Resistance Gene Analogues as a Tool for Rapid Identification and Cloning of Disease Resistance Genes in Plants 3 A Review

Sharma, Tilak Raj; Das, Ankita; Kumar, Sachin; Lodha, M. L.

doi:10.1007/bf03263289

Cited by 33 publications

(23 citation statements)

References 76 publications

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“…The CC-NB-LRR protein Rx of potato is found to interact with the Potato virus X (PVX) coat protein conferring resistance to PVX [13] and the Arabidopsis RPS4 gene is involved in resistance to Avr Rps4 of bacterial pathogens [14]. Based on the conserved domains of known R-genes across plant species, novel resistance gene analogues (RGAs) have been isolated from diverse plants by degenerate oligonucleotide primer based PCR approach [15]. This also serves as a tool for rapid isolation of full-length resistance genes.…”

Section: Introductionmentioning

confidence: 99%

Molecular cloning of a coiled-coil-nucleotide-binding-site-leucine-rich repeat gene from pearl millet and its expression pattern in response to the downy mildew pathogen

et al. 2016

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Section: Introductionmentioning

confidence: 99%

Molecular cloning of a coiled-coil-nucleotide-binding-site-leucine-rich repeat gene from pearl millet and its expression pattern in response to the downy mildew pathogen

et al. 2016

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“…The Nickel-CL agarose column (2.5 x 10.0 cm) was equilibrated with the equilibration buffer. The clarified lysate was loaded onto the column and the bound (His) 6 -tagged fusion protein was eluted. The eluted fractions were further analyzed for the recombinant protein by polyacrylamide gel electrophoresis.…”

Section: Affinity Purification Of Rga Rgpm 301 Expressed In Bl21 (De3)mentioning

confidence: 99%

“…The recombinant RGA RGPM 301 was expressed with (His) 6 -tag derived from the pRSET A expression system. The fusion protein was mainly obtained in the soluble fraction of E. coli cell lysate with a molecular mass of approximately 120 kDa resolved on a SDS-PAGE (Fig 4).…”

Section: Induction Of Rga Rgpm 301mentioning

confidence: 99%

“…The non-specifically bound proteins of recombinant RGA RGPM 301 were washed with wash buffer and removed. The (His) 6 tagged fusion protein bound to the agarose column was eluted and the fraction was analysed on SDS-PAGE for confirmation of the 120 kDa recombinant protein to near homogeneity (Fig 5A). The molecular mass of the purified protein corresponded with the .…”

Section: Purification Of His-tagged Recombinant Proteinmentioning

confidence: 99%

“…Plant R-genes with such domains have been isolated by following methods such as positional cloning, transposon tagging and homologous cloning [5]. Since specific domains in R-genes are conserved across plant species, novel resistance gene analogues (RGAs) have been isolated from various plants using degenerate oligonucleotide primer based polymerase chain reaction (PCR) approach [6]. These, RGAs are useful tools for the isolation of full-length Rgenes which provides vital information about the organization of these genes, their expression, roles in resistance mechanism and evolution [7].…”

Section: Introductionmentioning

confidence: 99%

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Cloning, expression and purification of resistance gene analogue RGPM 301 from pearl millet in Escherichia coli

Veena¹,

Melvin²,

Sekhar

et al. 2016

J App Biol Biotech

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Plants combat their pathogens with an array of defense responses. One of the key mechanisms involves products of resistance (R) genes which are responsible for recognition of effector molecules from pathogens and subsequent triggering of defense responses. Resistance gene analogues (RGAs) containing the specific conserved domains of R-genes are isolated from various plants using degenerate oligonucleotide primer based PCR approach. In an earlier study, RGPM 301 an RGA from pearl millet shown to be involved in resistance mechanism against downy mildew disease was isolated and characterized. In the present study, RGPM 301 containing an open reading frame (ORF) of 992 amino acids was cloned into pRSET A expression vector and expressed in Escherichia coli as a Hig-tag fusion protein. The recombinant RGA RGPM 301 was purified to near homogeneity using the Nickel-CL agarose column. Its molecular mass was found to be 120 kDa when separated on the SDS-PAGE which was confirmed by western blotting analysis using the anti-His antibody. The purified protein was subjected to in-gel trypsin digestion followed by mass spectrometric analysis for the confirmation of its identity. These findings facilitate further studies on the exact role of this RGA in the pearl millet downy mildew host pathogen system.

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Plant Disease Resistance Genes: From Perception to Signal Transduction

Padder

2013

Plant Signaling: Understanding the Molecular Crosstalk

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Resistance Gene Analogues as a Tool for Rapid Identification and Cloning of Disease Resistance Genes in Plants 3 A Review

Cited by 33 publications

References 76 publications

Molecular cloning of a coiled-coil-nucleotide-binding-site-leucine-rich repeat gene from pearl millet and its expression pattern in response to the downy mildew pathogen

Molecular cloning of a coiled-coil-nucleotide-binding-site-leucine-rich repeat gene from pearl millet and its expression pattern in response to the downy mildew pathogen

Cloning, expression and purification of resistance gene analogue RGPM 301 from pearl millet in Escherichia coli

Plant Disease Resistance Genes: From Perception to Signal Transduction

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