Phosphatidic Acid Produced by Phospholipase D Promotes RNA Replication of a Plant RNA Virus
Fig 5
BMV RNA replication requires PLD-derived PA in N. benthamiana protoplasts.
(A) An inhibitor of PLDs impairs BMV RNA replication in a single cell. N. benthamiana protoplasts were inoculated with in vitro transcribed BMV RNA1, RNA2, and RNA3. The inoculated protoplasts were incubated at 20°C for 18 hours in the presence of n-butanol. (B)-(C) Brome mosaic virus replication proteins 1a (B) and 2apol (C) interact with NbPLDβ. Appropriate combinations of capped transcripts were added to BYL. After in vitro translation at 25°C for 2 h, the extract was solubilized and mixed with a 10 μl bed volume of anti-FLAG M2 antibody agarose and incubated further at 4°C for 1 hours. After washing, proteins copurified with FLAG-tagged proteins were analyzed by immunoblotting with appropriate antibodies. (D) The effect of an exogenously supplied PA on the replication of BMV. N. benthamiana protoplasts were inoculated with BMV RNA1, RNA2, and RNA3 transcribed in vitro. The inoculated protoplasts were incubated at 20°C for 18 hours in the presence of progressively increasing concentrations of PA. Total RNA was analyzed by northern blotting using ribonucleotide probes that specifically recognize the 3’-UTR of BMV RNAs. Ethidium bromide-stained rRNA was used as a loading control and is shown below the northern blots. The numbers below the images represent the relative accumulation levels (means ± standard error) of viral RNAs (RNA1 + RNA2 + RNA3 + RNA4) using the Image Gauge program, which were calculated based on three independent experiments.