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OXR2 Increases Plant Defense against a Hemibiotrophic Pathogen via the Salicylic Acid Pathway - PubMed

Comparative Study

. 2020 Oct;184(2):1112-1127.

doi: 10.1104/pp.19.01351. Epub 2020 Jul 29.

Affiliations

Comparative Study

OXR2 Increases Plant Defense against a Hemibiotrophic Pathogen via the Salicylic Acid Pathway

Regina Mencia et al. Plant Physiol. 2020 Oct.

Abstract

Arabidopsis (Arabidopsis thaliana) OXIDATION RESISTANCE2 (AtOXR2) is a mitochondrial protein belonging to the Oxidation Resistance (OXR) protein family, recently described in plants. We analyzed the impact of AtOXR2 in Arabidopsis defense mechanisms against the hemibiotrophic bacterial pathogen Pseudomonas syringae oxr2 mutant plants are more susceptible to infection by the pathogen and, conversely, plants overexpressing AtOXR2 (oeOXR2 plants) show enhanced disease resistance. Resistance in these plants is accompanied by higher expression of WRKY transcription factors, induction of genes involved in salicylic acid (SA) synthesis, accumulation of free SA, and overall activation of the SA signaling pathway. Accordingly, defense phenotypes are dependent on SA synthesis and SA perception pathways, since they are lost in isochorismate synthase1/salicylic acid induction deficient2 and nonexpressor of pathogenesis-related genes1 (npr1) mutant backgrounds. Overexpression of AtOXR2 leads to faster and stronger oxidative burst in response to the bacterial flagellin peptide flg22 Moreover, AtOXR2 affects the nuclear localization of the transcriptional coactivator NPR1, a master regulator of SA signaling. oeOXR2 plants have increased levels of total glutathione and a more oxidized cytosolic redox cellular environment under normal growth conditions. Therefore, AtOXR2 contributes to establishing plant protection against infection by P. syringae acting on the activity of the SA pathway.

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Figures

Figure 1.
Figure 1.

AtOXR2 is induced both by SA treatment and by Pst DC3000 infection. A and B, AtOXR2 transcript levels were measured in Arabidopsis 10-d-old wild-type seedlings grown in one-half strength Murashige and Skoog (MS) medium and then flooded in mock solution or in a solution containing 50 μ

m

SA plus 0.025% (v/v) Silwet L-77 for 5 min. Samples were taken after 4 h of treatment. Alternatively, 10-d after sowing wild-type seedlings were flood inoculated for 5 min with Pst DC3000 at a concentration of 5 × 108 colony-forming units (CFU) mL−1 plus 0.025% (v/v) Silwet L-77. Mock-inoculated plants were flooded with sterile distilled water containing 0.025% (v/v) Silwet L-77. Samples were taken at 3 DAI. Transcript levels were measured by reverse transcription quantitative PCR (RT-qPCR) and referred to those of wild-type plants in mock treatment. Results are expressed as means ±

sd

of three independent experiments. Asterisks represent significantly different values by ANOVA, Tukey’s test (*P < 0.05 and **P < 0.01). C, Arabidopsis plants carrying a construct expressing the GUS reporter gene driven by the AtOXR2 promoter (Colombatti et al., 2019) were exposed to one-half-strength Murashige and Skoog (Mock) or to the same solution with 50 μ

m

SA for 1 h. Plants were incubated in 5-bromo-4-chloro-3-indolyl-β-glucuronic acid (X-gluc) staining solution for 3 h and then discolored in a 70% (v/v) ethanol solution to reveal GUS staining.

Figure 2.
Figure 2.

AtOXR2 modifies plant resistance to Pst DC3000. A, A bacterial population of Pst DC3000 was quantified at the beginning of the treatment (0 DAI) and 3 DAI. B, Disease phenotypes of Arabidopsis seedlings flood inoculated with Pst DC 3000 at a concentration of 5 × 108 CFU mL−1. Mock-inoculated plants were flooded with sterile distilled water containing 0.025% (v/v) Silwet L-77. Photographs were taken 3 DAI. Values represent means ±

sd

from five biological replicates. Asterisks indicate significant differences by ANOVA,

lsd

Fisher’s test (*P < 0.05) of the different genotypes in comparison with their respective wild type (Columbia-0 [Col-0] or Wassilewskija [WS]).

Figure 3.
Figure 3.

flg22-induced aROS is increased by AtOXR2 overexpression. A, Time-course kinetics of flg22-triggered aROS burst in wild-type (Wt) Col-0 plants, AtOXR2-overexpressing lines A and B, and the mutant oxr2.2. The aROS burst profiles of 24 leaf discs per genotype were averaged. Results are expressed as means ±

sd

. B, Quantitation of the area under the curve of flg22-induced ROS performed in the previously mentioned genotypes. Percentages were calculated relative to the wild-type levels. Values represent averages of independent experiments expressed as means ±

sd

. Asterisks indicate significant differences with regard to the wild type by

lsd

Fisher’s test (*P < 0.01). RLU, Relative light units.

Figure 4.
Figure 4.

AtOXR2 modifies basal SA levels and the expression of genes related to SA synthesis and response. A, Transcript levels for ICS1 and PR1, involved in SA synthesis and response. Expression levels were measured by RT-qPCR in 2-week-old plants grown under normal conditions. Transcript levels are referred to those of wild-type (Wt) plants of the same ecotype (OXR2-A and OXR2-B were referred to wild-type Col-0, and oxr2.1 was referred to wild-type WS). Results are expressed as means ±

se

. Asterisks indicate significant differences of three biological replicates by ANOVA,

lsd

Fisher’s test (*P < 0.05). B, Free SA content in plants with altered AtOXR2 expression. SA was measured in rosette leaves from 2-week-old plants by GC-MS. Results are expressed as means ±

sd

. Asterisks indicate significant differences of five biological replicates of each genotype by ANOVA,

lsd

Fisher’s test (*P < 0.05 and **P < 0.01). FW, Fresh weight.

Figure 5.
Figure 5.

Molecular analysis of plants with altered AtOXR2 expression during the course of infection with Pst DC3000. Transcript levels of ICS1 (A), NPR1 (B), NPR3 (C), and PR1 (D) were measured by RT-qPCR at different times during the infection. Expression levels are referred to those of wild-type plants of the same ecotype (Col-0 or WS) before the infection (0 DAI). Results are expressed as means ±

se

. Asterisks indicate significant differences compared with wild-type plants of the same ecotype at the same infection time by ANOVA,

lsd

Fisher’s test (*P < 0.05 and **P < 0.01).

Figure 6.
Figure 6.

AtOXR2 acts upstream of SID2 and NPR1. A, AtOXR2 expression levels measured by RT-qPCR in rosette leaves of wild-type (Wt), sid2, and npr1 plants, and the respective transformants obtained in the mutant backgrounds (sid2-OXR2A, sid2-OXR2B, npr1-OXR2A, and npr1-OXR2B). Results are expressed as means ±

sd

of five independent plants. Asterisks indicate significant differences and are referred to the levels of wild-type plants by ANOVA,

lsd

Fisher’s test (*P < 0.05). B, The enhanced resistance to Pst DC3000 due to AtOXR2 overexpression is lost in the sid2 and npr1 mutant backgrounds. Two-week-old plants were infected with Pst DC3000, and bacterial populations were quantified at 1 and 3 DAI. Results are expressed as means ±

sd

of five independent biological replicates. Asterisks indicate significant differences and are referred to the levels of wild-type plants at the correspondent DAI by ANOVA,

lsd

Fisher’s test (*P < 0.05). C, Analysis of transcript levels of pathogen response genes PR1 and PR5. Expression levels were measured by RT-qPCR in the cDNA samples used in A, prepared from plants before the pathogen infection (basal levels, 0 DAI). Results are expressed as means ±

sd

of five independent plants. Asterisks indicate significant differences referred to the levels of wild-type plants by ANOVA,

lsd

Fisher’s test (*P < 0.05).

Figure 7.
Figure 7.

AtOXR2 modifies the expression of several WRKY TFs related to the stress response. Transcript levels were measured by RT-qPCR at different times during the infection in 4-week-old plants from each genotype. Expression levels are referred to those of wild-type (Wt) plants of the same ecotype (Col-0 or WS) at basal conditions or before the infection (0 DAI). Asterisks indicate significant differences compared with wild-type plants of the same ecotype at the same infection time by ANOVA,

lsd

Fisher’s test (*P < 0.05 and **P < 0.01).

Figure 8.
Figure 8.

oeOXR2 plants show NPR1 nuclear accumulation when grown under control conditions. In A and C, oeOXR2, wild-type (WT), and oxr2.2 leaves expressing NPR1-GFP were imaged by CLSM. NPR1-GFP signal in green appears in the nucleus (marked with arrows), and chlorophyll fluorescence of chloroplasts appears in red. A, oeOXR2 plants showed stronger GFP signal than wild-type or oxr2.2 plants under basal conditions. Bars = 50 µm. B, Quantification of the number of nuclei with NPR1:GFP signal per field in different genotypes grown under control conditions in A. Values represent means ±

sd

(n = 5–10), and asterisks indicate significant differences by ANOVA,

lsd

Fisher’s test (**P < 0.05). C, After treatment with 0.5 m

m

SA, NPR1-GFP signals in all genotypes appeared equal. Bars = 50 µm. D, Western blot analysis of NPR1-GFP protein expression in plants with altered levels of AtOXR2. Detection of Actin was used as a loading control.

Figure 9.
Figure 9.

OXR2 plants show increased oxidation of cytosolic Grx1-roGFP2. A, Total GSH levels measured by a spectrophotometric assay on 4-week-old rosette leaves of plants with altered AtOXR2 expression. Values are expressed nanomole per gram fresh weight (FW) and represent means ±

sd

from five biological replicates. Asterisks indicate significant differences by ANOVA,

lsd

Fisher’s test (*P < 0.05). Wt, Wild type. B, 405/488 quantitation in 10-d after sowing plants with altered AtOXR2 expression grown in control conditions. Values represent means ±

sd

from 10 individual plants. The asterisk indicates a significant difference by ANOVA,

lsd

Fisher’s test (*P < 0.05). C, Degree of roGFP2 oxidation. D, Confocal images showing the 405/488 ratio. The color scale represents the redox state of the fluorescent sensor from the oxidized (ox.; in white) to the reduced (red.; in black) state. Bars = 50 µm.

Figure 10.
Figure 10.

Model of the role of AtOXR2 in plant defense. AtOXR2 is part of a loop for the activation of the SA-mediated plant defense pathway. By increasing ROS levels, AtOXR2 induces SA synthesis. In turn, SA, pathogens, and oxidative stress (ROS) stimulate the expression of AtOXR2. Increased AtOXR2 expression modifies the intracellular cytosolic redox environment, producing an increment in total GSH content and inducing the translocation of NPR1 to the nucleus, thus increasing defense and preparing plants against a potential pathogen attack. Black arrows correspond to connections known and established by other authors. Blue arrows represent results previously published by our group (Colombatti et al., 2019). Green-dotted arrows indicate original results presented in this work.

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