Plastid Mediated RNAi: A Novel Insecticidal Tool

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Pavan Kumar, Ph.D. Postdoctoral Researcher, Boyce Thompson Institute for Plant Research, Ithaca, New York.
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Recently, Zhang et al demonstrated a robust and promising way of controlling the devastating solanaceous crops insect pest, Colorado potato beetle (Leptinotarsa decemlineata; CPB), using a modified strategy for delivering plant mediated RNAi.

Colorado potato beetle.jpgIncreasing insect herbivores’ resistance to plant defense mechanisms and insecticides has demanded the development of newer and more effective ways of controlling insect pests. RNA interference (RNAi) has been widely anticipated in the control of insect pests by targeting their vital genes. However, promising results on RNAi-based insecticides development have not yet been  widely attained. The success of RNAi is dependent on the target gene, the dose and mode of  delivery of dsRNA, in addition to the endogenous RNAi machinery of the insect pest itself.

RNAi, a cellular phenomenon in eukaryotes, is known to down regulate target gene expression by disrupting their transcripts. When a long double-stranded RNA (dsRNA) complimentary to the target gene transcript enters the cells, cellular nucleases, known as DICERS, cleave such dsRNA into the small interfering RNA (siRNA) molecules typically 21-24 nucleotide long.These siRNA molecules are further recruited into the protein complex by cellular RNAi machinery and directed to bind the target mRNA based on the sequence similarity; eventually degrading the mRNAs.

http://www.sciencemag.org.proxy-um.researchport.umd.edu/content/347/6225/950/F1.large.jpg

from Whyard, Science 27 February 2015: Vol. 347 no. 6225 pp. 950-951   DOI:10.1126/science.aaa7722

However, siRNAs are reported to be less efficient compared to long dsRNA, suggesting that the formation of siRNAs dilutes the effectiveness of RNAi. Previously, in my work on Manduca sexta, we have shown that ‘the longer the dsRNA, the better the silencing’ by silencing the DICERS of transgenic Nicotiana attenuata plants expressing insect dsRNA; hence insects feeding on these plants ingest long dsRNA (Kumar et al 2012PLOS ONE 7, e31347).

RNAiIn the present study, Zhang et al cleverly overcome the limitations of long dsRNA retention in plants by generating transgenic plants that express long dsRNA in the chloroplast. Chloroplasts are devoid of the RNAi machinery and as a result transgenic plants expressing dsRNA in the chloroplasts will not be process dsRNA into siRNA and larvae feeding on these plants ingest long dsRNA.

First, Zhang et al confirmed that Nicotiana tabacum plants that were engineered to express dsRNA in chloroplasts accumulate long and intact dsRNA and that there is no siRNA generation. Furthermore, they tested whether accumulation and cellular stability of long, stem-loop, and hairpin forms of dsRNA are different. They did not notice any advantage in adding stem-loops or hairpins to dsRNA, hence Zhang et al restricted expression of long dsRNA of the target gene to the plant’s chloroplast.

Target gene selection is one of the key features for successful RNAi. Zhang et al targeted the two essential genes of CPB (1) ACT and (2) SHR.ACT encodes a cytoskeletal protein β-actin and SHR encodes Shrub, a subunit of a protein complex involved in membrane remodeling for vesicle transport.

The authors generated transgenic potato plants expressing dsRNA of ACT, SHR and ACT+SHR in chloroplasts and cytosol. CPB larvae fed on potato plants expressing ACT-dsRNA in their chloroplasts experienced 100% mortality by five days, whereas SHR-dsRNA expressing plants resulted in 70% mortality to CPB larvae after 9 days.

CPB Damage

CPB damage on detached leaves of wild-type potato plants and nuclear transgenic and transplastomic leaves expressing ACT dsRNA. Image from Zhang et. al.

However, ACT+SHR-dsRNA expressing plants were less efficient suggesting that expressing dsRNA (ACT+SHR), which two different target genes is not as effective as expressing dsRNA targeting single gene (Fig 1). Furthermore, the authors report that, plants that express ACT or SHR dsRNA in the cytoplasm did not result in any mortality, although they did reduce mean weight of CPB larvae, suggesting that nuclear expression of dsRNA is not effective, since plant DICERs process dsRNA into siRNA before they enter the CPB midgut.  The authors demonstrate by northern blot analysis that transplastomic plants express long dsRNA, not siRNA and larvae fed on these plants ingested long dsRNA.  In addition, this work also shows that mortality phenotype which was observed in CPB larvae is also observed in adult CPB as well and the mortality was due to the disorganization of actin filaments in CPB tissues.

Zhang et al presents an effective and groundbreaking approach to control the devastating insect pest (CPB) by expressing dsRNA of larval essential genes in chloroplast. The authors argue that their approach of plant mediated RNAi is insecticidal and alternative to the application of chemical pesticides to control CPB pest.

There is an urgent need to apply and test this approach to the other crop plants and insect pests, since not all the insect pests are amenable to RNAi like coleopteran insect CPB. However, increasing knowledge and novel tools will enable plant mediated RNAi to be the most effective and targeted way in the near future to control major insect pests.

Full crop protection from an insect pest by expression of long double-stranded RNAs in plastids.
Jiang Zhang, Sher Afzal Khan, Claudia Hasse1, Stephanie Ruf, David G. Heckel, Ralph Bock
Science 347 (991-994) 2015. DOI: 10.1126/science.1261680.

Whyard, S., 2015  Insecticidal RNA, the long and short of it. Science 347:  950-951doi: 10.1126/science.aaa7722.

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