Expression profiling and cross-species RNA interference (RNAi) of desiccation-induced transcripts in the anhydrobiotic nematode Aphelenchus avenae

Abstract

Background: Some organisms can survive extreme desiccation by entering a state of suspended animation known as anhydrobiosis. The free-living mycophagous nematode Aphelenchus avenae can be induced to enter anhydrobiosis by pre-exposure to moderate reductions in relative humidity (RH) prior to extreme desiccation. This preconditioning phase is thought to allow modification of the transcriptome by activation of genes required for desiccation tolerance.

Results: To identify such genes, a panel of expressed sequence tags (ESTs) enriched for sequences upregulated in A. avenae during preconditioning was created. A subset of 30 genes with significant matches in databases, together with a number of apparently novel sequences, were chosen for further study. Several of the recognisable genes are associated with water stress, encoding, for example, two new hydrophilic proteins related to the late embryogenesis abundant (LEA) protein family. Expression studies confirmed EST panel members to be upregulated by evaporative water loss, and the majority of genes was also induced by osmotic stress and cold, but rather fewer by heat. We attempted to use RNA interference (RNAi) to demonstrate the importance of this gene set for anhydrobiosis, but found A. avenae to be recalcitrant with the techniques used. Instead, therefore, we developed a cross-species RNAi procedure using A. avenae sequences in another anhydrobiotic nematode, Panagrolaimus superbus, which is amenable to gene silencing. Of 20 A. avenae ESTs screened, a significant reduction in survival of desiccation in treated P. superbus populations was observed with two sequences, one of which was novel, while the other encoded a glutathione peroxidase. To confirm a role for glutathione peroxidases in anhydrobiosis, RNAi with cognate sequences from P. superbus was performed and was also shown to reduce desiccation tolerance in this species.

Conclusions: This study has identified and characterised the expression profiles of members of the anhydrobiotic gene set in A. avenae. It also demonstrates the potential of RNAi for the analysis of anhydrobiosis and provides the first genetic data to underline the importance of effective antioxidant systems in metazoan desiccation tolerance.

Figure 1

Uversky plots (mean scaled hydropathy, <H>, against mean net charge, <R>, at neutral pH) as a predictor of disordered protein structure. Predicted protein sequences corresponding to AavLEA2 (EF026241) and LEA-like protein (EF026240) were analysed at http://www.pondr.com. The positions of test sequences are shown by a green diamond and a yellow triangle, respectively, in comparison to a set of disordered proteins (red circles) and a set of ordered proteins (blue squares). The boundary line shown between unfolded and folded space is empirically defined by the equation <H>_b = (<R> + 1.151)/2.785 [109].

Figure 2

A. avenae dsRNA uptake assay. C. elegans soaked in (A) unlabelled or (B) labelled dsRNA of GFP sequence. A. avenae soaked in (C, E) unlabelled or labelled (D, F) dsRNA of GFP sequence; in (E) and (F) 50 mM octopamine was also added.

Figure 3

Cross-species RNAi using A. avenae EST sequences to induce gene silencing in P. superbus. P. superbus larvae were subjected to RNAi for 11-13 days with selected A. avenae genes (GR463899, ggr-3; GR463913, col-14; GR463914, glutathione peroxidase; and GR463921, novel) identified in a preliminary screen. GFP was taken as a negative control for RNAi. The resulting mixed stage worms were subjected to desiccation at 10% RH for 24 h (negative control - black bar; other samples - grey bars). All samples were also subjected to mock desiccation at 100% RH (white bars). Mean values of triplicates, together with standard deviations, are shown; a one-way ANOVA with Tukey post-hoc test was performed on GR463914 and GR463921: *, significance at P < 0.05; **, significance at P < 0.01; ***, significance at P < 0.001; ns, not significant.

Figure 4

RNAi of cognate glutathione peroxidase transcripts in P. superbus reduces desiccation tolerance. Feeding of P. superbus with bacteria expressing dsRNA corresponding to A. avenae glutathione peroxidase sequence, GR463914, and P. superbus glutathione peroxidase sequences, GR881191 and GR881192, followed by assessment of desiccation tolerance. A bacterial feeder strain expressing dsRNA corresponding to GFP was used as a negative control. Mixed stage worms were subjected to desiccation at 10% RH for 24 h (negative control - black bar; other samples - grey bars). All samples were also subjected to mock desiccation at 100% RH (white bars). Mean values of triplicates, together with standard deviations, are shown; a one-way ANOVA with Tukey post-hoc test was performed: *, significance at P < 0.05; **, significance at P < 0.01; ***, significance at P < 0.001; ns, not significant.