FIG. 3.

Autoradiograms of Northern blot hybridizations of RNA samples isolated from both healthy and PLMVd-infected peach leaves. RNA samples were fractionated on either agarose (A to C) or polyacrylamide (D and E) gels and then blotted onto nylon filters. The polarity of the PLMVd riboprobe is indicated by the symbol (+) and (−) at the top of the panel. (A) RNA samples were isolated by various extraction procedures: RNeasy Plant mini kit (Qiagen) in the presence of EDTA (lanes 3 to 5); Tris-EDTA isolation (lanes 6 and 7); RNeasy Plant mini kit in the absence of additional EDTA (lane 8); and the PEG precipitation procedure (lanes 9 to 12). In lanes 1 and 2, nonradioactive synthetic PLMVd transcripts of plus (761 and 588 nt) and minus (745 and 462 nt) polarity, respectively, were loaded as controls. Lane 3 contains a sample of healthy GF-305 peach cultivar; lanes 4, 6, 8, 9, 11, and 12 contain RNA samples isolated from a PLMVd-infected RedGold peach cultivar. The samples in lanes 11 and 12 were subjected to either DNase treatment or alkaline hydrolysis prior electrophoresis. Lanes 5 and 7 contain to samples from a PLMVd-infected Redhaven cultivar, while lane 10 contains a sample from the leaves of the Agua cultivar. (B and C) The same filter following hybridization with either the plus- or minus-polarity riboprobe. All samples were isolated by the PEG precipitation procedure. Lanes 1 and 2 contain samples isolated from different leaves of a Redhaven peach. Lane 3 contains an RNA sample from a healthy Bailey cultivar. Lanes 4 and 5 contain RNA samples of PLMVd-infected Redhaven and Siberian C cultivars. (D and E) Filter following PAGE and blotting that was probed with both the plus- and minus-polarity riboprobes, respectively. In panel D, lanes 1 and 2 contain synthetic circular and linear PLMVd controls, while lanes 3 to 5 (which correspond to lanes 1 to 3 of panel E) contain samples from PLMVd-infected Redgold, Hardired, and Siberian C cultivars and lanes 6 and 7 (which correspond to lanes 4 and 5 of panel E) contain samples from healthy GF-305 and Bailey cultivars. Adjacent to the gel, the positions of several PLMVd transcripts are indicated as size references including both the circular (338C) and linear (338L) strands and the mixture of circular and linear molecules (PLMVd). The position of the unknown ∼1-kb species is also indicated in panels A and B.