Transcriptome Profiling Of Whitefly Midguts In Response To Begomovirus Infection And Screening Of Whitefly Proteins Interacting With Begomovirus

Geminiviruses have received great attention worldwide because of their tremendous economic impact.Geminiviruses are transmitted from infected to healthy plants mainly by insect vectors,and it is important to understand the mechanisms of geminivirus transmission by whiteflies.Tomato yellow leaf curl virus(TYLCV),one of the most devastating begomovirus,is transmitted exclusively by the whitefly Bemisia tabaci in a circulative,persistent,manner.The circulation of TYLCV in B.(abaci involves complex insect-virus interactions,whereas the molecular mechanisms of these interactions remain ambiguous.The midgut is a major barrier of geminivirus transmission in whiteflies and is critical in whitefly-geminivirus interactions.However,due to its tiny size,the interactions between whitefly midgut and TYLCV remain largely unknown.In addition,TYLCV can replicate in whitefly as least for a certain period of time,however the processes and mechanisms of TYLCV replication in whitefly are not clear.In this study,we investigated the transcriptional response of the midgut of B.tabaci Middle East-Asia Minor 1 species to TYLCV using Illumina sequencing.Then,to identify the whitefly proteins involved in TYLCV replication,yeast two hybrid was performed to screen whitefly cDNA library using non-structural proteins of TYLCV as baits.The results are summarized as follows:(1)Transcriptome responses of the whitefly midgut to TYLCV infectionA total of 5207 differentially expressed genes were identified(DEGs)in viruliferous whitefly midguts.Using enrichment analyses,we found that DEGs were significantly enriched in the gene ontologies of'Cargo receptor' and KEGG pathway of 'ATP-binding cassette(ABC)transporters',which might help the virus to cross gut barrier.TYLCV could perturb cell cycle and DNA repair as a possible result of its replication in the whitefly midgut.Our data also demonstrated that TYLCV activated whitefly defence responses,such as antimicrobial peptides and cell-membrane linked immune responses.Meanwhile,a number of genes involved in intracellular signaling molecules and pathways were activated by TYLCV.The significant induction of the immune system might be deleterious to virus survival in the whitefly midgut.(2)Yeast two hybrid(Y2H)screening of whitefly proteins interacts with non-structural proteins of TYLCVThe five non-structural protein genes of TYLCV:AC 1,AC2(fragment),AC3,AC4 and AV2 were constructed on pGBKT7 bait vector respectively,and transformed into Y2H Gold yeast strain to confirm non-toxicity and self-activation.The whitefly cDNA library was screened by sequential transformation method.The yeast cells were screened on SD/-Trp-Leu-His medium and then the yeast colonies were transferred to SD/-Trp-Leu-His-Ade(Containing X-a-gal and AbA toxin)medium.After Y2H screening,1 positive clone interacting with AC2,46 clones interacting with AC3,and 63 clones interacting with AC4 were obtained.Single plasmid was isolated and sequenced.Finally,18 whitefly proteins were confirmed to interact with non-structural proteins of TYLCV,including 1 with AC2,6 with AC3,and 11 with AC4.In summary,the data of transcriptome profiling reveal the complex insect-virus relationship in specific tissues and provide substantial molecular information for the role of whitefly midguts in virus transmission.The result of yeast two hybrid provides a valuable source of molecular information for the further research in TYLCV-whitefly interactions.