Infectious Cloning Of Cassava Common Mosaic Virus And Construction And Analysis Of Viral Expression Vecto

Cassava(Manihot esculenta Crantz),as the sixth largest food crop in the world is widely grown in South China.With increasing yields and technological innovations,cassava is also an industrial and biofuel crop for production of industrial starch and ethanol.However,Cassava common mosaic virus(Cs CMV,genus Potexvirus,family Alphaflexiviridae)is an emerging threat to cassava production in China.The generation of infectious viral clones have become an essential and powerful tool for understanding the pathogenesis of RNA viruses,and could help to unravel the complex process of viral infection and plant-virus-vector interactions.Here,an agroinfectioncompatible infectious Cs CMV c DNA clone designated p Cs CMV-HN were successfully constructed using the one-step In-Fusion Cloning based on the complete genome sequence of Cs CMV Hainan-DZ islolate(Cs CMV-HN).Furthermore,three fluorescent protein-tagged infectious c DNA clones of Cs CMV mosaic virus were developed to facilitate the tracking of virus infection.The main findings are as follows:(1)Construction of an agroinfectious full-length c DNA clone of Cs CMVThe full-length genomic complementary DNA(c DNA)of Cs CMV-HN was obtained by PCR and then cloned between the Ca MV 35 S promoter(35S P)and the poly(A)signal of T-DNA binary vector p Green II-35 S to generate p Cs CMV-HN using In-Fusion seamless cloning method.To test the infectivity of p Cs CMV-HN,the model plant Nicotiana benthamiana and cassava plants were infiltrated with agrobacterium-carrying p Cs CMV-HN.The result showed that the p Cs CMV-HN was infectious and induced mild mosaic symptoms of dark and light green patches in systemically infected cassava leaves at 10-14 days post inoculation.On day 45,The Cs CMV was detected in symptomatic plants agroinoculated with p Cs CMV-HN but not in mock-inoculated plants by RTPCR.Through RT-PCR detection and disease observation statistics,the infection efficiency of p Cs CMV-HN in the cuttings of Nicotiana benthamiana and cassava plants reached 100%.(2)Construction of two Cs CMV vector with a duplicated Cs CMV CP subgenomic m RNA promoter(SGP)for GFP expression in Nicotiana benthamiana and cassava plantsTwo full-length Cs CMV-GFP vectors,each carrying a duplicated Cs CMV CP subgenomic m RNA promoter(SGP)were designed.The GFP gene was positioned the upstream of CP gene.In p Cs CMV-GFP,the duplicated SGP included 90 bp upstream of the CP start codon,whereas in p Cs CMV-GFP-m1 it contained 171 bp upstream of the CP start codon.The Nicotiana benthamiana and cassava plants agroinfiltrated with p Cs CMV-GFP-m1 were observed more GFP fluorescence than those agroinfiltrated with p Cs CMV-GFP.GFP expression was confirmed by RT-PCR and Western blot analyses.(3)Construction of a Cs CMV vector including CP fusions with the Foot-And-Mouth Disease Virus(FMDV)2A catalytic peptide for GFP expression in Nicotiana benthamianaIn order to further improve the stability of the Cs CMV expression vector,we designed the p Cs CMV-GFP-2A,which was constructed based on GFP as a fusion with the CP via a FMDV 2A peptide.Following agroinfiltration,at 7-10 days the non-inoculated leaves can be observed to have GFP fluorescence under the irradiation of UV.Then,the GFP fluorescence intensity reaches the strongest on the 25 day.Furthermore,the GFP expression was detected in systemic leaves of p Cs CMV-GFP-2A-infected Nicotiana benthamiana but not in those of p Cs CMV-GFP-2A infected cassava plants by RT-PCR and western blot.