Abstract
Transplastomic potato plants expressing double-stranded RNA (dsRNA) targeted against essential genes of the Colorado potato beetle (CPB) can be lethal to larvae by triggering an RNA interference (RNAi) response. High accumulation levels of dsRNAs in plastids are crucial to confer an efficient RNAi response in the insects. However, whether length and sequence of the dsRNA determine the efficacy of RNAi and/or influence the level of dsRNA accumulation in plastids is not known. We compared the RNAi efficacy of different lengths of dsRNA targeted against the CPB β-Actin gene (ACT) by feeding in vitro-synthesized dsRNAs to larvae. We showed that, while the 60 bp dsRNA induced only a relatively low RNAi response in CPB, dsRNAs of 200 bp and longer caused high mortality and similar larval growth retardation. When the dsRNAs were expressed from the plastid (chloroplast) genome of potato plants, we found that their accumulation were negatively correlated with length. The level of dsRNA accumulation was positively associated with the observed mortality, suppression of larval growth, and suppression of target gene expression. Importantly, transplastomic potato plants expressing the 200 bp dsRNA were better protected from CPB than plants expressing the 297 bp dsRNA, the best-performing line in our previous study. Our results suggest that the length of dsRNAs is an important factor that influences their accumulation in plastids and thus determines the strength of the insecticidal RNAi effect. Our findings will aid the design of optimized dsRNA expression constructs for plant protection by plastid-mediated RNAi.
Highlights
RNA interference (RNAi) is a post-transcriptional gene silencing mechanism trigged by double-stranded RNA in eukaryotes. dsRNAs are cleaved by endoribonucleases of the Dicer family into 21-23 bp small interfering RNAs which are loaded into the RNA-induced silencing complex (RISC) to cleave and degrade cognate mRNAs (Gordon and Waterhouse 2007)
RNAi has emerged as a promising strategy for insect pest control
The factors influencing the efficiency of environmental RNAi in insects are not fully understood
Summary
RNA interference (RNAi) is a post-transcriptional gene silencing mechanism trigged by double-stranded RNA (dsRNA) in eukaryotes. dsRNAs are cleaved by endoribonucleases of the Dicer family into 21-23 bp small interfering RNAs (siRNAs) which are loaded into the RNA-induced silencing complex (RISC) to cleave and degrade cognate mRNAs (Gordon and Waterhouse 2007). Efficient delivery of insecticidal dsRNAs (targeting essential insect genes) is the key process in RNAi-based pest control. Injection of dsRNA into insects is suitable for laboratory research, but not for practical application towards pest control in the field. Strategies for oral delivery of dsRNA molecules have been explored in both the laboratory and the field, including the use of nanocarriers, detergents, polymers, engineered bacteria and yeasts, and transgenic plants (He et al, 2013; Murphy et al, 2016; Zhang et al, 2015; Zheng et al, 2019; Zhu et al, 2011). Since the costs of kilogram amounts of chemically synthesized dsRNA synthesis are very high and large-scale application to plants in the field is burdened with technical problems, expression of dsRNA in planta represents the method of choice for the control of herbivorous pest insects. The first plant-incorporated protectant (PIP) based on RNAi technology (targeting the western corn rootworm Diabrotica virgifera virgifera) was approved by the US Environmental
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