Deciphering Bacteriocyte-specific Cysteine-Rich protein structure and activity: a new potential BIOpesticide FAMILY alternative to conventional chemical treatments – BIOFAMILY

BIOFAMILY is a timely project, integrating the most recent scientific advances in the field of biopeptides. It engages a multidisciplinary approach that includes bioinformatics, insect physiology, biochemistry, synthetic chemistry, molecular biology and structural biology. Its originality and novelty reside at (i) creating a novel field of investigation: little is known about the diversity and properties of these small biological molecules considering the billions of sequences of DRPs yet identified, and the fast-increasing number of new sequences appearing in the recent literature; (ii) developing new methods: the proposal will integrate recent technical innovations on insect pests manipulation including RNA interference, flow cytometry methods adapted to study insect endosymbionts, whole mount immunohistochemistry and transmission electron microscopy approaches adapted to aphid bacteriocytes, and the state-of-the-art of the chemical synthesis of long disulfide rich peptides; (iii) designing new concepts: public concern over pesticide use and more stringent environmental regulations create the need for ecologically friendly innovative technologies involving new biopesticide molecules.

We have established that the BCR peptide family is specific to aphid species, making phylogenic research of this family very exciting. The BCR4 peptide exhibits acute insecticidal activity on aphids, making it a potential candidate for the development of a new insecticide family.

With the objective of developing the use of BCR peptides as bioinsecticides, the BIOFAMILY project aims at deciphering evolutionary and functional properties of this novel peptide family at different integration levels:
- Action of BCRs as entomocide agents and side effects of BCR on honey bees.
- Effect of BCRs on endosymbiont localization and bacteriocyte integrity
(induction of cellular swelling and mortality)
- Ability of BCRs to interfere with microbial survival and growth
- Binding activity & identification of the structural determinants responsible for BCR insecticidal activity
- Structure / Activity & Structure/evolution relationships

Da Silva P, 2019. Use of peptides as insecticidal agents. PCT n° : PCT/FR2019/051679.

Insect pests can cause important crop plant damages worldwide. Among them, aphids are serious pests in agriculture and cause significant losses by reducing plant growth through deprivation of nutrients, and, most importantly, by transmitting numerous plant viruses able to cause diseases in nearly all important crops. Up to now, most aphid pest control strategies have relied on the use of specific sets of systemic chemical pesticides. Those chemicals have been extensively used worldwide in the last decades, and increasingly stigmatized because of their negative impact on human health, their persistence in the environment, and their toxicity to non-target organisms. This creates the need for the development, of new eco-friendly technologies, which will undoubtedly benefit from the discovery of new biopesticide molecules that generate low non-target effects on the environment.
In this perspective, a new class of seven miniproteins, called Bacteriocyte-specific Cysteine Rich (BCR) peptides, has recently been identified in Acyrthosiphon pisum, commonly known as the pea aphid. BCR are small disulfide-bond rich proteins encoded by orphan genes and expressed exclusively in aphid bacteriocytes, the specialized insect cells that house endosymbiotic bacteria. Interestingly, all BCR-encoding genes are expressed in bacteriocyte cells all along aphid developmental life stages, suggesting that BCR proteins may be critical to endosymbiosis, presumably by interacting with endosymbiont population dynamics.
Remarkably, preliminary results from BF2i lab have uncovered that at least one member of this BCR family, the BCR4 peptide, exhibits an outstanding insecticidal activity against the pea aphid. In addition to their putative interaction of BCRs with symbiosis, this indicates that these peptides may potentially be used as biopesticides. The resolution of the BCR4 and BCR8 three-dimensional (3D) NMR structures by the CBM lab revealed that BCRs are a new structural family of antimicrobial peptides. The hypothesis of an antimicrobial activity of BCR4 and BCR8 was confirmed with the demonstration of a bactericidal activity suggesting a probable implication of the BCRs in the symbiosis regulation.
Our hypothesis is that BCRs target B. aphidicola cellular functions within bacteriocytes. The ingestion of exogenous BCR may be critical to endosymbiosis, presumably decreasing endosymbiont population and subsequently leading to the death of aphids. This indicates that these peptides may potentially be used as biopesticides
BIOFAMILY aims at providing a deep characterization of BCR peptides on both insect endosymbiosis disturbance and plant protection. More precisely, BIOFAMILY has three main objectives: (i) understanding the evolutionary context in which these natural toxins have evolved in the Aphididae family in order to draw evolutionary patterns and scenarios, potentially related to symbiotic relationships that BCR genes have undergone; (ii) tackling BCR functions by determining their subcellular expression and distribution and by analysing their impact on endosymbiont control and bacteriocyte differentiation and homeostasis resulting from BCR silencing (iii) characterising the 3D structure and the biochemical activity of BCRs in order to gain insights on their structure/activity and their evolution/structure relationships. This will pave the way to the identification of new insecticidal modes of action with novel molecular targets and design of more selective and eco friendly pesticides.
The strength of this project is to bring together highly skilled researchers and their complementary approaches around a single high value-added question: Structure and Activity of Bacteriocyte-specific Cysteine-Rich proteins: a new potential biopesticide family alternative to conventional chemical treatments.