Editing Of EIF4E In N.benthamiana Which Interacting With CBSV/UCBSV-VPg

Eukaryotie translation initiation factor 4E(eIF4E)is not only involved in the initiation of translation of intracellular proteins,but also its interaction with Virus genome linked protein(VPg)is necessary for the infection,replication and translation of Potyviridae.Currently,most of the recessive resistance genes found in plants are alleles of the eIF4E family.Using host factors such as transcription initiation factor eIF4E proteins as antiviral targets,antiviral breeding has broad application prospects.Cassava brown streak virus(CBSV)and Ugandan cassava brown streak virus(UCBSV)belong to the Ipomovirus of the Potyviridae family,which had caused serious damage for the production of cassava that regarded as the sixth largest grain crops in the world.Currently,there is no effective prevention and control method for CBSV/UCBSV,especially the lack of CBSV resistant breeding materials.So in this study,one of the hosts of CBSV/UCBSV--N.benthamiana was used as experimental material.First,we cloned the eight genes of the eIF4E family and found out the eIF4E gene interacted with CBSV/UCBSV-VPg using the LexA-Y2H yeast two-hybrid system and BiFC.Then,the eIF4E gene was further knocked out by utilizing CRISPR-Cas9 gene editing technology.The research result lays the foundation for investigateing whether blocking the interaction between CBSV/UCBSV-VPg and eIF4E results in the failure of virus replication and reproduction,which makes the N.benthamiana edited eIF4E gene obtain CBSV/UCBSV resistance,and exploring anti-CBSV/UCBSV virus molecular breeding in Cassava through gene editing host factors.The specific results are as follows:(1)Eight eIF4E genes were identified from bioinformatic analysis of N.benthamiana genomes,cloned by RT-PCR,specific primers with N.benthamiana as template.Temporarily named as Nb4El,Nb4E2,Nb4E3-1 Nb4E3-2,Nb4E4-1,Nb4E4-2,Nb4E5,Nb4E6.The comparison analysis of amino acid showed that the eight of eIF4E proteins were divided into three groups:Nb4E1,Nb4E2,Nb4E4-1,Nb4E4-2,Nb4E6 belonged to eIF4E protein;Nb4E3-1,Nb4E3-2 belonged to eIF(iso)4E isoform protein;Nb4E5 belongs to cap binding protein nCBP.(2)To investigate the interaction between CBSV-VPg,UCBSV-VPg and eIF4E family of N.benthamiana,an activation domain vector containing CBSV-VPg and UCBSV-VPg gene and a bait vector respectively containing eight eIF4Es gene were constructed,and there was no autoactivation and toxicity.The identified vectors were transformed into the yeast strain EGY48 and screened by auxotroph and X-galactosidase.The results of LexA-Y2HS showed that Nb4E3-2 interacts with CBSV-VPg and UCBSV-VPg,but the other seven proteins didn't interact with two VPg.(3)The bimolecular YC1-Nb4E3-1,YC1-Nb4E3-2,YN1-VPg and YNl-UVPg recombinant expression vectors were constructed and transformed into Agrobacterium cells for transient expression in N.benthamiana.Laser confocal microscope showed that the co-injection of CBSV-VPg or UCBSV-VPg with Nb4E3-2 could restored fluorescence,but there was no fluorescence when co-injected with Nb4E3-1,which further verified the interaction between Nb4E3-2 and CBSV/JCBSV-VPg.(4)To obtain genetically edited plants,we designed and constructed the gene editing vector pHSE-Nbiso4E-g1g2 based on CRISPR-Cas9 technology for the target Nb4E3-2,then editing vector was transferred into N.benthamiana by Agrobacterium-mediated method.Nb4E3-2 gene of twenty-seven strains of transgenic plants was amplified by PCR and sequenced analysis,which found that six gene edited strains with different gene mutation types.It remains to be further detected analysis and inoculated virus experiment.This study provides a theoretical basis for the further study of the resistance of CBSV/UCBSV associated with eIF4E-like factors,and also contributes an important reference for exploring new methods for resistanting CBSV/UCBSV virus of Cassava through gene editing eIF4E.