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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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Amino acid sequence alignment between P-loop and GLPL of TIR-NBS-LRR CaRGAs with the NBS domains of known R genes N (U15605), L6 (U27081) and M (U73916). The conserved domains are highlighted and indicated with an arrow. The alignment was performed using BioEdit 7.0.0 software. The threshold (%) for shading was set at 50. Similar amino acid residues are shaded grey and identical amino acid residues are shaded black.
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Figure 3: Amino acid sequence alignment between P-loop and GLPL of TIR-NBS-LRR CaRGAs with the NBS domains of known R genes N (U15605), L6 (U27081) and M (U73916). The conserved domains are highlighted and indicated with an arrow. The alignment was performed using BioEdit 7.0.0 software. The threshold (%) for shading was set at 50. Similar amino acid residues are shaded grey and identical amino acid residues are shaded black.

Mentions: The CaRGAs identified from pepper were obviously separated into non-TIR- and TIR-NBS-LRR subfamilies (Figure 1). Consequently, multiple sequence alignments of non-TIR- and TIR-NBS-LRR RGAs were performed separately. The sequence alignment between non-TIR-NBS-LRR RGAs and known disease R genes, including RPM1, Gpa2 and Prf, revealed six conserved motifs (i.e. P-loop, RNBS-A-non-TIR, kinase-2, RNBS-B, RNBS-C and GLPL) (Figure 2). Similarly, alignment analysis with known R genes N, M and L6 revealed that the TIR-NBS-LRR RGAs also contain six conserved motifs (i.e. P-loop, RNBS-A-TIR, kinase-2, RNBS-B, RNBS-C and GLPL) (Figure 3). The presence of these consensus motifs within the non-TIR- and TIR-NBS-LRR CaRGAs provides further evidence that the cloned sequences are NBS-LRR gene family members.


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)

Amino acid sequence alignment between P-loop and GLPL of TIR-NBS-LRR CaRGAs with the NBS domains of known R genes N (U15605), L6 (U27081) and M (U73916). The conserved domains are highlighted and indicated with an arrow. The alignment was performed using BioEdit 7.0.0 software. The threshold (%) for shading was set at 50. Similar amino acid residues are shaded grey and identical amino acid residues are shaded black.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: Amino acid sequence alignment between P-loop and GLPL of TIR-NBS-LRR CaRGAs with the NBS domains of known R genes N (U15605), L6 (U27081) and M (U73916). The conserved domains are highlighted and indicated with an arrow. The alignment was performed using BioEdit 7.0.0 software. The threshold (%) for shading was set at 50. Similar amino acid residues are shaded grey and identical amino acid residues are shaded black.
Mentions: The CaRGAs identified from pepper were obviously separated into non-TIR- and TIR-NBS-LRR subfamilies (Figure 1). Consequently, multiple sequence alignments of non-TIR- and TIR-NBS-LRR RGAs were performed separately. The sequence alignment between non-TIR-NBS-LRR RGAs and known disease R genes, including RPM1, Gpa2 and Prf, revealed six conserved motifs (i.e. P-loop, RNBS-A-non-TIR, kinase-2, RNBS-B, RNBS-C and GLPL) (Figure 2). Similarly, alignment analysis with known R genes N, M and L6 revealed that the TIR-NBS-LRR RGAs also contain six conserved motifs (i.e. P-loop, RNBS-A-TIR, kinase-2, RNBS-B, RNBS-C and GLPL) (Figure 3). The presence of these consensus motifs within the non-TIR- and TIR-NBS-LRR CaRGAs provides further evidence that the cloned sequences are NBS-LRR gene family members.

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