Screen Of Proteins Interacting With TYLCV And Analysis Of ABC Transporters In Bemisia Tabaci Q

The sweetpotato whitefly,Bemisia tabaci(Gennadius)(Hemiptera: Aleyrodidae),is a cosmopolitan pest that cause substantial damages through not only feeding plants but also transmitting plant viruses.B.tabaci has been demonstrated with a remarkable ability to develop resistance to insecticides in the field.All thses characteristics of B.tabaci support its strong fitness,which has been a serious threat in agriculture.Tomato yellow leaf curl virus(TYLCV),a typical member of the Begomoviruses,causes tomato yellow leaf curl disease which is one of the most seriouse tomato disease in the world.TYLCV is specially transmitted by B.tabaci.Study on the interaction between TYLCV and B.tabaci has become the key to uncover the mechanism of virus transmission.ABC transporter is one of the largest and ubiquitous groups of protein.Being as a new member in the metabolic resistance,insect ABC transporters have gained more attention in recent years.At present,mechanism of virus transmission by B.tabaci was poorlyunderstood meanwhile there's no report about the whole ABC transpoters in B.tabaci genome.Study on the interaction between TYLCV and B.tabaci as well as the identification and analysis of ABCs will help us understand the mechanism of virus transmission and insecticide resistance,which will also guide pest control.Split-ubiquitin yeast membrane system was used in this study.Firstly,we successfully constructed a B.tabaci Q cDNA library for yeast two hybrid.The coat proterin(CP)of TYLCV was used as a bait and 80 positive clones from cDNA library were successfully screened for the first time.Then interactions between these candidates and CP were verified by this yeast membrane hybrid system.At last,20 proteins in B.tabaci were demonstrated to interact with CP.Based on the sequence of a gene,putative antimicrobial knottin protein Btk-1,from yeast two hybrid,its full-length cDNA sequence was acquird from genome and transcriptom data.Interactin between CP and Btk-1 was verified by yeast two hybrid and bimolecular fluorescence complementation(BiFC).In addition,after analysis of transcriptom of B.tabaci with and without TYLCV,eight genes with over ten-fold changed expression level were discovered.A total of 55 ABC transporters containing all eight described subfamilies(A to H)were identified in the B.tabaci Q genome,including 8 ABCAs,3 ABCBs,6 ABCCs,2 ABCDs,1 ABCE,3 ABCFs,23 ABCGs and 9 ABCHs.In comparison to other species,subfamilies G and H in both phloem-and blood-sucking arthropods are expanded.The temporal expression profiles of these 55 ABC transporters throughout B.tabaci developmental stages and their responses to imidacloprid,a neonicotinoid insecticide,were investigated using RNA-seq analysis.Furthermore,the mRNA expression of ABC transporters was confirmed by the quantitative real-time PCR(RT-qPCR).Results showed that the mRNA expression levels estimated by RT-qPCR and RNA-seq analyses were significantly correlated(r=0.684,p<0.01).80% of the significantly up-regulated ABC transporters in imidacloprid-challenged B.tabaci were ABCGs.These combined results imply the potential contribution of ABCs especially ABCGs to the insecticide resistance of B.tabaci.