Overexpression of TiERF1 enhances resistance to sharp eyespot in transgenic wheat

Abstract

Wheat sharp eyespot, primarily caused by a soil-borne fungus Rhizoctonia cerealis, has become one of the most serious diseases of wheat in China. In this study, an ethylene response factor (ERF) gene from a wheat relative Thinopyrum intermedium, TiERF1, was characterized further, transgenic wheat lines expressing TiERF1 were developed, and the resistance of the transgenic wheat lines against R. cerealis was investigated. Southern blotting analysis indicated that at least two copies of the TiERF1 gene exist in the T. intermedium genome. Yeast one-hybrid assay indicated that the activation domain of TiERF1 is essential for activating the transcript of the reporter gene with the GCC-box cis-element. The TiERF1 gene was introduced into a Chinese wheat cultivar, Yangmai12, by biolistic bombardment. Results of PCR and Southern blotting analyses indicated that TiERF1 was successfully integrated into the genome of the transgenic wheat, where it can be passed down from the T0 to T4 generations. Quantitative reverse transcription-PCR analysis demonstrated that TiERF1 could be overexpressed in the stable transgenic plants, in which the expression levels of wheat pathogenesis-related (PR) genes primarily in the ethylene-dependent signal pathway, such as a chitinase gene and a beta-1,3-glucanase gene, were increased dramatically. Disease tests indicated that the overexpression of TiERF1 conferred enhanced resistance to sharp eyespot in the transgenic wheat lines compared with the wild-type and silenced TiERF1 plants. These results suggested that the overexpression of TiERF1 enhances resistance to sharp eyespot in transgenic wheat lines by activating PR genes primarily in the ethylene-dependent pathway.

Fig. 1.

Southern blotting analysis of genomic DNAs from T. intermedium (1) and wheat cv. Yangmai12 (2) digested with BglII, EcoRV, HindIII, and BamHI. Fragments were hybridized with an [α- ²P]dCTP-labelled probe from the TiERF1-specific fragment (nucleotides 407–747).

Fig. 2.

The transcriptional activation activity of TiERF1. (A) Scheme of the reporter and effector constructs. GAP, the promoter of the glyceraldehyde 3-phosphate dehydrogenase gene. (B) Yhe yeast cells selected in SD-Ura-Trp medium (plate) and quality assay of the β-galactosidase activity (lifted-filter) for four kinds of reporter and effector combinations. 1, Y-pYTiERF1/pGCC-LacZ; 2, Y-pYTiERF1/pLacZi; 3, Y-pYTiERF1/pmGCC-LacZ; 4, Y-pYTiERF1ΔAD/pGCC-LacZ. (This figure is available in colour at JXB online.)

Fig. 3.

(A) Scheme of the TiERF1 transformation vector pAHCTiERF1. (B) PCR analysis of the TiERF1 gene in T₃ plants of the transgenic lines. M, 100 bp ladders; P, pAHCTiERF1 plasmid; W, WT Yangmai12; 1–7, positive plants from the transgenic lines E111, E117, E199, E512, E765, E767, and E787; 8, silenced transgenic plant E314-3-3; 9, undetected transgenic plant E503-4-1. (C) Southern blotting of three positive PCR transgenic wheat lines. Genomic DNAs of T₃ plants from the transgenic lines E199, E767, and E787, and the wild-type Yangmai12 digested with BglII, EcoRV, and HindIII, were hybridized with the TiERF1-specific probe. M, λDNA/HindIII; P, pAHCTiERF1 plasmid. (D) RT-PCR analysis on TiERF1 expression in T₃ plants from the transgenic lines E111, E199, E765, E767, and E787, the silenced transgenic plant E314-3-3, and the wild-type Yangmai12. (E) Q-RT-PCR analysis on TiERF1 expression in T₄ plants from the transgenic lines E111, E117, E199, E231, E259, E334, E348, E512, E765, E767, E787, E314-3-3, and wild-type Yangmai12. The relative expression of the target gene is presented relative to average wild-type levels. The average and SE of three technical replicates are presented.

Fig. 4.

Sharp eyespot resistance of a TiERF1 transgenic wheat line E111 compared with wild-type Yangmai12. (This figure is available in colour at JXB online.)

Fig. 5.

Q-RT-PCR analysis of the expression of the PR genes ChiI (A), D-GLU (B), and PR10 (C) in T₄ progeny from the transgenic lines E199, E231, E259, E348, E765, E767, E787, and E314, and wild-type Yangmai12. The relative expression of the target gene is presented relative to average wild-type levels. The average and SE of three technical replicates are presented.

Fig. 6.

Q-RT-PCR analysis of the expression of the wheat ChiI (A), D-GLU (B), PR10 (C), and PR1 (D) genes in response to exogenous hormones. Seedlings of Yangmai12 were treated with ET, MeJA, ET plus MeJA, SA, CoCl₂ (an inhibitor of ET synthesis), and water (mock) for 24 h. The expression of the target gene is presented relative to average mock levels. The average and SE of three technical replicates are presented.

Fig. 7.

Q-RT-PCR analysis of the expression of TiERF1 (A), ChiI (B), D-GLU (C), PR10 (D), and PR1 (E) genes in T. intermedium in response to exogenous hormones. Seedlings of T. intermedium Z1146 were treated with ET, MeJA, SA, and water (mock) for 24 h. The expression of the target gene is presented relative to average mock levels. The average and SE of three technical replicates are presented.