Development of tobacco rattle virus-based platform for dual heterologous gene expression and CRISPR/Cas reagent delivery.

A large number of viral delivery systems have been developed for characterizing functional genes and producing heterologous recombinant proteins in plants, and but most of them are unable to co-express two fusion-free foreign proteins in the whole plant for extended periods of time. In this study, we modified tobacco rattle virus (TRV) as a TRVe dual delivery vector, using the strategy of gene substitution. The reconstructed TRVe had the capability to simultaneously produce two fusion-free foreign proteins at the whole level of Nicotiana benthamiana, and maintained the genetic stability for the insert of double foreign genes. Moreover, TRVe allowed systemic expression of two foreign proteins with the total lengths up to ∼900 aa residues. In addition, Cas12a protein and crRNA were delivered by the TRVe expression system for site-directed editing of genomic DNA in N. benthamiana 16c line constitutively expressing green fluorescent protein (GFP). Taker together, the TRV-based delivery system will be a simple and powerful means to rapidly co-express two non-fused foreign proteins at the whole level and facilitate functional genomics studies in plants.

2b ORFs encoded by subgroup IB strains of cucumber mosaic virus induce differential virulence on Nicotiana species.

Cucumber mosaic virus (CMV)-encoded 2b protein from subgroup IA or subgroup II was shown to be a determinant of virulence in many solanaceous hosts. In this study, the virulence of 2b proteins from subgroup IB strains was analysed using four intraspecies hybrid viruses, which were generated by precise replacement of the 2b open reading frame (ORF) in subgroup IA strain Fny-CMV with the 2b ORFs of four subgroup IB strains, Cb7-CMV, PGs-CMV, Rad35-CMV and Na-CMV, generating FCb7(2b)-CMV, FPGs(2b)-CMV, FRad35(2b)-CMV and FNa(2b)-CMV, respectively. FCb7(2b)-CMV was more virulent than Fny-CMV, and was similar in phenotype to its parental virus Cb7-CMV on the three Nicotiana species tested. FNa(2b)-CMV also was virulent on these host species, equivalent to Fny-CMV or Na-CMV. However, FRad35(2b)-CMV only caused mild mosaic or undetectable symptoms on all the host species tested, and was less virulent than Fny-CMV or Rad35-CMV. FPGs(2b)-CMV infected all the host species systemically, and induced either mosaic or barely visible symptoms, demonstrating that the inability of PGs-CMV to infect these three Nicotiana species was not due to its 2b protein. The diverse virulence was shown to be mediated by the 2b proteins rather than the C-terminal overlapping parts of the 2a proteins, and was associated with the level of viral progeny RNA accumulation in systemically infected leaves, but not with the rate of long-distance viral movement in host plants. Through analysis of encapsidation of viral RNAs, there was an apparent correlation between the virulence and the high level of encapsidated RNA 2 in virions of Fny-CMV, FCb7(2b)-CMV and FNa(2b)-CMV.

Quantitative determination of cucumber mosaic virus genome RNAs in virions by real-time reverse transcription-polymerase chain reaction.

A real-time RT-PCR procedure using the green fluorescent dye SYBR Green I was developed for determining the absolute and relative copies of cucumber mosaic virus (CMV) genomic RNAs contained in purified virions. Primers specific to each CMV ORF were designed and selected. Sequences were then amplified with length varying from 61 to 153 bp. Using dilution series of CMV genome RNAs prepared by in vitro transcription as the standard samples, a good linear correlation was observed between their threshold cycle (Ct) values and the logarithms of the initial template amounts. The copies of genomic RNA 1, RNA 2, RNA 3 and the subgenomic RNA 4 in CMV virions were quantified by this method, and the ratios were about 1.00:1.17:3.58:5.81. These results were confirmed by Lab-on-a-chip and northern blot hybridization assays. Our work is the first report concerning the relative amounts of different RNA fragments in CMV virions as a virus with tripartite genome.