Molecular Characterization And Defensive Functional Analysis Of MYB Transcription Factors TaMYB86 And TaRIM1 In Wheat

Common root rot and sharp eyespot are devastative diseases of wheat production in the world. Currently, breeding common root rot-/sharp eyespot-resistant wheat cultivars using conventional cross breeding and phenotypic selection methods is difficult since none of common root rot-/sharp eyespot-immune wheat cultivars/lines is available. Therefore, it is urgent to identify important genes in the defense response, clarify their defense roles and create wheat germplasm with resistance to common root rot/sharp eyespot. In order to defend attack of pathogens, plants have evolved comprehensive and complicated defense system. The MYB transcription factor families play an important role in the plants responses against different stresses. In this paper, MYB transcription factor genes TaMYB86 and TaRIM1 induced by pathogen were detected by molecular characteristics analysis and disease resistance identification. This research aims to identify important genes in the defense response and create wheat germplasm with resistance to common root rot/sharp eyespot.The main results are as follows:In order to develop and select new germplasm containing the TaMYB86 overexpression vector pUbi:MYC-TaMYB86 and the TaMYB86 transgenic wheat resistance to common root rot, we introduced TaMYB86 into Yangmai 16 via bombarding the particle lines on T0-T3 generations, and were detected by molecular characteristics analysis and disease resistance identification. The PCR results showed that the alien TaMYB86 was transferred and integrated stably into three transgenic wheat lines. The qRT-PCR results revealed the relative transcriptional level was apparently higher in transgenic wheat lines than the non-transformation Yangmai 16. As Western blotting results showed that the introduced MYC-TaMYB86 gene was translated into the MYC-TaMYB86 protein in these three overexpressing transgenic lines, but not in non-transformation Yangmai 16. Evaluating the effect of TaMYB86 overexpression on wheat defense response to B. sorokiniana, showed that the infection types and disease indexes of the three TaMYB86 transgenic wheat lines were significantly lower than those of non-transformation Yangmai 16(t-test, P < 0.01). The transcript levels of three wheat defense genes(PR10, PR17 c, and Chit1) were significantly elevated in these transgenic wheat lines compared to those in the non-transformation Yangmai 16. These results indicated that overexpression of TaMYB86 enhanced resistance to B. sorokiniana significantly in transgenic wheat lines and TaMYB86 played a positive role in defense response to B. sorokiniana.To explore whether TaRIM1 plays an important role in wheat resistance response against R. cerealis, we used BSMV-based VIGS method to down-regulate transcriptional levels of TaRIM1 in the resistant wheat line CI12633. The transcript levels of TaRIM1 were significantly reduced in CI12633 plants infected by BSMV:TaRIM1 compared to BSMV:GFP infected CI12633 plants(control plants), suggesting that TaRIM1 transcript was successfully down-regulated in BSMV:TaRIM1 infected plants. Then, the TaRIM1-silenced and BSMV:GFP infected CI12633 plants were further inoculated with R. cerealis, TaRIM1-silenced CI12633 plants showed more susceptible to the sharp eyespot disease caused by R. cerealis comparing with the BSMV:GFP infected CI12633 plants. These results suggested the down-regulation of TaRIM1 compromised the resistance to R. cerealis in CI12633, and that TaRIM1 is required for host resistance response to R. cerealis. The pUbi:MYC-TaRIM1 vector DNA was bombarded by gene gun into immature embryos of the spring wheat cultivar Yangmai 16 for generating transgenic wheat plants. The TaRIM1 transgenic wheat lines on generations of T0-T2 were detected by molecular characteristics analysis and disease resistance identification. The presence of TaRIM1 transgene cassette was detected by the PCR analysis and five stably transgenic lines containing Ubi:MYC-TaRIM1 transgene were selected. qRT-PCR assays showed that the transcriptional levels of TaRIM1 in these five TaRIM1-overexpressing transgenic lines were significantly elevated compared with non-transformed(wild type, WT) recipient wheat Yangmai 16. Western blotting analysis indicated that the introduced MYC-TaRIM1 gene was translated into the MYC-TaRIM1 protein in these five overexpressing transgenic lines, but not in non-transformed Yangmai 16. Following inoculation with R. cerealis for ~47 d, all the 5 TaRIM1-overexpressing wheat lines in successive two generations(T1-T2) showed significantly enhanced-resistance to sharp eyespot compared with susceptible WT wheat Yangmai 16. These results indicated that TaRIM1 positively contributed to wheat defense response to R. cerealis infection. The transcriptional levels of five wheat defense genes(Defensin, PR10, PR17 c, nsLTP1, and Chit1) were analyzed by qRT-PCR in TaRIM1-silencing and overexpression wheat plants as well their controls after the pathogen inoculation. These results indicated that TaRIM1 positively regulated, most likely activated, the expression of these defense genes in wheat and then enhance resistance to R. cerealis significantly in transgenic wheat lines.