Research On Promoter Editing Of MeSWEET10a Gene From Cassava

Cassava（Manihot esculenta Crantz）is not only an important starch crop,but also an important food crop in the tropics.Disease is one of the important biological factors to increase the yield of cassava.Cassava bacterial blight（CBB）is a bacterial disease caused by Xanthomonas axonopodis pv.Manihotis（Xam）.All the main cassava varieties in China are not resistant to CBB.Cultivating new cassava varieties with disease resistance is an urgent problem to be solved for the sustainable and healthy development of cassava industry.In this study,fluorescent quantitative PCR,double luciferase test,transgenic and gene editing techniques were used to verify the interaction between the susceptible gene MeSWEET10a of Chinese cassava variety Southern China 8（SC8）and Hainan isolated strain Xam11.The main results are as follows:1.The infection of Xam11 pathogen on the leaves of SC8 cassava proved that this variety was susceptible to CBB.Xam11 induced the expression of MeSWEET10a gene in SC8 cassava.2.The promoter of MeSWEET10a gene and the TAL20_Xam11 effector protein of Xam11strain were cloned.The double luciferase experiment showed that the TAL20_Xam11 effector protein activated the expression function of SC8 cassava MeSWEET10a gene by binding to the EBE region of MeSWEET10a gene promoter.3.The CRISPR/Cas9 editing vector of EBE region of MeSWEET10a promoter was constructed and the fragile embryogenic callus of SC8 cassava was transformed.The function and specificity of vector editing EBE region were verified by Sanger sequencing.4.Four cassava mutants with homozygous mutations in the EBE region of MeSWEET10a promoter were obtained.One mutant（line#1）inserted T in the EBE region,and the other three mutants（lines#2,#3,#4）all deleted TATA.Double luciferase experiments showed that after the insertion of T into EBE region,the activation ability of TAL20_Xam11 to p MeSWEET10a promoter decreased by 78.87%,while after the deletion of TATA in EBE region,TAL20_Xam11 had no effect on p MeSWEET10a promoter.5.The leaves of cassava seedlings of mutants#1,#2 and SC8 were inoculated with Xam11 pathogens.It was proved that the mutation in EBE region blocked the activation of MeSWEET10a gene expression by TAL20_Xam11 and enhanced the resistance of cassava to Xam11 pathogens.The results of this study proved that the pathogen of Xam11 could regulate the expression of MeSWEET10a gene and enhance the pathogenicity of the pathogen by binding the EBE region of the MeSWEET10a gene promoter of SC8 cassava variety through the EBE effector protein.The gene editing of EBE region by CRISPR/Cas9technology can block the binding process of TAL20_Xam11 effector protein to EBE region and improve the disease resistance of cassava.