FIG. 7.
ZYMV HCFRNK binds an siRNA-like duplex with a much greater affinity than ZYMV HCFINK. (A) A dilution series of N. benthamiana leaf extract expressing ZYMV HCFRNK was assayed in an EMSA binding reaction with 4.76 nM siRNA-like duplex. A representative EMSA gel is shown (inset). Samples were diluted in EV-infiltrated leaf extracts to the HC-Pro concentrations indicated. The Hill plot (line graph) was derived from the averages of four such experiments. Signals were quantified from phosphorimager data using ImageJ software. The Hill plot and biochemical values (top left) were generated using Origin 7.5 software. Bars show standard deviations for the four replicates. n, apparent Hill coefficient. (B) Electromobility shift competition assay. γ-32P-radiolabeled siRNA-like duplex was premixed to 4.54 nM with different concentrations of nonradioactive phosphorylated competitor duplex (cold) and incubated with N. benthamiana extracts expressing HA-tagged ZYMV HCFRNK or HCFINK. Competition assays were conducted with undiluted (2 μM) HCFINK-expressing extract (B) and at a 50-fold dilution (in uninoculated leaf extract) for HCFRNK (60 nM) (C). Extract from noninoculated leaves (neg) and RNA duplex without extract (dup) were run as controls. § marks a putative plant endogenous duplex-binding protein sometimes seen in EMSA.