Molecular characterization of a biotic and abiotic stress resistance-related gene RelA/SpoT homologue (PepRSH) from pepper

Tae Ho Kim; Sung Han Ok; Donghern Kim; Seok Cheol Suh; Myung Ok Byun; Jeong Sheop Shin

doi:10.1016/j.plantsci.2009.02.004

Molecular characterization of a biotic and abiotic stress resistance-related gene RelA/SpoT homologue (PepRSH) from pepper

Tae Ho Kim, Sung Han Ok, Donghern Kim, Seok Cheol Suh, Myung Ok Byun, Jeong Sheop Shin

Department of Life Sciences

Research output: Contribution to journal › Article › peer-review

20 Citations (Scopus)

Abstract

A gene encoding a putative guanosine 5′-diphosphate (or 5′-triphosphate) 3′-diphosphate ((p)ppGpp) synthetase, designated PepRSH (Pepper RelA/SpoT homologue), was isolated from hot peppers. PepRSH was found to contain five introns and six exons and a 2166-bp open reading frame encoding a protein of 721 amino acids; this protein displayed significant homology with other plant (p)ppGpp synthetases. A genomic DNA gel blot analysis revealed that the pepper genome has at least a single copy of PepRSH. PepRSH transcripts were highly accumulated in non-host resistance response-induced leaves and in leaves following induction with salicylic acid, methyl jasmonate, wounding, hydrogen peroxide, and ultraviolet-B. The expression of PepRSH was also influenced by abiotic stresses, such as flooding and high salinity. The deduced PepRSH protein has a putative chloroplast-targeting transit peptide at its N-terminus, and immunolocalization studies verified the translocation of PepRSH to the chloroplast. The predicted PepRSH protein is markedly similar to known plant and bacterial RSH proteins. Expression of a putative (p)ppGpp synthetase domain in an Escherichia coli single mutant (RelA^-SpoT⁺) complemented growth of the mutant but not of an E. coli double mutant (RelA^-SpoT^-), demonstrating that PepRSH has (p)ppGpp synthetase activity only in the (p)ppGpp synthetase domain. Site-directed mutagenesis of the conserved histidine and aspartic acid (HD) site in the putative HD domain of PepRSH revealed that the histidine and aspartic acid dual sites were critical residues for the (p)ppGpp synthetase activity of PepRSH protein. Mutation of the HD site limited the tolerance of bacteria to both salt and osmotic stress. Our results indicate that pepper plants have a (p)ppGpp regulatory system that is similar to that of bacteria and which may transduce stress-related signals through the regulation of (p)ppGpp by PepRSH localized in chloroplasts. Crown

Original language	English
Pages (from-to)	635-642
Number of pages	8
Journal	Plant Science
Volume	176
Issue number	5
DOIs	https://doi.org/10.1016/j.plantsci.2009.02.004
Publication status	Published - 2009 May

Bibliographical note

Funding Information:
This project was funded by a grant for Basic Research from the NIAB (National Institute of Agricultural Biotechnology) Research Program at the Rural Development Administration. This investigation was also supported by the Korea Research Foundation Grant funded by the Korean Government [KRF-2008-359-F00005]. We thank Dr. M. Cashel, National Institute of Health, USA, for providing E. coli mutants (CF1648, CF1652 and CF1693).

Keywords

(p)ppGpp synthetase
Biotic and abiotic stress
Pepper

ASJC Scopus subject areas

Genetics
Agronomy and Crop Science
Plant Science

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10.1016/j.plantsci.2009.02.004

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@article{8a472f9797e74e4e8074c924d97b5d54,

title = "Molecular characterization of a biotic and abiotic stress resistance-related gene RelA/SpoT homologue (PepRSH) from pepper",

abstract = "A gene encoding a putative guanosine 5′-diphosphate (or 5′-triphosphate) 3′-diphosphate ((p)ppGpp) synthetase, designated PepRSH (Pepper RelA/SpoT homologue), was isolated from hot peppers. PepRSH was found to contain five introns and six exons and a 2166-bp open reading frame encoding a protein of 721 amino acids; this protein displayed significant homology with other plant (p)ppGpp synthetases. A genomic DNA gel blot analysis revealed that the pepper genome has at least a single copy of PepRSH. PepRSH transcripts were highly accumulated in non-host resistance response-induced leaves and in leaves following induction with salicylic acid, methyl jasmonate, wounding, hydrogen peroxide, and ultraviolet-B. The expression of PepRSH was also influenced by abiotic stresses, such as flooding and high salinity. The deduced PepRSH protein has a putative chloroplast-targeting transit peptide at its N-terminus, and immunolocalization studies verified the translocation of PepRSH to the chloroplast. The predicted PepRSH protein is markedly similar to known plant and bacterial RSH proteins. Expression of a putative (p)ppGpp synthetase domain in an Escherichia coli single mutant (RelA-SpoT+) complemented growth of the mutant but not of an E. coli double mutant (RelA-SpoT-), demonstrating that PepRSH has (p)ppGpp synthetase activity only in the (p)ppGpp synthetase domain. Site-directed mutagenesis of the conserved histidine and aspartic acid (HD) site in the putative HD domain of PepRSH revealed that the histidine and aspartic acid dual sites were critical residues for the (p)ppGpp synthetase activity of PepRSH protein. Mutation of the HD site limited the tolerance of bacteria to both salt and osmotic stress. Our results indicate that pepper plants have a (p)ppGpp regulatory system that is similar to that of bacteria and which may transduce stress-related signals through the regulation of (p)ppGpp by PepRSH localized in chloroplasts. Crown",

keywords = "(p)ppGpp synthetase, Biotic and abiotic stress, Pepper",

author = "Kim, {Tae Ho} and Ok, {Sung Han} and Donghern Kim and Suh, {Seok Cheol} and Byun, {Myung Ok} and Shin, {Jeong Sheop}",

note = "Funding Information: This project was funded by a grant for Basic Research from the NIAB (National Institute of Agricultural Biotechnology) Research Program at the Rural Development Administration. This investigation was also supported by the Korea Research Foundation Grant funded by the Korean Government [KRF-2008-359-F00005]. We thank Dr. M. Cashel, National Institute of Health, USA, for providing E. coli mutants (CF1648, CF1652 and CF1693). ",

year = "2009",

month = may,

doi = "10.1016/j.plantsci.2009.02.004",

language = "English",

volume = "176",

pages = "635--642",

journal = "Plant Science",

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T1 - Molecular characterization of a biotic and abiotic stress resistance-related gene RelA/SpoT homologue (PepRSH) from pepper

AU - Kim, Tae Ho

AU - Ok, Sung Han

AU - Kim, Donghern

AU - Suh, Seok Cheol

AU - Byun, Myung Ok

AU - Shin, Jeong Sheop

N1 - Funding Information: This project was funded by a grant for Basic Research from the NIAB (National Institute of Agricultural Biotechnology) Research Program at the Rural Development Administration. This investigation was also supported by the Korea Research Foundation Grant funded by the Korean Government [KRF-2008-359-F00005]. We thank Dr. M. Cashel, National Institute of Health, USA, for providing E. coli mutants (CF1648, CF1652 and CF1693).

PY - 2009/5

Y1 - 2009/5

N2 - A gene encoding a putative guanosine 5′-diphosphate (or 5′-triphosphate) 3′-diphosphate ((p)ppGpp) synthetase, designated PepRSH (Pepper RelA/SpoT homologue), was isolated from hot peppers. PepRSH was found to contain five introns and six exons and a 2166-bp open reading frame encoding a protein of 721 amino acids; this protein displayed significant homology with other plant (p)ppGpp synthetases. A genomic DNA gel blot analysis revealed that the pepper genome has at least a single copy of PepRSH. PepRSH transcripts were highly accumulated in non-host resistance response-induced leaves and in leaves following induction with salicylic acid, methyl jasmonate, wounding, hydrogen peroxide, and ultraviolet-B. The expression of PepRSH was also influenced by abiotic stresses, such as flooding and high salinity. The deduced PepRSH protein has a putative chloroplast-targeting transit peptide at its N-terminus, and immunolocalization studies verified the translocation of PepRSH to the chloroplast. The predicted PepRSH protein is markedly similar to known plant and bacterial RSH proteins. Expression of a putative (p)ppGpp synthetase domain in an Escherichia coli single mutant (RelA-SpoT+) complemented growth of the mutant but not of an E. coli double mutant (RelA-SpoT-), demonstrating that PepRSH has (p)ppGpp synthetase activity only in the (p)ppGpp synthetase domain. Site-directed mutagenesis of the conserved histidine and aspartic acid (HD) site in the putative HD domain of PepRSH revealed that the histidine and aspartic acid dual sites were critical residues for the (p)ppGpp synthetase activity of PepRSH protein. Mutation of the HD site limited the tolerance of bacteria to both salt and osmotic stress. Our results indicate that pepper plants have a (p)ppGpp regulatory system that is similar to that of bacteria and which may transduce stress-related signals through the regulation of (p)ppGpp by PepRSH localized in chloroplasts. Crown

AB - A gene encoding a putative guanosine 5′-diphosphate (or 5′-triphosphate) 3′-diphosphate ((p)ppGpp) synthetase, designated PepRSH (Pepper RelA/SpoT homologue), was isolated from hot peppers. PepRSH was found to contain five introns and six exons and a 2166-bp open reading frame encoding a protein of 721 amino acids; this protein displayed significant homology with other plant (p)ppGpp synthetases. A genomic DNA gel blot analysis revealed that the pepper genome has at least a single copy of PepRSH. PepRSH transcripts were highly accumulated in non-host resistance response-induced leaves and in leaves following induction with salicylic acid, methyl jasmonate, wounding, hydrogen peroxide, and ultraviolet-B. The expression of PepRSH was also influenced by abiotic stresses, such as flooding and high salinity. The deduced PepRSH protein has a putative chloroplast-targeting transit peptide at its N-terminus, and immunolocalization studies verified the translocation of PepRSH to the chloroplast. The predicted PepRSH protein is markedly similar to known plant and bacterial RSH proteins. Expression of a putative (p)ppGpp synthetase domain in an Escherichia coli single mutant (RelA-SpoT+) complemented growth of the mutant but not of an E. coli double mutant (RelA-SpoT-), demonstrating that PepRSH has (p)ppGpp synthetase activity only in the (p)ppGpp synthetase domain. Site-directed mutagenesis of the conserved histidine and aspartic acid (HD) site in the putative HD domain of PepRSH revealed that the histidine and aspartic acid dual sites were critical residues for the (p)ppGpp synthetase activity of PepRSH protein. Mutation of the HD site limited the tolerance of bacteria to both salt and osmotic stress. Our results indicate that pepper plants have a (p)ppGpp regulatory system that is similar to that of bacteria and which may transduce stress-related signals through the regulation of (p)ppGpp by PepRSH localized in chloroplasts. Crown

KW - (p)ppGpp synthetase

KW - Biotic and abiotic stress

KW - Pepper

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U2 - 10.1016/j.plantsci.2009.02.004

DO - 10.1016/j.plantsci.2009.02.004

M3 - Article

AN - SCOPUS:61849151869

SN - 0168-9452

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SP - 635

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Molecular characterization of a biotic and abiotic stress resistance-related gene RelA/SpoT homologue (PepRSH) from pepper

Abstract

Bibliographical note

Keywords

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