Plant total rna isolation kit

Total RNA was isolated from tobacco (K326) flowers at stage 12 or different tissues of gerbera by using a plant total RNA isolation kit (Promega, USA) according to the manufacturer’s instructions, followed by reverse transcription by using a PrimeScript RT Reagent Kit with gDNA Eraser (Takara, Japan). SYBR Premix Ex Taq (Japan) was used for real-time quantitative PCR according to the manufacturer’s instructions. The amount of starting cDNA was adjusted according to the Ct value of the endogenous gene (18~20 cycles). Gene expression in the gerbera samples was normalized against the expression of the GhActin (AJ763915) gene as previously described^{29 (link)}; for tobacco, against that of NtActin (AB158612). The primers used are listed in Supplementary Table S2.

The emergence of typical mild mottle symptoms associated with CGMMV infection was regularly monitored up to 60 days post-infiltration (dpi) of the infectious clone. To assess the formation of virus particles in N. benthamiana plants inoculated with different binary constructs (refer to Supplementary Figure S1), leaf extracts were placed on grids and stained with 2% uranyl acetate (pH 4.2). The grids were examined using a transmission electron microscope (TEM, JEOL JEM-1011, Tokyo, Japan).
Leaf samples, whether symptomatic or asymptomatic, were harvested from all inoculated plants at various time intervals to confirm the presence of the virus, predict gene silencing, and assess recombinant gene expression in the plant system through RT-PCR using gene-specific primers. Total plant RNA was isolated using a plant total RNA isolation kit (Promega, Wisconsin, USA), and cDNA was synthesized using reverse transcriptase (Superscript III, Invitrogen, Carlsbad, USA) enzyme with CGMMV 3′ terminal primer (BM489R) following the manufacturer’s protocol (see Supplementary Table S1). The PCR reaction mixture and program were conditioned as per the PCR protocol described above.

Leaf samples from pBP4-infected plants were harvested at 7 days post-inoculation (DPI) to confirm virus infection. The total plant RNA was isolated from symptomatic leaves, using a plant total RNA isolation kit (Promega, Madison, WI, USA) following the manufacturer’s protocol. For the cDNA synthesis, the total RNA was used as a template for a reverse transcriptase reaction with Superscript III (Invitrogen, Waltham, MA, USA), using a CGMMV-specific 3′ terminal primer (BM489R) according to the manufacturer’s protocol. The infectivity of the wild-type virus was detected through RT-PCR using CP gene-specific primers (BM-1178F and BM-1179R). After wild-type CGMMV infection, the deletion mutant was infiltrated in the case of post-infiltration.