Fluorescent Localization Within The Vector Whitefly And Capsid Component Cloning And Expression Of CCYV And CYSDV

Virus diseases are the most disastrous type of plant diseases,known as “the cancer” of agricultural crop production causing severe losses due to lack of effective control measures.Management of plant virus diseases relies largely on blocking the key point during the process of virus transmission through the control of vector insects.Therefore,studies on virus-vector interactions will contribute not only to theoretical understanding of virus-vector-plant coevolution,behavioral and ecological adaptation,but also to shedding light on implementation of effective strategies for control of plant viruses and vector insects.The sweetpotato whitefly,Bemisia tabaci,is one of the most efficient vectors for plant virus transmission.It transmits 212 plant viruses in persistent,semipersistent and nonpersistent manners.CCYV(Cucurbit chlorotic yellows virus)and CYSDV(Cucurbit yellow stunting disorder virus),both transmitted semipersistently by B.tabaci,are causing heavy economic losses to cucurbit crops in many countries in the world.CCYV and CYSDV,reported in early 2010's and in 1990's,respectively,have just been paid attention to in recent years by scientists,and intensive studies on transmission mechanisms of the two viruses have not yet been reported for the time-being.Due to 75% in genetic similarity of CCYV and CYSDV,in the present research,we used the two virus species as materials to comparatively study mechanisms of virus-plant interactions in attempt to lay the theoretical basis for effective control of insect vectors and plant viruses.The contents and results of the present study are summarized as following:I.Establishment of CCYV and CYSDV system through whitefly transmissionA stable virus system is the basis for all virus researches.Vectors harboring semipersistent viruses can easily lose the viruses resulting in low transmission efficiency,so it is very necessary to develop infectious clone to facilitate studies on virus genomic functions,vector construction for virus over-expression and virus-induced gene silencing.In the present study,based on constructed full-length c DNA clones of CCYV(RNA1 and RNA2)fused to the T7 RNA polymerase promoter and the cauliflower mosaic virus 35 S promoter,agroinoculation with the infectious c DNA clones of CCYV resulted in systemic infection in the host plants of C.sativus.Thirty days after inoculation,B.tabaci adults were fed with the infected plants and then transferred to the healthy plants for virus transmission.The result showed that CCYV infectious c DNA clone system with agroinoculation can be efficiently transmitted by the whiteflies and be expressed stably in the plants.This provides basis for researches on interactions of viruses-vector insects,viruses-plants,or virus transmission mechanisms.CCYV and CYSDV are phloem-restricted viruses with low concentrations in the plants,so it is hopeful to obtain the virus system through transmission by insects from the diets containing extracted and concentrated virons.Whiteflies fed with CCYV or CYSDV particles through membrane-feeding were transferred to the healthy plants,PCR detection results indicated that the transmissions were not as we expected.Further experiments are needed to find out reasons or factors influencing transmission efficiency based on membrane-feeding.II.Localization of virus retention sites within the vector whitefly for CCYV and CYSDVVirus retention time,retention sites and circulative types within the vector determine the modes of virus transmission.So virus retention site is one of important aspects for determination of virus transmission types.Semipersistent transmission mode of CCYV and CYSDV,both transmitted by B.tabaci,are speculated mainly based on the transmission manner of other viruses within same genus as well as on some transmission characteristics of the two viruses,but direct evidence is lacking,especially information about retention sites within the vector.In the present study,whitefly adults were sequentially fed with artificial diets containing antibodies of anti-CCYV/CYSDV-CP and fluorescent Alexa Flour 488,then heads and foreguts of the whiteflies were dissected and observed under a laser confocal microscope.Merged image from two channels of the laser confocal microscope indicated that the foregut is the retention site for both CCYV and CYSDV.This first report on the retention site of CCYV and CYSDV provides the direct evidence for the semipersistent transmission mode of both viruses,and lays basis for studies of protein-protein interactions between vectors and viruses and for development of novel control measures targeting vector retention sites.III.Comparative analysis of male and female whitefly vectors on CYSDV transmission capacityLittle information is available on the role of female or male vectors in virus transmission.In this study,whitefly males and females were compared on their capacity for transmitting CYSDV virus by observation of fluorescent antibody-labeled viruses within the vector foregut under the laser confocal microscope and by transmission experiments.Whitefly adults were sequentially fed with artificial diets containing antibodies of anti-CYSDV-CP and fluorescent Alexa Flour 488,then heads and foreguts of the whiteflies were dissected and observed under a laser confocal microscope.The results showed that much more male individuals(25%~28%)were found to retain CYSDV than females(0.0%~2.4%).Similar results were obtained in transmission experiments.IV.Cloning and expression of genes of capsid component of CCYV and CYSDVMany proteins get involved in the process of virus transmission,for example,coat protein(CP)and minor coat protein(CPm)of CCYV and CYSDV are believed to play crucial roles in virus transmission.Here we cloned and expressed CP and CPm genes of CCYV and CYSDV so as to lay the basis for future studies on protein-protein interactions.The amplified CP and CPm genes were integrated into Gateway cloning system through p ENTR-D-TOPO reaction.Two types of recombinant vectors with FLAG-tag at C-and N-terminals respectively were agroinoculated to Nicotiana benthamiana.CP of CCYV or CYSDV with FLAG-tag at either terminal was expressed in the plants,but CPm was not detectable probably due to low expression or instability.