Fig. 4: Stable silencing after mating is associated with heritable silencing signals that can act in trans.

a Schematic showing piRNA-mediated activation of a positive feedback for production of secondary small RNAs (2° RNAs) upon mating-induced silencing. The secondary small RNAs made at one gene could potentially act on other homologous genes (trans silencing). b Animals expressing T were mated with iT animals that remained silenced for many generations (iT gen. number), and cross progeny were scored. The combined data from each cross is shown in Supplementary Fig. 7a. c T animals were mated with non-transgenic or hemizygous iT animals and cross progeny that inherited only T were scored. Schematic: parental presence of iT can be sufficient to silence T inherited through the other gamete, indicating the inheritance of a separable silencing signal (gray filling). d Requirement of hrde-1 for the activity of the silencing signal was tested by parental, maternal, and/or zygotic loss of HRDE-1. e Tcherry animals silenced for more than five generations upon initiation of mating-induced silencing were designated as iTcherry here. Males expressing Tcherry and Pgtbp-1::gtbp-1::gfp marker (gfp) were mated with iTcherry or Tcherry hermaphrodites overexpressing gpr-1 (gpr-1 oe). Expression of paternally inherited Tcherry in the germline was scored in cross progeny. f Silencing of T by the separable silencing signal or in trans by iT was assessed across generations. The remaining results of this cross showing the effect of trans silencing are shown in Supplementary Fig. 7c. g Males that express homologous (gfp) or non-homologous (mCherry^var, a synonymous mCherry variant or rfp) sequences fused to endogenous gtbp-1 expressed ubiquitously were mated with non-transgenic or iT hermaphrodites and fluorescence of GTBP-1::GFP, GTBP-1::mCherry^var or GTBP-1::RFP was quantified in cross progeny (left). Percentage of nucleotide identity and length of the longest continuous match shared by different fluorescent protein coding sequences in pairwise alignment using the Needleman–Wunsch algorithm (penalties for mismatch = 0.1, gap = −1, and gap extension = −0.2) with the mCherry or gfp sequence of T as reference are shown (right top). Schematic summary of homology-dependent trans silencing (right bottom). See Supplementary Data files 1–6 for each pairwise sequence alignment. Scoring of silencing (b–f) is as in Fig. 1c. Chromosomes with a dpy marker (blue font) and number of animals scored (n) are indicated. Asterisks indicate P < 0.05 using χ² test (b–d), Wilson’s estimates for proportions (e) or two-sided Student’s t-test (g), and ns indicates P > 0.05 using the same tests. Also see Supplementary Fig. 7 and ‘Genetic Crosses’ under Methods. Source data are provided as a Source Data file.