A Rice CPYC-Type Glutaredoxin OsGRX20 in Protection against Bacterial Blight, Methyl Viologen and Salt Stresses

Abstract

Glutaredoxins (GRXs) belong to the antioxidants involved in the cellular stress responses. In spite of the identification 48 GRX genes in rice genomes, the biological functions of most of them remain unknown. Especially, the biological roles of members of GRX family in disease resistance are still lacking. Our proteomic analysis found that OsGRX20 increased by 2.7-fold after infection by bacterial blight. In this study, we isolated and characterized the full-length nucleotide sequences of the rice OsGRX20 gene, which encodes a GRX family protein with CPFC active site of CPYC-type class. OsGRX20 protein was localized in nucleus and cytosol, and its transcripts were expressed predominantly in leaves. Several stress- and hormone-related motifs putatively acting as regulatory elements were found in the OsGRX20 promoter. Real-time quantitative PCR analysis indicated that OsGRX20 was expressed at a significantly higher level in leaves of a resistant or tolerant rice genotype, Yongjing 50A, than in a sensitive genotype, Xiushui 11, exposed to bacterial blight, methyl viologen, heat, and cold. Its expression could be induced by salt, PEG-6000, 2,4-D, salicylic acid, jasmonic acid, and abscisic acid treatments in Yongjing 50A. Overexpression of OsGRX20 in rice Xiushui 11 significantly enhanced its resistance to bacterial blight attack, and tolerance to methyl viologen and salt stresses. In contrast, interference of OsGRX20 in Yongjing 50A led to increased susceptibility to bacterial blight, methyl viologen and salt stresses. OsGRX20 restrained accumulation of superoxide radicals in aerial tissue during methyl viologen treatment. Consistently, alterations in OsGRX20 expression affect the ascorbate/dehydroascorbate ratio and the abundance of transcripts encoding four reactive oxygen species scavenging enzymes after methyl viologen-induced stress. Our results demonstrate that OsGRX20 functioned as a positive regulator in rice tolerance to multiple stresses, which may be of significant use in the genetic improvement of rice resistance.

Keywords: OsGRX20; bacterial blight; genetic transformation; glutaredoxin; methyl viologen; rice; salt stress.

FIGURE 1

Analysis of the subcellular localization of OsGRX20 protein using a tobacco transient transformation system in tobacco epidermal cells. The vector 35S:GFP without any pieces of OsGRX20 sequence as control under fluorescence channel (A), bright-field channel (B), and overlay (C). 35S:OsGRX20:GFP construct (D) and 35S:RFP without OsGRX20 sequence (E) under fluorescence channel. Merge of 35S:OsGRX20:GFP and 35S:RFP channels (F). Agrobacterium carrying different vectors were infiltrated into N. benthamiana leaves. At 2 days post infiltration, the leaf epiderm was cut and examined by confocal laser scanning microscopy. The white bar represents 25 μm. This experiment was repeated three times with similar results.

FIGURE 2

Spatial expression patterns of OsGRX20. Expression of GUS driven by the OsGRX20 promoter of transgenic rice plant in root (A), leaf (B), stem (C), flower (D) and germinated seed (E). Scale bars are 1 mm (A–C) or 3 mm (D,E). (F) Expression pattern revealed by qPCR. The expression level was firstly normalized using β-actin as an internal reference, and then made relative to the amount of corresponding mRNAs in leaf sample of Xiushui 11 variety. Bars represent means (three replicates) ± SD.

FIGURE 3

Expression of the OsGRX20 gene in leaves in response to stress. Expression patterns of OsGRX20 in two contrasting genotypes in response to Xoo strain-Zhe173 infection (A), MeV treatments (B), cold (C) or heat (D). The transcript level was firstly normalized using β-actin as an internal reference, and then made relative to the amount of corresponding mRNAs in 0 h sample. Bars represent means (three replicates) ± SD. The asterisks indicate that a significant difference was detected between two genotypes (^∗p < 0.05; ^∗∗p < 0.01).

FIGURE 4

Overexpression of OsGRX20 enhanced rice resistance to Xoo infection. (A) Schematic diagram of the OsGRX20-pCAMBIA1301 overexpression construct. (B) The transcript level of OsGRX20 in T₁ transgenic lines is relative to that in wild-type Xiushui 11 (XS11). Bars represent means (three to five replicates) ± SD. Lesion phenotype (C) and lesion area (D) in OsGRX20-overexpressing lines OE7 and OE8 compared with wild-type Xiushui 11 (XS11). The flag leaves at the booting stage were inoculated with Xoo strain-PXO66 for 21 days. The black bar represents 2 cm. The double asterisks indicate that a significant difference (p < 0.01) in the lesion area was detected between transgenic plants and the respective wild -type. Data represent mean ± SD from three independent biological replicates.

FIGURE 5

Interfering of OsGRX20 increased susceptibility to Xoo infection. (A) Schematic diagram of the OsGRX20-RNAi construct. (B) The transcript levels of OsGRX20 in T₂ transgenic plants are relative to that in the wild-type Yongjing 50A (YJ50). Bars represent means (three to five replicates) ± SD. (C) Lesion phenotype. (D) Reduced resistance to Xoo in OsGRX20-interfering lines SE3 and SE5 compared with wild-type Yongjing 50A (YJ50). The flag leaves at the booting stage were inoculated with Xoo strain-PXO66 for 21 days. The black bar represents 2 cm. The double asterisks indicate that a significant difference (p < 0.01) in the lesion area was detected between transgenic plants and the respective wild-type. Data represent mean ± SD from three independent biological replicates.

FIGURE 6

The role of OsGRX20 in rice tolerance to MeV. (A) Growth of wild-type XS11 and OsGRX20-overexpression plants after 10 μM MeV treatment for 7 days. (B) Relative fresh weight and chlorophyll content of OsGRX20-overexpression and control plants. The seedlings at three-leaf stage were transferred to solution supplemented with 10 μM MeV for 7 days. All values are means (±SD) from two independent experiments (10 seedlings per experiment). The asterisks indicate that a significant difference (^∗p < 0.05; ^∗∗p < 0.01) was detected between transgenic plants and wild-type. (C) Changes of plant growth and leaf wilting in OsGRX20-overexpressed and control plants after sprayed with 100 μM MeV for 10 days. Seedlings grown in four rows in a plastic tank from left to right are wild-type Xiushui11 (XS11), expressing an empty vector (EV), overexpressing lines OE7 and OE8. (D) Phenotype of wild-type YJ50 and OsGRX20-RNAi plants after application of 10 μM MeV through roots for 7 days. (E) Relative fresh weight and chlorophyll content of OsGRX20-RNAi and control plants. (F) Changes of plant growth and leaf wilting in OsGRX20-RNAi and wild-type YJ50 plants are distinguishable after sprayed with 100 μM MeV for 10 days. Seedlings grown in four rows in a plastic tank from left to right are the wild-type Yongjing 50A (YJ50), expressing an empty vector (EV), RNAi lines SE3 and SE5.

FIGURE 7

Effects of OsGRX20 overexpression and RNAi on the growth and wilting degree under salt stress. The seedlings at five-leaf stage were transferred to 150 mM NaCl for 0 day (left) and for 8 days (right). Seedlings grown in four rows in a plastic tank from left to right are the wild-type Xiushui11 (XS11), expressing an empty vector (EV), overexpressing lines OE7 and OE8 (top); wild-type Yongjing 50A (YJ50), expressing an empty vector (EV), RNAi lines SE3 and SE5 (bottom).

FIGURE 8

Modulating OsGRX20 expression influenced levels of superoxide radicals, AsA/DHA and gene transcripts associated with ROS detoxification after MeV treatment. Five-leaf plants from wild-type Xiushui11 (XS11) and Yongjing 50A (YJ50), OsGRX20-overexpression lines OE7 and OE8, and RNAi lines SE3 and SE5 were incubated in 10 μM MeV for 24 h, and the aerial tissue was subjected to analysis. (A) Relative superoxide radical level was calculated by comparison to the non-treated seedlings of individual genotypes. (B) The AsA/DHA ratio under control condition and after 24-h treatment. (C) Relative mRNA levels of four genes associated with ROS detoxification in OsGRX20-overexpression line OE7 and wild-type Xiushui 11. Data represent mean ± SD from three independent biological replicates, and an asterisk indicates a significant difference between wild-type vs. transgenic plants (^∗p < 0.05; ^∗∗p < 0.01).