Construction And Application Of Agrobacterium-mediated Foxtail Mosaic Virus Infectious CDNA Clones Vector

Foxtail mosaic virus (FoMV) has a broad host range, infecting56species of the Poaceae and atleast35dicot species including rice, wheat, and corn and its genome structure resembles that ofPotato virus X (PVX), the type species of the genus Potexvirus, and the gene functions arepresumed to be similar as well. Over the past few years, PVX was used to express a largenumber of foreign proteins and developed as virus-induced gene silencing vector in theseedding of tobacco, tomato and potato plants by Agrobacterium infiltration-mediated transientexpression. Vector using FoMV have been reported less until now.In this study, the complete sequence of FoMV cDNAs was cloned into vector pCB301which carrying duplicated35S promoter. Get the right recombinant plasmid and transformedinto Agrobacterium GV3101, it was then infiltrated into Nicotiana benthamiana andN.benthamiana RDR6. Observe the symptoms and determine the activity ofAgrobacterium-mediated infectious clone. The virus inoculated to Hordeum vulgare viasap-mechaniclly in four varieties. At14post-inoculation days, there are symptoms similar aslight mosaic and line pattern greenness showed on maturing leaves. Constructe theAgrobacterium-mediated infectious clone successfully by the molecular detetion results withthe viral RNA were detected via TBE electrophoresis result of the total RNA and RT-PCR. Theresult showed that the virus accumulation in N. benthamiana was lower than N.benthamianaRDR6; the pCB301-FoMV-wt inoculated via agroinfection four varieties including H. vulgareGolden Promise, Ingrid, Movex, Pallas, but no viral RNA was detected and symptoms showedon the plant. In corresponding, the mechanically inoculated test, H. vulgare Ingrid, Movex,Pallas was infected successfully except H. vulgare Golden Promise.On the basis of obtaining Agrobacterium infection clone named pCB301-FoMV-wt. Usingmolecular biology methods, to renovate and build new clone containing2FoMV CPsubgenomic promoter.The2CP subgenomic promoter forward adjacent direct repeat, and themultiple cloning sites were inserted between2CP sub-genomic promoter. The downstream ofrestriction sites contains the complete CP gene and its promoter, and this recombinant clone asa candidate VIGS vector (pCB301-FoMV-VIGS). Followed, the GFP ORF fragment andendogenous PDS fragment from H. vulgare Movex were inserted into the pCB301-FoMV-VIGS,and were named pCB301-FoMV-PDS, pCB301-FoMV-GFP. The clone pCB301-FoMV-VIGS, pCB301-FoMV-PDS, pCB301-FoMV-GFP recombinant plasmids were transformed intoAgrobacterium GV3101by electroporation, followed inoculated to the N.benthamiana RDR6by infiltration inoculation, but the plant did not show symptoms, and molecular test resultsshowed that the infection failed also.Via inoculating H. vulgare Movex with extracts from N.benthamiana leaves infected withdifferent kinds of recombinant virus, we found that three recombinant virus inoculated leavesshowed similar phenotypes to pCB301-FoMV-wt inoculated leaves, only in which we detectedviral genomic RNA using RT-PCR, and there is no GFP detected in leaves inoculated with threerecombinant virus under UV light, the phenomenon reminded us that there is no gene silencinghappened in these inoculated leaves. We concluded that virus got gene recombination ininoculated leaves is responsible for the loss of inserted-fragment， which may causereversemutation of the three recombinant virus. Also, the length of inserted-fragment and thechoice of MCS may afferts the result.