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Identification of a novel salicylic acid inducible endogenous plant promoter regulating expression of CYR1, a CC-NB-LRR type candidate disease resistance gene in Vigna mungo

Abstract

In the present study, the upstream regulatory region of CYR1, a CC-NBS-LRR type candidate disease resistance gene of Vigna mungo has been characterized. PLACE and PlantCARE search revealed presence of some biotic and abiotic stress responsive cis-elements namely, wound/pathogen inducible W-box, salicylic acid (SA) inducible TCA element, sugar inducible pyrimidine box abscisic acid/drought responsive MYB etc. in this upstream region. The 877 bp long upstream/putative-promoter region (Cyr1P) was segmented into six different fragments, Cyr1P1-Cyr1P6 and coupled with GUS-reporter gene. Their ability to express the GUS in different plants like tobacco, spinach, onion and Vigna, individually were investigated both transiently and transgenetically. Among these, Cyr1P4 (−572 to +1) and Cyr1P5 (−472 to +1) showed capability to drive expression of GUS in all above plant systems. EMSA and site directed mutagenesis study confirmed effective binding of tobacco nuclear factors to the regulatory region 1 (−673 to −573, RR1) and 2 (−472 to −371, RR2) of Cyr1P promoter. Histochemical and biochemical GUS assay of transgenic tobacco tissues expressing GUS under the control of Cyr1P4 and Cyr1P5 promoter fragments demonstrated that they are near-constitutive type of promoters. The expression level of GUS driven by Cyr1P4 and Cyr1P5 promoter was enhanced in presence of exogenous SA and NaCl. The inducible R gene promoters like Cyr1P4 and Cyr1P5 may become powerful tools in developing MYMIV-resistance in susceptible Vigna and use of such promoters coupled with R genes could strengthen our understanding regarding the molecular events of plant pathogen interaction.

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Abbreviations

GUS:

β-Glucuronidase

SA:

Salicylic acid

EMSA:

Electrophoretic mobility shift assay

MYMIV:

Mungbeen yellow mosaic India virus

MUG:

4-Methylumbelliferyl-beta-d-glucuronide

X-gluc:

5-Bromo-4-chloro-3-indolyl-beta-d-glucuronic acid

bp:

Base pair

h:

Hour/s

NaCl:

Sodium chloride

X-gal:

5-Bromo-4-chloro-3-indolyl-β-d-galactopyranoside

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Acknowledgments

We are thankful to the Director, Institute of Life Sciences for providing us all the lab facilities and providing core fund to carry out the work. We are thankful to Mr. Abhimanyu Das for his kind help in the study.

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Correspondence to Nrisingha Dey.

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Soumitra Maiti and Sunita Patro have contributed equally to this article.

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Maiti, S., Patro, S., Pal, A. et al. Identification of a novel salicylic acid inducible endogenous plant promoter regulating expression of CYR1, a CC-NB-LRR type candidate disease resistance gene in Vigna mungo . Plant Cell Tiss Organ Cult 120, 489–505 (2015). https://doi.org/10.1007/s11240-014-0616-z

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  • DOI: https://doi.org/10.1007/s11240-014-0616-z

Keywords

  • Endogenous promoter
  • SA inducible
  • Disease resistance gene
  • Vigna mungo