Overexpression of NPR1 in Brassica juncea Confers Broad Spectrum Resistance to Fungal Pathogens

Abstract

Brassica juncea (Indian mustard) is a commercially important oil seed crop, which is highly affected by many biotic stresses. Among them, Alternaria leaf blight and powdery mildew are the most devastating diseases leading to huge yield losses in B. juncea around the world. In this regard, genetic engineering is a promising tool that may possibly allow us to enhance the B. juncea disease resistance against these pathogens. NPR1 (non-expressor of pathogen-related gene 1) is a bonafide receptor of salicylic acid (SA) which modulates multiple immune responses in plants especially activation of induced and systemic acquired resistance (SAR). Here, we report the isolation and characterization of new NPR1 homolog (BjNPR1) from B. juncea. The phylogenetic tree constructed based on the deduced sequence of BjNPR1 with homologs from other species revealed that BjNPR1 grouped together with other known NPR1 proteins of Cruciferae family, and was nearest to B. napus. Furthermore, expression analysis showed that BjNPR1 was upregulated after SA treatment and fungal infection but not by jasmonic acid or abscisic acid. To understand the defensive role of this gene, we generated B. juncea transgenic lines overexpressing BjNPR1, and further confirmed by PCR and Southern blotting. The transgenic lines showed no phenotypic abnormalities, and constitutive expression of BjNPR1 activates defense signaling pathways by priming the expression of antifungal PR genes. Moreover, BjNPR1 transgenic lines showed enhanced resistance to Alternaria brassicae and Erysiphe cruciferarum as there was delay in symptoms and reduced disease severity than non-transgenic plants. In addition, the rate of disease spreading to uninfected or distal parts was also delayed in transgenic plants thus suggesting the activation of SAR. Altogether, the present study suggests that BjNPR1 is involved in broad spectrum of disease resistance against fungal pathogens.

Keywords: Alternaria; Brassica juncea; NPR1; Powdery mildew; jasmonic acid; salicylic acid; systemic acquired resistance.

FIGURE 1

Phylogenetic analysis of BjNPR1 with other NPR1 proteins from different plant species. The deduced amino acid sequences of BjNPR1 was retrieved from NCBI GenBank and were further aligned with ClustalW using MEGA7.1 bioinformatic tool. The tree was generated using Maximum-Likelihood (ML) method with 1000 bootstrap replicates. GenBank IDs of each NPR1 protein sequence are given in the brackets behind the species names.

FIGURE 2

A multiple sequence alignment and in silico analysis of BjNPR1 protein sequence. (A) Alignments of the deduced amino acid sequences of BjNPR1 (accession no. ABC94642) revealed 92% identity with BnNPR1 (accession no. XP013725724), 78% with BolNPR1 (accession no. XP013605797), 78% with BrNPR1 (accession no. XP009109186), and 66% with AtNPR1 (accession no. ABR46023). (B) The conserved domains (BTB-ANK) of the BjNPR1 protein after analyzed by Pfam.

FIGURE 3

Expression analysis of BjNPR1 gene after hormonal treatment and fungal infection. Forty days old B. juncea plants were treated with different defense stimulators (SA, JA, and ABA) and also infected with fungal pathogens (A. brassicae and E. cruciferarum). Leaf samples were harvested at different time points for RNA isolation. Control plants for each treatment were treated with sterile distilled water containing equal amount of solvent used for hormone preparation. (A) Expression analysis of BjNPR1 after SA (B) JA (C) ABA (D) Alternaria infection and (E) E. cruciferarum, respectively. SE for each bar is shown. The asterisks indicate statistically significant difference relative to control and was calculated by student’s t-test (^∗P < 0.05; ^∗∗P < 0.01).

FIGURE 4

Development of 35S-BjNPR1 transgenic lines and molecular analysis. (A) Schematic representation of BjNPR1-pBi121 construct and T-DNA map of pBi121 binary vector. (B) T-DNA integration in 10 transgenic lines was confirmed by PCR amplification, using 35S promoter (forward) and BjNPR1 (reverse) gene specific primers. (C) Real-time PCR quantification of BjNPR1 in wild type and 35S-NPR1 overexpressed B. juncea plants. The asterisks indicate statistically significant differences between control (non-transgenic) and BjNPR1 transgenic plants (^∗P < 0.05; ^∗∗P < 0.01). (D) Southern blotting analysis showing BjNPR1 transgene integration, DNA was digested with HindIII restriction enzyme and hybridized with DIG labeled 35S-NPR1 probe. (E) Southern blotting analysis showing copy number in selected highly expressed transgenic line (L2, L5, L7, and L8), respectively, DNA was digested with Sac1 and hybridized with DIG labeled NPTII gene probe.

FIGURE 5

Quantification of mRNA levels of SA- and JA-dependent PR genes in wild type and BjNPR1 transgenic plants. Expression analysis of PR1, PR2, PR3, PR5, PR12, and PR13 in control and BjNPR1 transgenic plants. The relative expression levels of SA and JA dependent BjPR genes in transgenic and wild-type plants were compared with that of a control alpha tubulin gene. The data are the mean ± SE of three biological replicates. SE for each bar is shown. The asterisks indicate statistically significant differences between the BjNPR1 transgenic and control (non-transgenic) plants (^∗P < 0.05; ^∗∗P < 0.01).

FIGURE 6

Agronomic traits of wild type and BjNPR1 transgenic plants. Different agronomic traits such as size and shape of leaf, silique, flower, number of pods, number of seeds as well plant height were studied in BjNPR1 transgenic and control plants. BjNPR1 transgenic lines did not show any phenotypic abnormalities. Scale bar 1 cm (A,B), 0.1 cm (C,D), 0.5 cm (E,F), 0.1 cm (G,I), and 0.05 cm (H,J).

FIGURE 7

Overexpression of BjNPR1 improves disease resistance in B. juncea. (A) Disease resistance screening of BjNPR1 transgenic lines (L2 and L5) after Alternaria infection. L2 and L5 showed delay and reduction in disease severity than wild-type plants. (B) Lesions diameter of control (wild type), L2 and L5 BjNPR1 lines after Alternaria infection at different dpi. Bar = 40 mm. (C) Evaluation of disease resistance to Alternaria in BjNPR1 transgenic lines using six-point disease severity index. (D) Alternaria disease severity was monitored in BjNPR1 transgenic lines and control plants at 15th dpi based on total leaf area infected. Three biological replicates were used for infection. SD are the means of three biological replicates and asterisks shows statistically significant difference (^∗P < 0.05; ^∗∗P < 0.01) between BjNPR1 transgenic lines and wild-type plants.

FIGURE 8

Analysis of cell death and fungal biomass in BjNPR1 transgenic and wild-type plants using trypan blue staining. (A) Microscopic examination of Alternaria mediated cell death in BjNPR1 lines and wild-type plants are shown with white color arrows. (B) Trypan blue staining of BjNPR1 transgenic and control plants after Alternaria infection. Alternaria spore load or biomass in BjNPR1 transgenic lines and wild-type plants at various dpi are shown with black color arrows. BjNPR1 transgenic lines showed reduced cell death and spore count as compared to wild-type plants. Bar = 40 μm.

FIGURE 9

Screening of BjNPR1 transgenic lines for powdery mildew disease resistance. Forty days old wild-type plants and BjNPR1 transgenic plants were infected with E. cruciferarum and disease scoring was done at different time intervals. (A,B) BjNPR1 transgenic lines (L2 and L5) showed reduced number of E. cruciferarum colonies than wild-type plants at 7th, 12th, and 18th dpi. (C) E. cruciferarum disease severity in BjNPR1 transgenic lines and wild-type plants. Bar = 35 μm. The asterisks indicate statistically significant differences between the BjNPR1 transgenic and control (non-transgenic) plants after powdery mildew infection (^∗P < 0.05; ^∗∗P < 0.01).

FIGURE 10

Microscopic examination of cell death and fungal biomass in BjNPR1 transgenic and wild-type plants using trypan blue staining. (A) Microscopic examination of E. cruciferarum mediated cell death in BjNPR1 lines and wild-type plants are shown with bold white arrows. (B) E. cruciferarum spore load or biomass in BjNPR1 transgenic lines and wild-type plants at various dpi after trypan blue staining are highlighted with bold black arrows. Bar = 30 μm.