Abstract
Genetically modified (GM) crops producing double-stranded RNAs (dsRNAs) are being investigated largely as an RNA interference (RNAi)-based resistance strategy against crop insect pests. However, limitations of this strategy include the sensitivity of dsRNA to insect gut nucleases and its poor insect cell membrane penetration. Working with the insect pest cotton boll weevil (Anthonomus grandis), we showed that the chimeric protein PTD-DRBD (peptide transduction domain—dsRNA binding domain) combined with dsRNA forms a ribonucleoprotein particle (RNP) that improves the effectiveness of the RNAi mechanism in the insect. The RNP slows down nuclease activity, probably by masking the dsRNA. Furthermore, PTD-mediated internalization in insect gut cells is achieved within minutes after plasma membrane contact, limiting the exposure time of the RNPs to gut nucleases. Therefore, the RNP provides an approximately 2-fold increase in the efficiency of insect gene silencing upon oral delivery when compared to naked dsRNA. Taken together, these data demonstrate the role of engineered RNPs in improving dsRNA stability and cellular entry, representing a path toward the design of enhanced RNAi strategies in GM plants against crop insect pests.
Highlights
The Cotton boll weevil (Anthonomus grandis) is an economically important crop insect pest that attacks cotton fields, in South America
We report to what extend peptide transduction domain (PTD)-dsRNA binding domain (DRBD) binds to long double-stranded RNAs (dsRNAs) by performing an electrophoretic mobility shift assay (EMSA)
PTD-eGFP binding to dsRNA was detected as a slight but continuous smear starting from the free probe
Summary
The Cotton boll weevil (Anthonomus grandis) is an economically important crop insect pest that attacks cotton fields, in South America (de Lima et al, 2013). The larvae live and feed off the cotton flowers and buds, causing serious damage to cotton yields This endophytic habit is incompatible with chemical pesticide treatments, and no simple or efficient alternatives have been discovered to date (Ribeiro et al, 2010; Neves et al, 2014). Developing resistant cotton plants is considered the best alternative for preventing boll weevil attacks For this purpose, the use of the RNA interference (RNAi) has been applied to generate genetically modified (GM) plants resistant to different pests (Baum et al, 2007; Mao et al, 2011; Zhang et al, 2015). The generation of GM plants producing dsRNA could be a sustainable, efficient and specific resistance strategy against crop insect pests
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