Contents

    Overview

           The plastid-mediated RNAi tobacco PLASTID PESTICIDES™ constructed can be divided into four parts: Engineered bacteria construction, Insect feeding and bioassays, and Plastid transformation, Insect feeding and bioassays. The biosafety module will be introduced into the engineering safety form plastid-free pollen.

    Engineered Bacteria Construction

    Selection of Backbones

           Five backbones were selected in the literature: At-miR159(At, Arabidopsis thaliana), Dm-ba (Dm, Drosophila melanogaster), Dv-miR279 (Dv, Diabrotica virgifera), Dv-miR1 and Tc-ba (Tc, Tribolium castaneum). The literature shows that the effect of the insect backbones is better than that of the plant backbones. The results of the five backbones compared with the wild type are as follows:

    design-1.png

           By comparing the rate of death and pupation failure：greatest inhibition Tc-ba > Dv-miR279 > Dv-miR1 > Dm-ba > At-miR159, so PLASTID PESTICIDES™ finally chose the Tc-ba backbone.

    Screening of Lethal RNAi Target Genes

           Chitin is one the main components of the insect cuticle, and chitin synthase (CHS) is an important enzyme required for chitin formation.

           Previous experimental results suggest that SlCHS1 might play an important role during S. litura larval molting, while SlCHS2 has no significant effect on S. litura molting. Therefore, PLASTID PESTICIDES™ have chosen SlCHS1 for further study.

    design-2.png

    Target Fragment Construction

           A vector for amiCHS1 with Tc-ba as the backbone and SlCHS1 as the target gene was designed.

    Vector Construction

           Due to the long growth cycle of tobacco and the short growth cycle of E. coli, PLASTID PESTICIDES™ planned to test the effectiveness of the experiment with E. coli first, and designed 3 different types of engineered bacteria.

    Engineering Bacteria I (T7: Tc-ba-miR-CHS1): To detect whether amiRNA-CHS1 affects the expression level of SlCHS1 gene.

    Engineering Bacteria II (T7: 2xCP): To detect whether MS2-VLP affects the expression level of SlCHS1 gene.

    Engineering Bacteria III (Tc-ba-miR-CHS1 & T7：2xCP): To detect whether amiRNA-CHS1 encapsulated with MS2-VLPs protein affects the expression level of SlCHS1 gene.

    Insect Feeding and Bioassays

           Prepare a small plastic box, use a pinhole to prick a hole in the lid of the plastic box to make it breathable, put the artificial feed of S. litura in a 50mL syringe, squeeze the artificial feed into each small plastic box, squeeze the feed about 1cm long each time and press it flat, use a pipette to absorb 200μL of bacterial solution on the feed, open the lid and air dry naturally for 30min~1h, and wait for the artificial feed to completely absorb the bacterial solution before the biological test.

           The hatched larvae were placed in a small feed box (10 S. litura were placed in each box), and the data were weighed on the 3rd, 5th, 7th, 9th, and 11th days of receiving the insects, and the growth status was recorded, and the RNA was extracted for reverse transcription, and the relative expression of its target gene SlCHS1 was detected by qPCR. The sixth-instar larvae of S. litura are placed on sandy soil (sandy soil should maintain a certain amount of moisture). On about the twenty-fifth day, S. litura, the pupae of S. litura were photographed and their weight measured, and they began to pupate into moths in about a week, during which the pupation rate, deformity rate, and mortality rate were counted.

    Tobacco Plastid Transformation

    Construction of Vectors or Fragmented DNA for Plastid Transformation

           The expression of amiRNA in the detection bacteria can effectively interfere with the growth and development of S. litura, and then perform plastid transformation.

    Engineering Bacteria IV (Prrn: Tc-ba-miR-CHS1-1 ): To detect whether amiRNA-CHS1 expressed in plastid affects the expression level of SlCHS1 gene.

    Engineering Bacteria V (Prrn:Tc-ba-miR-CHS1-1 & Prrn-SD：2xCP ): To detect whether MS2-VLP expressed in plastid affects the expression level of SlCHS1 gene.

    Plastid Transformation

           Using the endogenous homologous recombination mechanism of plant plastids, the plastid expression vector was transformed into tobacco by gene gunning. Cut the leaves bombarded by the gene gun into small pieces and transfer to callus induction medium containing spectinomycin for screening.

    Homogenization of Screening

           After obtaining the resistance callus, the buds were induced on the regeneration medium containing spectinomycin until the resistant shoots were obtained, and finally the resistant shoots were transferred to the rooting medium to induce rooting, and the amiRNA plastid transformation tobacco was obtained.

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    Insect feeding and bioassays

    The feed was changed to genetically modified tobacco leaves, and the previous bioassay process was repeated.

    The Module of Biosafety

    No Plastids in the Pollen

           Plastids exhibit maternal inheritance, meaning they are not present in pollen, which prevents the spread of transgenes via pollen and mitigates environmental safety issues. Another attraction of PM‐RNAi approach for insect management is the exclusion of plastids from the sperm cells of pollen in most angiosperm species. Therefore, pollinators and pollen‐eating insects are not exposed to substantial amounts of plastid‐produced amiRNA.

    Highly targeted

           Compared to siRNAs, amiRNA-based tactics are thought to be more effective due to their high specificity and minimal off-target effect. By contrast, artificial microRNA has high sequence specificity and only recognizes a target sequence with less than five mismatches.


    Reference

    [1] Yu HZ, Li NY, Xie YX, Zhang Q, Wang Y, Lu ZJ. Identification and Functional Analysis of Two Chitin Synthase Genes in the Common Cutworm, Spodoptera litura. Insects. 2020 Apr 17;11(4):253.

    [2] Bock R, Khan MS. Taming plastids for a green future. Trends Biotechnol. 2004 Jun;22(6):311-8.

    [3] Bally J, Fishilevich E, Doran RL, Lee K, de Campos SB, German MA, Narva KE, Waterhouse PM. Plin-amiR, a pre-microRNA-based technology for controlling herbivorous insect pests. Plant Biotechnol J. 2020 Sep;18(9):1925-1932.

    [4] Xu W, Li S, Bock R, Zhang J. A heat-inducible expression system for external control of gene expression in plastids. Plant Biotechnol J. 2024 Apr;22(4):960-969.

    [5] Arakane, Y.; Specht, C.A.; Kramer, K.J.; Muthukrishnan, S.; Beeman, R.W. Chitin synthase are required for survival, fecundity and egg hatch in the red flour beetle, Tribolium castaneum. Insect Biochem. Mol. Biol. 2008, 38, 959–962.

    [6] Xu W, Zhang M, Li Y, He W, Li S, Zhang J. Complete protection from Henosepilachna vigintioctopunctata by expressing long double-stranded RNAs in potato plastids. J Integr Plant Biol. 2023 Apr;65(4):1003-1011.

    [7] Wu M, Zhang Q, Dong Y, Wang Z, Zhan W, Ke Z, Li S, He L, Ruf S, Bock R, Zhang J. Transplastomic tomatoes expressing double-stranded RNA against a conserved gene are efficiently protected from multiple spider mites. New Phytol. 2023 Feb;237(4):1363-1373.

    [8] Chen, H.; Yin, Y.P.; Li, Y.; Mahmud, M.S.; Wang, Z.K. Identification and analysis of genes differentially expressed in the Spodoptera litura fat body in response to the biocontrol fungus, Nomuraea rileyi. Comp. Biochem. Phys. B 2012, 163, 203–210.

    Plastid

    Plastids are a group of organelles found in plant and algal cells. They are involved in the synthesis and storage of food, as well as in the production of pigments. The most well-known plastid is the chloroplast, which is responsible for photosynthesis, converting light energy into chemical energy stored in glucose. Other types of plastids include chromoplasts, which store pigments such as carotenoids, and leucoplasts, which store starch, lipids, and proteins. Plastids have their own DNA, known as the plastome, and they can replicate independently of the cell's nucleus.

    Pollen
    • Pollen Pollen is a fine, powdery substance consisting of microscopic grains (pollen grains) that are produced by the male parts of seed plants, specifically the anthers of angiosperms (flowering plants) and the microsporangia of gymnosperms (cone-bearing plants). Each pollen grain contains the male gametophyte, which includes the male reproductive cells necessary for fertilization. Pollen plays a critical role in the sexual reproduction of plants, and it is dispersed by various means, including wind, water, and animals, to reach the female part of the flower (the stigma) where it can germinate and initiate the formation of a pollen tube, leading to fertilization.
    Backbone (in Genetic Engineering)

    In genetic engineering, a backbone refers to the core structure of a vector, such as a plasmid, viral vector, or bacteriophage, into which foreign DNA fragments can be inserted. The backbone provides essential elements for the propagation and maintenance of the construct within host cells, including origin of replication (ori), antibiotic resistance genes for selection, and sometimes regulatory elements. By selecting appropriate backbones, researchers can control the expression of the inserted genes and ensure that the genetic material is stably inherited during cell division.

    Chitin

    Chitin is a long-chain polymer of N-acetylglucosamine, a derivative of glucose. It is a structural polysaccharide that forms a key component of the exoskeletons of arthropods, such as insects, crustaceans, and arachnids, as well as the cell walls of fungi. Chitin is second only to cellulose in abundance among naturally occurring organic polymers. Its properties, such as strength, flexibility, and resistance to many chemicals, make it an important material in nature. In biotechnology, chitin and its derivatives, like chitosan, have various applications, including in wound healing, drug delivery, and water treatment.

    Chitin Synthase (CHS)

    Chitin synthase is an enzyme that catalyzes the formation of chitin from its monomer, N-acetylglucosamine (NAG). This enzyme is crucial for the development and maintenance of the exoskeleton in arthropods and the cell walls in fungi. There are multiple isoforms of chitin synthase, each with specific roles in different stages of development and in response to environmental conditions. In pest management, chitin synthase is a target for the development of insecticides, as inhibiting this enzyme can disrupt the growth and survival of pests.