Characterization Of Cytokinin Synthesis Gene OsIPT9 In Brown Planthopper Resistance Of Rice

Rice is one of the most important food crops in China.Rice production is directly related to the food security and farmers’ income.Brown planthopper（BPH）is the largest and most severe pest in rice production.Despite the annual efforts to spray pesticides for control,BPH still causes heavy losses.Cultivating rice varieties resistant to brown planthopper is internationally recognized as the most economical,effective and environment-friendly green control measure against brown planthopper.In previous studies,we isolated rice brown planthopper resistance gene Bph6.It was found that Bph6 up regulated the level of cytokinin（CK）in rice to exert insect resistance.CK plays an important role in plant growth and development,apical dominance,aging regulation of organs and grain yield.In recent years,the mechanism of CK in plant defense responses to various biological and abiotic stresses has been more and more favored by researchers.Cis-zeatin（CZ）is the highest content of CK in rice.Exogenous application of CZ significantly improved the resistance of Bph6 plants to brown planthopper.However,there is no direct genetic evidence to show the role of CK,especially CZ,in rice resistance to brown planthopper.This study revealed the important role of OsIPT9 and CZ in BPH resistance by analyzing the role of isopentenyl transferase gene OsIPT9,the key rate limiting enzyme in CK synthesis,in the insect resistance mediated by Bph6.The main results are as follows:1.It was confirmed that the increase of CK synthesis gene expression in Bph6-NIL after brown planthopper infection was the reason for the increase of CK content.The contents of cytokinins such as CZ,IP,IPR and Czr increased significantly in Bph6-NIL after brown planthopper infection.It was found that the expression of CK synthesis genes OsIPT7、OsIPT9、OsIPT10 and LOGL9 increased significantly by Q-PCR.2.It was found that the expression of OsIPT9 protein in Bph6-NIL also increased significantly after brown planthopper infection.We expressed and purified OsIPT9 protein by using E.coli system,then prepared antibodies.With the result of western blot,it was found that OsIPT9 protein content in Bph6-NIL increased gradually with brown planthopper infection.3.We confirmed that OsIPT9 is a functional tRNA-IPT protein.The results of phylogenetic tree suggest that OsIPT9 is a functional tRNA-IPT protein.We demonstrated that conclusion by using the functional complementation experiment with defective yeast MT-8.4.We revealed the important role of OsIPT9 in rice resistance to brown planthopper.We constructed CRISPR/Cas9 vectors of OsIPT9 and OsIPT10,and obtained knockout lines of OsIPT9 and OsIPT10 by transgenic.Compared with Bph6-NIL,OsIPT9 homozygous knockout lines showed a significant decrease in insect resistance identification at seedling stage,and a significant increase in Honeydew amount,brown planthopper weight gain and percentage of insect weight gain.We identified the brown planthopper resistance of OsIPT9 homozygous knockout lines.It was found that knockout of OsIPT9 gene significantly reduced the resistance of Bph6-NIL to brown planthopper,revealing the important role of OsIPT9 in rice resistance to brown planthopper.This study confirmed the function of CZ in rice insect resistance in genetics,and also provided a basis for the analysis of the insect resistance molecular mechanism of Bph6 gene.The results will provide important theoretical support for the study of rice insect resistance mechanism and the interaction between plants and insects,and provide an important practical basis for the breeding and improvement of rice resistance to brown planthopper...