Isolation And Functional Analysis Of Disease-Resistance Related Genes TiERF1 And TiDPK1 From Thinopyrum Intermedium

In recent years, the production of wheat in China has been seriously affected by wheat sharp eyespot caused by a soilborne fungus Rhizoctonia cerealis. Because the genetics of wheat resistance to sharp eyespot 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. Some ethylene response factor (ERF) transcription factors in plants have been evidenced to play important roles in regulating defense response to various pathogens. In this study, an 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 Th. intermedium genome. Yeast one-hybrid assay indicated that the activation domain of TiERFl 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, Yangmail2, 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. Quantitative reverse transcriptional-PCR analysis demonstrated that TiERF1 could be overexpressed in the stable transgenic plants, in which the transcript levels of wheat pathogenesis-related (PR) genes primarily in the ethylene-/jasmonic acid-dependent signal pathway, such as a chitinase gene and aβ-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 ET/JA-dependent pathway.Barley yellow dwarf virus, which was spreaded by aphids, can cause one of the most serious virus diseases of small-grain cereals, i.e. wheat, worldwide. Up to now, there is no effective resistance gene in Triticum. Thinopyrum intermedium, a wheat relative, shows a high level of resistance to BYDV. It possesses Bdv2 resistance gene which locates on the long arm of 7X(7Ai#1) chromosome of Th. intermedium and has high resistance to several isolates of BYDV. Through biotechnology and distant crossing methods, the 7Ai#1 long arm fragment of Th. intermedium that possesses Bdv2 has been introduced into wheat, and developed some wheat-Th. intermedium translocation lines with BYDV resistance, for example YW642. However, the alien translocation chromosome may possess disadvantageous characteristics. It is necessary to isolate BYDV resistance gene in 7Ai#1L for unraveling BYDV resistance mechanism and carrying out engineering breeding for disease resistance. In our previous study, we isolated 14 novel genetic expression sequences specific to the Bdv2 region in wheat-Th. intermedium T7DS.7DL-7Ai#1L translocation lines YW642 based on comparing genomics and chromosome mapping. Out of the expression sequences, one has homology with a rice protein kinase. This study was carried out with the gene sequence of that protein kinase as the initiation sequence, and obtained the following results:(1) Knocking down the transcript of the protein kinase gene in BYDV-resistant wheat-Th. intermedium translocation line YW642 by virus-induced gene silencing compromised the Bdv2-mediated resistance to BYDV. This result revealed that the protein kinase gene was an important BYDV-resistant gene in the 7Ai#1L region. (2) The full-length cDNA containing complete ORF, which encodes a dual-specificity phosphatase potentially capable of acting on substrate Ser/Thr and Tyr residues, was isolated from Th. intermedium leave cDNA by RACE and RT-PCR, and temporarily named as TiDPK1 (Thinopyrum intermedium Dual-specificity Protein Kinase 1). TiDPK1 contains three distinctive domains of receptor-like kinase (RLK):(a) an extracellular domain; (b) a membrane-spanning domain; and (c) a protein kinase domain that contains all of the 11 subdomains conserved among protein kinases. (3) The protein kinase domain of recombinant TiDPK1 showed strong autophosphorylation. (4)The fusion protein of TiDPK1-green fluorescent protein was targeted to cytomembrane, suggesting that TiDPK1 may contribute to signal transduction. (5) Southern hybridization analysis revealed that TiDPK1 was located in the region of 7Ai#1L that possesses the BYDV resistance gene Bdv2. Additionally, homologous copies were both present in the genomes of wheat and Th. intermedium. This result revealed that TiDPK1 was a member of a small multigene family. (6) Q-RT-PCR analysis showed that TiDPK1 was expressed predominantly in Bdv2-harboring materials and upregulated by BYDV infection in a time-dependent manner. The activation of TiDPK1 mRNA expression is obvious at 12 to 24h post BYDV infection. At the young seedling untreated, the expression of TiDPK1 did not show tissue-specific. At 40 days post BYDV infection, at the adult stage, TiDPK1 transcript was tissue-specific, the transcript level was high in leaves (the disease mainly occurred tissues), moderate in stems (BYDV transmitted tissues), and low in immature spikes and roots, implying that TiDPK1 expression may correspond to its resistant function. Additionally, the transcript level of TiDPK1 in the seedlings was significantly induced by treatment for 12 h with exogenous BR and SA hormones. (7) The yeast two-hybrid system was used to investigate the interaction of TiDPK1 with the coat protein or RdRp of BYDV. The results showed that TiDPK1 could interact with BYDV coat protein but not with RdRp. (8) The function of TiDPK1 gene was studied through functional complementary experiments by means of loss-of-function and gain-of-function. Comparing to the transgenic receptor of susceptible wheat Zhong8601, transgenic wheat lines expressing TiDPK1 exhibited good resistance to BYDV, in which the resistance was similar to the gene donor YW642. However, transgenic wheat plants silencing TiDPK1 expression by RNA interfering broke Bdv2 mediated-resistance in the recipient-wheat YW642. Based on these findings, TiDPK1 should be an important gene during the host resistance to BYDV, and may facilitate the wheat breeding with BYDV resistance.