Construction of stable infectious full-length and eGFP-tagged cDNA clones of Mirabilis crinkle mosaic virus via In-Fusion cloning

Mirabilis crinkle mosaic virus (MiCMV) was tentatively classified as a new member of the genus Potyvirus in the family Potyviridae in 2019. However, it was considered to be basella rugose mosaic virus based on the sequence similarity of the coat protein (CP) region. In this study, infectious MiCMV cDNA clones under the control of the 35S promoter were constructed with an In-Fusion cloning method. Systemically infected leaves of Mirabilis jalapa and Nicotiana benthamiana plants inoculated with pMiCMV and pMiCMV-NIb/eGFP had mosaic symptoms by 5 dpi. Infections were confirmed by a western blot analysis, electron microscopy, RT-PCR, and the inoculation of N. benthamiana seedlings with progeny virions. Systemic infections were not observed after Nicotiana glutinosa leaves were similarly inoculated, with eGFP fluorescence detected only in the inoculated leaves. Interestingly, the symptoms induced by pMiCMV and pMiCMV-NIb/eGFP were not similar to those caused by the wild-type MiCMV in Basella rubra plants. Moreover, RT-PCR analyses of B. rubra plants with virus-specific primers (MicpF and MicpR) indicated that a non-target fragment corresponding to the MiCMV CP coding region was amplified. This is the first report of the construction of a biologically active, full-length cDNA copy of the MiCMV RNA genome.

Keywords: In-Fusion; Infectious clone; Mirabilis crinkle mosaic virus; Potyvirus.