Overexpression of ATP Synthase Subunit Beta (Atp2) Confers Enhanced Blast Disease Resistance in Transgenic Rice

Abstract

Previous research has shown that the pathogenicity and appressorium development of Magnaporthe oryzae can be inhibited by the ATP synthase subunit beta (Atp2) present in the photosynthetic bacterium Rhodopseudomonas palustris. In the present study, transgenic plants overexpressing the ATP2 gene were generated via genetic transformation in the Zhonghua11 (ZH11) genetic background. We compared the blast resistance and immune response of ATP2-overexpressing lines and wild-type plants. The expression of the Atp2 protein and the physiology, biochemistry, and growth traits of the mutant plants were also examined. The results showed that, compared with the wild-type plant ZH11, transgenic rice plants heterologously expressing ATP2 had no significant defects in agronomic traits, but the disease lesions caused by the rice blast fungus were significantly reduced. When infected by the rice blast fungus, the transgenic rice plants exhibited stronger antioxidant enzyme activity and a greater ratio of chlorophyll a to chlorophyll b. Furthermore, the immune response was triggered stronger in transgenic rice, especially the increase in reactive oxygen species (ROS), was more strongly triggered in plants. In summary, the expression of ATP2 as an antifungal protein in rice could improve the ability of rice to resist rice blast.

Keywords: ATP synthase subunit beta (Atp2); Magnaporthe oryzae (M. oryzae); reactive oxygen species (ROS); rice; transgenic.

Figure 1

Expression level of ATP2 in rice; (A) PCR analysis of plants transformed with ATP2 using specific primers for the hpt gene; the DNA samples in lanes 1–30 were obtained from the 30 T1 transgenic plant lines used in the PCR analysis, representatively (M: DL2000 DNA markers, CK: the blank control, N: the negative control, and P: the positive control). (B) qRT-PCR analysis of ATP2 expression in transformed plants relative to that of positive plant #21; (C) Expression levels of the Atp2 protein in wild-type ZH11 and two ATP2-overexpressing lines. Asterisks (**) show a significant difference between the wild-type plants used as controls and the transgenic plant (p < 0.01).

Figure 2

Effects of ATP2 overexpression on rice blast resistance in rice seedling leaves; (A) Wild-type ZH11 and two ATP2-overexpressing lines; fourteen-day-old rice seedlings were sprayed with 1 × 10⁵/mL conidial suspension of the Guy11 strain. Inoculated rice seedling leaves were photographed at 7 dpi. (B) The lesion density was quantified by the number of lesions per unit leaf area. Asterisks (**) show a significant difference between the wild-type plants used as controls and the transgenic plants (p < 0.01). Three independent experiments were conducted.

Figure 3

Phenotypes of the wild-type ZH11 and two ATP2-overexpressing lines; (A,B) The bud growth and bud length, respectively, of wild-type ZH11 and two ATP2-overexpressing lines at 3 days; (C–E) Root length, and plant height, respectively, of transgenic and wild-type plant seedlings at 14 days old; (F) 1000-grain weight of transgenic and wild-type plant in rice paddies; Asterisks (**) show a significant difference between the wild-type plants used as controls and the transgenic plants (p < 0.01). Three independent experiments were conducted.

Figure 4

The expression levels of PAD4, LOX1, PBZ1, PR1a, and CHT1 in the infected rice were assayed using qPCR. RNA samples were extracted from rice plants at 0 hpi, 24 hpi, and 48 hpi with the conidial suspension of M. oryzae strain Guy11. The average cycle threshold (Ct) values for triplicate reactions were normalized to the EF1α gene in rice. Significant differences can be seen when different letters are used for each treatment (p < 0.05). The data from three separate assays are provided as the mean ± SD.

Figure 5

(A–D) Effects of ATP2 overexpression on the activity of antioxidative enzymes, including SOD, CAT, POD, and chlorophyll a and b contents, respectively, in rice seedling leaves at 0 hpi, 24 hpi, and 48 hpi using a conidial suspension of the M. oryzae strain Guy11. Significant differences can be seen when different letters are used for each treatment (p < 0.05). The data from three separate studies are provided as the mean ± SD.

Figure 6

Effects of ATP2 overexpression on the accumulation of ROS in rice leaves infected with the causal agent conidia of the M. oryzae strain Guy11; (A) NBT staining demonstrating the production and accumulation of O²⁻ in rice seedling leaves, and DAB staining demonstrating the production and accumulation of H₂O₂ in rice seedling leaves; (B) Quantitative determination of ROS accumulation in rice seedling leaves infected with conidial suspension of the M. oryzae strain Guy11, a causal agent of rice blast disease. Significant differences can be seen when different letters are used for each treatment (p < 0.05). The data from at least three separate studies are provided as the means ± SD. (C) Chitin (0.1 μg/mL) activates the ROS burst in rice leaves. H₂O served as a negative control. The values are the means ± SD (n = 3). RLU, relative light units.