Construction of Full-Length Infectious cDNA Clones of Citrus Mosaic Virus RNA1 and RNA2 and Infection of Citrus Seedlings by Agrobacterium-Mediated Vacuum-Infiltration

Abstract

The development of full-length infectious cDNA clones for plant RNA viruses is important for studying their molecular biological characteristics, functional genomics, pathogenesis, and vectorization applications. Citrus mosaic virus (CiMV), a member of the genus Sadwavirus, is of economic importance to the citrus industry and comprises a bipartite, positive-sense, single-stranded RNA genome encapsidated in icosahedral virions. In the present study, full-length cDNA clones of CiMV RNA1 and RNA2 were constructed based on a ternary yeast− Escherichia coli− Agrobacterium tumefaciens shuttle vector, pTY, using transformation-associated recombination (TAR) strategy. Infectivity of cDNA clones of CiMV RNA1 and RNA2 was examined in multiple citrus varieties via Agrobacterium-mediated vacuum-infiltration (AVI) through symptom observation, RT-PCR, and virion detection with an electron microscope. Furthermore, the genome-sized RT-PCR fragments of RNA1 and RNA2 were obtained from symptomatic Jinchengyou ( Citrus grandis) plants infected by the cloned virus (CiMV211). In addition, CiMV211 produced typical symptoms of wild-type CiMV in cowpea ( Vigna angularis) plants inoculated by Agrobacterium-mediated injection. This is the first report of infectious cDNA clones of CiMV, which may lay the foundation for research on the pathogenesis and vectorization of the virus.