Large-scale analysis of NBS domain-encoding resistance gene analogs in Triticeae

Abstract

Proteins containing nucleotide binding sites (NBS) encoded by plant resistance genes play an important role in the response of plants to a wide array of pathogens. In this paper, an in silico search was conducted in order to identify and characterize members of NBS-encoding gene family in the tribe of Triticeae. A final dataset of 199 sequences was obtained by four search methods. Motif analysis confirmed the general structural organization of the NBS domain in cereals, characterized by the presence of the six commonly conserved motifs: P-loop, RNBS-A, Kinase-2, Kinase-3a, RNBS-C and GLPL. We revealed the existence of 11 distinct distribution patterns of these motifs along the NBS domain. Four additional conserved motifs were shown to be significantly present in all 199 sequences. Phylogenetic analyses, based on genetic distance and parsimony, revealed a significant overlap between Triticeae sequences and Coiled coil-Nucleotide binding site-Leucine rich repeat (CNL)-type functional genes from monocotyledons. Furthermore, several Triticeae sequences belonged to clades containing functional homologs from non Triticeae species, which has allowed for these sequences to be functionally assigned. The findings reported, in this study, will provide a strong groundwork for the isolation of candidate R-genes in Triticeae crops and the understanding of their evolution.

Keywords: NBS domain; Triticeae; data mining; phylogeny; plant resistance genes.

Figure 1

Logo created with LogoMat-M program, illustrating the 176-positions HMM profile, obtained from the alignment of 422 intact-NBS sequences derived from PSIBLAST.

Figure 2

Data mining pipeline and integration of all non redundant sequences from different searches, into a primary compilation.

Figure 3

Neighbor-Joining phylogenetic comparison of Triticeae and non Triticeae NBS-encoding genes and RGAs. The numbers on the branches indicate the percentage of 1000 bootstrap replicates that support the node with only values > 50% reported. The evolutionary distances were computed using the Dayhoff matrix based method (Schwarz and Dayhoff, 1979). The rate variation among sites was modelled with a gamma distribution (shape parameter = 8). All positions containing gaps and missing data were eliminated. The analysis involved 243 amino acid sequences (in red: reference TNLs, in green: reference CNLs, in black: 199 core NBS representing the diversity of NBS-encoding RGAs in Triticeae). 62 clades are shown and TIR-NBS taxa are distinguished from CC-NBS ones.

Figure 4

Maximum parsimony phylogenetic comparison of Triticeae and non Triticeae NBS-encoding genes and RGAs. The numbers on the branches indicate the percentage of 1000 bootstrap replicates that support the node with only values > 50% reported. The MP tree was obtained using the Close-Neighbor-Interchange algorithm (Nei and Kumar, 2000). All positions containing gaps and missing data were eliminated. The analysis involved 243 amino acid sequences (in red: reference TNLs, in green: reference CNLs, in black: 199 core NBS representing the diversity of NBS-encoding RGAs in Triticeae). 89 clades are shown and TIR-NBS taxa are distinguished from CC-NBS ones.

Figure 5

Identification of 11 patterns of organization for the six major motifs of the NBS domain (P-loop, RNBS-A, Kinase-2, Kinase-3 and RNBS-C and GLPLA) in 199 Triticeae core NBS sequences, representing the diversity of core NBS in this tribe. Only motifs with an E-value of 0.0001 and that do not overlap other, more significant ones, are represented.