Team:NYMU-Taipei/Design
Overview
Through the utilization of differing gene expression and location of M. anisopliae cells during different stages of its infection cycle alongside with light-induced phototoxic fluorescent proteins, we aim to design entomopathogenic-fungi-specific killswitch that can greatly mitigate the safety concerns of the genetic modified fungal insecticides in hope that it will increase the feasibility of wide spread use of these enhanced bioinsecticides.
Design
What is Metarhizium anisopliae?
Metarhizium. anisopliae serves as our chassis organism in this project due to its status as a model organism for various biological insect control studies and wide spread use as an fungal bioinsecticide[1]. The fungus also have been the target for many genetic modification projects aimed to amplify its insecticidal activities(2)(3). In our project, we will be utilizing Pmcl 1,the insect hemolymph-inducible promoter of M. anisopliae, as the controlling element for the production of the phototoxic protein, KillerRed.
Metarhizium-collogen-like promoter, or Pmcl1, is the promoter that controls the production of Metarhizium-collagen-like-proteins(3) in wildtype Metarhizium anisopliae, the transcripts of the Mcl1 gene could be detected within 20 minutes of the fungus contacting the hemolymph, but could not be detected when the fungus is cultured in any other medium(4). Utilizing Pmcl 1 allows KillerRed production to be strong and time specific.
KillerRed:KillerRed protein is a red fluorescent protein with excitation and emission maxima at 585 and 610 nm respectively. When the protein comes in contact with light of wavelengths of 520-590 nm, it produces reactive oxygen species (ROS) along with intense photobleaching. The phototoxicity induced by KillerRed generated ROS is 1,000times greater than those produced by common fluorescent proteins5. The increase concentrations of ROS in the cytoplasm will disrupt normal cellular functions and impede fungal growth and, in the best case scenario, induce programed necrosis6.
Circuit
Kill Switch Circuit:
The kill switch circuit contains the hemolymph-induced promoter, Pmcl, followed by the KillerRed gene (BBa_K1184000) and a trpC terminator, TtrpC. This sets the mass production of KillerRed proteins to the hemolymph infection phase of M. anisopliae’s life cycle.
Our kill switch circuit is contained within pBARGPE1 fungi-specific vectors. This backbone grants transformed M. anisopliae phosphinothricin and glufosinate ammonium resistance with the phosphinothricin acetyltransferase, or BlpR, gene.
mcl
Reference
Sudakin D.L. Biopesticides. Toxicol. Rev. 2003;22:83–90. doi: 10.2165/00139709-200322020-00003.
St Leger, R., Joshi, L., Bidochka, M. J., & Roberts, D. W. (1996). Construction of an improved mycoinsecticide overexpressing a toxic protease. Proceedings of the National Academy of Sciences of the United States of America, 93(13), 6349–6354.
Wang CS, St Leger RJ (2007) A scorpion neurotoxin increases the potency of a fungal insecticide. Nat Biotechnol 25: 1455–1456.
Wang, C., & St. Leger, R. J. (2006). A collagenous protective coat enables Metarhizium anisopliae to evade insect immune responses. Proceedings of the National Academy of Sciences of the United States of America, 103(17), 6647–6652.
Pletnev, S., Gurskaya, N. G., Pletneva, N. V., Lukyanov, K. A., Chudakov, D. M., Martynov, V. I., … Pletnev, V. (2009). Structural Basis for Phototoxicity of the Genetically Encoded Photosensitizer KillerRed. The Journal of Biological Chemistry, 284(46), 32028–32039.
Breitenbach, M., Weber, M., Rinnerthaler, M., Karl, T., & Breitenbach-Koller, L. (2015). Oxidative Stress in Fungi: Its Function in Signal Transduction, Interaction with Plant Hosts, and Lignocellulose Degradation. Biomolecules,5(2), 318–342.
