Xixi Zheng, Zijin Weng, Han Li, Zichun Kong, Zaihui Zhou, Fei Li, Weihua M, Yongjun Lin, Hao Chen

Plant Biotechnological Journal; 27 October 2020 (Please cite this article as doi: 10.1111/PBI.13504)

Figure: Rice Striped Stem Borer (Chilo suppressalis)

Abstract

RNA interference (RNAi) is a promising approach for developing insect‐resistant crops. In the first two proof‐of‐concept studies, DNA fragments derived from essential insect genes were constructed into plant expression cassettes as inverted repeats, enabling long double‐stranded RNAs (dsRNAs) to be transcribed in host plants (Baum et al., 2007; Mao et al., 2007). After ingestion, dsRNAs overexpressed in planta suppressed target gene expression via small interference RNA (siRNA)‐mediated RNAi in western corn rootworm (Diabrotica virgifera virgifera) (Baum et al., 2007) and cotton bollworm (Helicoverpa armigera) (Mao et al., 2007), and thereby reduced their viability. Subsequent studies have mostly employed a similar approach and attempted to develop RNAi crops against various insect species (Liu et al., 2020). However, except for coleopterans that are generally susceptible to RNAi, most insects (e.g., lepidopterans, dipterans, hymenopterans, and hemipterans) exhibit unpredictable responses to dsRNA‐induced RNAi, and this has become a hurdle for ubiquitous adaption of this strategy (Cooper et al., 2019). Potential factors influencing RNAi efficacy in insects involve dsRNA stability, cellular dsRNA absorption, core RNAi machinery integrity, systemic RNAi spread, and target gene amenability (Cooper et al., 2019).

Our results demonstrated that amiRNA-induced RNAi is more applicable for controlling SSB than dsRNA-induced RNAi. Because endogenous miRNAs naturally regulate their gene targets in insects, their use theoretically avoids certain factors that cause inefficient RNAi, such as refractory target genes and relevant RNAi machinery deficiency (Cooper et al., 2019). Moreover, critical siRNA and miRNA components associated with their biogenesis and function are significantly different (Cooper et al., 2019), potentially causing variations among insects in terms of small RNA stability, cellular uptake, and intercellular transport between siRNA and miRNA. These factors could explain why SSB is resistant to dsRNA-induced RNAi but relatively susceptible to miRNA-induced RNAi. Taken together, amiRNA-induced RNAi can be an alternative approach for controlling these insect pests insensitive to dsRNA-induced RNAi, which would broaden the applicability of RNAi-mediated insect pest management.

See: https://onlinelibrary.wiley.com/doi/10.1111/pbi.13504

Figure.1 Overexpressing amiRNA of csu-novel-260 confers high resistance against striped stem borer (SSB). (a) The biosynthetic pathway of ecdysteroids and putative target site of csu-novel-260 in 5′ UTR of dib (modified from He et al., 2017). (b) Five-day stemcutting feeding assay. (c) Thirty-five day consecutive stemcutting feeding assay. (d) Small RNA sequencing of csu260-16 and csu260-18 rice. The bottom sequence is the amiRNA precursor of csu-novel-260. The red and blue bases in the amiRNA precursor are the positions of amiRNA and amiRNA*, respectively. Sequences above the amiRNA precursor of csu-novel-260 are the sequenced amiRNAs (threshold of read number ≥ 10 in csu260-16) matching the amiRNA precursor. The two sequences in pink are the two most abundant amiRNA sequences. The three numbers at the end of each amiRNA sequence are their length, and read counts of csu260-16 and csu260-18. (e) Agomir feeding assay. (f) Field assessment of SSB-resistance in the tillering stage (deadheart rate) and the mature stage (whitehead rate) under manual infestation conditions. (g) Field performance under manual infestation in the maturity stage. Left: csu260-18; right: Zh11 control. (h) Agronomic assessment of csu260-16 and csu260-18 under field conditions. Data in (b), (c), (e), and (f) are presented as mean ± SE. * and ** indicate statistically significant difference at P < 0.05 and P < 0.01, respectively, compared with the WT control, according to Student’s t-test.