The effect of the 5′UTR Py-rich stretch motif on MYVB promoter activity and MYVB replication. (A) Fluorometric GUS activity analysis of the mutant promoter construct in N. benthamiana leaves. pINT121 and pBINGUS were used as positive and negative controls, respectively, and GUS activity of pINT121 was arbitrarily assigned as 100%. The number of replicates of each sample is indicated in parentheses. (B) Southern blot of total nucleic acid extracts of tobacco leaf co-inoculated with infectious clones of MYVV plus MYVB (lanes 1 and 3), or MYVV plus MYVBΔUTR (lanes 2 and 4). Blots were probed with either the CP gene sequence of MYVV (lanes 1 and 2) or the full-length MYVB sequence (lanes 3 and 4). Photographs of the ethidium bromide-stained gels are shown below the blot as an indication of loading control. (C) Densitometric quantification of band intensities. The value obtained from MYVV plus MYVB was arbitrarily assigned as 100%. Standard deviation values are based on three independent experiments.