Host generated siRNAs attenuate expression of serine protease gene in Myzus persicae

Abstract

Background: Sap sucking hemipteran aphids damage diverse crop species. Although delivery of ds-RNA or siRNA through microinjection/feeding has been demonstrated, the efficacy of host-mediated delivery of aphid-specific dsRNA in developing aphid resistance has been far from being elucidated.

Methodology/principal findings: Transgenic Arabidopsis expressing ds-RNA of Myzus persicae serine protease (MySP) was developed that triggered the generation of corresponding siRNAs amenable for delivery to the feeding aphids. M. persicae when fed on the transgenic plants for different time intervals under controlled growth conditions resulted in a significant attenuation of the expression of MySP and a commensurate decline in gut protease activity. Although the survivability of these aphids was not affected, there was a noticeable decline in their fecundity resulting in a significant reduction in parthenogenetic population.

Conclusions/significance: The study highlighted the feasibility of developing host based RNAi-mediated resistance against hemipteran pest aphids.

Figure 1. Expression analysis of MySP during nymph developmental stages of M. persicae.

(A) Expression profiles of MySP at different developmental stages of M. persicae ranging from early (1–2 day-old new-born nymph), median (3–5 day-old nymph) to late (6–7 day-old apterous adult) was determined by semi-quantitative RT-PCR. cDNA, prepared from 5 µg of total RNA in each sample, was PCR amplified with MySP specific primers (lanes 2–4; upper panel) and 18SrRNA (lanes 2–4; lower panel). The latter was used as endogenous control showing similar RNA concentrations in aphid samples collected at different developmental stages. (B) Integrated density values (IDV) of the PCR products of MySP in the nymphs collected at different development stages as described in (A).

Figure 2. Dendrogram illustrating the relatedness of serine protease gene sequence of M. persicae (MySP) with those of different Indian pollinators.

Phylogenetic relationship of MySP and its counterpart from Indian pollinators was conducted using MEGA 4.0.2 which is shown along with branch lengths. DNA sequences were aligned using CLUSTAL W and a tree was constructed by neighbour-joining program from a similarity matrix of pairwise comparisons. The Nucleotide/ p-distance option was selected in substitution model section. Bootstrap values (*) were assessed with 1000 replicates and are shown at the dendrogram nodes. The scale bar represents sequence divergence.

Figure 3. Detection of MySP-specific siRNAs in different SP transgenic lines.

Total RNA (50 µg) was extracted from the control and different SP transgenic lines (SP8, SP17, and SP20), run on 15% denaturing acrylamide gel, and hybridized with MySP-specific RNA probes. Northern hybridization detected 21–24 nt siRNAs in transgenic samples (upper panel). Loading of equal amounts of RNA was confirmed by ethidium bromide staining (lower panel).

Figure 4. siRNA-mediated suppression of MySP transcript and gut protease activity in aphid nymphs fed on SP transgenic lines.

(A) Aphids were collected from the control and SP-transgenic line at different time points after their release and also those developed parthenogenetically for quantitative Real-time PCR analysis of the relative expression profiles of MySP. 18S rRNA was used as an internal control, and values for the control plants (inoculated nymphs and parthenogenetic progenies) were normalized to 1. Data presented are means of two independent biological replicates with three technical replicates of each ± SD. Asterisks * and ** indicate reductions in the expression of MySP by ≤ 0.5 fold and > 0.5 fold, respectively in the aphids that were fed on the SP-transgenics as compared to those on the control plants. (B) Gut protease activity was estimated by EnzChek protease assay performed with 10 µg of total protein extracted from M. persicae collected from the control and SP transgenics 7 days after the release of the aphids. Protease activity was expressed as equivalent to trypsin (ng/ml). Values are mean ± SE; n = 4. Different letters on the histograms indicate that the means differ significantly (P ≤ 0.05).

Figure 5. Population increase and fecundity of aphids released on control and MySP-silenced transgenic lines.

Nymphs (3–5 day-old) of M. persicae were released on 21day- old control and SP-transgenic plants (SP8, SP17, and SP20). The inoculated plants were reared in a growth chamber under controlled conditions (24°C ± 2°C, 80–85% relative humidity and a 16 h light / 8 h dark photoperiod). Data are presented for the control and SP transgenics showing (A) mean increase in the total number of aphids (counted manually) on 8^th and 15^th day after their release, and (B) the fecundity rate of aphid nymphs. Values for (A) are mean ± SE; n = 6, whereas for (B) it represent average number of nymphs produced per 10 aphids ± SE; n = 3. Different letters on the histograms (A and B) indicate that the means differ significantly (P ≤ 0.05).