Using CRISPR-Cas9 Technology To Edit TaMKP1 Gene To Create Wheat Disease-Resistant Materials

Wheat is an important grain crop in China.However,it faces a serious threat from wheat stripe rust,a disease caused by Puccinia striiformis f.sp.tritici(Pst)that can spread rapidly and unpredictably.This disease poses a significant risk to wheat production and even national food security.The high variability of wheat stripe rust can quickly lead to the loss of disease resistance in wheat varieties.Therefore,it is urgent to explore and utilize genes related to disease resistance.Previous studies in our laboratory have shown that TaMKP1 negatively regulates wheat resistance to stripe rust.To further analyze the mechanism of TaMKP1 in resistance to stripe rust,this study employed CRISPR-Cas9 gene editing technology to target and edit TaMKP1 in the wheat genome.Mutant plants were screened using bar gene detection,PCR test,and sequencing.The resistance of both wild-type wheat Fielder and mutant plants to stripe rust at the seedling and adult stages was evaluated.Subsequently,related agronomic traits were statistically investigated and analyzed.The main results are as follows:1.The results of wheat stripe rust resistance identification at the seedling stage showed that after editing TaMKP1,the resistance of wheat to stripe rust was significantly enhanced compared with the wild-type Fielder.The Pst biomass was significantly decreased,and the resistance of each mutant plant was different.The three-copy mutant showed the strongest resistance to stripe rust,and the Pst biomass was only 10% of the control.In contrast,the A-copy mutant had relatively poor resistance to stripe rust,and the Pst biomass was about 50% of the control.These findings suggest that the three copies of TaMKP1 contribute differently to resistance to stripe rust in wheat.DAB staining and trypan blue staining were performed on TaMKP1 mutant plants after inoculation with Pst.A large area of reactive oxygen species burst and cell necrosis were observed on the leaves of TaMKP1 mutant plants,but not in wild-type wheat.By infecting the TaMKP1 mutant plants at the adult stage with a mixed strain,the results showed that the three-copy mutant of TaMKP1 gene had the strongest resistance to wheat stripe rust,and the identification results were consistent with the seedling stage.Subsequently,the TaMKP1 gene-edited wheat was inoculated with powdery mildew,and the improvement of wheat resistance to powdery mildew was observed,indicating that in the actual production process,the editing of the TaMKP1 may increase the resistance of wheat to various diseases.2.Previous studies have shown that TaMKP1 is located in the nucleus.In this study,the function of nuclear localization was determined by constructing a nuclear output signal vector.The results showed that TaMKP1 with nuclear localization signal tag(NLS)was localized in the nucleus,and TaMKP1 with nuclear export signal(NES)was localized in the cell membrane and cytoplasm.Subsequently,it was inoculated with S.sclerotiorum 1980 to observe the incidence,and trypan blue staining was used to observe cell necrosis.The results showed that the lesion diameter and cell necrosis area of tobacco leaves when TaMKP1 was located on the nucleus were significantly larger than those when TaMKP1 was located outside the nucleus,indicating that the process of TaMKP1 participating in gene expression regulation may be carried out in the nucleus.3.To determine whether wheat with TaMKP1 mutation has potential for agricultural production and application,the main agronomic traits were measured in this experiment.The results of agronomic traits investigation showed that after TaMKP1 editing,except for flag leaf width,there was significant difference between TaMKP1 editing and wild type,the main agronomic traits such as plant height,flag leaf length,grain size and 1000-grain weight of wheat changed to varying degrees.Among them,the agronomic traits of the three-copy-edited plants were the best,with an average plant height of 10 cm higher than the control,an average flag leaf length of1.4 cm longer than the control,and an average thousand-grain weight of 6.8 g heavier than the control,indicating that editing out TaMKP1 can not only improve wheat resistance to stripe rust,but also increase wheat yield.In summary,the negative regulation of wheat stripe rust resistance by TaMKP1 is inseparable from its localization changes.TaMKP1 mutation can significantly improve the resistance of wheat to stripe rust,and can also increase wheat yield.It provides an important theoretical basis and practical application ideas for using a single gene in wheat to achieve disease control and yield increase at the same time.