The Analysis Of Knock Out VviEDR1 And VviEDR2 Gene By Crispr/Cas9 Gene Editing To Improve Powdery Mildew Resistance In Grapevine

Grapevine is one of the most economically important fruits crops all over the world.The European cultivated grapevine species(Vitis vinifera L)with a desirable fruits quality and nutritional value is widely cultivated and utilized.In grapevine production,however,it is seriously harmed by grapevine powdery mildew.Therefore,the research about the function of genes related to powdery mildew resistance in grapevine is vital to reveal the mechanism of resistance to powdery mildew and to breed new cultivars resisting to grapevine powdery mildew.Since 2013,the development of genome editing was very rapid,especially CRISPR/Cas9 system.Compared with the traditional breeding methods,the genome editing technology is able to directly modify the target gene,to improve the accuracy of modifying the target traits and to speed up the breeding process.Here,the CRISPR/Cas9 technology was used to improve resistance to powdery mildew in European grapevine Vitis vinifera L cv.Thompson Seedless.As PDS gene deficiency leads to a visible bleaching phenotype,the VviPDS1,phytoene desaturase in grapevine,was selected as the target gene for CRPSPR/Cas9-mediated genome editing in this study.And it was used to verify whether the CRISPR/Cas9 system allows genome editing in grapevine.In addition,Enhanced Disease Resistance 1(EDR1)and Enhanced Disease Resistance 2(EDR2)play a negative role in the defense response against powdery mildew in Arabidopsis thaliana.The open reading frames of VviEDR1 and VviEDR2 were cloned from the c DNA of European grapevine Vitis vinifera cv.Thompson Seedless.The VviEDR1 gene and VviEDR2 gene were knocked out using the CRISPR/Cas9 system to obtain mutants.It will provide an important information to reveal the molecular mechanism of the grapevine resistance to powdery mildew and the breeding of new disease-resistant cultivars.This research obtained main results as follows:1.VviPDS1 gene was selected as the target gene and a target site was selected in the fifth exon of the VviPDS1 gene.The CRISPR/Cas9 knock-out vector was constructed.Different types of mutations,such as substitution,insertion and deletion,were detected in grapevine protoplasts by transient transformation using protoplasts from grapevine leaves.In addition,the vector was transformed to the embryogenic callus of Vitis vinifera cv.Thompson Seedless through Agrobacterium-mediated transformation.Twenty lines with different types of mutations at the target site were obtained and the editing efficiency was37.74%.Among them,nine lines with biallelic mutations were homozygous mutants.Different types of mutations,such as substitutions,insertions and deletions occurred.The types of deletions and insertions were common,and only one line was substituted by 6nucleotides.These biallelic mutations were shown different albino phenotypes.These indicated that the CRISPR/Cas9 system allows genome editing in the transient expression and stable expression of grapevine cells,which can produce homozygous knockout in transgenic lines of grapevine.2.The open reading frame of VviEDR1 was cloned from the c DNA of Vitis vinifera cv.Thompson Seedless.The sequence similarity of the kinase domain in the C-terminal is high.Here,three different target sites were selected to knock out the VviEDR1 gene.Among them,the T1 target site is located before the kinase domain.The T2 and T3 targets are located in the kinase domain.And single-target and double-target CRISPR/Cas9knock-out vectors were constructed.Different types of mutations,such as substitution,insertion and deletion,were detected in grapevine protoplasts by transient transformation of protoplasts from grapevine leaves.The types of deletions were common.Surprisingly,a large region nucleotide deletion(about 162 bp)between T2 and T3 targets was detected.These vectors were transformed to the embryogenic callus of Vitis vinifera cv.Thompson Seedless through Agrobacterium-mediated transformation.Thirty-two mutation lines were obtained.Among them,different types of editing occurred in the target sites T2 and T3,and the editing efficiency were 20% and 27.96%,respectively.Among them,four lines were homozygous mutants and twenty-eight lines were heterozygous mutants.Mutant lines with insertion of one nucleotide were common.The resistance to the powdery mildew of mutant lines that the VviEDR1 gene was knocked out was evaluated based on trypan blue staining of leaf sections.Appressorium of the fungal appeared to be collapsed in mutant lines leaves inoculated with En NAFU1 at 3 dpi and 5 dpi.The development of primary hypha and secondary hypha was slowly and the callose was accumulated in mutant lines leaves.However,the fungal grow healthily in leaves of Vitis vinifera cv.Thompson Seedless.3.A target site was selected to target the VviEDR2 gene,and a single-target knoc-kout vector was constructed.Knock-out vector was transformed to embryogenic callus of Vitis vinifera cv.Thompson Seedless through Agrobacterium-mediated transformation.Eight mutant lines were obtained and all the mutant lines were homogeneous.The editing efficiency was 30.77%.The resistance to the powdery mildew of mutant lines was evaluated based on trypan blue staining of leaf sections.The results showed that the VviEDR2 knock-out mutants exhibited improved disease resistance than Vitis vinifera cv.Thompson Seedless.