Comparison of low molecular weight RNA from PSTVd-infected and non-infected tomato and from Nicotiana benthamiana transformed with a [³⁵S]GFP transgene that is undergoing PTGS. All samples were run on the same gel and blotted onto the same membrane. The lanes containing tomato RNA were hybridised with a full length PSTVd(–) RNA transcript. The uninfected control contains an unspecific cross hybridisation signal detected under these low stringency conditions. The lane containing N.benthamiana RNA was hybridised with a full length GFP(–) RNA transcript. Markers were ³²P-labelled mGFP5 RNA, transcribed in vitro and digested with RNase T1. Sizes are indicated to the left of the gel. PTGS-specific RNA is indicated by an arrow to the right of the gel.

Northern analysis for detection of PTGS-typical small RNAs. Samples were hybridised with a full length PSTVd(–) RNA transcript. (A) Lane 1, healthy control, contains an unspecific cross-hybridisation signal detected under these low stringency conditions (compare with Fig. 1); lane 2, 23mer DNA marker (lower arrowhead); lane 3, 25mer DNA marker (upper arrowhead); lane 4, crude extract of tomato plants inoculated with PSTVd isolate KF440-2; lane 5, LiCl-soluble fraction of the extract shown in lane 4; lane M, mixture of synthetic RNA transcripts, consisting prominently of lengths between 27 and 39 nt. (B) A close-up of the relevant section of lanes 2–5 in different exposure times, detailing the signals originating from the small RNAs. The positions corresponding to 22–25 nt are indicated by arrowheads.

Northern analysis for detection of PTGS-typical small RNAs. Tomato plants were inoculated with PSTVd isolates KF440-2 (440-2), KF5 and RG1, and leaves were harvested at days 30 and 62 post-inoculation to prepare extracts that were fractionated by LiCl. Hybridisation probes were as described under each panel. H, healthy control; M, transcription marker. The position of the small RNAs is indicated by an arrowhead. The cross hybridisation signal obtained with the sense probe is also indicated (upper arrow).

Northern analysis for detection of PTGS-typical small RNAs from LiCl-fractionated extracts from leaves of tomato plants infected with three different PSTVd isolates. The hybridisation probe was the sense probe (Fig. 3, right panel). The positions of the two relevant signals are indicated with arrowheads.