Characteristics And Mechanism Of Vector Transmission Of Geminiviruses As Affected By Viral Genomic Recombination

Genomic recombination is common for plant viruses.Some studies have explored the pathogenicity and ecological significance of recombinant viruses,but whether recombination could imbue a vector-associated selective advantage to viruses has been barely explored.Plant diseases caused by begomoviruses(family Geminiviridae)have caused substantial losses to agricultural production all over the world,and begomoviruses are transmitted in a persistent circulative manner by whiteflies of the Bemisia tabaci cryptic species complex.Tomato yellow leaf curl Shuangbai virus(TYLCSbV),a newly begomovirus collected from tomato plants in Yunnan,China,is likely a recombinant.Previous studies have shown that begomoviruses are encapsulated in vesicle structures during the transport through whiteflies bodies,and the coat protein(CP)of a begomovirus is the only protein that is known to be involved in its transmission.In this study,by comparing the biological characteristics of TYLCSbV and its parent viruses,we analyzed the pathogenicity and vector transmission specificity of TYLCSbV,and explored the relationship between the recombinant virus and the populations distribution of different B.tabaci cryptic species in the field.The physiological mechanism affecting the differential transmission of TYLCSbV and its parent virus among different B.tabaci cryptic species was analyzed,and the molecular mechanism underlying the transmission of TYLCSbV was further investigated from the perspective of CP sequences and B.tabaci proteins that interacted with CP.(1)The genomic recombination characteristic of TYLCSbV affects its pathogenicity and B.tabaci transmission specificityThe recombination and sequence analysis indicated that TYLCSbV is likely originated from recombination of AYVCNV C1-C4 sequences and Tb CSV intergenic region(IR),V1-V2 sequences.Agrobacterium-mediated inoculation showed that the infectivity of TYLCSbV to tomato and tobacco plants is comparable to that of AYVCNV,but is significantly higher than that of Tb CSV.These together with the sequence alignment results indicated that TYLCSbV might have “inherited” pathogenicity factors contained in the AYVCNV genome to achieve efficient infection.Although TYLCSbV and AYVCNV exhibit similar infectivites to host plants,they have different vector specificity in transmission.That is,TYLCSbV is efficiently transmitted by the whitefly B.tabaci Mediterranean(MED)rather than by the whitefly B.tabaci Middle East-Asia Minor 1(MEAM1),a vector transmission specificity is similar to that of Tb CSV.In contrast,AYVCNV is more efficiently transmitted by MEAM1.Moreover,field surveys showed that MED had displaced MEAM1 in some regions where TYLCSbV was collected.Viral competition assays indicated that TYLCSbV outcompeted AYVCNV when the transmission was done by MED,while the outcome of competition between the two viruses reversed when the transmission was done by MEAM1.(2)The contrasted transmission of TYLCSbV and AYVCNV between MED and MEAM1 was related to accumulation of the virus in whitefly body After acquisition of TYLCSbV or AYVCNV by the two species of whitefly,we quantified the virus in the whole body and honeydew of the whiteflies by quantitative real-time polymerase chain reaction(qRT-PCR).The data showed that the quantity of TYLCSbV in MED whole body was significantly higher than that in MEAM1,whereas the quantity of AYVCNV in MEAM1 whole body was significantly higher than that in MED;however,the quantities of the two viruses in honeydew between the two species of whitefly were opposite to those in whole body.These results suggested that virus transmission efficiency via B.tabaci is positively correlated with the virus quantity in whitefly body,which is determined by the ability of the species in retaining the virus.The quantification and immunofluorescence results also indicate that the contrasted transmission efficiency of TYLCSbV and AYVCNV between MED and MEAM1 is positively correlated with the accumulation of the viruses in whitefly organs/tissues.(3)Key CP sites that determine the transmission of TYLCSbV and AYVCNV First,through phylogenetic tree and sequence alignment analysis of CP sequences,we predicted that the key amino acids that determine the contrasted transmission specificities among TYLCSbV and AYVCNV might at positions 40 or 147-256.By comparing the quantity of mutant viruses in whitefly whole body and midgut,we found that changing of the 40 th amino acid of TYLCSbV CP did not affect the accumulation of virus in the two whitefly species.When the amino acids of TYLCSbV CP between147-256 was substituted by those of AYVCNV CP,virus accumulation in the two whitefly species was altered to become consistent with that of AYVCNV;that is,the virus accumulation in MEAM1 was significantly higher than that in MED.(4)B.tabaci vesicle-associated membrane protein 2 interact with virus CP and positively regulate virus acquisitionWe used Co-IP coupled with LC-MS/MS assay to identify the whitefly proteins that interacted with the CP of begomovirus.Vesicle-associated membrane protein 2(VAMP2),a protein involved in vesicle-mediated transport pathway,was selected for further analysis.Using Co-IP and GST pull-down assays,we found that VAMP2 could interact with the CPs of three species of begomoviruses including TYLCSbV.When the combination of CP and VAMP2 protein in whitefly body was interfered by VAMP2 antibody,the quantity of virus in the whitefly whole body was significantly decreased.These results suggested that B.tabaci VAMP2 protein can interact with viruses CP and promote virus acquisition.In conclusion,our results showed that the recombinant TYLCSbV and its parent virus AYVCNV exhibit similar infectivities on a range of plant species and cultivars,whereas the transmission specificity of TYLCSbV by different B.tabaci cryptic species is different from AYVCNV but similar to its aother parent virus Tb CSV.This ingenious recombination of pathogenicity and vector specificity from two parent viruses provides TYLCSbV a selective advantage in transmission,and the replacement of different B.tabaci cryptic species populations in the field potentially could have influenced the virus evolution towards an extended transmission.Our study also shows that the transmission efficiencies of TYLCSbV and AYVCNV are positively correlated with the accumulations of the viruses in B.tabaci whole bodies and organs/tissues,and the key CP amino acids that determine their accumulation are between 147-256.In addition,we also identified a conserved vesicle-associated protein,VAMP2,that is conducive to the acquisition of begomoviruses.These findings support the view that recombination can facilitate the adaptation of viruses and provide new insight into the understanding of the selection pressure exerted by vector in the adaptive evolution of viruses.In addition,these findings are of reference value for further analysis of the virus transmission mechanism via vectors and for the development of plant disease prevention and control technology.