The Ectopic Expression of the MpDIR1(t) Gene Enhances the Response of Plants from Arabidopsis thaliana to Biotic Stress by Regulating the Defense Genes and Antioxidant Flavonoids

Abstract

The Defective in Induced Resistance 1 (DIR1) gene, a member of the lipid transferase proteins (LTPs), plays a crucial role in plant defense against pathogens. While previous transcriptomic studies have highlighted the significant expression of citrus LTPs during biotic stress, functional annotations of LTPs in the Citrus genera remain limited. In this study, we cloned the Murraya paniculata DIR1 (MpDIR1(t)) gene and overexpressed it in Arabidopsis thaliana to evaluate its stress response mechanisms against biotic stress. The transgenic Arabidopsis lines showed fewer disease symptoms in response to Pseudomonas syringae (Pst DC3000) compared to wild-type Arabidopsis. Defense and pathogenesis-responsive genes such as PR1, PR4, PR5, and WRKY12 were significantly induced, showing a 2- to 12-fold increase in all transgenic lines compared to the wild type. In addition, the Pst DC3000-infected transgenic Arabidopsis lines demonstrated elevated levels of flavonoids and salicylic acid (SA), along with higher expression of SA-related genes, compared to the wild type. Moreover, all transgenic lines possessed lower reactive oxygen species levels and higher activity of antioxidant defense enzymes such as superoxide dismutase, peroxidase, and catalase under Pst DC3000 stress compared to the wild type. The up-regulation of defense genes, activation of the SA pathway, accumulation of flavonoids, and reinforcement of antioxidant defense mechanisms in transgenic Arabidopsis lines in response to Pst DC3000 underscore the critical role of MpDIR1(t) in fortifying plant immunity. Thus, MpDIR1(t) constitutes a promising candidate gene for improving bacterial disease resistance in commercial citrus cultivars.

Keywords: Arabidopsis; biotic stress; citrus; gene expression; salicylic acid; stress response mechanisms.

Figure 1

Gene expression and amino acid sequence analysis of DIR1 gene. (A) Our gene expression pattern of the DIR1 gene in six different citrus species (with and without CLas inoculation); (B) an amino acid sequence analysis of the DIR1 homologous genes cloned from Citrus reticulata (CsDIR1), Atalantia buxifolia (AbDIR1), and Citrus maxima (CmDIR1); (C) the MpDIR1(t) amino acid sequence compared with its homologous genes from other plants (MR: Morella rubra, AT: Arabidopsis thaliana) Five-pointed star * in (B,C) marks every 10th amino acid for sequence counting. Healthy control: without CLas; CLas-infected: 4 weeks post inoculation with CLas bacteria. Each value is the mean of three biological replicates. A Student’s t-test was used to compare the gene expression of healthy and CLas-infected citrus at ** p < 0.01.

Figure 2

Phylogenetic analysis of the deduced protein sequence of the MpDIR1(t) gene with its homolog genes from Arabidopsis thaliana.

Figure 3

Inoculation of Pst DC3000 bacteria and gene expression analyses in the wild-type and transgenic lines. (A) Gene expression of the Pst DC3000 bacterial pathogen in Arabidopsis at different time points after inoculation, (B) Expression of the MpDIR1(t) gene in Arabidopsis at different time intervals after Pst DC3000 inoculation. CK: healthy control; WT-I: infected plants from the transgenic wild type (control); TG1-I: infected plants from transgenic line 1; TG3-I: infected plants from transgenic line 3; TG6-I: infected plants from transgenic line 6. Each value is the mean of three biological replicates. A Student’s t-test was used to compare transgenic Arabidopsis expressing MpDIR1(t)-TG and WT at * p < 0.05 and ** p < 0.01.

Figure 4

Expression pattern of pathogen-responsive and SA-mediated defense genes. (A) Protease inhibitors 1, AtPI1; (B) pathogenesis-related protein 1, AtPR1; (C) pathogenesis-related protein 2, AtPR2; (D) pathogenesis-related protein 4, AtPR4; (E) pathogenesis-related protein 5, AtPR5; (F) pathogenesis-related protein 10, AtPR10; (G) AtWRKY12; (H) phenylalanine ammonia lyase AtPAL. At: Arabidopsis thaliana; CK: healthy control; WT-I: infected plants from the wild type; TG1-I: infected plants from transgenic line 1; TG3-I: infected plants from transgenic line 3; TG6-I: infected plants from transgenic line 6. Each value is the mean of three biological replicates. A Student’s t-test was used to compare transgenic Arabidopsis expressing MpDIR1(t)-TG and WT at * p < 0.05 and ** p < 0.01.

Figure 5

Salicylic acid and antioxidant enzymatic activities of Arabidopsis leaves injected with Pst DC3000. (A) Salicylic acid; (B) superoxide dismutase, SOD; (C) peroxidase, POD; (D) catalase, CAT; (E) antioxidant capacity (mM Trolox/100 mg); (F) antioxidant activity (%). CK: healthy control; WT-I: infected plants from the wild type; TG1-I: infected plants from transgenic line 1; TG3-I: infected plants from transgenic line 3; TG6-I: infected plants from transgenic line 6. Each value is the mean of three biological replicates. A Student’s t-test was used to compare transgenic Arabidopsis expressing MpDIR1(t)-TG and WT at * p < 0.05 and ** p < 0.01.

Figure 5

Salicylic acid and antioxidant enzymatic activities of Arabidopsis leaves injected with Pst DC3000. (A) Salicylic acid; (B) superoxide dismutase, SOD; (C) peroxidase, POD; (D) catalase, CAT; (E) antioxidant capacity (mM Trolox/100 mg); (F) antioxidant activity (%). CK: healthy control; WT-I: infected plants from the wild type; TG1-I: infected plants from transgenic line 1; TG3-I: infected plants from transgenic line 3; TG6-I: infected plants from transgenic line 6. Each value is the mean of three biological replicates. A Student’s t-test was used to compare transgenic Arabidopsis expressing MpDIR1(t)-TG and WT at * p < 0.05 and ** p < 0.01.

Figure 6

Arabidopsis leaf flavonoids infected with Pst DC3000 bacteria. (A) Hierarchical cluster analysis (HCA), where the columns signify Arabidopsis WT and transgenic lines and the rows represent flavonoid compounds (rows were normalized). (B) Principal component analysis (PCA). (*) means isomers of compound; WT-I: infected plants from the wild type; TG1-I: infected plants from transgenic line 1; TG3-I: infected plants from transgenic line 3; TG6-I: infected plants from transgenic line 6.

Figure 7

Biochemical variables of Arabidopsis leaves infected from Pst DC3000. (A) Superoxide radicals (SOR), (B) hydrogen peroxide (H₂O₂) contents, (C) reactive oxygen species (ROS), (D) electrolytic leakage (%), (E) malondialdehyde (MDA). CK: healthy control; WT-I: infected plants from the wild type; TG1-I: infected plants from transgenic line 1; TG3-I: infected plants from transgenic line 3; TG6-I: infected plants from transgenic line 6. Each value is the means of three biological replicates. A Student’s t-test was used to compare transgenic Arabidopsis expressing MpDIR1(t)-TG and WT at * p < 0.05 and ** p < 0.01.

Figure 7

Biochemical variables of Arabidopsis leaves infected from Pst DC3000. (A) Superoxide radicals (SOR), (B) hydrogen peroxide (H₂O₂) contents, (C) reactive oxygen species (ROS), (D) electrolytic leakage (%), (E) malondialdehyde (MDA). CK: healthy control; WT-I: infected plants from the wild type; TG1-I: infected plants from transgenic line 1; TG3-I: infected plants from transgenic line 3; TG6-I: infected plants from transgenic line 6. Each value is the means of three biological replicates. A Student’s t-test was used to compare transgenic Arabidopsis expressing MpDIR1(t)-TG and WT at * p < 0.05 and ** p < 0.01.