Silencing an aphid-specific gene SmDSR33 for aphid control through plant-mediated RNAi in wheat

Abstract

Grain aphid (Sitobion miscanthi) is one of the most dominant and devastating insect pests in wheat, which causes substantial losses to wheat production each year. Engineering transgenic plants expressing double strand RNA (dsRNA) targeting an insect-specific gene has been demonstrated to provide an alternative environmentally friendly strategy for aphid management through plant-mediated RNA interference (RNAi). Here we identified and characterized a novel potential RNAi target gene (SmDSR33) which was a gene encoding a putative salivary protein. We then generated stable transgenic wheat lines expressing dsRNA for targeted silencing of SmDSR33 in grain aphids through plant-mediated RNAi. After feeding on transgenic wheat plants expressing SmDSR33-dsRNA, the attenuated expression levels of SmDSR33 in aphids were observed when compared to aphids feeding on wild-type plants. The decreased SmDSR33 expression levels thus resulted in significantly reduced fecundity and survival, and decreased reproduction of aphids. We also observed altered aphid feeding behaviors such as longer duration of intercellular stylet pathway and shorter duration of passive ingestion in electroneurography assays. Furthermore, both the surviving aphids and their offspring exhibited decreased survival rates and fecundity, indicating that the silencing effect could be persistent and transgenerational in grain aphids. The results demonstrated that SmDSR33 can be selected as an effective RNAi target for wheat aphid control. Silencing of an essential salivary protein gene involved in ingestion through plant-mediated RNAi could be exploited as an effective strategy for aphid control in wheat.

Keywords: RNA interference (RNAi); aphid control; grain aphid (Sitobion miscanthi); salivary protein; wheat (Triticum aestivum L).

Figure 1

Characterization of SmDSR33. (A) Multiple sequence alignment of SmDSR33 protein and orthologs from other aphid species. The deduced amino acid sequences from eleven aphid species include Acyrthosiphon pisum (NM_001163178.1), Aphis glycines (AG000929-RA), Aphis gossypii (XM_027996540.1), Diuraphis noxia (XM_015509488.1), Melanaphis sacchari (XM_025349621.1), Myzus cerasi (Mca00769.t1), Myzus persicae (XM_022311485.1), Pentalonia nigronervosa (g3912.t1), Rhopalosiphum maidis (XM_026949227.1). Rhopalosiphum padi (Rpa07522.t1), and Sipha flava (XM_025560312.1). Black shades indicate identical amino acids. Pink shades indicate similar amino acid, and blue shades include the sequences with identical and similar residues. Signal peptide of SmDSR33 is highlighted with blue box. (B) Phylogenetic tree of SmDSR33 and its homologs from other aphid species constructed with the maximum likelihood method. Bootstrap supporting values (1000 replicates) are shown at the branch nodes. (C) The expression profile of SmDSR33 in grain aphid at different development stages. The expression profiles of SmDSR33 at different developmental stages of grain aphids fed on wheat. Values and error bars represent the mean and SEM of three independent biological replicates, each with a pool of 15 individual aphids.

Figure 2

RNAi induced silencing of SmDSR33 gene in wheat. (A) The encoding sequence of SmDSR33 and its deduced amino acid sequence. The sequences selected for construction of the RNAi vector are highlighted in yellow. (B) A schematic show of the SmDSR33 expression cassette and position of Ssp I restriction enzyme. (C) Southern blot analysis of the transgenic wheat lines. Genomic DNA was digested with SspI and hybridized with a SmDSR33 gene fragment with the expression cassette digested with Ssp I as a positive control. (D) Relative expression levels of SmDSR33 of grain aphid fed on wild-type and transgenic wheat lines. The expression level of SmDSR33 in the adult aphids fed on wild-type and different transgenic wheat lines after inoculation of one-day-old newborn nymphs, respectively. Values and error bars represent the mean and SEM of three independent biological replicates, each with a pool of 15 surviving individual aphids (Student’s t-test, ** P<0.01).

Figure 3

Fitness analysis of aphids fed on transgenic plants. (A) Mortality of aphids fed on wild-type and transgenic wheat lines. The mortality of aphids fed on wild-type and dsSmDSR33 expression transgenic wheat lines. Twenty synchronous one-day-old nymphs were put into clip cages individually on transgenic and wild-type wheat plants. All experiments were repeated three times. Values and bars represent the mean ± SEM (Student’s t-test, * P<0.05, ** P<0.01). (B) The longevity of different stages, adult preoviposition period (APOP) and total preoviposition period (TPOP) of aphids fed on transgenic lines and wild-type control. (C) The adult longevity, fecundity and the total longevity of aphids fed on transgenic wheat lines and wild-type control. (D) The reproduction of aphids fed on transgenic wheat lines and the wild-type control. All experiments were repeated three times, each with 20 synchronous one-day-old nymphs. Values and bars represent the mean ± SEM (Student’s t-test, * P<0.05, ** P<0.01).

Figure 4

Effects of SmDSR33 silencing on feeding behavior of grain aphid based on EPG recordings. (A–F) Representative parameters of aphid feeding behavior. Non-probing (np), stylet probing (C), intracellular stylet puncture (pd), phloem salivation (E1), and phloem ingestion (E2). Data shown are mean ± SEM. Asterisks above bars indicate significant differences between controls and treatments (Student’s t-test, * p < 0.05; ** p < 0.01). (G) Representative EPG waveforms of grain aphids feeding on dsGFP wheat plants and 33-592 transgenic wheat plants.

Figure 5

Transgenerational effect of SmDSR33 silencing. The one-day-old newborn nymphs of aphids feeding on transgenic wheat lines were transferred to fresh wild-type wheat plants and subsequently allowed to reproduce on wild-type. (A) The SmDSR33 transcript levels of adult aphids were determined in fourth successive aphid generations. (B) The mortality of the first generation of the offspring of aphids fed on transgenic lines at different time points after being switched to wild-type plants. Values and bars represent the mean ± SEM (Student’s t-test, * P<0.05, ** P<0.01).