| In recent years, the production of wheat in China has been seriously affected by wheat sharp eyespot caused by a soilborne fungus Rhizoctonia cerealis, and scab (FHB) caused by a necrotrophic fungal pathogen Fusarium graminearum. Because the genetics of sharp eyespot and FHB resistance is not fully understood, and progress of the corresponding traditional breeding is slowly, it is urgent to unravel the wheat defense mechanisms against R. cerealis and F. gramineaum. Some ERF and MYB transcription factors in plants have been evidenced to play important roles in regulating defense response to various pathogens. Our intention is to identify defense-related ERF and MYB transcription factor genes using a two-step strategy: firstly to identify transcription factor genes that show altered transcript levels during the early stage of the defence response; and, secondly, to perform functional analysis of these candidate genes. In this study, we isolated and analyzed systemically the characteristics and defense function of two novel pathogen-induced ERF and MYB genes from wheat, named TaPIEP1 and TaPIMP1, which will lay theoretical foundation for illuminating molecular defense mechanisms against wheat sharp eyespot and scab, and provide 2 potential genes for improving the fungal resistance in wheat by gene engineering method.The study made the following results:1. Two pathogen-induced transcription factor full-length genes TaPIEP1 and TaPIMP1 were isolated from wheat. (1) Using the homologous cloning strategy, RT-PCR and RACE methods, the full-length cDNA sequence of a ERF transcription factor gene TaPIEP1 in wheat was isolated from cDNA of a resistant wheat cv. Sumai3 inoculated with F. gramineaum. Sequencing analysis indicated that TaPIEP1 does not contain intron and encodes an ERF type transcription factor TaPIEP1. TaPIEP1 is consisted of 280 amino acids and possesses the typical structure characteristics associated with ERFs, which includes a singal AP2/ERF DNA-binding domain, an acidic transactivation domain, a serine-rich region and 2 nuclear localization signal sites. The entire sequence of TaPIEP1 has a limited identity with those of other ERF proteins, for example, sharing only 36.46% identity with its highest homologous ERF protein OsERF1. The above results suggest that TaPIEP1 is a novel member of plant ERF transcription factor family. TaPIEP1 belongs to the B-3c or IX subgroup of plant ERF family. (2) Using the homologous cloning strategy, RT-PCR and RACE methods, the full-length cDNA sequence of a MYB transcription factor gene TaPIMP1 was isolated from cDNAs of Sumai3 inoculated with F. gramineaum. Comparing the cDNA sequence with the genomic DNA sequence of TaPIMP1, there is a 260-bp intron between the 233 nucleotide (G) and the 234 nucleotide (C) of the encoding sequence of TaPIMP1. TaPIMP1 encodes a R2R3 type MYB transcription factor, TaPIMP1, consisting of 323 amino acids. The entire sequence of TaPIMP1 has a limited identity with those of other MYB proteins, even sharing 43.69% identity to its highest homologous MYB protein OsJAMYB. The above results suggest that TaPIMP1 is a novel member of plant MYB transcription factor family. Analysis of the phylogenetic tree indicated that TaPIMP1 is more closely related with rice OsJAMYB (with 43.69% identity) and Arabidopsis AtMYB108 (with 40.11% identity), whereas distant from other wheat MYB proteins in NCBI (only with identity from 17.32% to 28.48%).2. The expression pattern of TaPIEP1 was characterized. The results of Q-RT-PCR analysis indicated that the transcript of TaPIEP1 could be induced by R. cerealis, F. gramineaum and Blumeria graminis, and the transcript induction of TaPIEP1 in three resistant cultivars was higher than that in susceptible ones in all the inoculated time points. Furthermore, the transcript induction of TaPIEP1 by R. cerealis or F. gramineaum was faster than that by B. graminis, suggesting that TaPIEP1 was mainly involved in early defense response to R. cerealis and F. gramineaum. The transcript of TaPIEP1 could also be induced by treatments with ethylene (ET), methyl jasmonic acid (MeJA) and salicylic acid (SA), in which the transcript induction of TaPIEP1 by ET and MeJA was faster than that by SA. Moreover, ET treatment could accelerate and enhance the induction of TaPIEP1 transcript in response to F. graminearum infection, suggesting that TaPIEP1 was mainly involved in regulating defense responses to R. cerealis and F. gramineaum via ET/JA signaling pathway, and it may be also involved in defense response to B. graminis via SA signaling pathway.3. The expression pattern of TaPIMP1 was characterized primarily. The results of semi-quantity RT-PCR indicated that the transcript of TaPIMP1 could be induced by R. cerealis, F. gramineaum and exogenous phytohormones ET or MeJA. It was primarily speculated that TaPIMP1 might be involved in modulating wheat defense response to R. cerealis and F. gramineaum via ET/JA signaling pathway.4. TaPIEP1 was demonstrated to be a nucleur localizing, GCC box-binding and transactivation type ERF transcription factor. The results of subcellular localization indicated that, in the transformed onion epidermal cells, the TaPIEP1-GFP fusion protein was localized in the nucleus, whereas the control GFP was distributed throughout the cell, demonstrating that TaPIEP1 was targeted to the nucleus. The result of electrophoretic mobility shift assay indicated that TaPIEP1 could specifically bind to the GCC box cis-element, but not to mGCC box or DRE box cis-element, and the GCCGCC sequence was the core sequence of the GCC box cis-element. Results of yeast-one-hybrid indicated that TaPIEP1 could bind with the GCC box in vivo, and activate effectively the expression of the report gene LacZ, confirming that TaPIEP1 is an activation-type ERF transcription factor. Furthermore, deletion of the putative activation domain of TaPIEP1 could reduce mostly but not abrogated completely the transactivation activity of TaPIEP1, suggesting that the putative activation domain did is a main transactivation domain, but another one may exist in TaPIEP1.5. Defense functions of TaPIEP1 and TaPIMP1 to R. cerealis and Ralstonia solanacearum were confirmed. (1) TaPIEP1 was transformed into wheat cultivar Yangmai12 via microprojectile bombardment. PCR test was performed to select the positive transgenic wheat individuals. Identification of sharp eye-spot resistance indicated that the positive TaPIEP1 transgenic wheat individuals in the T1 generation showed enhanced resistance to sharp eye-spot (the infection degeree is 0) compared with that of the untransformed Yangmai12 (the disease degeree is 3 to 4), suggesting that overexpression of TaPIEP1 could enhance wheat resistance to R. cerealis, and that TaPIEP1 regulates positively wheat defense to R. cerealis. (2) TaPIMP1 was transformed into tobacco cv. W38 background by Agrobacterium-mediated transformation. Identification of disease resistance indicated that some positive TaPIMP1 transgenic tobacco individuals showed enhanced resistance to Ralstonia solanacearum, suggesting that the gene does positively regulate plant defense response to some pathogens.
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