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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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Expression profiles of 13 pepper NBS-LRR CaRGAs. A) Expression patterns obtained from semi-quantitative reverse transcriptase PCR experiments in different tissues and in response to the exogenous application of hormones. B) Relative transcript levels obtained from quantitative real-time PCR experiments in different tissues and in response to the exogenous application of hormones. Representative images are shown. R: Root, S: Stem and L: Leaf. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a reference gene for the expression analysis of pepper CaRGA genes [65].
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Figure 6: Expression profiles of 13 pepper NBS-LRR CaRGAs. A) Expression patterns obtained from semi-quantitative reverse transcriptase PCR experiments in different tissues and in response to the exogenous application of hormones. B) Relative transcript levels obtained from quantitative real-time PCR experiments in different tissues and in response to the exogenous application of hormones. Representative images are shown. R: Root, S: Stem and L: Leaf. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a reference gene for the expression analysis of pepper CaRGA genes [65].

Mentions: To analyze the expression levels of CaRGAs in the different organs of pepper, two representatives of each class were randomly used for expression analysis using reverse transcription (RT)-PCR. CaRGA14 and CaRGA15 were the only two members of Class CaRGAIV. Given that the sequence similarity among these RGAs was high, only one pair of specific primers was designed using Primer 5.0 software. A total of thirteen pairs of specific primers were obtained. As shown in Figure 6A, thirteen CaRGAs were expressed in different plant organs, but their expression levels were different. Among these, CaRGA04, CaRGA13 and CaRGA38 were expressed in the leaves, stems and roots at low levels, whereas CaRGA01, CaRGA03, CaRGA14 and CaRGA18 were expressed at relatively high levels. The remaining CaRGAs were expressed at intermediate levels. No expression of CaRGA44 and CaRGA49 genes was detected in the roots. The CaRGAV and II subgroups showed similar expression patterns.


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)

Expression profiles of 13 pepper NBS-LRR CaRGAs. A) Expression patterns obtained from semi-quantitative reverse transcriptase PCR experiments in different tissues and in response to the exogenous application of hormones. B) Relative transcript levels obtained from quantitative real-time PCR experiments in different tissues and in response to the exogenous application of hormones. Representative images are shown. R: Root, S: Stem and L: Leaf. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a reference gene for the expression analysis of pepper CaRGA genes [65].
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 6: Expression profiles of 13 pepper NBS-LRR CaRGAs. A) Expression patterns obtained from semi-quantitative reverse transcriptase PCR experiments in different tissues and in response to the exogenous application of hormones. B) Relative transcript levels obtained from quantitative real-time PCR experiments in different tissues and in response to the exogenous application of hormones. Representative images are shown. R: Root, S: Stem and L: Leaf. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a reference gene for the expression analysis of pepper CaRGA genes [65].
Mentions: To analyze the expression levels of CaRGAs in the different organs of pepper, two representatives of each class were randomly used for expression analysis using reverse transcription (RT)-PCR. CaRGA14 and CaRGA15 were the only two members of Class CaRGAIV. Given that the sequence similarity among these RGAs was high, only one pair of specific primers was designed using Primer 5.0 software. A total of thirteen pairs of specific primers were obtained. As shown in Figure 6A, thirteen CaRGAs were expressed in different plant organs, but their expression levels were different. Among these, CaRGA04, CaRGA13 and CaRGA38 were expressed in the leaves, stems and roots at low levels, whereas CaRGA01, CaRGA03, CaRGA14 and CaRGA18 were expressed at relatively high levels. The remaining CaRGAs were expressed at intermediate levels. No expression of CaRGA44 and CaRGA49 genes was detected in the roots. The CaRGAV and II subgroups showed similar expression patterns.

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