Overexpression of the Arabidopsis MACPF Protein AtMACP2 Promotes Pathogen Resistance by Activating SA Signaling

Abstract

Immune response in plants is tightly regulated by the coordination of the cell surface and intracellular receptors. In animals, the membrane attack complex/perforin-like (MACPF) protein superfamily creates oligomeric pore structures on the cell surface during pathogen infection. However, the function and molecular mechanism of MACPF proteins in plant pathogen responses remain largely unclear. In this study, we identified an Arabidopsis MACP2 and investigated the responsiveness of this protein during both bacterial and fungal pathogens. We suggest that MACP2 induces programmed cell death, bacterial pathogen resistance, and necrotrophic fungal pathogen sensitivity by activating the biosynthesis of tryptophan-derived indole glucosinolates and the salicylic acid signaling pathway dependent on the activity of enhanced disease susceptibility 1 (EDS1). Moreover, the response of MACP2 mRNA isoforms upon pathogen attack is differentially regulated by a posttranscriptional mechanism: alternative splicing. In comparison to previously reported MACPFs in Arabidopsis, MACP2 shares a redundant but nonoverlapping role in plant immunity. Thus, our findings provide novel insights and genetic tools for the MACPF family in maintaining SA accumulation in response to pathogens in Arabidopsis.

Keywords: MACP2; indole glucosinolates; membrane attack complex/perforin-like protein; pathogen infection; salicylic acid signaling.

Figure 1

Characterization of T-DNA insertional mutants and transgenic overexpression lines in MACP2. (A) Schematic depicting the MACP2 gene, indicating the T-DNA insertion site of MACP2-KO-1 and MACP2-KO-2. (B) Identification of MACP2-KOs via PCR. The full-length MACP2 was amplified with primer pair XS2591 and XS2592. The length-contained T-DNA sequence was amplified via primer pair LBa1 and XS2592 in KO-1, and KO-2. (C) Semiquantitative PCR of MACP2 in WT and MACP2-KOs. The full-length MACP2 was amplified with primer pair XS2591 and XS2592. The ACTIN2 was amplified with primer pair ACTIN2-F and ACTIN2-R. (D) Identification in DNA level of MACP2-YFP transgenic plants. MACP2 CDS was cloned into pFGC-RCS binary vector then the expression cassette of MACP2-YFP was inserted into the Arabidopsis genome. UBQ10 and XS2489 were derived from the pFGC-RCS plasmid and MACP2 CDS, respectively. (E) Identification in RNA level of MACP2-YFP transgenic plants. Transcriptional level of MACP2 in MACP2-OE-1 and MACP2-OE-2 upregulated 3–5 times as that in wild-type. The data represent means from three independent repeats. Statistical differences were identified using Student’s t test. ** p < 0.01. (F) Identification in protein level of MACP2-YFP transgenic plants. Anti-GFP was used to recognize the specific YFP tag. CBB represented Coomassie blue staining.

Figure 2

Overexpression of MACP2 showed accelerated cell death in rosettes. Trypan blue staining (A) and DAB staining (B) of wild-type, MACP2-KO mutants, and MACP2-OE rosettes after 4, 5, and 6-week development. MACP2-OEs obtained more cell death lesions and higher levels of H₂O₂, indicated by the brown color, than wild-type in the 5th and 6th weeks, while MACP2-KOs obtained less cell death and lower levels of H₂O₂ than wild-type. Bar = 1 mm.

Figure 3

MACP2-strengthened plant resistance relying on SA pathway to the bacterial pathogen. (A) Phenotypes of wild-type, MACP2-KOs, and MACP2-OEs in response to Pst DC3000 infection. Four-week-old wild-type, MACP2-KOs, and MACP2-OEs were infected with Pst DC3000 on leaf surface and photographed 5 days after treatment. (B) Bacterial populations at 5 days postinoculation in wild-type, MACP2-KOs, and MACP2-OEs leaves. The data represent means from three independent repeats. Statistical differences were identified using Student’s t test. ** p < 0.01. (C) SA contents detection of wild-type, MACP2-KO mutants, and MACP2-OEs adult plants during Pst DC3000 infection. The contents of SA and SAG were measured by LC-MS. The “g” in “ng/g” represents the fresh weight. The experiments were biologically repeated three times with similar results. Error bars represent SD (n = 3 biological replicates). * p < 0.05, ** p < 0.01 by Student’s t test. (D) Phenotypes of leaves from 4-week-old wild-type, MACP2-OE, eds1-22, and MACP2-OE eds1-22 leaves in response to Pst DC3000 infection. (E) Bacterial populations at 5 days postinoculation in wild-type, MACP2-OE, eds1-22, and MACP2-OE eds1-22 leaves. The data represent means from three independent repeats. Statistically significant differences were identified using Student’s t test. * p < 0.05, ** p < 0.01.

Figure 4

MACP2-weakened plant resistance depending on SA pathway to fungal pathogen. (A) Phenotypes of leaves from 4-week-old wild-type, MACP2-KO mutants, and MACP2-OEs plants in response to B. cinerea infection. Added B.c. on leaf surface and photographed 3 days after treatment. (B) Relative lesion size of wild-type, MACP2-KOs, and MACP2-OEs leaves after 3 days of B. cinerea infection. The lesion size was calculated by ImageJ and relative lesion size was calculated by comparing the values from treated leaves versus mock leaves. Asterisks indicate significant differences from the wild-type. ** p < 0.01 by Student’s t test. (C) SA contents detection of wild-type, MACP2-KO mutants, and MACP2-OEs adult plants during B. cinerea infection. The contents of SA and SAG were measured by LC-MS. The “g” in “ng/g” represented the fresh weight. The experiments were biologically repeated three times with similar results. Error bars represent SD. n = 3 biological replicates. ** p < 0.01 by Student’s t test. (D) Phenotypes of leaves from 4-week-old wild-type, MACP2-OE, eds1-22, and MACP2-OE eds1-22 leaves in response to B. cinerea infection. (E) Relative lesion size of wild-type, MACP2-OE, eds1-22, and MACP2-OE eds1-22 leaves after 3 days of B. cinerea infection. Asterisks indicate significant differences from the wild-type. ** p < 0.01 by Student’s t test.2.5. MACP2 Differentially Modulates Plant Sensitivities to Fungal and Bacterial Pathogens via the SA Signaling Pathway.

Figure 5

MACP2 differentially modulate plant sensitivities to fungal and bacterial pathogens via SA signaling pathway. Heatmaps show the fold change of key regulators in SA and JA signaling pathways in wild-type, MACP2-KO mutants, and MACP2-OEs plants after infection with Pst DC3000 (A) and B. cinerea (B). The transcriptional profiles of relative gene expression values were analyzed using the TB tools.

Figure 6

AS of MACP2 responded to fungal and bacterial pathogens. (A) Classification of three AS of MACP2 gene. The blue represents exons and white represents introns. (B) Semiquantitative PCR detection of full-length MACP2 and specific sequence of each AS (T1, T2, T3) in 4-week-old rosettes of wild-type. The full-length of MACP2 was amplified with primer pair XS2591 and XS2592. The specific sequences of AS were amplified with primer pairs MACP2-1 F/MACP2-1 R, MACP2-2 F/MACP2-2 R, and MACP2-3 F/MACP2-3 R, respectively. (C) qRT-PCR detection of common sequence (MACP2) and specific sequence of each AS (T1, T2, T3) in 4-week-old rosettes of wild-type. The common sequence of three AS was amplified with primer pair MACP2-F and MACP2-R. The specific sequences of AS were amplified with primer pair mentioned in (B). The ACTIN2 was amplified with primer pair ACTIN2-F and ACTIN2-R. Asterisks indicate significant differences from the wild-type. ** p < 0.01 by Student’s t test. (D) qRT-PCR detection of AS responding to Pst DC3000 and B. cinerea after 3 days of infection on 4-week-old rosettes of wild-type. Asterisks indicate significant differences from the wild-type. * p < 0.05, ** p < 0.01 by Student’s t test.

Figure 7

Indolic GS contributed to bacteria resistance of MACP2-OE. (A) Heatmaps show the fold change of key regulators in GS biosynthesis, containing MYB34, MYB51, and MYB122 in wild-type, MACP2-KO mutants, and MACP2-OEs plants after Pst DC3000 infection. The transcriptional profiles of relative gene expression values were analyzed using the TB tools. (B) Indolic GS contents of wild-type, MACP2-KO mutants, and MACP2-OEs plants after Pst DC3000 infection. The contents of indolic GS were measured by LC-MS. The experiments were biologically repeated three times with similar results. Error bars represent SD. n = 3 biological replicates. Asterisks indicate significant differences from the wild-type. * p < 0.05, ** p < 0.01 by Student’s t test.