HIGS-mediated crop protection against cotton aphids

Aphids are sap-sucking insects of the order Hemiptera and are considered major agricultural pests owing to their direct feeding damage and transmission of plant viruses (Yu et al., 2016). The rapid development of insecticide-resistant pest biotypes and strong dispersal capacity cause significant economic losses in a wide range of plant hosts (Yu et al., 2016; Powell et al., 2006). Plants expressing Bacillus thuringiensis (Bt) toxins have been successful against lepidopteran and coleopteran pests (Wu et al., 2008). However, aphids have evolved into the most abundant pests in Bt crop fields, including in the Bt cotton growing area in China (Lu et al., 2010; Yu et al., 2016).

RNA interference (RNAi) regulates gene expression in a sequence-specific manner in most eukaryotes (Zhao and Guo, 2022). In recent years, RNAi-mediated pest control has been achieved via the production of double-stranded RNA (dsRNA) in transgenic plants, a technology referred to as host-induced gene silencing (HIGS), exhibit retarded growth and reduced fecundity or mortality of the corresponding pest species (Dong et al., 2024; Mao et al., 2011; Zhang et al., 2022). However, the effect of RNAi on aphid resistance in cotton plants has not been reported.

In this study, to construct the cotton aphid (Aphis gossypii)-specific dsRNA, a gene encoding polyprenyl diphosphate synthase (PDSS) was selected. PDSSs play a critical role in the formation of the prenyl side-chain tail of ubiquinone. Two subunits of aphid long-chain PDSSs designated AgDPPS1 and AgDPPS2, were characterized in Aphis gossypii (Zhang and Li, 2013). A 541-bp A. gossypii-specific DPPS1 (KC431243.1) fragment was used to create an RNAi construct for cotton plant transformation (Figure 1a). Southern blot analysis revealed that two individual cotton transformants, AgDPPSi-1 and AgDPPSi-2, each with a single insertion, were obtained (Figure 1b). Small RNA hybridization detected the production of sRNAs in both AgDPPSi lines but not in wild-type (WT) cotton plants (Figure 1c). The offspring of AgDPPSi-1 and AgDPPSi-2, which accumulate sRNAs (Figure S1a), were used for bioassays with cotton aphids. Aphids collected from cotton leaves growing in the natural field were fed on leaves of AgDPPSi and WT cotton plants (Figure S1b). Equal numbers of aphids were fed on the leaves in one plate (Figure 1d). Compared to that at 1 day post-feeding (dpf), the number of total aphids on WT leaves at 3 dpf increased significantly (Figures 1d and S1c). In contrast, the number of total aphids on either AgDPPSi leaf was lower than that at 1 dpf (Figures 1d and S1c). On occasion, aphids moved away quickly, leading to inaccurate numbers of aphids as the initial feeding on the leaves at 1 dpf (Figure S1c). The increased aphid numbers on WT leaves probably resulted from the reproduction of feeding aphids, but from those fed on the AgDPPSi leaves might not be ruled out. Nevertheless, a few dead aphids were observed on AgDPPSi leaves at 3 dpf (Figures 1d and S1c). To more precisely, we repeated the bioassay with leaves separately placed in plates. The total aphid number significantly increased at 3 dpf upon feeding on WT leaves, with more nymphs observed than at 1 dpf (Figures 1e and S1d). A reduced number of surviving aphids accompanied by increased numbers of dead aphids were observed upon feeding on either AgDPPSi leaves with few nymphs (Figures 1e and S1d). We took 72 h of time-lapse images to record the actual aphid performance on the WT and AgDPPSi-2 leaves (Figure S2a). Each leaf was fed 40 aphids and began shooting, and the video was shortened to approximately 100 s. Very active movement was observed for aphids on either leaf on day 1 (Figure S2a, 0–33 s), which wore off on day 2 (Figure S2a, ~33–66 s). Neonatal nymphs were first observed on day 1 on the WT leaf and increased in number on day 3 (Figure S2b, red circles). In contrast, nymphs were rarely observed on AgDPPSi-2 leaf. Moreover, aphids look likely unvital and ultimately die on AgDPPSi-2 leaf (Figure S2a,b). These results demonstrate that AgDPPSi plants effectively reduced aphid survival and impaired their fecundity. Aphids on five individual leaves of the WT and AgDPPSi-1 were counted. The results confirmed an increase in aphids that fed on WT leaves at 3 dpf compared with 1 dpf (Figure 1f) but a decrease in those that fed on AgDPPSi-1 leaves (Figure 1f), indicating that AgDPPSi decreased the survival rates and fecundity and induced significant mortality (~50%) in aphids. Similar mortality rates of aphids fed on leaves of AgDPPSi-2 were detected. We then analysed the expression levels of the AgDPPS1 gene in aphids collected from WT and AgDPPSi leaves at 3 dpf via RT-qPCR analysis. In agreement with the bioassay results, compared with those of aphids fed WT leaves, the expression levels of the AgDPPS1 gene were significantly lower in aphids fed either AgDPPSi-1 or AgDPPSi-2 leaves (Figure 1g).

Figure 1

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Development of transgenic cotton lines with resistance against cotton aphids. (a) A diagram of the RNAi construct, 35S-AgDPPS1i. (b) Southern blot detection of transgenic cotton plants. Two transgenic cotton lines, AgDPPS-1 and -2, each with a single insertion, were detected. (c) Northern blot detection of AgDPPSi-derived sRNAs. (d, e) Feeding assays of cotton aphid on WT and AgDPPSi-1 cotton leaves. (f) Changes of aphid numbers on WT and AgDPPSi-1 leaves. (g) Relative expression of AgDPPS1 gene in aphids fed on leaves at 3 dpf. (h) Examination of the aphid resistance of AgDPPSi-2 cotton plants in a natural cotton-growing area. An overall view of the cotton plants and several close-up pictures with corresponding positions in the overall view are shown. (i) Changes of aphid numbers on WT and AgDPPSi-2 plants. (j) Detection of AgDPPS1 expression by Northern blotting. * indicates P < 0.05.

Next, we examined the aphid resistance of AgDPPSi cotton plants in a natural cotton-growing area. WT and AgDPPSi-2 cotton seeds were sown in an experimental cotton growing field. There was no phenotypic difference between the WT and AgDPPSi-2 plants (Figure S3a). No insecticide was applied during the entire cotton-growing season, and severe aphid infestation occurred as always. The cotton plants were then covered with a net to provide a relatively constant environment for aphid eruption and behaviour analysis (Figure S3b). An overall view of the cotton plants is shown in Figure 1h. Several close-up pictures were taken. Pictures are enlarged, and the corresponding positions are labelled (Figure 1h). In general, the AgDPPSi-2 plants were much cleaner and healthier than the WT plants were. Sticky and dusty leaves on many of the WT plants were observed (Figure 1h). Many adult aphids and nymphs were observed on the petioles and abaxial sides of the WT cotton leaves (Figure 1h). While most AgDPPSi-2 plants were clean and almost aphid free, a number of aphids were also observed on some abaxial surfaces of the AgDPPSi-2 leaves; however, they were not as dense and dusty as aphid-infected WT leaves were. Five aphid-infected plants from the WT and AgDPPSi-2 plants were randomly selected for counting aphid numbers. The average number of aphids on AgDPPSi-2 plants (~764 per plant) was significantly lower than that on WT plants (~2034 per plant) on July 5, 2023, a severe aphid infestation period (Figure 1i). The number of aphids decreased on July 23, 2023, after days of rain (Figure 1i). The average number of aphids on AgDPPSi-2 plants remained lower (~198 per plant) than that on WT plants (~846 per plant). Aphids were then collected from leaves after counting for aphid RNA extraction. Total RNAs were isolated from mixed aphids collected from WT leaves or AgDPPSi-2 leaves. Northern blot detection revealed that AgDPPS1 mRNA was degraded in aphids on AgDPPSi-2 leaves compared with WT cotton leaves (Figure 1j), indicating that AgDPPSi effectively silenced AgDPPS1 mRNA in aphids on AgDPPSi-2 plants, resulting in a reduction in aphid viability. Taken together, our data demonstrate the aphid resistance of AgDPPSi cotton plants in natural cotton fields.

In conclusion, we developed transgenic cotton plant lines via the expression of an RNAi construct targeting the A. gossypii-specific DPPS1 gene. DPPS1 silencing impedes the formation of ubiquinone. Consequently, aphids that fed on or infected AgDPPSi plants exhibited reduced survival and fecundity in bioassays both indoors and in natural cotton-growing fields.