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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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Schematic representation of plant NBS-LRR R gene structure. (A) Characteristic plant R gene domains. (B) Conserved motifs within the NBS domain. The solid arrow indicates the position and direction of the designed degenerate forward and reverse primers (P-loop and GLPL conserved motifs, respectively). The degenerate primer sequences are shown in Additional file 5.
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Figure 7: Schematic representation of plant NBS-LRR R gene structure. (A) Characteristic plant R gene domains. (B) Conserved motifs within the NBS domain. The solid arrow indicates the position and direction of the designed degenerate forward and reverse primers (P-loop and GLPL conserved motifs, respectively). The degenerate primer sequences are shown in Additional file 5.

Mentions: Two pairs of degenerate primers were selected for isolating the RGA sequences in pepper (Additional file 5). The positions of the degenerate primers were identified in the two conserved domains (P-loop and GLPL, respectively) in the plant R genes (Figure 7). PCR amplifications were performed using a PTC-100 thermal cycler. PCR amplification reactions were conducted in a total volume of 25 μL containing 20 ng of template DNA, 2 μL of 10× PCR Buffer, 1.5 μL of 25 mmol MgCl2, 2 μL of dNTPs (2 mmol/L), 1 μL of Primer-F (10 μmol/L), 1 μL of Primer-R (10 μmol/L), 1 unit of ExTaq DNA (5 U/μL) and 15.3 μL of ddH2O. The PCR reaction cycle was as follows: denaturation at 94°C for 4 min, followed by 35 cycles of denaturation at 94°C for 30 s, annealing at 55°C for 30 s and extension at 72°C for 60 s, followed by a final extension at 72°C for 5 min.


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)

Schematic representation of plant NBS-LRR R gene structure. (A) Characteristic plant R gene domains. (B) Conserved motifs within the NBS domain. The solid arrow indicates the position and direction of the designed degenerate forward and reverse primers (P-loop and GLPL conserved motifs, respectively). The degenerate primer sequences are shown in Additional file 5.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 7: Schematic representation of plant NBS-LRR R gene structure. (A) Characteristic plant R gene domains. (B) Conserved motifs within the NBS domain. The solid arrow indicates the position and direction of the designed degenerate forward and reverse primers (P-loop and GLPL conserved motifs, respectively). The degenerate primer sequences are shown in Additional file 5.
Mentions: Two pairs of degenerate primers were selected for isolating the RGA sequences in pepper (Additional file 5). The positions of the degenerate primers were identified in the two conserved domains (P-loop and GLPL, respectively) in the plant R genes (Figure 7). PCR amplifications were performed using a PTC-100 thermal cycler. PCR amplification reactions were conducted in a total volume of 25 μL containing 20 ng of template DNA, 2 μL of 10× PCR Buffer, 1.5 μL of 25 mmol MgCl2, 2 μL of dNTPs (2 mmol/L), 1 μL of Primer-F (10 μmol/L), 1 μL of Primer-R (10 μmol/L), 1 unit of ExTaq DNA (5 U/μL) and 15.3 μL of ddH2O. The PCR reaction cycle was as follows: denaturation at 94°C for 4 min, followed by 35 cycles of denaturation at 94°C for 30 s, annealing at 55°C for 30 s and extension at 72°C for 60 s, followed by a final extension at 72°C for 5 min.

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