Cloning And Functional Analysis Of TaNRH In Leaf Rust Resistance In Wheat

Puccinia triticina is a highly specialized living nutrient fungus.In the incompatible combination of wheat and Puccinia triticina,the host cells resist the infection of Puccinia triticina by a hypersensitive reaction(HR).The NB-LRR resistance protein is an important R protein and is divided into two subclasses,TIR-NB-LRR and CC-NB-LRR,depending on the N-terminal conserved domain.In recent years,there have been more and more reports on the CC-NB-LRR proteins in plants resistance.In the early stage of the laboratory,the wheat near-isogenic line TcLr26 and its recurrent parent Thatcher(Tc)were incompatible with the leaf rust physiological race 260(with HR)and compatible combination(no HR),respectively,and transcriptome sequencing was performed to screen for the important genes involved in HR induction and regulation.In this study,by analysing the transcriptome data,a new member of the CC-NB-LRR gene family was screened and the mechanism of this gene in HR induction of wheat resistance to leaf rust was explored.The main results are as follows:1.By analyzing the transcriptome database of interaction between wheat and Puccinia triticina,we screened and obtained a new member of the CC-NB-LRR gene family.The expression of this gene in the affinity combination was almost 0,but it was constitutive expression in the incompatible combination.so it was speculated that this gene may be associated with the occurrence of host HR in the incompatible combination and named it TaNRH(NB-LRR gene required for HR).Bioinformatics analysis revealed that the full-length CDS of this gene is 2874 bp,encoding 957 amino acids,with an Rx-CC-like domain at the N-terminus,a NB-ARC domain at the center,and an LRR domain at the C-terminus.2.It was confirmed by PCR amplification that TaNRH was specifically present in the TcLr26 genome and was not present in Tc.RT-qPCR was used to detect the expression level of TaNRH gene in incompatibility group at different time after inoculation.The results showed that in the incompatible combination,the expression of TaNRH gene did not change significantly with the inoculation time,that is TaNRH gene was constitutively expressed.3.The p-Super1300::TaNRH-GFP vector was constructed,and transient transformation of tobacco revealed that TaNRH protein was localized in the cytoplasm.4.The TaNRH gene was silenced on TcLr26 by VIGS and RNAi technology,respectively,and then inoculated with leaf rust race 260.The HR area of TaNRH gene silencing plants was significantly increased and the number of HMC was significantly increased compared with the control plants.It is proved that TaNRH plays a positive regulatory role in the HR occurrence of wheat resistance to Puccinia triticina infection.The TaNRH gene was overexpressed on Tc,and after inoculation with Puccinia triticina,it was found that overexpression of TaNRH could open the signal pathway for inducing HR,and HR could be induced without the induction of Puccinia triticina.5.The yeast two-hybrid vectors pGADT7-TaNRH,pGBKT7-TaRanGAP2 and pGBKT7-TaNRH were constructed,then pGADT7-TaNRH was co-transformed with pGBKT7-TaRanGAP2 and pGBKT7-TaNRH,respectively.The results showed that TaNRH cannot be self-associated,but TaNRH and TaRanGAP2 could interact in the cell.6.The BiFC vectors pSPYCE(M)-TaNRH,pSPYNE(R)173-TaRanGAP2 and pSPYNE(R)173-TaNRH were constructed.After transient transformation of tobacco revealed that TaNRH cannot be self-associated in tobacco cells,but TaNRH and TaRanGAP2 could interact in tobacco cells.