubiquitination of proteins is directed by diverse families of ubiquitin-protein ligases (or E3s) in plants. One important type uses Cullin-3 as a scaffold to assemble multisubunit E3 complexes containing one of a multitude of bric-a-brac/tramtrack/broad complex (BTB) proteins that function as substrate recognition factors. We previously described the 80-member BTB gene superfamily in Arabidopsis thaliana. Here, we describe the complete BTB superfamily in rice (Oryza sativa spp japonica cv Nipponbare) that contains 149 BTB domain-encoding genes and 43 putative pseudogenes. Amino acid sequence comparisons of the rice and Arabidopsis superfamilies revealed a near equal repertoire of putative substrate recognition module types. However, phylogenetic comparisons detected numerous gene duplication and/or loss events since the rice and Arabidopsis BTB lineages split, suggesting possible functional specialization within individual BTB families. In particular, a major expansion and diversification of a subset of BTB proteins containing Meprin and TRAF homology (MATH) substrate recognition sites was evident in rice and other monocots that likely occurred following the monocot/dicot split. The MATH domain of a subset appears to have evolved significantly faster than those in a smaller core subset that predates flowering plants, suggesting that the substrate recognition module in many monocot MATH-BTB E3s are diversifying to ubiquitinate a set of substrates that are themselves rapidly changing. Intriguing possibilities include pathogen proteins attempting to avoid inactivation by the monocot host.

Figure 1.

Grouping of the BTB Proteins Based on the Nature of Additional Motifs Flanking the BTB Domain, along with a Protein Composition Diagram of Representative Members. Ank, ankyrin; Arm, armadillo; CC, coiled-coil; Pent, pentapeptide; CaM BD, calmodulin binding domain; TPR, tetratricopeptide repeat.

Figure 2.

Phylogenetic Trees of the Complete BTB Protein Superfamilies from Rice and Arabidopsis. Alignments of the ∼110–amino acid BTB domains were used to generate midpoint rooted NJ trees. The subfamilies identified from the phylogenetic analysis are marked on the bottom. Individual members of the tree are color-coded by the nature of the domains appended to the BTB domain. Closed circles indicate rice-specific subfamilies. The closed diamond indicates the Arabidopsis-specific E2 subfamily. Arrowheads identify BTB proteins confirmed to directly interact with CUL3. Asterisks indicate motifs found to be associated with BTB domains only in plants. Boxes on the nodes of the phylogenetic trees indicate moderate (≥65%, gray) or strong (≥90%, black) bootstrap support from 1000 replicates. Expanded views of the trees with the branches labeled with sequence identifiers and bootstrap values are in Supplemental Figure 10 online.

Figure 3.

Phylogenetic Trees of 135 MATH-BTB Proteins from Representative Land Plant Species. Alignments of the ∼110–amino acid BTB domains or the ∼110–amino acid MATH domains were used to generate NJ phylogenetic trees. The core group is highlighted by the gray ovals. Numbers at the internal branches leading to the core group indicate percentage of bootstrap support from 1000 replicates. Expanded views of the trees with the branches labeled with sequence identifiers are in Supplemental Figure 11 online as well as the color code for the species. (A) BTB domain tree color-coded by the species. (B) MATH domain tree color-coded by the species. (C) BTB domain tree color-coded by the number of introns.

Figure 4.

Gene Structure and Expression of the Core and Expanded MATH-BTB Groups in Land Plants. (A) Gene diagrams for representative MATH-BTB genes. Black or gray boxes denote exons, white boxes untranslated regions, and solid lines indicate introns. Dashed lines indicate homologous exons. At, Arabidopsis thaliana; Mt, Medicago truncatula; Os, Oryza sativa; Pp, Physcomitrella patens; Pot, Populus trichocarpa; Sb, Sorghum bicolor; and Sm, Selaginella moellendorffi. (B) Expression analysis for rice MATH-BTB core and expanded groups and rice A1 subfamily pseudogene loci. The value in each box indicates the number of different tissues in which full-length cDNAs (FL-cDNAs) or ESTs were identified, the number of libraries in which significant expression was detected in the rice MPSS database, or the RICEATLAS whole-genome oligoarray expression data set. Shade of the boxes denotes level of expression (see Methods). A question mark indicates that significant expression was detected but that tag sequence (MPSS) or oligonucleotide (RICEATLAS) matched multiple locations in the genome. A slash indicates that a locus was not represented in the data set.

Figure 5.

Chromosomal Clustering of the Expanded MATH-BTB Genes and Related Pseudogenes. The clusters include 54 of the 70 functional expanded MATH-BTB and MATH-related-BTB genes and 32 of 36 pseudogenes based on The Institute for Genomic Research (TIGR) rice pseudomolecules (Osa1, release 4). The positions of the segments in the rice chromosomes 8, 10, and 11 are indicated on the left. An expanded view of this figure with sequence identifiers for each locus is in Supplemental Figure 12 online.

Figure 6.

Interaction of Rice Expanded Group MATH-BTB Proteins with CUL3 Proteins. (A) Y2H analyses of rice Os04g53410, Os08g13070, Os10g29110, and Os10g29310 with truncated Os CUL3b and full-length At CUL3a and At CUL3b by growth selection at 23°C for 4 d on 7 mM 3-amino-1′,2′,3′-triazole. Specificity was confirmed by Y2H with At CUL1. pGBKT7 and pGADT7 are the AD and BD vectors without inserts. pGBKT7/p53 and pGADT7/COIL express unrelated proteins and are included as negative controls. (B) Diagram of the CUL3 proteins used in the Y2H analysis. The positions of the various signature domains are indicated. aa, amino acids.

Figure 7.

Inference of Positively Selected Sites in Core and Expanded MATH Domains. Alignments of 31 core and 104 expanded ∼110–amino acid MATH domains were generated in ClustalW and displayed with MacBoxshade using a threshold of 55% sequence identity (see Supplemental Figure 8 online). Top panels: Representative sequences from the alignments. Conserved and similar amino acids are shown in black and gray boxes, respectively. Dots denote gaps. Bottom panels: Histograms showing the maximum likelihood K_A/K_S ratios calculated for each gap-free position in the alignments. The dotted lines indicate a K_A/K_S ratio of 1.0. Sites under likely positive selection (K_A/K_S ≥ 1.0, P < 0.1) are marked with asterisks. The arrowhead indicates a highly conserved Trp residue located within a region containing positions under positive selection in both monocot and C. elegans MATH domains (Thomas, 2006).

Figure 8.

K_A/K_S Ratio Analysis of Plant Core and Monocot Expanded Group MATH and BTB Domains. (A) Phylogenetic trees of phylogenetically clustered MATH-BTB sequences were constructed, and the ratio of nonsynonymous (K_A) to synonymous (K_S) distance was calculated in each branch. Closed circles and squares represent the estimated MATH and BTB domain K_A/K_S ratios for one branch in the trees for the expanded and core group, respectively. The solid line indicates 1-to-1 relationships between ratios. (B) Distribution of MATH domain K_A/K_S ratios minus BTB domain K_A/K_S ratios for each branch for the core (black boxes) and expanded (gray boxes) groups. Nonparametric U-test (P < 0.000055) indicates that the relationship of selective pressures between the MATH and BTB domains is significantly different between core and expanded groups.