Diversity in nucleotide binding site-leucine-rich repeat genes in cereals

Abstract

The diversity of the largest group of plant disease resistance genes, the nucleotide binding site-leucine-rich repeat (NBS-LRR) genes, was examined in cereals following polymerase chain reaction (PCR) cloning and database mining. NBS-LRR genes in rice are a large and diverse class with more than 600 genes, at least three to four times the complement of Arabidopsis. Most occur in small families containing one or a few cross-hybridizing members. Unlike in Arabidopsis and other dicots, the class of NBS-LRR genes coding for a Toll and mammalian interleukin-1 receptor (TIR) domain were not amplified during the evolution of the cereals. Genes coding for TIR domains are present in the rice genome, but have diverged from the NBS-LRR genes. Most cereal genes are similar in structure to the members of the non-TIR class of dicots, although many do not code for a coiled-coil domain in their amino termini. One unique class of cereal genes, with ~50 members, codes for proteins similar to the N-termini and NBS domains of resistance genes but does not code for LRR domains. The resistance gene repertoire of grasses has changed from that of dicots in their independent evolution since the two groups diverged. It is not clear whether this reflects a difference in downstream defense signaling pathways.

Figure 1

Estimation of genomic copy number of different nucleotide binding site–leucine-rich repeat (NBS–LRR) families in rice. Different cloned NBS-coding sequences were used as probes on gel-blot hybridizations to DNAs of four rice lines digested with four different restriction enzymes. The top blot is probed with rNBS79 and the bottom blot is probed with rNBS72, a predicted single-copy gene.

Figure 2

Cladistic analysis of the nucleotide binding site (NBS) region in rice NBS–leucine-rich repeat (LRR) genes. A total of 354 rice sequences and four other cereal R gene sequences were used for generating a neighbor-joining tree. The 354 rice sequences were selected so that none exhibited >75% sequence identity to any others in the NBS region. The tree is a representative tree in which groups with >75% bootstrapping value (% of trees of 1000 generated) are represented by a single branch with an average branch length for that group. The complete tree can be viewed from the online supplementary material section. The members of the individual groups are shown in the tree for the smaller groups or listed as follows: Group 1 = rNBS57, In4386-3, In3256-1, In4208-2, In4208-2B, rNBS24, In1277-2, In10933-1, In8374-4, In32618-1, rNBS58, AP003345, In 6313-2; group 2 = rNBS15, rNBS12, rNBS17, rNBS6, rNBS3, rNBS97, In37394-1, In9848-1, AB019186, In6575-2; group 3 = rNBS77, rNBS7, rNBS13, In7573-1, In18748-1, In37595-1; group 4 = rNBS112, rNBS8, OSM13267, In2847-2, In7323-1, AC74283; group 5 = Pita, In20736-1, In1586-1, In10315-1, In20846-1, In3014-1, In785-4B, In10722-1; group 6 = rNBS96, AP001073-c, AP001073-a, AP003848, In22736-1, In5389-2; group 7 = AC092548, AP003859, In4930-1, In3292-1, In16749-1, In3436-1, In10436-2; group 8 = rNBS25, rNBS92, AP003918, In31789-1, In40443-1; group 9 = rNBS11, rNBS26, rNBS27, rNBS38, rNBS39, In10362-1; group 10 = AC092388, AC097277-2, AC097277-1, In7217-2, In20247-1, In15637-1, In5597-3; group 11 = rNBS84, AP003568, AP004092, In19349-1, In13218-2, In2456-1, In47000-1, In31232-1, 5790-1; group 12 = rNBS32, rNBS2, rNBS16, rNBS59, rNBS49, rNBS31, rNBS34, rNBS35, rNBS78, AB022164, In7980-1, In10136-1, In2646-2, In10063-1, In340-3; group 13 = rNBS9, rNBS37, In16096-1, In18642-1, In36703-1, In14463-1; group 14 = Rp1, rNBS33, rNBS86, rNBS108, AP004061, AP003368-c, AL606992, AL606616-2, OSM140512, In7903-1, In9457-1, In20413-1, In20680-1; group 15 = Xa1, rNBS10, rNBS19, rNBS42, rNBS52, rNBS53, AL606660-2, AL606660-5, OSM15716, OSM12815, In12431-1; group 16 = rNBS48, rNBS83, rNBS85, rNBS41, rNBS62, AC098834, AP004010, AP003621-b, OSM15362, OSM14953, OSM11901, In46824-1, In30175-1, In3589-3, In27999-1, In4754-2; group 17 = rNBS68, rNBS44, AL513004-1, AL513004-2, OSM129685, In271-4, In47803-1, In7388-1, In11951-1, In17986-1, In3169-2, In271-2, In3639-1, In9038-1; group 18 = rNBS87, rNBS36, AP003930-e, AP003930-b, AP003827-d, In1964-1. Gene designations are as in Table 1.

Figure 3

Structures and approximate numbers of nucleotide binding site–leucine-rich repeat and related genes in rice. Open boxes represent domains with no significant database similarity except for similar genes from other plant species.

Figure 4

Consensus sequence of the rice nT-nucleotide binding site (NBS) class genes. The consensus was generated from an alignment of 47 nT-NBS genes by HMMER 2.2g. Conserved regions in the N-terminal domain are underlined. Sequences in bold correspond to sequences conserved in NBS-coding domains; the P-Loop, kinase-2, RNBSB, and GLPL motifs (Table 3). More conserved amino acid positions are represented by upper-case letters.

Figure 5

Consensus sequence of three predicted rice TIR-nucleotide binding site (NBS) genes with two related Arabidopsis proteins. The region showing similarity to the TIR domain of TIR–NBS–leucine-rich repeat genes is underlined. Sequences in bold correspond to sequences conserved in NBS-coding domains, the P-Loop, kinase-2, RNBSB, and GLPL motifs (Table 3). Serine residues in the serine-rich N terminus and leucine residues in the C terminus are highlighted. More conserved amino acid positions are represented by upper-case letters.

Figure 6

Phylogenetic analysis of nucleotide binding site-leucine-rich repeat (NBS–LRR) and related genes from rice and dicot species. Rice NBS–LRR sequences were selected from different clades identified in Figure 1. Other designations correspond to characterized resistance genes or GenBank, Monsanto, or Indica database accession numbers and the species designations are indicated after the clone designation: O.s., rice; Z.m. maize; S.t. potato; A.t. Arabidopsis; L.e. tomato; L.u. flax; N.g. tobacco, and H.s. human. The 48 nT-NBS sequences are all from rice, while two of the TIR–NBS sequences are from Arabidopsis and the other three are from rice.