Isolation and characterization of systemic acquired resistance marker gene PR1 and its promoter from Brassica juncea

Abstract

Systemic acquired resistance (SAR) is an inducible defense response in plants that provides enhanced resistance against a variety of pathogens. In this regard, SAR marker gene PR1 (pathogenesis-related gene 1) was isolated from Brassica juncea and was named as BjPR1. The amino acid sequence of BjPR1 protein showed 99, 92, and 78% similarity with known PR1 proteins of Brassica rapa, Brassica napus, and Arabidopsis thaliana, respectively. Quantitative real-time PCR (qRT-PCR) analysis showed increased expression of BjPR1 gene both in local (infected) and distal (non-infected) leaves of B. juncea after Alternaria brassicae infection, whereas mechanical wounding showed expression only in local (wounded) leaves but not in distal (unwounded) leaves. Moreover, BjPR1 gene was strongly induced by salicylic acid (SA), whereas no such induction was observed following jasmonic acid (JA) or abscisic acid (ABA) treatments. To further elucidate gene regulation pattern of BjPR1, 2 kb promoter region of BjPR1 was isolated and subjected to in silico analysis which identified many potential cis-regulatory elements associated with plant defense as well as signaling pathways. The transient GUS expression analysis showed strong expression of GUS gene driven by BjPR1 promoter after SA treatment, while as ABA and JA downregulates GUS gene expression compared to control. In addition, BjPR1 promoter was significantly induced by wounding at local tissues. Hence, these results highlight the multiple role of BjPR1 gene in response to biotic and abiotic stresses. In addition, the present study also reported BjPR1 promoter as stress-specific inducible promoter that can be ideal candidate for controlling the expression of biotic stress response genes in transgenic plants.

Keywords: Abscisic acid; Alternaria brassicae; Brassica juncea; Jasmonic acid; Pathogenesis-related proteins; Salicylic acid; Systemic acquired resistance.

Fig. 1

Structural analysis of BjPR1 nucleotide and amino acid sequences a Nucleotide and amino acid sequences of BjPR1 with putative phosphorylation sites like serine and tyrosine are shown in bold italics b 3D structure of BjPR1 protein c Conserved domain of the BjPR1 protein. The predicted BjPR1 protein contained a conserved motif at residues 30–161 aa that belonged to the SCP-PR1 like super family d Multiple sequence alignment of the BjPR1 protein sequence with other plant PR1 proteins. Comparison of deduced amino acid sequence of BjPR1 with other plant PR1 s from B. rapa, B.napus, B. oleracea, A. thaliana, C. sativa, O. sativa, and Z. mays. Conserved residues are shown with shaded colours

Fig. 2

Phylogenetic relationship of BjPR1 with homologs of other plant species, constructed using the MEGA 7.0 program. Bootstrap values denote the divergence of each branch and the scale indicates branch length. BjPR1 is highlighted as black colour circular marker

Fig. 3

In vivo infection of B. juncea with A. brassiace a A. brassiace culture grown on root radish medium b Microscopic identification of A. brassicae fungus (Conidia under 100X microscope) c Uninfected B. juncea leaf as control d‒e B. juncea leaves after Alternaria infection

Fig. 4

Local and systemic expression of BjPR1 gene after Alternaria infection and wounding: a expression of BjPR1 in local (infected) leaves at various time points; b expression of BjPR1 in distal (non-infected) leaves; c relative expression of BjPR1 in local (wounded) leaves; d relative expression of BjPR1 in distal (unwounded) leaves. SE for each bar is shown. The relative expression was calculated using ΔΔCt method. The asterisks indicate statistically significant differences between the control and treated B. juncea plants (*P < 0.05; **P < 0.01)

Fig. 5

Transcriptional studies of BjPR1 under various hormonal stresses by qRT-PCR analysis. B. juncea plants were treated with 2-mM SA, 100-μM MeJA, and 50-μM ABA, respectively. Housekeeping gene alpha tubulin was used as internal control. All data are represented as means of three replicates (n = 3) ± SE. The asterisks indicate statistically significant differences between the control and hormone treated B. juncea plants (*P < 0.05; **P < 0.01)

Fig. 6

Transient expression analysis of BjPR1 promoter in tobacco leaves: a schematic representation of BjPR1 promoter cloned in pORER2 vector (promoter less GUS reporter vector) at Pst1 and BamH1 sites for studying promoter inducibility; b healthy N. benthamiana plants for transient expression analysis. After 24 h of agroinfiltration, plants were treated with sterile water (control), ABA, JA, and SA, respectively. Wounding was carried out with sterile needle. Leaf samples were harvested from control and treated plants after 24 h of treatment for GUS staining; c Promoter less GUS reporter vector as negative control; d GUS gene expression driven by BjPR1 promoter without treatment; e effect of ABA on the expression of GUS gene driven by BjPR1 promoter; f effect of JA on the expression of GUS gene driven by BjPR1 promoter in tobacco leaf; g Effect of SA on the expression of GUS gene driven by BjPR1 promoter; h wound-induced GUS accumulation in tobacco leaf