Cytoplasmic H2O2 prevents translocation of NPR1 to the nucleus and inhibits the induction of PR genes in Arabidopsis

Abstract

Plants activate a number of defense reactions in response to pathogen attack. One of the major pathways involves biosynthesis of Salicylic Acid (SA), which acts as a signaling molecule that regulates local defense reaction at the infection site and in induction of systemic acquired resistance (SAR). SA is sensed and transduced by NPR1 protein, which is a redox sensitive protein that acts as a central transcription activator of many pathogenesis related and defense related genes. In its uninduced state NPR1 exists as an oligomer in the cytoplasm. Following pathogen attack and SAR induction, cells undergo a biphasic change in cellular redox, resulting in reduction of NPR1 to a monomeric form,which moves to the nucleus. Recently, it was shown that pathogen attack or SA treatment cause S-nitrosylation of NPR1, promoting NPR1 oligomerization and restricting it in the cytoplasm. We used A. thaliana mutants in cytosolic ASCORBATE PEROXIDASE, apx1, and plants expressing antisense CATALASE gene, as well as the CATALASE inhibitor 3-amino-1,2,4-triazole, to examine the effect of H2O2 on the pathogen-triggered translocation of the NPR1 to the nucleus. Our results show that the pathogen-triggered or SA-induced nuclear translocation is prevented by accumulation of H2O2 in the cytosol. Moreover, we show that increased accumulation of cytoplasmic ROS in apx1 mutants reduced the NPR1-dependent gene expression. We suggest that H2O2 has a signaling role in pathogenesis, acting as a negative regulator of NPR1 translocation to the nucleus, limiting the NPR1-dependent gene expression.

Figure 1

The effect of suppression of catalase activity on the localization of NPR1-GFP protein in the nucleus. (A–C) Roots of eight day-old NPR1-GFP (A and B) or NPR1-GFP plants that were crossed with antisense Catalase (C) were inoculated with P. putida (B and C) or left intact (A). Nuclear localization of NPR1 was analyzed 24 hours after inoculation, using DAPI stain. Brightfield light image (left part), UV filter (central part), narrow-pass GFP filter (right part). Bar = 25 µm. (D) Seedlings of wild-type, mutants of ASCORBATE PEROXIDASE1 (apx1) and antisense CATALASE (CAT AS) transgenics were infected with P. putida. ROS levels were analyzed 24 hpi as described in Methods. (E) Percentage of NPR1 localization in the nucleus 24 h.p.i. with P. putida. One hundred root hairs from 6 seedlings of NPR1-GFP and NPR1-GFP crossed with antisense CATALASE were scored in each treatment. Error bars indicate standard deviation of the mean.

Figure 2

The effect of absence of catalase on the NPR1-nuclear localization. Eight-day-old NPR1-GFP (A and B), or NPR1-GFP crossed with antisense CATALASE (C and D) plants were exposed to Salicylic acid for 24 hours and analyzed for NPR1 nuclear localization in leaves (A and C) and roots (B and D) by confocal microscopy using DAPI stain. Brightfield light (BF), DAPI (nuclei, UV filter), GFP (narrow-pass GFP filter), RED (Chlorophyl autofluorescence, RED filter). Right column shows merged pictures. Note the greenish color in the NPR1-GFP plants and the lack of green color in plants crossed with antisense CATALASE. The experiment was repeated three times with very similar results. Quantification of the results is shown in Supplemental Figure S1. Bar = 20 µm.

Figure 3

The effect of inhibition of catalase on the NPR1-nuclear localization. Eight-days-old NPR1-GFP seedlings were transferred to medium containing 20 µM 3ATA and after 2 hours either infected with P. putida or exposed to Salicylic acid. After another 24 hours the roots were analyzed for NPR1 nuclear localization, using DAPI staining as described in Materials and Methods. The assay was repeated three times, each with four repeats, with very similar results. Bar = 40 µm.

Figure 4

The effect of ROS accumulation on NPR1-dependent gene expression. (A) Wild-type and apx1 mutants were infected with P. putida (P.p) and RNA was isolated after 24 hours. Gene expression was analyzed by semi-quantitative RT-PCR with PR1 primers. Bands were quantified using ImagePro Plus software package. Error bars indicate standard deviation of the mean from repeats (N = 5). (B) eight days old wild-type (WT) and ASCORBATE PEROXIDASE1 (apx1) mutants were treated with 0.5 mM SA for 24 hours. Gene expression was analyzed by Real-Time PCR or semi-quantitative PCR (inset) using primers for PR1 or Wall Associated Kinase1 (WAK1). Error bars indicate standard deviation of the mean from 6 repeats (N = 6). RNA samples for all assays were normalized according to the expression of the β-actin gene. All experiments were repeated 3 times with very similar results. (C) Eight days old wild-type (WT) and antisense CATALASE1(CAT) plants were treated with 0.5 mM SA or left intact. RNA from roots was isolated from the roots 24 hours later and analyzed by quantitative Real-Time PCR using WAK1 primers. Error bars indicate standard deviation of the mean of 6 repeats (N = 6).