Transgenic Expression of dsRNA Targeting the Pentalonia nigronervosa acetylcholinesterase Gene in Banana and Plantain Reduces Aphid Populations

Abstract

The banana aphid, Pentalonia nigronervosa, is the sole insect vector of banana bunchy top virus (BBTV), the causal agent of banana bunchy top disease. The aphid acquires and transmits BBTV while feeding on infected banana plants. RNA interference (RNAi) enables the generation of pest and disease-resistant crops; however, its effectiveness relies on the identification of pivotal gene sequences to target and silence. Acetylcholinesterase (AChE) is an essential enzyme responsible for the hydrolytic metabolism of the neurotransmitter acetylcholine in animals. In this study, the AChE gene of the banana aphid was targeted for silencing by RNAi through transgenic expression of AChE dsRNA in banana and plantain plants. The efficacy of dsRNA was first assessed using an artificial feeding assay. In vitro aphid feeding on a diet containing 7.5% sucrose, and sulfate complexes of trace metals supported aphid growth and reproduction. When AChE dsRNA was included in the diet, a dose of 500 ng/μL was lethal to the aphids. Transgenic banana cv. Cavendish Williams and plantain cvs. Gonja Manjaya and Orishele expressing AChE dsRNA were regenerated and assessed for transgene integration and copy number. When aphids were maintained on elite transgenic events, there was a 67.8%, 46.7%, and 75.6% reduction in aphid populations growing on Cavendish Williams, Gonja Manjaya, and Orishele cultivars, respectively, compared to those raised on nontransgenic control plants. These results suggest that RNAi targeting an essential aphid gene could be a useful means of reducing both aphid infestation and potentially the spread of the disease they transmit.

Keywords: RNA interference; acetylcholinesterase; artificial diet; banana; banana aphid; plantain; sugars.

Figure 1

Survivability of banana aphids at day 7 post-feeding on various diets supplemented with different concentrations of sucrose. Data are presented as mean percentage survivability (%) and error lines indicate the standard deviation.

Figure 2

Banana aphid survivability curve at varying dsRNA concentrations and time intervals at day 2 (D2), day 3 (D3), and day 7 (D7). Data are presented as mean percentage survivability (%) and error lines indicate the standard deviation.

Figure 3

Transformation, selection, and regeneration of putative transgenic events. (A) Proliferating embryogenic cell suspension, (B) developing embryos on transformed embryogenic cells on selection medium, (C) developing embryos on control untransformed embryogenic cells on nonselective medium, (D,E) germinating embryos, (F) fully regenerated putative transgenic plant. Scale bar = 1 cm.

Figure 4

PCR amplification using AChE specific primers to show the presence of transgene in the transgenic banana and plantain events: (A) Cavendish Williams, (B) Orishele, (C) Gonja Manjaya. M—molecular marker 1 kb ladder; NTC—nontemplate control; CN—control nontransgenic plant; +—plasmid as positive control, numbers on lanes of the image (A–C) indicate the independent transgenic events of Cavendish Williams, Orishele, and Gonja Manjaya, respectively.

Figure 5

Southern blot analysis to confirm the integration of AChE transgene in selected transgenic events representing plantain (Orishele and Gonja Manjaya) and banana (Cavendish Williams) cultivars. M—molecular marker; CN—control nontransgenic plant; P—plasmid DNA as positive control.

Figure 6

RT-PCR analysis to confirm expression of dsRNA-AChE in selected transgenic events. (A) Cavendish Williams, (B) Orishele, (C) Gonja Manjaya, (D) Musa 25S housekeeping gene in selected transgenic plants as an internal control. M—molecular marker 1 kb ladder; CN—control nontransgenic plant; NTC—nontemplate control.

Figure 7

Relative change in aphid populations on dsRNA-AChE transgenic plants compared to nontransgenic control plants upon challenge with aphids. (A) Cavendish Williams, (B) Orishele, (C) Gonja Manjaya. The different number of transgenic events indicates the independent events generated for the three cultivars tested. Data are presented as LSmean values and standard error (SE).

Figure 8

Mean live aphid population growing on nontransgenic plants of various cultivars of banana and plantain. AG—Agbagba, CW—Cavendish Williams, GM—Gonja Manjaya, OB—Obino l’Ewai, and OR—Orishele. Data are presented as means with standard error.

Figure 9

A graphical representation of the T-DNA region of the pNXT-35S-ACE hp plasmid.

Figure 10

Evaluation of transgenic banana and plantains expressing AChE-dsRNA by challenging with aphids. (A–C) Aphids rearing on the leaves of a nontransgenic banana plant and (D) aphid-challenged transgenic plants in an insect-proof cage within a controlled environment chamber.