DCL-dependent generation of 21 nt sRNAs, and AGO involvement in 32 nt sRNA generation.

<p><b>A</b>. <i>Copia3-LTR</i> 21 nt sRNAs in <i>PiDcl1</i> (D1t7, D1t8) and <i>PiRnh5</i> (D2t1) silenced lines. An upward mobility shift to approximately 24/25 nt was observed for the most silenced <i>PiDcl1</i> line, D1t8. <b>B</b>. <i>PITG_22969</i> (CRN) 21 nt sRNAs are abolished upon silencing of <i>PiDcl1</i> (D1t7, D1t8) or <i>PiRnh5</i> (D2t1). <b>C</b>. <i>Crypton6</i> 32 nt sRNAs in <i>PiDcl1</i> (D1t7, D1t8) and <i>PiRnh5</i> (D2t1) silenced lines. Silencing of these genes had no effect on the accumulation of 32 nt sRNAs. <b>D</b>. <i>PiAvrblb1</i> 32 nt sRNAs in <i>PiDcl1</i> (D1t7, D1t8) and <i>PiRnh5</i> (D2t1) silenced lines. Silencing of these genes had no effect on the accumulation of 32 nt sRNAs. <b>E</b> and <b>F</b>. <i>PiAvrblb1</i> 32 nt sRNAs in <i>PiAgo1</i>, <i>4</i>, or <i>5</i> (A1b, A4a, A5c) silenced lines. The accumulation of 32 nt sRNAs is strongly reduced in <i>PiAgo4</i> or <i>5</i> lines, while over-accumulation occurs for <i>PiAgo1</i>. Silenced lines used were <i>PiDcl1</i> (Dicer-like; D1t8, t7), <i>PiRnh5</i> (Dicer-like helicase; D2t1), <i>PiAgo1</i> (Argonaute 1; A1b) <i>PiAgo4</i> (Argonaute 4; A4a), and <i>PiAgo5</i> (Argonaute 5; A5c). Loading controls (U4 spliceosomal RNA) are shown below each autoradiograph.