The Fd-GOGAT1 mutant gene lc7 confers resistance to Xanthomonas oryzae pv. Oryzae in rice

Abstract

Disease resistance is an important goal of crop improvement. The molecular mechanism of resistance requires further study. Here, we report the identification of a rice leaf color mutant, lc7, which is defective in chlorophyll synthesis and photosynthesis but confers resistance to Xanthomonas oryzae pv. Oryzae (Xoo). Map-based cloning revealed that lc7 encodes a mutant ferredoxin-dependent glutamate synthase1 (Fd-GOGAT1). Fd-GOGAT1 has been proposed to have great potential for improving nitrogen-use efficiency, but its function in bacterial resistance has not been reported. The lc7 mutant accumulates excessive levels of ROS (reactive oxygen species) in the leaves, causing the leaf color to become yellow after the four-leaf stage. Compared to the wild type, lc7 mutants have a broad-spectrum high resistance to seven Xoo strains. Differentially expressed genes (DEGs) and qRT-PCR analysis indicate that many defense pathways that are involved in this broad-spectrum resistance are activated in the lc7 mutant. These results suggest that Fd-GOGAT1 plays an important role in broad-spectrum bacterial blight resistance, in addition to modulating nitrogen assimilation and chloroplast development.

Figure 1. Phenotype of the lc7 mutant.

(A) Plant at the seedling stage. (B) Plant at the seedling stage grown in the field for two months. (C) Plant at the tillering stage. (D) Plant at maturity. (E–G). The investigation of agronomic traits, including grain weight per plant, tiller number seed-setting ratio, and thousand-grain weight. Asterisks indicate the significance of differences between wild-type and lc7 mutant plants as determined by Student’s t-test. These data were obtained from three independent replicates, **P < 0.01 (t-test). Each scale bar is indicated.

Figure 2. The ultrastructure and physiological-biochemical analysis of chloroplasts.

(A,C) The chloroplast ultrastructure of wild-type and lc7 at maturity. (B,D) The chloroplast structure of the lc7 mutant at maturity. (E) The pigment contents of chla, chlb, and chla+chlb; the rate of chla/chlb; and the contents of carotenoids. (F) The photosynthetic rate of the wild-type and lc7 at the four-leaf stage. These data were obtained from three independent replicates. *P < 0.05, **P < 0.01 (t-test).

Figure 3. Active oxygen-scavenging enzyme activity in the lc7 mutant.

(A) DAB and Trypan blue staining for H₂O₂ accumulation and cell death. Leaves were treated at 30 days after sowing. (B) The assay of oxygen scavenging enzyme activity in the lc7 mutant. SOD, superoxide dismutase; MDA, malondialdehyde; CAT, catalase; GST, glutathione-S-transferase. These data were obtained from three independent replicates. *P < 0.05, **P < 0.01 (t-test).

Figure 4. The lc7 mutants showed high broad-spectrum resistance to Xoo.

(A) Resistance phenotype of the lc7 mutants 14 days after inoculation with seven Xoo strains. (B) Average lesion length of the lc7 mutants 14 days after inoculation with seven Xoo strains. (C) RT-PCR analysis of pathogen-related genes (PRs) in the lc7 mutant, OsActin was used as an internal control. These data were obtained from three independent replicates. The scale bar is indicated.

Figure 5. Map-based cloning of the lc7 gene.

(A) The lc7 gene was mapped to a 78-kb region between the markers S16 and S22 on the long arm of chromosome 7 with nine candidate genes. The LOC_Os07g46460 gene encoding Fd-GOGAT1 in the lc7 mutant with a single-base substitution at the 983^rd position is the best candidate gene of lc7. (B,C) PCR amplification of Hyg and LC7 from the control and transgenic plants. M = Trans2k plus DNA Marker (http://www.transgen.com.cn). (D,E) The relative expression level of Fd-GOGAT1 in transgenic plants. (F) The expression patterns of Fd-GOGAT1 in different tissues as analyzed by qRT-PCR. (G) The Fd-GOGAT activity in wild-type, lc7 and complemented lc7 plants. (H) The phenotypes of T₁ transgenic plants and their parents at the booting stage. (I) The lesion length of lc7 mutant and complemented lc7 plants 14 days after inoculation with Xoo strain PXO99. Nip-V and lc7-V are transgenic plants from the Nipponbare and lc7 mutant transformed with the pCAMBIA1301 vector, respectively; lc7-C is the function-complemented lc7 plant; and NRi is the OsFd-GOGAT1 RNAi plant. The scale bar is indicated.