Blocking tombusvirus replication through the antiviral functions of DDX17-like RH30 DEAD-box helicase
Fig 9
Confocal microscopy shows co-localization of RH30 with the viral repRNAs in whole plants infected with CNV.
(A-B) Most of RH30 is re-targeted into the replication compartment where RNA synthesis takes place. The viral (-)repRNA and (+)repRNA carried six copies of the MS2 phage RNA hairpin (MS2hp) recognized by MS2 CP fused with RFP. The replication compartment was also marked by the BFP-tagged p33 replication protein in N. benthamiana. Note that RFP-MS2CP contains a weak nuclear localization, therefore this protein ends up in the nucleus in the absence of target RNAs in the cytosol. 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, GFP-RH30, RFP-MS2CP, repRNA(-)MS2hp or repRNA(+)MS2hp and the helper virus CNV20KSTOP gRNA. The repRNA(+)MS2hp consists of the repRNA(+) carrying six copies of cis-MS2 hairpin, which can be bound by RFP-MS2CP to show the subcellular localization of repRNA(+). The repRNA(-)MS2hp consists of repRNA(+) carrying six copies of trans-MS2 hairpin, which can only be recognized by RFP-MS2CP when viral RNA replication produces the complimentary strand repRNA(-) by the helper virus CNV20KSTOP. The absence of transient expression of GFP-RH30, repRNA(+)/(-)MS2hp or CNV20KSTOP were used as controls. The agro-infiltrated leaves were collected for confocal microscopy imaging 3.5 days post infiltration. Scale bars represent 10 μm. Each experiment was repeated.