PRX34 mediates the oxidative burst in Arabidopsis. Here we characterized two additional Arabidopsis prx34 null mutants (prx34-2, prx34-3), besides the well-studied prx34-1. Due to a decrease in corresponding peroxidase, the activity that generates reactive oxygen species (ROS) was significantly lower in cell wall extracts of prx34-2 and prx34-3 plants. Consistently, the prx34-2 and prx34-3 exhibited reduced accumulation both of ROS and callose in Flg22-elicitor-treated leaves, leading to enhanced susceptibility to bacterial and fungal pathogens. In contrast, ectopic expression of PRX34 in the wild type caused enhanced resistance. PRX34 is thus a component for disease resistance in Arabidopsis.
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We acknowledge Prof. Dr. Shinji Tsuyumu (Laboratory of Plant Pathology, Faculty of Agriculture, Shizuoka University, Shizuoka, Japan) for generously providing Pectobacterium carotovorum subsp. carotovorum strain Pc1. This research was supported in part by the Grants-in-Aid for Scientific Research (18K05645) from the Japan Society for Promotion of Science (JSPS).
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Fig. S1 Response of prx34-1 mutants to fungal and bacterial pathogens. a Symptoms and area of lesions induced by Botrytis cinerea (MAFF712189), Colletotrichum higginsianum (MAFF305635) and Pectobacterium carotovorum subsp. carotovorum strain Pc1. Inoculations: B. cinerea and C. higginsianum, 5-μl drop of 2 × 105 conidia/ml in distilled water or 1% Sabouraud maltose broth was placed on each side of the mid vein of detached leaves of 4-week-old wild type or prx34-1; P. carotovorum subsp. carotovorum strain Pc1, 5-μl drop of 1×105 cfu/ml was placed on wounded leaves. All inoculated leaves were incubated for 2 or 3 days at 22 °C, then photographed, and lesions were measured. Data are the average ± SD of 10 leaves from five independent plants. No significant difference was observed between the wild type and mutant prx34-1, as revealed by Dunnett’s test. b Flg22-elicted ROS generation in Arabidopsis leaves. Mature leaves of 4-week-old seedlings of the wild type or prx34-1 were infiltrated with approximately 0.1 ml of 100 nM Flg22 or water (as control). The leaves were stained with 3,3′-diaminobenzidine (DAB) at 2 h after treatment to detect H2O2. Experiments were repeated three times with similar results. c Flg22-elicited callose detected by aniline blue staining. Mature leaves were infiltrated with approximately 0.1 ml of 100 nM Flg22 or water (as control) as described above. Leaves were stained with aniline blue 24 h after treatment to detect callose. The number of callose deposits was calculated using ImageJ software. Data are the average ± SD of 10 leaves from five independent plants. Asterisks indicate significant difference (Dunnett’s test; ***p < 0.001).
Fig. S2 Genotyping of three prx34 mutants. PCR was performed with a Phire Plant Direct PCR Kit (Thermo Fisher Scientific) using genomic DNAs of mutant prx34-1 (SALK_051769) (a), prx34-2 (GABI_728F08) (b) or prx34-3 (SALK_112466C) (c). Control amplification was carried out with primers supplied with the kit that amplifies a 297-bp fragment of a highly conserved region of chloroplast DNA. Wild type Col-0 was used as the control. The T-DNA border primer (BP), left (LP) and right (RP) genomic primers for each mutant line were designed using T-DNA Primer Design (http://signal.salk.edu/tdnaprimers.2.html) and are listed in Table S1. The RP was always on the side of the flanking sequence (the 3′ end of the insertion). Therefore, by using the three primers (BP+LP+RP), the wild type (no insertion) yielded an amplicon of about 1100 bp (from LP to RP). For homozygous insertion lines, the PCR amplified a distinct band of 410 + N bp (from RP to insertion site 300 + N bases, plus 110 bases from BP to the left border of the vector).
Fig. S3 Induction of PRX34 transcripts in Arabidopsis in response to Flg22 or chitoheptaose. Mature leaves of 4-week-old wild type seedlings were infiltrated with approximately 0.1 ml of 100 nM Flg22 (GenScript, Piscataway, NJ, USA), 100 μg/ml chitoheptaose (GLU437; Elicityl, Crolles, France) or water (as control). Leaves were harvested 6, 12 and 24 h after infiltration, then subjected to quantitative RT-PCR using a Shimadzu GVP-9600 Gene Detection System (Shimadzu, Kyoto, Japan) and primers in Supplementary Table 2. Expression level of PRX34 was normalized using EF1-α (At5g60390) and expressed relative to the water-treated control sample. Data are the average ± standard deviation (SD) from three independent plants. Asterisks indicate statistically significant difference (Dunnett’s test; *p < 0.05; ***p < 0.001).
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Zhao, L., Phuong, L.T., Luan, M.T. et al. A class III peroxidase PRX34 is a component of disease resistance in Arabidopsis. J Gen Plant Pathol 85, 405–412 (2019). https://doi.org/10.1007/s10327-019-00863-9
- Apoplastic oxidative burst
- Cell wall
- Class III peroxidase
- Reactive oxygen species (ROS)