The effect PSTVd R+ and PSTVd var1 mutations on the interaction of VirP1 with PSTVd RNA. (A) Primary and secondary structure of the mutated area of PSTVd. The nucleotides that vary from the wild-type sequence are in lower case characters. (B) Electrophoretic mobility shift assay (EMSA). The picture shown here is representative of four such experiments. Radiolabeled WTR34Taq, R+R34Taq, and var1R34Taq RNA transcripts were incubated with (+) and without (−) purified his-tagged VirP1. The experiment is duplicated with boiled and snap-cooled RNA transcripts. (C) The three-hybrid test. The WTR34Taq, R+R34Taq, and var1R34Taq RNAs are contained in the RNA hybrids MS2-R34WT, MS2-R34R+, and MS2-R34var1, respectively. Each one of those RNA hybrids, and MS2 as well, were tested with the protein hybrid AD-VirP1ct. In each case, two independent double transformants of the S. cerevisiae strain L40-coat were assayed for β-gal activity.
