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

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.