The apoplastic oxidative burst peroxidase in Arabidopsis is a major component of pattern-triggered immunity

Abstract

In plants, reactive oxygen species (ROS) associated with the response to pathogen attack are generated by NADPH oxidases or apoplastic peroxidases. Antisense expression of a heterologous French bean (Phaseolus vulgaris) peroxidase (FBP1) cDNA in Arabidopsis thaliana was previously shown to diminish the expression of two Arabidopsis peroxidases (peroxidase 33 [PRX33] and PRX34), block the oxidative burst in response to a fungal elicitor, and cause enhanced susceptibility to a broad range of fungal and bacterial pathogens. Here we show that mature leaves of T-DNA insertion lines with diminished expression of PRX33 and PRX34 exhibit reduced ROS and callose deposition in response to microbe-associated molecular patterns (MAMPs), including the synthetic peptides Flg22 and Elf26 corresponding to bacterial flagellin and elongation factor Tu, respectively. PRX33 and PRX34 knockdown lines also exhibited diminished activation of Flg22-activated genes after Flg22 treatment. These MAMP-activated genes were also downregulated in unchallenged leaves of the peroxidase knockdown lines, suggesting that a low level of apoplastic ROS production may be required to preprime basal resistance. Finally, the PRX33 knockdown line is more susceptible to Pseudomonas syringae than wild-type plants. In aggregate, these data demonstrate that the peroxidase-dependent oxidative burst plays an important role in Arabidopsis basal resistance mediated by the recognition of MAMPs.

Figure 1.

Downregulation of PRX33 and PRX34 Transcripts in Peroxidase T-DNA and Antisense Knockdown Lines. (A) Quantitative RT-PCR analysis of PRX33 and PRX34 transcripts in 23- to 25-d old rosette leaves. (B) Quantitative RT-PCR analysis of PRX33 and PRX34 transcripts in 4-week-old rosette leaves 2 h after infiltration of 0.5 μM Flg22 of Elf26. Data represent the average ± sd. All quantitative gene expression measurements were performed using technical and biological triplicates. The experiments were repeated at least two times with similar results. Values for the peroxidase T-DNA and antisense knockdown lines were found to be significantly different from rbohD with a P value < 0.05 as determined by Student’s t test (A). Values for the peroxidase T-DNA and antisense knockdown lines treated with Flg22 were found to be significantly different from Col-0, WS, and rbohD with a P value < 0.05 as determined by Student’s t test (B). prx34, prx34:T-DNA; prx33, prx33:T-DNA; prx33-34-, prx33:T-DNA;prx34:RNAi.

Figure 2.

Hydrogen Peroxide Production Detected by DAB Staining in Mature Arabidopsis Leaves. Hydrogen peroxide production detected by DAB staining was quantified as the percentage and intensity of leaf area stained. Approximately 0.1 mL of 0.5 μM Flg22, 0.5 μM Elf26, 100 μg/mL FoCWE, 100 μg/mL OG, or 100 μg/mL PGN were infiltrated into mature rosette leaves of 23- to 25-d-old Arabidopsis plants. The infiltrated leaves were detached at 2 h after MAMP treatment and then stained with DAB as described in Methods. At least six independent plants were used as biological replicates, and three rosette leaves were sampled from each plant. The experiment was repeated at least two times. Representative leaves for each condition were selected for Supplemental Figures 3 to 5 online, and these leaves were used for the quantitative analysis shown in this figure. A combination of tools from Adobe Photoshop and Cell Profiler (Carpenter et al. 2006) was used to establish the threshold of DAB staining in the leaves and distinguish the staining from the background. The final measurement used to quantify the DAB staining was the area of the stain divided by the total area of the selected representative leaf. Col-0, prx34, asFBP1.1, and rbohD (A); WS, prx33 and prx33-34- (B); Col-0, WS, asFBP1.1, rbohD, prx33, prx34, prx33-34- (C) (see Supplemental Figures 3 to 5 online). prx34, prx34:T-DNA; prx33, prx33:T-DNA; prx33-34-, prx33:T-DNA;prx34:RNAi.

Figure 3.

Growth of P. syringae in Mature Leaves of the Peroxidase T-DNA and Antisense Knockdown Lines. As described in Methods, ?0.1 mL of a P. syringae tomato strain DC3000 suspension at OD600 = 0.0005 was infiltrated into mature rosette leaves of 23- to 25-d-old Arabidopsis plants, leaves were harvested at 2, 24, 48, and 72 h after infiltration, and bacterial counts were determined. Data represent the average ± sd. Values for all four lines at 72 h after infiltration were found to be significantly different from each other except Col-0 and prx34:T-DNA with a P value < 0.05 as determined by Student’s t test (A). Values for prx33:T-DNA and prx33:T-DNA;prx34:RNAi at 72 h after infiltration were found to be significantly different from WS, with a P value < 0.05 as determined by Student’s t test (B). At least four biological replicates from independent plants were used for each time point, and the entire experiment was repeated with similar results. prx34, prx34:T-DNA; prx33, prx33:T-DNA; prx33-34-, prx33:T-DNA;prx34:RNAi. cfu, colony-forming units.

Figure 4.

MAMP-Elicited Callose Accumulation Detected by Aniline Blue Staining in Mature Arabidopsis Leaves. Approximately 0.1 mL of 0.5 μM Flg22, 0.5 μM Elf26, or 100 μg/mL FoCWE were infiltrated into mature rosette leaves of 23- to 25-d-old Arabidopsis plants. At least six independent plants were used as biological replicates, and three rosette leaves were sampled from each plant. The experiment was repeated at least two times. Representative leaves are shown. Infiltrated plants were incubated at high humidity for 16 to 20 h, and then leaves were harvested and stained for callose as described in Methods. See Supplemental Figure 8 online for quantitation of the data. prx34, prx34:T-DNA. Bar = 20 μM.

Figure 5.

Quantitative RT-PCR Analysis of MAMP-Elicited Genes in Peroxidase-Downregulated Plants. (A) Basal levels of CYP71A12, CYP79B2, CYP81F2, and MYB51 mRNAs were measured in 23- to 25-d-old plants as described. (B) Levels of CYP71A12, CYP79B2, CYP81F2, and MYB51 mRNAs were measured at 2 h after infiltration of 0.1 mL of 0.5 μM Flg22 or Elf26. Data represent the average ± sd. All quantitative gene expression measurements were performed using technical and biological triplicates. The experiments were repeated at least two times with similar results. Values for CYP71A12 expression in rbohD and prx33 were found to be significantly different from all other expression values, with a P value < 0.05 as determined by Student’s t test (A). Values for the gene expression levels of each of the four genes in Col-0 and WS, and all genes except CYP79B2 in rbohD, were found to be significantly different from the gene expression levels of all four genes in the peroxidase knockdown lines, with a P value < 0.05 as determined by Student’s t test (B). prx34, prx34:T-DNA; prx33, prx33:T-DNA; prx33-34-, prx33:T-DNA;prx34:RNAi.

Figure 6.

Exogenous H₂O₂ Application Rescues Callose Accumulation in asFBP1.1 and prx34. Approximately 0.1 mL of 0.5 μM Flg22 was infiltrated into mature rosette leaves of 23- to 25-d-old Arabidopsis plants. At least six independent plants were used as biological replicates, and three rosette leaves were sampled from each plant. The experiment was repeated at least two times. Representative leaves are shown. The infiltrated leaves were detached at 2 h after MAMP treatment and then placed in 12-well sterile plates overnight in 1 mL solution containing either 5 μM, 15 μM H₂O₂, or sterile dH₂O. The leaves were then harvested and stained for callose as described in Methods. Hydrogen peroxide was applied in solution at the time of Flg22 treatment. prx34, prx34:T-DNA. Bar = 20 μM.