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Curcuma longa Mitogen-Activated Protein Kinase 6 (ClMPK6) Stimulates the Defense Response Pathway and Enhances the Resistance to Necrotrophic Fungal Infection

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Abstract

Mitogen-activated protein kinase (MAPK) cascades are highly conserved signaling modules that transduce the externally perceived signals and play crucial role in plant defense against pathogen attack. In the present study, a turmeric (Curcuma longa L.) complementary DNA (cDNA) encoding a MAPK gene responsive to Pythium aphanidermatum infection was isolated using rapid amplification of cDNA ends (RACE)-PCR. It was designated as ClMPK6 based on its high homology with Arabidopsis AtMPK6. The full-length cDNA of 1484 bp length carried an open reading frame (ORF) of 1176 bp encoding a 391 amino acid polypeptide. ClMPK6 protein contains Thr-Glu-Tyr (TEY) motif on its activation loop with a common docking (CD) domain at the C-terminal end and belong to subgroup A of MAPK family. Southern hybridization revealed single copy of ClMPK6 in turmeric genome, and its intron-exon composition showed highly conserved nature of these signaling kinases across different species. Quantitative RT-PCR showed high expression of ClMPK6 in rhizome tissues of mature turmeric plants. Analysis of temporal expression revealed significant induction of ClMPK6 transcript in response to defense signaling molecules and pathogen attack at the early stages. Ectopic overexpression of ClMPK6 in Arabidopsis plants resulted in enhanced resistance to Botrytis cinerea and constitutively high expression of defense responsive genes like PDF1.2, PAD3, AOS, ACS2, ACS6, etc. Our results suggest that ClMPK6 substantiate the characteristics of AtMPK6 orthologs in defense against necrotrophic infection in plants.

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Acknowledgments

SN is grateful to Siksha O Anusandhan University for providing financial support under the institutional PhD fellowship programme. ER is grateful to Science and Engineering Research Board (SERB), Dept. of Science and Technology (DST), Govt. of India for financial support in form of Junior Research Fellowship. The work is funded by research grant (REGR/2289/SOAU) from Siksha O Anusandhan University, Bhubaneswar, India. The authors are thankful to DST-FIST, Govt. of India, for the facilities provided to Centre of Biotechnology, Siksha O Anusandhan University.

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  1. Raj Kumar Joshi

    Present address: Department of Agricultural, Food and Nutritional Sciences, Faculty of Agricultural, Life & Environmental Sciences, University of Alberta, Edmonton, AB, Canada, T6G 2H1

Authors and Affiliations

  1. Functional Genomics Laboratory, Centre of Biotechnology, Siksha O Anusandhan University, Bhubaneswar, 751003, Odisha, India

    Satyabrata Nanda, Ellojita Rout & Raj Kumar Joshi

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  1. Satyabrata Nanda
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Figure S1

Agarose gel electrophoresis for the cloning of ClMPK6 from turmeric. a Isolation of 646 bp fragment from 5′ region obtained through 5′ RACE-PCR. b Isolation of 1026 bp fragment from 3′ region obtained through 3′ RACE-PCR. c Amplification of 1484 bp fragment representing the full-length cDNA of ClMPK6 gene. M, 100 bp ladder plus; + positive control with expected amplification of ≈300 bp fragment from turmeric Actin 1 gene; − denotes negative control having turmeric RNA as template. (JPG 113 kb)

Figure S2

a SOPMA analysis of deduced ClMPK6 protein: α helix, extended strand, β turn, and random coil are indicated with the blue, red, green, and pink vertical lines, respectively. b Hydropathicity analysis of ClMPK6. The vertical axis indicates the average hydropathicity and the horizontal axis indicates the individual amino acids. Higher negative values indicate hydrophobic nature of ClMPK6. (JPG 401 kb)

Figure S3

qPCR analysis of defense responsive genes in wild type (WT) and transgenic Arabidopsis line ClMPK6#4. Expression levels are shown as relative those of the control plants (0 h), which were set to 1. Standard error bars for the fold changes determined by qRT-PCR are shown. ANOVA test was used to determine the significance of difference between infected and mock samples. Asterisk indicates significant difference at a P value <0.05. (JPG 375 kb)

Figure S4

qPCR analysis of defense responsive genes in wild type (WT) and transgenic Arabidopsis line ClMPK6#31. Expression levels are shown as relative those of the control plants (0 h), which were set to 1. Standard error bars for the fold changes determined by qRT-PCR are shown. ANOVA test was used to determine the significance of difference between infected and mock samples. Asterisk indicates significant difference at a P value <0.05. (JPG 376 kb)

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Nanda, S., Rout, E. & Joshi, R.K. Curcuma longa Mitogen-Activated Protein Kinase 6 (ClMPK6) Stimulates the Defense Response Pathway and Enhances the Resistance to Necrotrophic Fungal Infection. Plant Mol Biol Rep 34, 886–898 (2016). https://doi.org/10.1007/s11105-015-0972-9

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