Skip to main content
. 2019 May 28;15(5):e1007771. doi: 10.1371/journal.ppat.1007771

Fig 3. Confocal microscopy shows the retargeting of the mostly nuclear RH30 into the large replication compartment in plant protoplasts and whole plants infected with CNV.

Fig 3

(A) Most of RH30 is re-targeted into the replication compartment marked by the BFP-tagged p33 replication protein (pointed by arrows) in N. benthamiana protoplasts. Second panel: in the absence of viral components, GFP-tagged RH30 is mostly present in the nucleus, as marked by the histone protein (RFP-H2B). Third panel: The re-targeted GFP-RH30 is present in the viral replication compartment, marked by p33-BFP replication protein and RFP-SKL peroxisomal matrix marker. Arrows point at the viral replication compartment. Fourth panel: RH30 is not co-localized to the peroxisomes in the absence of tombusvirus replication. Fifth panel: The re-targeted GFP-RH30 is partially co-localized with the ER marker within the viral replication compartment, marked by p33-BFP replication protein. The leaves of N. benthamiana plants (transgenic plants expressing nucleus marker RFP-H2B or ER marker RFP-ER) were agro-infiltrated to express p33-BFP, GFP-RH30, and CNV20KSTOP gRNA. Leaves without the expression of p33-BFP and CNV20KSTOP gRNA were used as controls. The agro-infiltrated leaves were collected to isolate protoplasts for confocal imaging 2.5 days post agro-infiltration. Scale bars represent 10 μm. (B) Confocal microscopy images show co-localization of TBSV p33-BFP or CIRV p36-BFP replication proteins and the GFP-RH30 in planta. The large replication compartment was visualized via expression of TBSV p33-BFP or CIRV p36-BFP. Expression of the above proteins from the 35S promoter was done after co-agroinfiltration into N. benthamiana leaves. The leaves of N. benthamiana plants were agro-infiltrated to express TBSV p33-BFP or the CIRV p36-BFP, GFP-RH30, and CNV20KSTOP or CIRV gRNAs. Leaves without the expression of p33-BFP or p36-BFP and the viral RNAs were used as controls. The agro-infiltrated leaves were collected for confocal imaging 2.5 days post agro-infiltration. Scale bars represent 10 μm. Each experiment was repeated.