Inducibility of the CaDRRG-Pro promoter from chickpea by defense hormones and Ascochyta rabiei elicitors in stable transgenic tobacco as a model plant

Naeimi, Sara; Mamarabadi, Mojtaba; Shokouhifar, Farhad

Inducibility of the CaDRRG-Pro promoter from chickpea by defense hormones and Ascochyta rabiei elicitors in stable transgenic tobacco as a model plant

Articles in Press

Document Type : Original Article

Authors

Sara Naeimi

Mojtaba Mamarabadi

Farhad Shokouhifar

Department of Plant Protection, Faculty of Agriculture, Ferdowsi University of Mashhad, Mashhad, Islamic Republic of Iran.

Abstract

Analysis of early responsive gene promoters provides tools for engineering plant disease resistance. Ascochyta blight, caused by the fungus Ascochyta rabiei, is a major constraint to chickpea cultivation. The CaDRRG gene was previously identified as an early responsive gene showing more than 11-fold increased expression within 3 hours of A. rabiei inoculation. In this study, the CaDRRG-Pro promoter was functionally characterized using stable transgenic tobacco plants. The binary vector pCaDRRG-Pro-Gi, containing a 772 bp CaDRRG-Pro fragment fused to an intron-containing GUS reporter gene, was transferred to Agrobacterium tumefaciens. Stable transgenic lines were generated, and a hierarchical GUS assay identified a high-expressing line (code 1-5) from 30 regenerated plantlets with 63% savings in assays. The promoter was rapidly induced by methyl jasmonate and salicylic acid, with detectable GUS activity at 1 hour and maximum induction within 6 hours, reaching levels comparable to the constitutive CaMV 35S promoter. The promoter also responded to crude mycelial extracts from A. rabiei pathotypes, with clear induction at 3 hours. Direct fungal inoculation resulted in delayed induction (120 hours) with vascular-associated expression patterns. No basal expression was observed in stable transgenic lines under non-induced conditions. The intron-containing GUS reporter prevented prokaryotic expression in Agrobacterium, ensuring specificity. These results provide functional validation of the CaDRRG-Pro promoter, demonstrating its rapid inducibility by defense hormones and fungal elicitors, and position it as a valuable tool for pathogen-inducible expression of resistance genes in chickpea breeding programs.

Keywords

Ascochyta blight

Hierarchical GUS assay

Methyl jasmonate

Pathogen-inducible promoter

Salicylic acid

Transgenic tobacco

Subjects

Plant Disease Management

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