Hypersensitive response-like lesions 1 codes for AtPPT1 and regulates accumulation of ROS and defense against bacterial pathogen Pseudomonas syringae in Arabidopsis thaliana

Abstract

Aims: Plants employ both basal and resistance gene (R gene)-mediated defenses in response to pathogens. Reactive oxygen species (ROS) are widely reported to play a central role in both basal and R gene-mediated defense; however, the nature of ROS has been less well established for basal defense. In addition, spatial distribution of redox moieties and mechanisms of plant responses during basal defense are poorly understood. We investigated redox signaling in Arabidopsis thaliana in response to virulent bacterial pathogen, focusing on the role of the mitochondria in balancing energy demands against generation of physiologically relevant ROS.

Results: Positional cloning of an Arabidopsis lesion mimic mutant identified a polyprenyl transferase involved in the biosynthesis of Coenzyme Q10 (CoQ), which leads to novel insights into physiological ROS levels and their role in basal resistance. Gain- and loss-of-function studies identified Coenzyme Q10 redox state to be a key determinant of ROS levels. These Coenzyme Q10 redox state-mediated ROS levels had a direct bearing on both response against pathogen and ability to thrive in high oxidative stress environments.

Innovation: We demonstrate that Coenzyme Q10 redox state generates an ROS threshold for a successful basal resistance response. Perturbation of the Coenzyme Q10 redox state has the potential to disrupt plant defense responses against bacterial pathogens.

Conclusions: Coenzyme Q10 redox state is a key regulator of Arabidopsis basal resistance against bacterial pathogens.

FIG. 1.

Positional cloning of the HRL1 gene. Diagram representing the HRL1 region of Arabidopsis after mapping using CAPS markers. Single nucleotide polymorphisms and Insertion Deletions (InDels) were utilized to narrow down the HRL1 genetic region to a smaller part of the lower arm of the fourth chromosome. The identified 34 kb region in BAC F9D16 was then digested into overlapping fragments, and complementation analysis was performed. Genetic markers covering the HRL1 locus on the lower arm of chromosome 4 are indicated. Arabidopsis BAC clones around the HRL1 region are indicated.

FIG. 2.

Complementation of the hrl1 mutant phenotype. (A) Phenotype of line 84 at 5 weeks (a), line 84 at 3 weeks (b), 5-week-old hrl1 mutant (c), 5-week-old line 110 (d) and 5-week-old wild-type Col-0 (e), genomic fragment used for complementation in line 84 (f), genomic fragment used for complementation in line 110 (g). Arrows indicate lesions. (B) Rosette leaves of 4-week-old plants were infiltrated with virulent Psm ES4326 (Psm) and Pst DC3000 (Pst) at a titer of 5×10⁵ cfu/ml. For each genotype, nine plants were tested individually. Two leaf discs from each plant were collected at day 0 and 3 dpi (days post infiltration). Bacterial growth is presented as cfu/leaf disc and represents the mean and SD of nine independent plants. The entire experiment was repeated at least twice more with plants grown at different times, and similar results were obtained. Asterisks indicate statistically significant differences (*p<0.001, Mann–Whitney test). (C) Rosette leaves of 4-week-old plants were infiltrated with avirulent Pst DC3000 (avrRpm1) and Pst DC3000 (avrRpt2) at a titer of 5×10⁵ cfu/ml. For each genotype, nine plants were tested individually. Two leaf discs from each plant were collected at day 0 and 3 dpi. Bacterial growth is presented as cfu/leaf disc and represents the mean and SD of nine individual plants. The entire experiment was repeated at least twice more with plants grown at different times, and similar results were obtained. Asterisks indicate statistically significant differences (*p<0.001, Mann–Whitney test). To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars

FIG. 3.

HRL1 overexpression leads to compromised defense. (A) Rosette leaves of 4-week-old plants of HRL1 overexpressing lines and wild-type Col-0 were infiltrated with Pst DC3000 at a titer of 5×10⁵ cfu/ml, and disease development (chlorosis- and water-soaked lesions) was monitored over a period of 5 days. Plants were photographed at 3 dpi. (B) Rosette leaves of 4-week-old plants were infiltrated with virulent Psm ES4326 (Psm) and Pst DC3000 (Pst) at a titer of 5×10⁵ cfu/ml. For each genotype, nine plants were tested individually. Two leaf discs from each plant were collected at day 0 and 3 dpi. Bacterial growth is presented as cfu/leaf disc and represents the mean and SD of nine individual plants. The entire experiment was repeated at least twice more with plants grown at different times, and similar results were obtained. Asterisks indicate statistically significant differences (*p<0.001, Mann–Whitney test). (C) Rosette leaves of 4-week-old plants were infiltrated with avirulent Pst DC3000 (avrRpm1) and Pst DC3000 (avrRpt2) at a titer of 5×10⁵ cfu/ml. For each genotype, nine plants were tested individually. Two leaf discs from each plant were collected at day 0 and 3 dpi. Bacterial growth is presented as cfu/leaf disc and represents the mean and SD of nine individual plants. The entire experiment was repeated at least twice more with plants grown at different times, and similar results were obtained. Asterisks indicate statistically significant differences (*p<0.001, Mann–Whitney test). To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars

FIG. 4.

HRL1 expression affects ratio of reduced/oxidized ubiquinone. (A) Total Coenzyme Q₁₀ (CoQ) content in wild type (Col-0), transgenic lines (HRL1 OX1, HRL1 OX2), and mutant (hrl1) plants. Asterisks indicate statistically significant differences (*p<0.001, Mann–Whitney test). The entire experiment was repeated at least twice more with plants grown at different times, and similar results were obtained. (B) Percentage distribution of ubiquinone (oxidized CoQ) and ubiquinol (reduced CoQ) in wild type (Col-0), transgenic lines overexpressing HRL1 (HRL1 OX1, HRL1 OX2) and hrl1 mutant plants.

FIG. 5.

Oxygen utilization by mitochondrial isolates from different genotypes. Rate of substrate oxidation in mitochondria isolated from plants of indicated genotypes. Arrow indicates time of ascorbate/TMPD (A), and succinate (B) addition. The entire experiment was repeated at least twice more with plants grown at different times, and similar results were obtained. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars

FIG. 6.

HRL1 overexpression makes plants less sensitive to reactive oxygen species (ROS). (A) Seedlings of HRL1 overexpression lines and wild-type Col-0 were grown in normal Peters media (JR PETERS, Inc., Allentown, PA) under a 16 h light/8 h dark cycle for 14 days. Response to a high ROS environment was analyzed by transferring these seedlings to either a 23 h light/1 h dark cycle or Peters media with 150 mM NaCl or Peter's media with 1 μM paraquat. Seedlings were photographed after 1 week of exposure to a high ROS environment. The entire experiment was repeated at least twice more with plants grown at different times, and similar results were obtained. (B) Staining for hydrogen peroxide (DAB staining) and superoxide (NBT staining) levels in Col-0 and HRL1 overexpression lines under different nonbiotic stresses. Seedlings were harvested for staining after 48 h of high ROS environment. The entire experiment was repeated at least twice more, and plants grown at different times and similar results were obtained. (C) Gene expression analysis of AtrbohF and Aox1d by real-time PCR in Col-0 and HRL1 overexpression lines under different abiotic stresses. Seedlings were harvested for RNA isolation after 48 h of high ROS environment. The entire experiment was repeated at least twice more with plants grown at different times, and similar results were obtained. Asterisks indicate statistically significant differences (*p<0.001, Mann–Whitney test). To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars

FIG. 7.

Proposed model for the role of HRL1 in defense signaling. (A) In wild-type plants, HRL1 is involved in Coenzyme Q₁₀ (CoQ) biosynthesis. CoQ acts in the mitochondrial electron transport chain (ETC) as an electron carrier by getting cyclically reduced (ubiquinol) and oxidized (ubiquinone). This electron carrier function ensures loss of minimal electrons during the oxidation/reduction process that contributes toward generation of ROS. The physiological ROS pool creates a threshold ROS level that enables effective oxidative bursts during defense against pathogens. (B) In hrl1 mutants, reduced levels of CoQ compromise effective quenching of free electrons, thereby increasing electrons available for ROS production. These enhanced ROS levels contribute to activation of constitutive defense against pathogens along with a free radical-induced lesion phenotype, both of which are hallmarks of hrl1 mutant plants. (C) In HRL1 overexpression plants, increased levels of CoQ effectively quench free electrons, thereby reducing electrons available for ROS production. These reduced ROS levels lead to sub-optimal oxidative bursts required for effective defense against pathogens and thus the enhanced susceptibility against pathogens in these HRL1 overexpressing plants. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars