Overexpression of the pathogen-inducible wheat TaWRKY45 gene confers disease resistance to multiple fungi in transgenic wheat plants

Abstract

Recently we cloned and characterized the gene for the wheat transcription factor TaWRKY45 and showed that TaWRKY45 was upregulated in response to benzothiadiazole (BTH) and Fusarium head blight (FHB) and that its overexpression conferred enhanced resistance against F. graminearum. To characterize the functional role of TaWRKY45 in the disease resistance of wheat, in the present study we conducted expression analyses of TaWRKY45 with inoculations of powdery mildew and leaf rust and evaluated TaWRKY45-overexpressing wheat plants for resistance to these diseases. TaWRKY45 was upregulated in response to infections with Blumeria graminis, a causal fungus for powdery mildew, and Puccinia triticina, a causal fungus for leaf rust. Constitutive overexpression of the TaWRKY45 transgene conferred enhanced resistance against these two fungi on transgenic wheat plants grown under greenhouse conditions. However, the expression of two resistance-related genes, Pm3 and Lr34, was not induced by the inoculation with powdery mildew in TaWRKY45-overexpressing wheat plants. These results suggest that TaWRKY45 is involved in the defense responses for multiple fungal diseases in wheat but that resistance involving TaWRKY45 differs from at least Pm3 and/or Lr34-related resistance. Our present and previous studies indicate that TaWRKY45 may be potentially utilized to improve a wide range of disease resistance in wheat.

Keywords: TaWRKY45; leaf rust; multiple resistance; overexpression; powdery mildew; wheat.

Fig. 1

Transcription profiles of TaWRKY45, Pm3 and Lr34 genes in the leaves of Bobwhite before inoculation (0 h) and 3 days (3 d) and 8 days (8 d) after inoculation with B. graminis. Five plants at each time point were used as replicates, and plant number is indicated above the gel image.

Fig. 2

Transcription profiles of the TaWRKY45 gene in the leaves of Bobwhite after inoculation with P. triticina at 0 hours (0 h), 3 h, 24 h and 7 days (7 d) after inoculation. Control, non-inoculated seedlings; Inoculated, those seedlings inoculated with the fungus. Five plants at each time point were used as replicates, and plant number is indicated above the gel image.

Fig. 3

Transcription profiles of TaWRKY45, Pm3 and Lr34 genes in leaves after BTH application at 0 hours (0 h), 3 h, 24 h and 4 days (4 d). C and BTH represent samples treated with the solvent only and with BTH, respectively. Three plants at each time point were used as replicates, and plant number is indicated above the gel image.

Fig. 4

Evaluation of TaWRKY45-overexpressing wheat plants for resistance to powdery mildew. (A and B) Results of genomic PCR (A) and RT-PCR (B) showing the segregation of the transgene inheritance and expression, respectively. (C) Primary leaves of transgenic wheat plants (lines 1, 3, 6, 7 and 10) and controls including non-transgenic Bobwhite (Bw), Norin 4 (N4), Selkirk (Sk) and Norin 29 (N29) at 7 dai with powdery mildew. The number above each leaf indicates the recorded infection type (IT). The number above each line name indicates the plant number. P indicates a plasmid control. The vertical bar on the left represents 1 cm scale.

Fig. 5

Expression of the TaWRKY45 transgene and Pm3 and Lr34 in the T₁ progeny of transgenic lines 1, 5, 7 and 13. The number below each line name indicates the plant number. P and Bw represent the plasmid construct of the transgene and Bobwhite as controls, respectively. (A) Transcription profile of the TaWRKY45 transgene in the T₁ progenies of transgenic lines 1, 5, 7 and 13 before inoculation with powdery mildew. (B) Transcription profiles of the Pm3 and Lr34 genes in the leaves before (0 h) and 3 days (3 d) after inoculation with powdery mildew.

Fig. 6

Evaluation of TaWRKY45-overexpressing wheat plants for resistance to leaf rust. (A and B) Results of genomic PCR (A) and RT-PCR (B) showing the segregation of the transgene inheritance and expression, respectively. (C) Leaves of transgenic plants (lines 7, 8, 12 and 13) and controls, including Kitakami-komugi (Kt), Hokushin (Hk), Norin 61 (N61) and non-transgenic Bobwhite (Bw), at 7 dai with leaf rust. The number above each leaf indicates the recorded infection type. The number above each line name indicates the plant number. P indicates a plasmid control. The vertical bar on the left represents 1 cm scale.