PK-profiling method for identifying the expression of resistance-associated genes in partially resistant oats to crown rust
Abstract Background Protein kinases play a key role in plant cell homeostasis and the activation of defense mechanisms. Partial resistance to fungi in plants is interesting because of its durability. However, the variable number of minor loci associated with this type of resistance hampers the relia...
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doaj-59957e88987f487381bf512e5b61647b2020-11-25T02:22:13ZengBMCBMC Plant Biology1471-22292018-12-0118111310.1186/s12870-018-1604-yPK-profiling method for identifying the expression of resistance-associated genes in partially resistant oats to crown rustYolanda Loarce0Pilar Dongil1Araceli Fominaya2Juan M. González3Esther Ferrer4Department of Biomedicine and Biotechnology, University of Alcalá, Campus UniversitarioDepartment of Biomedicine and Biotechnology, University of Alcalá, Campus UniversitarioDepartment of Biomedicine and Biotechnology, University of Alcalá, Campus UniversitarioDepartment of Biomedicine and Biotechnology, University of Alcalá, Campus UniversitarioDepartment of Biomedicine and Biotechnology, University of Alcalá, Campus UniversitarioAbstract Background Protein kinases play a key role in plant cell homeostasis and the activation of defense mechanisms. Partial resistance to fungi in plants is interesting because of its durability. However, the variable number of minor loci associated with this type of resistance hampers the reliable identification of the full range of genes involved. The present work reports the technique of protein kinase (PK)-profiling for the identification of the PK genes induced in the partially resistant oats line MN841801–1 following exposure to the fungus Puccinia coronata. This is the first time this technique has been used with cDNA (complementary DNA) from a suppression subtractive hybridization library obtained after the hybridization of cDNAs from inoculated and mock-inoculated plants. Results Six degenerate primers based on the conserved domains of protein kinases were used in a PK-profiling assay including cDNA from mock-inoculated leaves and subtracted cDNA. Of the 75.7% of sequences cloned and sequenced that showed significant similarity to resistance genes, 76% were found to code for PKs. Translation and ClustalW2 alignment of each sequence cloned with the complete sequences of the most similar B. distachyon PKs allowed those of the partially resistant oat line to be deduced and characterized. Further, a phylogenetic study carried out after alignment of these B. distachyon PK sequences with the most similar protein sequences of related species also allowed to deduce different functions for the PK cloned. RT-qPCR (Reverse Transcription-quantitative PCR) was analyzed on nine representative sequences to validate the reliability of the employed PK-profiling method as a tool for identifying the expression of resistance-associated genes. Conclusions PK-profiling would appear to be a useful tool for the identification of the PKs expressed in oats after challenge by P. coronata, and perhaps other pathogens. Most of the PKs studied are related to receptor-like protein kinases expressed shortly after infection. This is in agreement with previous studies indicating a close relationship between partial resistance and the first layer of defense against pathogen used by plants.http://link.springer.com/article/10.1186/s12870-018-1604-yPartial resistancePK-profilingSubtraction libraryProtein kinases |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Yolanda Loarce Pilar Dongil Araceli Fominaya Juan M. González Esther Ferrer |
spellingShingle |
Yolanda Loarce Pilar Dongil Araceli Fominaya Juan M. González Esther Ferrer PK-profiling method for identifying the expression of resistance-associated genes in partially resistant oats to crown rust BMC Plant Biology Partial resistance PK-profiling Subtraction library Protein kinases |
author_facet |
Yolanda Loarce Pilar Dongil Araceli Fominaya Juan M. González Esther Ferrer |
author_sort |
Yolanda Loarce |
title |
PK-profiling method for identifying the expression of resistance-associated genes in partially resistant oats to crown rust |
title_short |
PK-profiling method for identifying the expression of resistance-associated genes in partially resistant oats to crown rust |
title_full |
PK-profiling method for identifying the expression of resistance-associated genes in partially resistant oats to crown rust |
title_fullStr |
PK-profiling method for identifying the expression of resistance-associated genes in partially resistant oats to crown rust |
title_full_unstemmed |
PK-profiling method for identifying the expression of resistance-associated genes in partially resistant oats to crown rust |
title_sort |
pk-profiling method for identifying the expression of resistance-associated genes in partially resistant oats to crown rust |
publisher |
BMC |
series |
BMC Plant Biology |
issn |
1471-2229 |
publishDate |
2018-12-01 |
description |
Abstract Background Protein kinases play a key role in plant cell homeostasis and the activation of defense mechanisms. Partial resistance to fungi in plants is interesting because of its durability. However, the variable number of minor loci associated with this type of resistance hampers the reliable identification of the full range of genes involved. The present work reports the technique of protein kinase (PK)-profiling for the identification of the PK genes induced in the partially resistant oats line MN841801–1 following exposure to the fungus Puccinia coronata. This is the first time this technique has been used with cDNA (complementary DNA) from a suppression subtractive hybridization library obtained after the hybridization of cDNAs from inoculated and mock-inoculated plants. Results Six degenerate primers based on the conserved domains of protein kinases were used in a PK-profiling assay including cDNA from mock-inoculated leaves and subtracted cDNA. Of the 75.7% of sequences cloned and sequenced that showed significant similarity to resistance genes, 76% were found to code for PKs. Translation and ClustalW2 alignment of each sequence cloned with the complete sequences of the most similar B. distachyon PKs allowed those of the partially resistant oat line to be deduced and characterized. Further, a phylogenetic study carried out after alignment of these B. distachyon PK sequences with the most similar protein sequences of related species also allowed to deduce different functions for the PK cloned. RT-qPCR (Reverse Transcription-quantitative PCR) was analyzed on nine representative sequences to validate the reliability of the employed PK-profiling method as a tool for identifying the expression of resistance-associated genes. Conclusions PK-profiling would appear to be a useful tool for the identification of the PKs expressed in oats after challenge by P. coronata, and perhaps other pathogens. Most of the PKs studied are related to receptor-like protein kinases expressed shortly after infection. This is in agreement with previous studies indicating a close relationship between partial resistance and the first layer of defense against pathogen used by plants. |
topic |
Partial resistance PK-profiling Subtraction library Protein kinases |
url |
http://link.springer.com/article/10.1186/s12870-018-1604-y |
work_keys_str_mv |
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