Insecticide Pro Max
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    • Description

    Project Description

    1. The inspiration of the team engaged in this project

    The willow leaf beetle Plagiodera versicolora(Coleoptera: Chrysomelidae) is one of the most important forest pests worldwide, which primarily feeds the leaves of Salicacease plants1. At present, chemical control is the main control method for P. versicolora, but it poses a great threat to the environment and human health2. Although the introduction of Bacillus thuringiensis (Bt) toxin into plants is an effective approach for controlling P. versicolora3,4, Bt toxin-resistance also could be evolved by the beetle5,6. In recent years, novel and environment-friendly strategies have been developed for the management of P. versicolora, such as microbial pest control strategy and RNA interference (RNAi) technology7-9.

    As a kind of good biocontrol bacteria, Pseudomonas has been widely used in the control of plant diseases and insect pests10,11. P. fluorescens strains CHA0 and Pf-5 was showed to kill the larvae of Manduca sexta and Galleria mellonella12. Expressing an insecticidal protein (IPD072Aa) derived from P. chlororaphis in maize showed great protection from Diabrotica virgifera virgifera 13. Pioneer Hi-Bred Canada Company has developed a trait-pyramid maize line (DP23211) to control D. v. virgifera through the expression of DvSSJ1 dsRNA and IPD072Aa protein14. PIP-47Aa, another insecticidal protein isolated from P. mosseii, can kill multiple insects, including D. v. virgifera, Diabrotica barberi, D. undecimpunctata howardii, D. speciosa and Phyllotreta cruciferae15. Recently, we have isolated a P. chloroaphis B3-3G strain that exhibits certain resistance to P. versicolora. To improve its anti-insect effect, we propose to express insecticidal double-stranded (dsRNAs) in P. chloroaphis B3-3G.

    RNAi is a gene silencing mechanism mediated by dsRNAs to induce the degradation of complementary mRNAs in most eukaryotes. Since its first discovery in the nematode, Caenorhabditis elegans16, RNAi technology has been widely utilized as a powerful reverse genetics tool for gene function study and recently developed as a promising approach for pest control17-19. Upon entry into a target cell, long dsRNA is processed by Dicer into small interfering RNAs of 20–25 nucleotide long. Subsequently, Argonaute proteins assemble these siRNAs to form an RNA-induced silencing complex that mediates the degradation of the endogenous mRNA complementary to its guide strand20. Our previous study showed that dsRNA of SRP54 and actin generated in ribozyme III (RNase III)-deficient Escherichia coli HT115(DE3) caused significant mortality of P. versicolora8. Moreover, suppression of the immune genes of P. versicolora through E. coli-produced dsRNA also promoted the biological control of the P. versicolora by Beauveria bassiana7. Whether P. chloroaphis B3-3G could be used to produce sufficient insecticidal dsRNAs thus controlling P. versicolora merits investigation.

    2.Purpose and significance of the research project

    The purpose of this project is to pyramid anti-insect abilities of Pseudomonas and RNAi technology to control P. versicolora. It will open up a new way for the management of P. versicolora, and provide the basis for engineering resistance against other insects.

    3. Research contents

    1.To avoid the possible degradation of dsRNA, ribozyme III encoding gene (rnc) of P. chloroaphis B3-3G is knockout (P. chloroaphis B3-3G-Δrnc) via homologous recombination mediated by pK18.

    2.Construct dsRNA expression vector.

    3.Transform dsRNA expression vector into P. chloroaphis B3-3G-Δrnc and detect the expression levels of dsRNA.

    4.Determine the insecticidal effects of dsRNA-producing bacteria on P. versicolora, including insect mortality rate and suppression of target gene.

    4. Anticipated achievements

    To obtain a dsRNA-producing Pseudomonas that has highly insecticidal activity against P. versicolora.

    5. References

    1. Utsumi, S., Ando, Y., Roininen, H., Takahashi, J. & Ohgushi, T. Herbivore community promotes trait evolution in a leaf beetle via induced plant response. Ecol. Lett. 16, 362-70 (2013).

    2. Demirci, M., Sevim, E., Demir, İ. & Sevim, A. Culturable bacterial microbiota of Plagiodera versicolora (L.) (Coleoptera: Chrysomelidae) and virulence of the isolated strains. Folia Microbiol. 58, 201–210 (2013).

    3. Xu, S. et al. Plastid-expressed Bacillus thuringiensis (Bt) cry3Bb confers high mortality to a leaf eating beetle in poplar. Plant Cell Rep. 39, 317–323 (2020).

    4. Yang, R.L. et al. Genetic transformation and expression of transgenic lines of Populus × euramericana with insect-resistance and salt-tolerance genes. Genet. Mol. Res. 15, gmr.15028635 (2016).

    5. Tabashnik, B.E., Brevault, T. & Carriere, Y. Insect resistance to Bt crops: lessons from the first billion acres. Nat. Biotechnol. 31, 510–521 (2013).

    6. Jurat-Fuentes, J.L., Heckel, D.G. & Ferre, J. Mechanisms of resistance to insecticidal proteins from Bacillus thuringiensis. Annu. Rev. Entomol. 66, 121-140 (2021).

    7. Tu, C. et al. Enhanced toxicity of entomopathogenic fungi Beauveria bassiana with bacteria expressing immune suppressive dsRNA in a leaf beetle. Pestic. Biochem. Physiol. 193, 105431 (2023).

    8. Zhang, Y., Xu, L., Li, S. & Zhang, J. Bacteria-mediated RNA interference for management of Plagiodera versicolora (Coleoptera: Chrysomelidae). Insects 10, 415 (2019).

    9. Li, Y. et al. RNA interference of vATPase subunits A and E affects survival of larvae and adults in Plagiodera versicolora (Coleoptera: Chrysomelidae). Pestic. Biochem. Physiol. 188, 105275 (2022).

    10. Kupferschmied, P., Maurhofer, M. & Keel, C. Promise for plant pest control: root-associated pseudomonads with insecticidal activities. Front. Plant Sci. 4, 287 (2013).

    11. Keel, C. A look into the toolbox of multi-talents: insect pathogenicity determinants of plant-beneficial pseudomonads. Environ Microbiol 18, 3207-3209 (2016).

    12. Péchy-Tarr, M. et al. Molecular analysis of a novel gene cluster encoding an insect toxin in plant-associated strains of Pseudomonas fluorescens. Environ Microbiol 10, 2368-86 (2008).

    13. Schellenberger, U. et al. A selective insecticidal protein from Pseudomonas for controlling corn rootworms. Science 354, 634-637 (2016).

    14. Anderson, J.A. et al. Agronomic and compositional assessment of genetically modified DP23211 maize for corn rootworm control. GM Crops Food 11, 206-214 (2020).

    15. Wei, J.Z. et al. A selective insecticidal protein from Pseudomonas mosselii for corn rootworm control. Plant Biotechnol. J. 16, 649-659 (2018).

    16. Fire, A. et al. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 391, 806–811 (1998).

    17. Kim, D.S. & Zhang, J. Strategies to improve the efficiency of RNAi-mediated crop protection for pest control. Entomol. Gen. 43, 5–19 (2023).

    18. Zhu, K.Y. & Palli, S.R. Mechanisms, applications, and challenges of insect RNA interference. Annu. Rev. Entomol. 65, 293–311 (2020).

    19. Li, S., Kim, D. & Zhang, J. Plastid-mediated RNA interference: a potential strategy for efficient insect pest control. Plant Cell Environ. doi.org/10.1111/pce.14652, (2023).

    20. Wilson, R.C. & Doudna, J.A. Molecular mechanisms of RNA interference. Annu. Rev. Biophys. 42, 217–239 (2013).