Screening And Validation Of Interacting Proteins Of Sitobion Miscanthi With Proteins Of Barley Yellow Dwarf Virus

Wheat yellow dwarf disease caused by Barley yellow dwarf virus(BYDV)is an important virus disease for wheat production in various wheat regions in China.GAV is the main strain in the feild of China,which is transmitted by Sitobion miscanthi(It was mistakenly identified as Sitobion avenae in China,and it is still widely used)in a persistent non-propagative manner.Moreover,based on field investigation,BYDV-GAV and S.miscanthi often occur together,which aggravates the loss of wheat yield and quality.There are two barriers and immune responses in vector aphids,and these barriers must be overcome if the virus is to spread.Then,it must be involving the specific recognition and interaction between the proteins of BYDV-GAV and the receptor proteins in S.miscanthi.Previous studies have found that cp and atp of BYDV-GAV are both important for the virus to break through the barriers in the mediator,but it is still unclear which proteins in the mediator aphids participate in the interaction process.Therefore,in this study,BYDV-GAV and its mediator S.miscanthi were used as the research objects to study the proteins interacting with BYDV-GAV cp and atp in S.miscanthi by yeast two-hybrid,and verified by yeast two-hybrid and BiFC.It is of great significance to understand the interaction mechanism between BYDV-GAV and S.miscanthi,and to prevent and control wheat yellow dwarf disease by blocking the transmission of BYDV.The main results were as follows:1.For GAL4 yeast two hybrid system screen,the bait plasmids p GBKT7-cp and p GBKT7-atp were successfully constructed by inserting the BYDV-GAV coat protein gene and aphid transmission protein gene into the bait vector p GBKT7.Then,the two bait plasmids were tested for toxicity and self-activation.The results show that their protein products were correctly expressed in Y2 H Gold yeast cells,no toxinity to yeast growth,and no autoactication.So both of these two bait plasmids were suitbale for library screening.2.A total of 300 S.miscanthi which were newly emerged with BYDV-GAV and a total of 300 S.miscanthi without BYDV-GAV were collected,and a c DNA plasmid library of S.miscanthi was constructed based on the total RNA of the whole aphids.The co-transfer screening procedures were performed for library screening of two baits: p GBKT7-cp got fifteen prey ptoteins,and p GBKT7-atp got eleven prey ptoteins,Among them,lwr,ata and ccq were screened by both PGKBT7-cp and p GKBT7-atp.3.Five proteins lwr,ata,ccq,tra and tph screened by PGKBT7-cp and 8 proteins lwr,ata,ccq,ntf,tcs,sia,ugt and nap screened by PGKBT7-atp were selected according to the number of repeats and the function of homologous genes in NCBI.The 10 candidate proteins was verified one-on-one with bait proteins by yeast two-hybrid.Seven prey plasmids were successfully constructed by inserting the candidate proteins’ gene into the prey vector p GADT7.Seven prey plasmids with their corresponded bait plasmids were transferred into Y2 H Gold yeast cells respectively.Two prey proteins lwr and ntf were shown that they had strong interactions with both cp and atp.Two prey proteins ccq and nap owned weak interaction relationship with both cp and atp.4.Bimolecular fluorescence complementation was used to further verify the interaction between the two bait proteins and the candidate proteins.Five fluorescent expression vector plasmids were successfully(The fluorescent expression vector plasmids of nap was failing)constructed by inserting the candidate proteins’ gene into p NC-BIFC-vcn,and bait proteins cp and atp into p NC-BiFC-vnn.The plasmid was transformed into GV3101 of Agrobacterium tumefaciens and injected into Nicotiana benthamiana.The results showed that the candidate proteins lwr and ntf could interact with BYDV-GAV both cp and atp,but ccq could not interact with both cp and atp.5.The phylogenetic tree constructed by MEGA 6.0 showed that lwr and ntf of S.miscanthi had the highest homology with Acyrthosiphon pisum.The results of SMART analysis showed that lwr had UBCC domain.The results of TMHMM and Signal P 5.0showed that bothlwr and ntf were non-transmembrane and non-secretory proteins.In conclusion,two candidate proteins lwr and ntf that have strong interactions relationship with both BYDV-GAV cp and p GKBT7-atp were identified in this study.This study will lay a foundation for further understanding the transmission mechanism of S.miscanthi and the control of wheat yellow dwarf disease.