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Analysis of TIR- and non-TIR-NBS-LRR disease resistance gene analogous in pepper: characterization, genetic variation, functional divergence and expression patterns.

Wan H, Yuan W, Ye Q, Wang R, Ruan M, Li Z, Zhou G, Yao Z, Zhao J, Liu S, Yang Y - BMC Genomics (2012)

Bottom Line: The plant nucleotide-binding site (NBS)-leucine-rich repeat (LRR) gene family is the largest class of known disease resistance genes (R genes) effective against such pathogens.Therefore, the isolation and identification of such R gene homologues from pepper will provide a critical foundation for improving disease resistance breeding programs.The findings of this study provide a better understanding of the evolutionary mechanisms that drive the functional diversification of non-TIR- and TIR-NBS-LRR R genes in pepper.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Vegetables, Zhejiang Academy of Agricultural Sciences, Hangzhou, People's Republic of China.

ABSTRACT

Background: Pepper (Capsicum annuum L.) is one of the most important vegetable crops worldwide. However, its yield and fruit quality can be severely threatened by several pathogens. The plant nucleotide-binding site (NBS)-leucine-rich repeat (LRR) gene family is the largest class of known disease resistance genes (R genes) effective against such pathogens. Therefore, the isolation and identification of such R gene homologues from pepper will provide a critical foundation for improving disease resistance breeding programs.

Results: A total of 78 R gene analogues (CaRGAs) were identified in pepper by degenerate PCR amplification and database mining. Phylogenetic tree analysis of the deduced amino acid sequences for 51 of these CaRGAs with typically conserved motifs ( P-loop, kinase-2 and GLPL) along with some known R genes from Arabidopsis and tomato grouped these CaRGAs into the non-Toll interleukin-1 receptor (TIR)-NBS-LRR (CaRGAs I to IV) and TIR-NBS-LRR (CaRGAs V to VII) subfamilies. The presence of consensus motifs (i.e. P-loop, kinase-2 and hydrophobic domain) is typical of the non-TIR- and TIR-NBS-LRR gene subfamilies. This finding further supports the view that both subfamilies are widely distributed in dicot species. Functional divergence analysis provided strong statistical evidence of altered selective constraints during protein evolution between the two subfamilies. Thirteen critical amino acid sites involved in this divergence were also identified using DIVERGE version 2 software. Analyses of non-synonymous and synonymous substitutions per site showed that purifying selection can play a critical role in the evolutionary processes of non-TIR- and TIR-NBS-LRR RGAs in pepper. In addition, four specificity-determining positions were predicted to be responsible for functional specificity. qRT-PCR analysis showed that both salicylic and abscisic acids induce the expression of CaRGA genes, suggesting that they may primarily be involved in defence responses by activating signaling pathways.

Conclusion: The identified CaRGAs are a valuable resource for discovering R genes and developing RGA molecular markers for genetic map construction. They will also be useful for improving disease resistance in pepper. The findings of this study provide a better understanding of the evolutionary mechanisms that drive the functional diversification of non-TIR- and TIR-NBS-LRR R genes in pepper.

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Phylogenetic tree analysis based on alignment of the deduced amino acid sequences of pepper CaRGAs with known R genes. The tree was constructed by the neighbor-joining method using MEGA 5.0 software. The 51 CaRGA sequences and 2 resistance genes were grouped into seven subgroups: I–VII. I–IV are part of the non-TIR-NBS-LRR family, whereas V–VII belong to the TIR-NBS-LRR family. Bootstrap values (1000 replicates) are given below the branches. The known R genes with the NBS domain that were used were N (U15605), L6 (U27081), M (U73916), Prf (U65391), Gpa2 (AF195939) and RPM1 (X87851).
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Figure 1: Phylogenetic tree analysis based on alignment of the deduced amino acid sequences of pepper CaRGAs with known R genes. The tree was constructed by the neighbor-joining method using MEGA 5.0 software. The 51 CaRGA sequences and 2 resistance genes were grouped into seven subgroups: I–VII. I–IV are part of the non-TIR-NBS-LRR family, whereas V–VII belong to the TIR-NBS-LRR family. Bootstrap values (1000 replicates) are given below the branches. The known R genes with the NBS domain that were used were N (U15605), L6 (U27081), M (U73916), Prf (U65391), Gpa2 (AF195939) and RPM1 (X87851).

Mentions: To investigate the evolutionary relationships among the NBS-LRR CaRGAs of pepper, phylogenetic trees were constructed based on the region between the P-loop and GLPL motifs using Molecular Evolutionary Genetics Analysis (MEGA) 5.0 software [43]. The aforementioned four CaRGAs were excluded from subsequent analyses because they lacked conserved P-loop and GLPL motifs. Thus, a total of fifty-three CaRGAs of pepper were used for phylogenetic tree construction. Six known R genes (RPM1, Gpa2, L6, M, N and Prf), downloaded from the GenBank database, were also included in the analysis. The phylogenetic tree is shown in Figure 1. Two primary groups, non-TIR- and TIR-NBS-LRR, were obviously distinguished, which is consistent with previously described patterns [9,15]. The non-TIR-NBS-LRR subfamily included thirty-eight members (CaRGA13–CaRGA43, CaRGA45–CaRGA48, CaRGA51, BS2 and CaMi), whereas only fifteen members (CaRGA01–CaRGA12, CaRGA49–CaRGA50 and CaRGA44) belonged to the TIR-NBS-LRR subfamily. These results support the view that both TIR-NBSLRR and non-TIR-NBS-LRR R genes occur in dicot species [15]. Within the non-TIR-NBS-LRR subfamily, CaRGAs could be separated into four subgroups, which were designated CaRGA I–IV. In the TIR-NBS-LRR subfamily, the CaRGAs could be divided into three subgroups, which were designated CaRGA V–VII. Each of these seven subgroups included different numbers of CaRGA members. The CaRGA III, CaRGA IV, CaRGA V and CaRGA VI subgroups contained two to four members, whereas the CaRGA I, CaRGA II and CaRGA VII subgroups consisted of eight to nineteen members. This result reflects a difference in the abundance of these RGA subgroups in the pepper genome (Figure 1). The characteristics of these non-TIR- and TIR-NBS CaRGA sequences from pepper are shown in Table 1.


Analysis of TIR- and non-TIR-NBS-LRR disease resistance gene analogous in pepper: characterization, genetic variation, functional divergence and expression patterns.

Wan H, Yuan W, Ye Q, Wang R, Ruan M, Li Z, Zhou G, Yao Z, Zhao J, Liu S, Yang Y - BMC Genomics (2012)

Phylogenetic tree analysis based on alignment of the deduced amino acid sequences of pepper CaRGAs with known R genes. The tree was constructed by the neighbor-joining method using MEGA 5.0 software. The 51 CaRGA sequences and 2 resistance genes were grouped into seven subgroups: I–VII. I–IV are part of the non-TIR-NBS-LRR family, whereas V–VII belong to the TIR-NBS-LRR family. Bootstrap values (1000 replicates) are given below the branches. The known R genes with the NBS domain that were used were N (U15605), L6 (U27081), M (U73916), Prf (U65391), Gpa2 (AF195939) and RPM1 (X87851).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3472223&req=5

Figure 1: Phylogenetic tree analysis based on alignment of the deduced amino acid sequences of pepper CaRGAs with known R genes. The tree was constructed by the neighbor-joining method using MEGA 5.0 software. The 51 CaRGA sequences and 2 resistance genes were grouped into seven subgroups: I–VII. I–IV are part of the non-TIR-NBS-LRR family, whereas V–VII belong to the TIR-NBS-LRR family. Bootstrap values (1000 replicates) are given below the branches. The known R genes with the NBS domain that were used were N (U15605), L6 (U27081), M (U73916), Prf (U65391), Gpa2 (AF195939) and RPM1 (X87851).
Mentions: To investigate the evolutionary relationships among the NBS-LRR CaRGAs of pepper, phylogenetic trees were constructed based on the region between the P-loop and GLPL motifs using Molecular Evolutionary Genetics Analysis (MEGA) 5.0 software [43]. The aforementioned four CaRGAs were excluded from subsequent analyses because they lacked conserved P-loop and GLPL motifs. Thus, a total of fifty-three CaRGAs of pepper were used for phylogenetic tree construction. Six known R genes (RPM1, Gpa2, L6, M, N and Prf), downloaded from the GenBank database, were also included in the analysis. The phylogenetic tree is shown in Figure 1. Two primary groups, non-TIR- and TIR-NBS-LRR, were obviously distinguished, which is consistent with previously described patterns [9,15]. The non-TIR-NBS-LRR subfamily included thirty-eight members (CaRGA13–CaRGA43, CaRGA45–CaRGA48, CaRGA51, BS2 and CaMi), whereas only fifteen members (CaRGA01–CaRGA12, CaRGA49–CaRGA50 and CaRGA44) belonged to the TIR-NBS-LRR subfamily. These results support the view that both TIR-NBSLRR and non-TIR-NBS-LRR R genes occur in dicot species [15]. Within the non-TIR-NBS-LRR subfamily, CaRGAs could be separated into four subgroups, which were designated CaRGA I–IV. In the TIR-NBS-LRR subfamily, the CaRGAs could be divided into three subgroups, which were designated CaRGA V–VII. Each of these seven subgroups included different numbers of CaRGA members. The CaRGA III, CaRGA IV, CaRGA V and CaRGA VI subgroups contained two to four members, whereas the CaRGA I, CaRGA II and CaRGA VII subgroups consisted of eight to nineteen members. This result reflects a difference in the abundance of these RGA subgroups in the pepper genome (Figure 1). The characteristics of these non-TIR- and TIR-NBS CaRGA sequences from pepper are shown in Table 1.

Bottom Line: The plant nucleotide-binding site (NBS)-leucine-rich repeat (LRR) gene family is the largest class of known disease resistance genes (R genes) effective against such pathogens.Therefore, the isolation and identification of such R gene homologues from pepper will provide a critical foundation for improving disease resistance breeding programs.The findings of this study provide a better understanding of the evolutionary mechanisms that drive the functional diversification of non-TIR- and TIR-NBS-LRR R genes in pepper.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Vegetables, Zhejiang Academy of Agricultural Sciences, Hangzhou, People's Republic of China.

ABSTRACT

Background: Pepper (Capsicum annuum L.) is one of the most important vegetable crops worldwide. However, its yield and fruit quality can be severely threatened by several pathogens. The plant nucleotide-binding site (NBS)-leucine-rich repeat (LRR) gene family is the largest class of known disease resistance genes (R genes) effective against such pathogens. Therefore, the isolation and identification of such R gene homologues from pepper will provide a critical foundation for improving disease resistance breeding programs.

Results: A total of 78 R gene analogues (CaRGAs) were identified in pepper by degenerate PCR amplification and database mining. Phylogenetic tree analysis of the deduced amino acid sequences for 51 of these CaRGAs with typically conserved motifs ( P-loop, kinase-2 and GLPL) along with some known R genes from Arabidopsis and tomato grouped these CaRGAs into the non-Toll interleukin-1 receptor (TIR)-NBS-LRR (CaRGAs I to IV) and TIR-NBS-LRR (CaRGAs V to VII) subfamilies. The presence of consensus motifs (i.e. P-loop, kinase-2 and hydrophobic domain) is typical of the non-TIR- and TIR-NBS-LRR gene subfamilies. This finding further supports the view that both subfamilies are widely distributed in dicot species. Functional divergence analysis provided strong statistical evidence of altered selective constraints during protein evolution between the two subfamilies. Thirteen critical amino acid sites involved in this divergence were also identified using DIVERGE version 2 software. Analyses of non-synonymous and synonymous substitutions per site showed that purifying selection can play a critical role in the evolutionary processes of non-TIR- and TIR-NBS-LRR RGAs in pepper. In addition, four specificity-determining positions were predicted to be responsible for functional specificity. qRT-PCR analysis showed that both salicylic and abscisic acids induce the expression of CaRGA genes, suggesting that they may primarily be involved in defence responses by activating signaling pathways.

Conclusion: The identified CaRGAs are a valuable resource for discovering R genes and developing RGA molecular markers for genetic map construction. They will also be useful for improving disease resistance in pepper. The findings of this study provide a better understanding of the evolutionary mechanisms that drive the functional diversification of non-TIR- and TIR-NBS-LRR R genes in pepper.

Show MeSH
Related in: MedlinePlus