FIG 6.

BMV RNA1 accumulated to higher levels than BMV RNA2 early in infection. (A) Minus-strand BMV RNA1 accumulated to higher levels than did RNA2 in wheat seedlings. Wheat seedlings were inoculated with 50 μg/ml of B1 or the B2.3/4 virions or an equal mixture of the two. Total RNAs were extracted with TRIzol reagent and quantified as described in Materials and Methods. Comparable amounts of input were confirmed by photospectrometry and gel electrophoresis. Abundances of the BMV RNAs were quantified using RT-PCR from three independent samples of the total RNA extracted from wheat seedlings inoculated with either B1, B2.3/4, or an equal molar ratio of the two sets of BMV virions. Total RNAs were harvested 24 h postinoculation (hpi). The range for one standard deviation is shown above the bars, as is the P value. The input RNA and performance of the primers against known amounts of BMV DNA were adjusted to allow accurate quantification of the BMV RNAs. (B) Plus-strand RNA1 replicates to a higher abundance than RNA2 after inoculation. The Northern blots were performed with total RNAs extracted from wheat seedlings after the number of hours postinoculation shown above the gel. rRNA served as a loading control in the RNA gel for Northern blot analysis. An image of the ethidium bromide-stained gels used for the Northern blot analysis is shown below the autoradiograms. The full-length BMV RNAs were identified by RNAs extracted from WT BMV virions. The bands highlighted with asterisks likely correspond to prematurely terminated RNA3 (37). (C) Quantification of the accumulation of plus-strand BMV RNA1 and RNA2 over time. The amount of RNA present was normalized to the amount present in the inoculum. The same results were observed in two independent experiments.