Construction of an agroinfectious clone of bean rugose mosaic virus using Gibson Assembly.
Construction of agroinfectious viral clones usually requires many steps of cloning and sub-cloning and also a binary vector, which makes the process laborious, time-consuming, and frequently susceptible to some degree of plasmid instability. Nowadays, novel methods have been applied to the assembly of infectious viral clones, and here we have applied isothermal, single-step Gibson Assembly (GA) to construct an agroinfectious clone of Bean rugose mosaic virus (BRMV) using a small binary vector. The procedure has drastically reduced the cloning steps, and BRMV could be recovered from agroinfiltrated common bean twenty days after inoculation, indicating that the infectious clone could spread in the plant tissues and efficiently generate a systemic infection. The virus was also recovered from leaves of common bean and soybean cultivars mechanically inoculated with infectious clone two weeks after inoculation, confirming the efficiency of GA cloning procedure to produce the first BRMV agroinfectious clone to bean and soybean.
Universidade Estadual de Maringá, 5790 Av. Colombo, Maringá, PR, Brazil.
Taise
T
Bijora
Universidade de Brasília, Brasília, DF, Brazil.
Rosana
R
Blawid
Universidade Estadual de Maringá, 5790 Av. Colombo, Maringá, PR, Brazil.
Danielle K T
DKT
Costa
Embrapa Recursos Genéticos e Biotecnologia, Brasília, DF, Brazil.
Francisco J L
FJL
Aragão
Universidade Estadual de Maringá, 5790 Av. Colombo, Maringá, PR, Brazil. ersouto@uem.br.|Department of Agronomy, Center of Agricultural Sciences, Universidade Estadual de Maringá, Maringá, PR, 87020-900, Brazil. ersouto@uem.br.
Eliezer R
ER
Souto
Universidade de Brasília, Brasília, DF, Brazil. tatsuya@unb.br.|Department of Cellular Biology, Biological Sciences institute, Universidade de Brasília, Brasília, DF, 70910-900, Brazil. tatsuya@unb.br.
Tatsuya
T
Nagata
D016960:Agrobacterium tumefaciens / Q000235:genetics*; D003001:Cloning, Molecular / Q000379:methods*; D017798:Comovirus / Q000235:genetics*; D018076:DNA, Complementary / Q000235:genetics; D007887:Fabaceae / Q000821:virology; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics; D016679:Genome, Viral; D010802:Phylogeny; D010935:Plant Diseases / Q000821:virology; D018515:Plant Leaves / Q000821:virology; D010957:Plasmids; D012367:RNA, Viral / Q000235:genetics; D013025:Glycine max / Q000821:virology; D014170:Transformation, Genetic
D018076:DNA, Complementary; D012367:RNA, Viral
Bean rugose mosaic virus; Comovirus; Gibson assembly; Infectious clone; Isothermal reaction