Mechanisms Of Begomoviruses Replication And Their Immune Escape In The Whitefly Vector

The whitefly Bemisia tabaci,herbivory insect with sucking mouthparts,is currently considered as a cryptic species complex containing dozens of cryptic species.The genus Begomovirus is the largest genus of the plant virosphere and currently contains more than 400 accepted species.Begomoviruses have caused a great loss of vegetables and staple food crops production worldwide and are exclusively transmitted by whiteflies in a circulative manner.Recently,more and more evidences demonstrate that some begomoviruses are able to replicate and transcribe in whiteflies,but the underlying molecular mechanism is unknown.In order to be transmitted,many viruses have evolved to inhibit the innate immunity system of insect vectors through complex interactions between virus and vector components.Nevertheless,it is not clear about the effect of begomoviruses on whitefly innate immunity.In this study,working with tomato yellow leaf curl virus(TYLCV),tomato yellow leaf curl China virus(TYLCCNV)and papaya leaf curl China virus(Pa LCu CNV),we focused on these important questions,and the results are summarized as follows:1)The effect of TYLCCNV intergenic region on virus replication in whitefliesBegomoviral intergenic region is crucial for virus replication and transcription in plants.Previous studies have demonstrated that TYLCCNV is able to replicate in whiteflies,whereas Pa LCu CNV is unable to.To investigate the role of intergenic region in begomovirus replication in vector whiteflies,we replaced the intergenic region of TYLCCNV with that of Pa LCu CNV,constructing a mutant TYLCCNV(m TYLCCNV).The results showed that both TYLCCNV and m TYLCCNV could be acquired and transmitted by whiteflies.When whiteflies were first given a 6 h AAP on virus-infected plants and then transfered onto cotton,a non-host plant of the virus,the amount of TYLCCNV in whiteflies increased at 2 days after the transfer and then decreased.However,the amount of m TYLCCNV decreased rapidly upon the transfer and remained low thereafter,suggesting that m TYLCCNV is unable to replicate in whiteflies.Furthermore,the transmission efficiency of m TYLCCNV decreased dramatically compared with that of TYLCCNV,indicating that virus replication promotes virus transmission by the whitefly vector.Taken together,these results indicate that the intergenic region of begomovirus genome plays a significant role in virus replication in insect vectors.2)Identification of whitefly proteins interacting with TYLCCNV intergenic regionIn consideration of the important role of begomovirus intergenic region in its replication in whiteflies,we speculated that some whitefly proteins may participate in virus transcription and replication in whiteflies by interacting with TYLCCNV intergenic region.The virion-sense sequence of the right part and the complementarysense sequence of the left part of TYLCCNV intergenic region were used to construct the bait plasmids for yeast one-hybrid screening of whitefly proteins.We identified 52 whitefly proteins that interacted with the intergenic region of TYLCCNV.We then selected 32 proteins for further verification and 12 of them were confirmed to bind to TYLCCNV intergenic region.Then five proteins: hairy and enhancer of split homolog-1(HES1),max-like protein X(MLX),nucleolar protein 11(NOL11),transcriptional adapter 3-A(ADA3-A)and heat shock factor(HSF)that function in gene transcription regulation were selected for further analysis via RNA interference(RNAi).After knocking down the expression of Bt HES1,Bt NOL11,Bt ADA3-A or Bt HSF,the amount of viral DNA in whiteflies was decreased when compared with that of the control,indicating these genes may be involved in virus replication in whiteflies.3)The mechanisms of begomovirus replication in whitefliesAfter silencing Bt HES1 or Bt HSF,the virus loads in whiteflies decreased significantly.To reveal the detailed molecular mechanisms underlying begomovirus replication in whiteflies,we investigated the role of these two genes in virus replication.We found that Bt HES1 specifically bound to CACGTG motif in the right part of TYLCCNV intergenic region and activated downstream gene transcription.Silencing of Bt HES1 decreased viral transcription,replication and transmission.These results suggest that the transcription factor Bt HES1 binds to the intergenic region of TYLCCNV to activate the transcription of viral genes and,thus,facilitate virus replication and transmission.In addition,Bt HSF interacted with the left part of TYLCCNV intergenic region,and RNAi-mediated knockdown of Bt HSF suppressed TYLCCNV replication and transmission.More importantly,we found that Bt HSF also bound to the intergenic region of TYLCV and facilitated TYLCV replication in whiteflies.4)The interactions between TYLCV and whitefly JAK/STAT pathwayOur previous studies have isolated whitefly STAT(BtSTAT)as a putative TYLCV coat protein(CP)interacting protein using a yeast two-hybrid system.Here,we firstly confirmed the interaction between TYLCV CP and whitefly STAT.Next,we characterized the whitefly DOME,JAK and STAT,which are the core components of JAK/STAT pathway.Moreover,we identified four BtSTAT downstream genes:Bta03341,Bta9750,Bta12654 and Bta12955.After the suppression of JAK/STAT pathway by feeding whiteflies with ds RNA or inhibitor SH-4-54,the amount of viral DNA and CP increased in whiteflies,suggesting that the whitefly JAK/STAT pathway functions as an antiviral mechanism against TYLCV accumulation in whiteflies.In addition,the virus loads in whiteflies increased after knocking down the expression of Bta03341 or Bta9750,indicating that these two genes are the downstream antiviral factors of whitefly JAK/STAT pathway.More importantly,we found that the infection of TYLCV inhibited the whitefly JAK/STAT pathway,which was achieved through hijacking of BtSTAT by TYLCV CP.Taken together,we demonstrated that the intergenic region of TYLCCNV plays a critical role in virus replication in whiteflies.Whitefly proteins participate in virus replication through binding to the intergenic region of viral genome.In addition,we proved that the whitefly JAK/STAT pathway plays an antiviral role against TYLCV accumulation in whiteflies.However,the virus has evolved to suppress the JAK/STAT pathway through hijacking of the core transcription factor STAT with its CP,which facilitating its transmission by the whitefly vector.These findings reveal a relationship of coevolved adaptations between begomoviruses and whiteflies and provide important insights into the understanding of virus-insect vector interactions,in addition to giving valuable clues for the development of new strategies to block virus transmission.