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.
This is a preview of subscription content, access via your institution.
Buy single article
Instant access to the full article PDF.
Price excludes VAT (USA)
Tax calculation will be finalised during checkout.
Electrophoretic mobility shift assay
Mungbeen yellow mosaic India virus
Basak J, Kundagrami S, Ghose TK, Pal A (2005) Development of yellow mosaic virus (YMV) resistance linked DNA marker in Vigna mungo from populations segregating for YMV-reaction. Mol Breed 14:375–383. doi:10.1007/s11032-005-0238-6
Beerhues L, Kombrink E (1994) Primary structure and expression of mRNAs encoding basic chitinase and 1, 3-beta-glucanase in potato. Plant Mol Biol 24:353–367
Belkhadir Y, Subramaniam R, Dangl JL (2004) Plant disease resistance protein signaling: nBS-LRR proteins and their partners. Curr Opin Plant Biol 7:391–399. doi:10.1016/j.pbi.2004.05.009
Cao Y, Ding X, Cai M et al (2007) The expression pattern of a rice disease resistance gene Xa3/Xa26 is differentially regulated by the genetic backgrounds and developmental stages that influence its function. Genetics 177:523–533. doi:10.1534/genetics.107.075176
Chen H, Nelson RS, Sherwood JL (1994) Enhanced recovery of transformants of Agrobacterium tumefaciens after freeze-thaw transformation and drug selection. Biotechniques 16(664–668):670
Chen G, Pan D, Zhou Y et al (2007) Diversity and evolutionary relationship of nucleotide binding site-encoding disease-resistance gene analogues in sweet potato (Ipomoea batatas Lam.). J Biosci 32:713–721
Dellagi A, Helibronn J, Avrova AO et al (2000) A potato gene encoding a WRKY-like transcription factor is induced in interactions with Erwinia carotovora subsp. atroseptica and Phytophthora infestans and is coregulated with class I endochitinase expression. Mol Plant-Microbe Interact 13:1092–1101. doi:10.1094/MPMI.2000.13.10.1092
Dey N, Maiti IB (1999) Structure and promoter/leader deletion analysis of mirabilis mosaic virus (MMV) full-length transcript promoter in transgenic plants. Plant Mol Biol 40:771–782
Escobar C, Aristizéabal F, Navas A et al (2012) Isolation of active DNA-binding nuclear proteins from tomato galls induced by root-knot nematodes. Plant Mol Biol Rep 19:375–376. doi:10.1007/BF02772837
Eulgem T (2005) Regulation of the arabidopsis defense transcriptome. Trends Plant Sci 10:71–78. doi:10.1016/j.tplants.2004.12.006
Eulgem T, Rushton PJ, Schmelzer E et al (1999) Early nuclear events in plant defence signalling: rapid gene activation by WRKY transcription factors. EMBO J 18:4689–4699. doi:10.1093/emboj/18.17.4689
Eulgem T, Rushton PJ, Robatzek S, Somssich IE (2000) The WRKY superfamily of plant transcription factors. Trends Plant Sci 5:199–206
Eulgem T, Tsuchiya T, Wang XJ et al (2007) EDM2 is required for RPP7-dependent disease resistance in Arabidopsis and affects RPP7 transcript levels. Plant J Cell Mol Biol 49:829–839. doi:10.1111/j.1365-313X.2006.02999.x
Flor HH (1971) Current status of the gene-for-gene concept. Annu Rev Phytopathol 9:275–296. doi:10.1146/annurev.py.09.090171.001423
Gang DR, Costa MA, Fujita M et al (1999) Regiochemical control of monolignol radical coupling: a new paradigm for lignin and lignan biosynthesis. Chem Biol 6:143–151. doi:10.1016/S1074-5521(99)89006-1
Gu K, Yang B, Tian D et al (2005) R gene expression induced by a type-III effector triggers disease resistance in rice. Nature 435:1122–1125. doi:10.1038/nature03630
Jefferson RA, Kavanagh TA, Bevan MW (1987) GUS fusions: beta-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J 6:3901–3907
Jones JDG, Dangl JL (2006) The plant immune system. Nature 444:323–329. doi:10.1038/nature05286
Jones D, Jones J (1996) The roles of leucine-rich repeats in plant defenses. Adv Bot Res 89–167
Katagiri F (2004) A global view of defense gene expression regulation—a highly interconnected signaling network. Curr Opin Plant Biol 7:506–511. doi:10.1016/j.pbi.2004.07.013
Kiernan JM, Wu FC, Goldberg KB et al (1993) Transformation in Nicotiana edwardsonii. In: Bajaj YPS (ed) Plant Protoplasts Genet Eng III. Springer, Berlin Heidelberg, pp 294–307
Kumar D, Patro S, Ranjan R et al (2011) Development of useful recombinant promoter and its expression analysis in different plant cells using confocal laser scanning microscopy. PLoS ONE 6:e24627. doi:10.1371/journal.pone.0024627
Kundu S, Chakraborty D, Pal A (2011) Proteomic analysis of salicylic acid induced resistance to mungbean yellow mosaic India virus in Vigna mungo. J Proteomics 74:337–349. doi:10.1016/j.jprot.2010.11.012
Lebel E, Heifetz P, Thorne L et al (1998) Functional analysis of regulatory sequences controlling PR-1 gene expression in Arabidopsis. Plant J Cell Mol Biol 16:223–233
Maiti S, Basak J, Kundagrami S et al (2011) Molecular marker-assisted genotyping of mungbean yellow mosaic India virus resistant germplasms of mungbean and urdbean. Mol Biotechnol 47:95–104. doi:10.1007/s12033-010-9314-1
Maiti S, Paul S, Pal A (2012) Isolation, characterization, and structure analysis of a non-TIR-NBS-LRR encoding candidate gene from MYMIV-resistant Vigna mungo. Mol Biotechnol 52:217–233. doi:10.1007/s12033-011-9488-1
Meyers BC, Kozik A, Griego A et al (2003) Genome-wide analysis of NBS-LRR-encoding genes in Arabidopsis. Plant Cell 15:809–834
Nimchuk Z, Eulgem T, Holt BF, Dangl JL (2003) Recognition and response in the plant immune system. Annu Rev Genet 37:579–609. doi:10.1146/annurev.genet.37.110801.142628
Oldroyd GE, Staskawicz BJ (1998) Genetically engineered broad-spectrum disease resistance in tomato. Proc Natl Acad Sci 95:10300–10305
Puzio Lausen, Heinen Grundler (2000) Promoter analysis of pyk20, a gene from Arabidopsis thaliana. Plant Sci Int J Exp Plant Biol 157:245–255
Riechmann JL, Meyerowitz EM (1998) The AP2/EREBP family of plant transcription factors. Biol Chem 379:633–646
Rushton PJ, Somssich IE (1998) Transcriptional control of plant genes responsive to pathogens. Curr Opin Plant Biol 1:311–315
Schardl CL, Byrd AD, Benzion G et al (1987) Design and construction of a versatile system for the expression of foreign genes in plants. Gene 61:1–11
Sparkes IA, Runions J, Kearns A, Hawes C (2006) Rapid, transient expression of fluorescent fusion proteins in tobacco plants and generation of stably transformed plants. Nat Protoc 1:2019–2025. doi:10.1038/nprot.2006.286
Staiger D, Kaulen H, Schell J (1989) A CACGTG motif of the Antirrhinum majus chalcone synthase promoter is recognized by an evolutionarily conserved nuclear protein. Proc Natl Acad Sci 86:6930–6934
Stokes TL, Kunkel BN, Richards EJ (2002) Epigenetic variation in Arabidopsis disease resistance. Genes Dev 16:171–182. doi:10.1101/gad.952102
Vlot AC, Klessig DF, Park SW (2008) Systemic acquired resistance: the elusive signal(s). Curr Opin Plant Biol 11:436–442. doi:10.1016/j.pbi.2008.05.003
Yu D, Chen C, Chen Z (2001) Evidence for an important role of WRKY DNA binding proteins in the regulation of NPR1 gene expression. Plant Cell 13:1527–1540
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.
Soumitra Maiti and Sunita Patro have contributed equally to this article.
Electronic supplementary material
Below is the link to the electronic supplementary material.
About this article
Cite this article
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