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

Veena, M. A.; Melvin, Prasad; Prabhu, S.; Sekhar, Shailasree; Shetty, H. Shekar; Kini, K. Ramachandra

doi:10.1007/s11033-016-3944-8

Cited by 13 publications

(8 citation statements)

References 43 publications

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“…Transcripts for resistant gene analogs (RGA) were also up regulated in the resistant genotype. The role of RGA, belonging to non-TIR NBS LRR group has been indicated in pearl millet-downy mildew interaction (Veena et al, 2016 ).…”

Section: Resultsmentioning

confidence: 99%

De novo Transcriptome Sequencing to Dissect Candidate Genes Associated with Pearl Millet-Downy Mildew (Sclerospora graminicola Sacc.) Interaction

et al. 2016

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Understanding the plant-pathogen interactions is of utmost importance to design strategies for minimizing the economic deficits caused by pathogens in crops. With an aim to identify genes underlying resistance to downy mildew, a major disease responsible for productivity loss in pearl millet, transcriptome analysis was performed in downy mildew resistant and susceptible genotypes upon infection and control on 454 Roche NGS platform. A total of ~685 Mb data was obtained with 1 575 290 raw reads. The raw reads were pre-processed into high-quality (HQ) reads making to ~82% with an average of 427 bases. The assembly was optimized using four assemblers viz. Newbler, MIRA, CLC and Trinity, out of which MIRA with a total of 14.10 Mb and 90118 transcripts proved to be the best for assembling reads. Differential expression analysis depicted 1396 and 936 and 1000 and 1591 transcripts up and down regulated in resistant inoculated/resistant control and susceptible inoculated/susceptible control respectively with a common of 3644 transcripts. The pathways for secondary metabolism, specifically the phenylpropanoid pathway was up-regulated in resistant genotype. Transcripts up-regulated as a part of defense response included classes of R genes, PR proteins, HR induced proteins and plant hormonal signaling transduction proteins. The transcripts for skp1 protein, purothionin, V type proton ATPase were found to have the highest expression in resistant genotype. Ten transcripts, selected on the basis of their involvement in defense mechanism were validated with qRT-PCR and showed positive co-relation with transcriptome data. Transcriptome analysis evoked potentials of hypersensitive response and systemic acquired resistance as possible mechanism operating in defense mechanism in pearl millet against downy mildew infection.

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Section: Resultsmentioning

confidence: 99%

De novo Transcriptome Sequencing to Dissect Candidate Genes Associated with Pearl Millet-Downy Mildew (Sclerospora graminicola Sacc.) Interaction

et al. 2016

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“…Of the clones obtained, RGPM 301 (GenBank accession number: AY117410) which contained a partial sequence was 83 % similar to rice R-proteins [13]. To identify and characterize the protein encoded by this partial sequence the full-length gene was isolated using rapid amplification of cDNA ends PCR approach [14]. This RGA, was 3552 bp long comprising an ORF of 2979 bp (GenBank accession number: KP226586) coding for 992 amino acids.…”

Section: Tir-nbs-lrr (Tnl) or Coiled Coil-nbs-lrr (Cnl/cc-nbs-lrr)mentioning

confidence: 99%

“…This RGA, was 3552 bp long comprising an ORF of 2979 bp (GenBank accession number: KP226586) coding for 992 amino acids. Based on the conserved domains and motifs this CC-NBS-LRR gene was grouped as a non-TIR NBS LRR containing RGA [14]. Quantitative real time-polymerase chain reaction (qRT-PCR) studies revealed higher expression of transcripts of RGPM 301 in pearl millet cultivar resistant to downy mildew upon infection with the pathogen S. graminicola, as well as upon treatment with biotic and abiotic elicitors [14].…”

Section: Tir-nbs-lrr (Tnl) or Coiled Coil-nbs-lrr (Cnl/cc-nbs-lrr)mentioning

confidence: 99%

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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“…Meanwhile, there are few studies describing R-gene induction when unusual perturbations occur, because they are specific to each interaction; this case, the expression of RGAs has been analyzed in response to elicitor molecules [15,22]. On the other hand, in susceptible cultivars that exhibited a low RGA expression, without pathogen challenge, the exogenous application of JA and SA inductors increased the expression levels of this gene [23].…”

Section: Introductionmentioning

confidence: 99%

Elicitor Induced JA-Signaling Genes Are Associated with Partial Tolerance to Hemibiotrophic Pathogen Phytophthora capsici in Capsicum chinense

et al. 2022

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Phytophthora capsici causes root and stem rot disease in Capsicum. However, molecular mechanisms underlying this pathosystem are little known. The use of elicitors as tools that trigger defense responses to biotic stresses to study molecular plant defense has increased. In this study, early defense induced in the susceptible cultivar C. chinense using three elicitors to assess its role during interaction with hemibiotrophic P. capsici. The response to infection by phenotypic analyses across the time during disease development in seedlings treated with elicitors was compared. Likewise; defense-gene expression were investigated by qRT-PCR. A total of five resistance genes were used as markers of signaling pathways mediated by jasmonate/ethylene (JA/ET) and salicylic acid (SA). Further, six R genes analogs (CcRGAS) related to oomycete-defense were employed. The results showed that elicitors MeJA and b-aminobutyric acid (BABA) slightly reduced disease symptoms. Moreover, MeJA or BABA treatments followed by challenge with P. capsici up-regulated the expression level of genes related to the JA/ET signaling pathway (CcLOX2, CcPDF1 and CcETR1). Furthermore, MeJA treatment followed by challenge triggered a significant induction of de CcRGAS and CcRPP13 expression within 24 h of inoculation. This suggests that in the early defense mechanisms against P. capsici JA signaling plays an important role.

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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

Cited by 13 publications

References 43 publications

De novo Transcriptome Sequencing to Dissect Candidate Genes Associated with Pearl Millet-Downy Mildew (Sclerospora graminicola Sacc.) Interaction

De novo Transcriptome Sequencing to Dissect Candidate Genes Associated with Pearl Millet-Downy Mildew (Sclerospora graminicola Sacc.) Interaction

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

Elicitor Induced JA-Signaling Genes Are Associated with Partial Tolerance to Hemibiotrophic Pathogen Phytophthora capsici in Capsicum chinense

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