Contract number

J4-1772

Department:

Department of Biology

Type of project

ARRS projects

Type of project

Basic research project

Role

Lead

Duration

01.07.2019 - 30.06.2022

Total

1.30 FTE

Project manager at BF

Sepčić Kristina

ABSTRACT

To meet increasing food demands and to comply with stricter environmental demands, agriculture must increase food production and quality while decreasing its detrimental ecological impact. These strategies can also be directed towards efficient control of pests, by replacing the use of first‐generation chemical pesticides with novel, safer, and environmentally friendly biopesticides.

Ostreolysin A6 (OlyA6), pleurotolysin A2 (PlyA2), and erylysin A (EryA) are proteins from the mushroom genus Pleurotus which belong to the aegerolysin family. OlyA6, PlyA2 and EryA have recently been found to specifically bind ceramide phosphoethanolamine (CPE), a membrane sphingolipid that is specific to invertebrates, especially insects. In concert with pleurotolysin B (PlyB) with the membrane‐attack‐complex/perforin domain which is also produced by Pleurotus mushrooms, these aegerolysins can form multimeric bicomponent pore complexes in artificial and biological membranes containing physiologically relevant concentrations of CPE. These characteristics make these aegerolysin/PlyB protein complexes interesting as tools for for selective suppression of organisms whose cell membranes contain the aegerolysin molecular lipid target: the CPE.

The proposed project is based on encouraging results that were obtained within the recently concluded national research Project J4‐7162, in which we demonstrated, by feeding assays, the potential of the OlyA6/PlyB, PlyA2/PlyB and EryA/PlyB protein complexes to selectively kill larvae and adults of two economically most important coleopteran pests, the western corn rootworm (Diabrotica v. virgifera) and the Colorado potato beetle (Leptinotarsa decemlineata). The toxicities of these protein complexes are comparable to, and in the case of CPB even greater than, that of the proteinaceous Cry toxins from Bacillus thuringiensis, which have been introduced into genetically modified crops to control western corn rootworm, Colorado potato beetle, and other insect pests. Cry toxins exert their insecticidal action through binding to (glyco)protein receptors in the insect midgut, and as a consequence, insects are continuously developing resistance against these toxins. Due to the interactions of OlyA6/PlyB, PlyA2/PlyB and EryA/PlyB with their specific insect membrane lipid receptor and not with pest proteins that can be prone to mutation, the chances of evolving resistance to these insecticidal aegerolysin-based protein complexes should be extremely small.

Within the proposed project, we aim to further evaluate the potential of OlyA6/PlyB, PlyA2/PlyB and EryA/PlyB to act as biopesticides against Colorado potato beetle, through the development of genetically modified potatoes that can produce these insecticidal complexes. We will also further elucidate the molecular mechanisms of insecticidal activity of these protein complexes, by exploring the mechanism of the pore formation on CPE – containing membranes, and by searching for putative additional protein and lipid receptor(s) in the insect midgut. Finally, we will further explore the toxicity of aegerolysin/PlyB complexes against additional agriculturally important pests, as well as against non‐target insects (e.g. the bees), and rodents. The proposed study will complete the missing parts in the molecular mechanisms of ac on of these novel lipid‐binding biopes cides, and will pave the way for the development of new environment‐friendly and long‐term effective insecticides.


THE PHASES OF THE PROJECT AND THEIR REALIZATION

We are currently optimizing the OlyA, PlyA2, EryA and PlyB sequences to be used for transfection in plants. We have performed preliminary experiments with cryoelectron microscopy to determine the formation of OlyA/PlyB pores on artificial lipid vesicles containing CPE, and have performed preliminary lipidomic analyses of the colorado potato beetle. Toxicity studies on non-target organisms will be performed in spring 2020.  

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