Co-opting the fermentation pathway for tombusvirus replication: Compartmentalization of cellular metabolic pathways for rapid ATP generation
Fig 12
Knockdown of the cellular Pdc1 fermentation enzyme inhibits ATP accumulation within the tombusvirus replication compartment in N. benthamiana.
(A) A scheme of the FRET-based detection of cellular ATP within the replication compartment. The enhanced ATP biosensor, ATeamYEMK was fused to TBSV p33 replication protein. (B) Knock-down of Pdc1 mRNA level by VIGS in N. benthamiana was done using a TRV vector. Twelve days latter, expression of p33-ATeamYEMK was done in upper N. benthamiana leaves by agroinfiltration. The YFP signal was generated by mVenus in p33-ATeamYEMK via FRET 1.5 days after agro-infiltration. The FRET signal ratio is shown in the right panels. The more intense FRET signals are white and red (between 0.5 to 1.0 ratio), whereas the low FRET signals (0.1 and below) are light blue and dark blue. We also show the average quantitative FRET values (obtained with ImageJ) for 10–20 samples on the graph. Note that N. benthamiana plants were mock-inoculated. (C-D) Comparable experiments with p33-ATeamYEMK in the Pdc1 knockdown N. benthamiana plants infected with the peroxisomal TBSV and CNV tombusviruses. See further details in panel B.