Transgenerational inheritance of an acquired small RNA-based antiviral response in C. elegans

Abstract

Induced expression of the Flock House virus in the soma of C. elegans results in the RNAi-dependent production of virus-derived, small-interfering RNAs (viRNAs), which in turn silence the viral genome. We show here that the viRNA-mediated viral silencing effect is transmitted in a non-Mendelian manner to many ensuing generations. We show that the viral silencing agents, viRNAs, are transgenerationally transmitted in a template-independent manner and work in trans to silence viral genomes present in animals that are deficient in producing their own viRNAs. These results provide evidence for the transgenerational inheritance of an acquired trait, induced by the exposure of animals to a specific, biologically relevant physiological challenge. The ability to inherit such extragenic information may provide adaptive benefits to an animal.

Figure 1. Inheritance of an antiviral RNAi response

(A) Schematic presentation of the FHV genome and the FR1gfp transgene used in this study (Lu et al., 2009). The RNA2 transcript that produces the capsid is not shown. (B) Replication of FHV was monitored by recording the expression of GFP in rde-1 and rde-4 mutant worms containing the FR1gfp transgene 48 hours after heat-shock induction of viral replication. The animals were RNAi-deficient for more than 5 generations. (C) A scheme depicting the cross that tests the requirement for rde-1 and rde-4 in generating virus-silencing viRNAs. Animals shown in the F2 and later generation were all selected to contain the FR1gfp transgene as assessed by the co-injection marker rol-6. rde-1(−/−) animals were scored in the F2 generation for whether they express or do not express GFP after heat-shock induction. Since rde-1(−/−) animals (or rde-4 mutant animals) are not capable of initiating an RNAi response, the FR1gfp; rde-1(−/−) F2 generation is either not able to protect itself against viral propagation (hence heat-shocked animals would be GFP(+) as indicated schematically with a green animal); or the F2 generation inherits antiviral viRNAs that were produced by earlier generations and can therefore protect itself against the virus (hence heat-shocked animals would be GFP(−) as indicated schematically with a dark animal). As shown in panel D, the latter is the case. (D) DIC and GFP images of heat-shocked rde-1(−/−) and rde-4(−/−) worms that have been homozygous mutant for rde-1 or rde-4 for several generations (as indicated in panel C) and contain FR1gfp as assessed by the rol-6 transgene marker. The rde-1 and rde-4 genotypes were assessed through a linked unc marker.

Fig.2. Genetic analysis of the transgenerational inheritance of an antiviral response

Animals of all the genotypes schematically shown in this figure were tested for whether they express GFP after heat shocking adult animals (see Experimental Procedures), as a measure to assess viral silencing; dark animals do not express GFP after heat shock, green animals do. Numbers are shown for the most relevant genotypes. The X-linked FR1gfp transgene is present only when specifically indicated. (A) Reconstitution of the RNAi machinery re-silences viral production. (B) The antiviral RNA silencing can pass through the sperm and is independent of the presence of the viral template. Because the FR1gfp array is on the X chromosome, F1 males originating from the cross of male FR1gfp/0 (indicating hemizygosity); rde-1(−/−) with rde-1(−/−) hermaphrodites will not contain the array. (C) Long-term silenced worms contain a non-chromosomally encoded dominant spreading signal. Crossed animals carry the same genotype in regard to the transgene and rde locus, yet one strain is long-term silenced, while the other has lost its ability to silence. The long-term silenced strain is able to silence the non-silent strain in trans. Viral silencing was always assessed by heat-shock treatment to induce the gfp-tagged viral transcript. If the FR1gfp transgene was not present in a strain, it is not shown in the genotype.

Fig.3. The RdRP amplification by rrf-1 is required only for long-term silencing

Animals of all the genotypes schematically shown in this figure were tested for whether they express GFP after heat shocking adult animals (see Experimental Procedures), as a measure to assess viral silencing; dark animals do not express GFP after heat shock, green animals do. Numbers are shown for the most relevant genotypes. FR1gfp(+) indicates that animals contain the FR1gfp array but it was not tested whether it is contained in the homozygous (FR1gfp(+/+)) or heterozygous (FR1gfp(+/−)) state. If the FR1gfp transgene was not present in a strain, it is not shown in the genotype. (A) RNAi competent grandparents initiate an RNAi response that is sufficient for viral silencing for at least 2 generations in the absence of rrf-1. (B) The virus escapes long-term silencing (GFP/Virus(+)) when rrf-1 is neutralized in otherwise GFP/Virus(−) >F5 generation rde-1(−/−) animals.

Fig.4. viRNA match to specific epitopes in the viral genome

The number and strandedness of 20–30nt viRNAs are shown with respect to their alignment to the FHV genome. The thickness of lines, which indicate the location of individual reads, is proportional to number of reads. Note that the transmitted viRNA reads match to the main two epitopes of the virus. Clustering of epitopes to the 3' end of FHV have been noted before (Parameswaran et al., 2010). See Experimental Procedures and Table 2 for more details on the reads.