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http://www.paca.inra.fr/institut-sophia-agrobiotech_eng/

Thesis defense - Dries AMEZIAN

Jeudi 20 Janvier - 9:00 - Visioconférence par ZOOM

Thesis defense
Dries AMEZIAN : "Use of the cell model of the fall armyworm, Spodoptera frugiperda, Sf9 cells to study the role of transcription factors on the expression of detoxification genes in response to xenobiotics."

En présentiel (limité) ou via Zoom :  https://inrae-fr.zoom.us/j/2943306815

ID de réunion : 294 330 6815

Président/te du jury :

  • Dr. Christine COUSTAU, DR CNRS, Sophia Antipolis

Rapporteurs/trices :

  • Pr. Emeritus Philip BATTERHAM, University of Melbourne, Australia
  • Dr. Jean-Philippe DAVID, CR CNRS, LECA, Grenoble

Examinateurs/trices :

  • Pr. Thomas VAN LEEUWEN, University of Ghent, Belgium

Directeur/trice de Thèse :

  • Dr. Ralf NAUEN, Dinstinguished Science Fellow, Bayer AG, Monheim, Germany
  • Dr. Gaëlle LE GOFF, CR INRAE, ISA, Sophia Antipolis

Abstract :

The fall armyworm (FAW), Spodoptera frugiperda (Lepidoptera: Noctuidae) is a polyphagous pest feeding on numerous host-plants including important crops such as maize, rice and sorghum. It is one of the world’s most destructive pests which only recently invaded the eastern hemisphere incl. Asia. It provides exceptional economic damage in many crops across continents each year. Controlling this insect pest largely relies on the application of insecticides resulting in the development of resistance to many classes of synthetic insecticides. FAW has developed sophisticated adaptative mechanisms to eliminate xenobiotics (plant secondary metabolites and insecticides), among them, upregulation and duplication of genes expressing detoxification enzymes. They are often expressed at low basal level and induced when the insect is exposed to xenobiotics. While the role of these enzymes is well characterized in several pest insects, the transcription factors controlling their expression remain largely unexplored. The aim of my thesis was to determine the role of Cap'n'collar isoform C (CncC) and musculoaponeurotic fibrosarcoma (Maf) in S. frugiperda adaptation to xenobiotics employing an Sf9 cell model.

I used the cell model of S. frugiperda, the Sf9 cells and showed that CncC, Maf and several detoxification enzymes are induced after exposure to indole 3-carbinol (I3C), a glucosinolate found in Brassicaceae such as cabbage and broccoli, and methoprene (Mtp), a juvenile hormone (JH) mimic insecticide. I showed that transient overexpression of CncC and Maf in Sf9 cells was followed by overexpression of several detoxification genes. In order to characterize the role of these transcription factors in response to xenobiotics two types of stably transformed cell lines were established. The first cell lines overexpress CncC, Maf or both genes while the second were mutated for CncC (Knock-Out, KO) using the CRISPR/Cas9 technique. I performed cell viability assays (MTT) and used molecular probes in High Content Screening (HCS) to test whether the modification of the CncC:Maf pathway affected the ability of Sf9 cells to cope with toxic stress. The OE cell lines were more tolerant to I3C and Mtp than the control (wildtype Sf9 cell line), whereas the KO cell lines were more sensitive to these xenobiotics. The activities of some detoxification enzymes, carboxylesterases (CEs) and glutathione S-transferases (GSTs) toward model substrates were also increased in OE cell lines, whereas they were decreased in KO cell lines. Recent studies have suggested that activation of the CncC:Maf pathway is mediated by the production of reactive oxygen species (ROS) upon toxic stress. I therefore measured ROS production in Sf9 cells treated with I3C and Mtp. Both xenobiotics triggered in-cell ROS pulses although at limited levels in OE lines, unlike to KO lines for which ROS levels were more prominent. The use of an antioxidant suppressed the ROS pulses and restored tolerance of KO cells to I3C and Mtp. Finally, I compared the differentially expressed genes in the OE and KO cell lines in a transcriptomic analysis using RNA-seq. This allowed me to identify genes potentially controlled by CncC and Maf, most of them being detoxification genes with a role in insecticide resistance and metabolism of plant compounds as demonstrated in several studies. In conclusion, I present here new data in designed model Sf9 cell lines suggesting that the CncC:Maf signaling pathway plays a central role in FAW adaptation to toxic environmental compounds and insecticides. This knowledge helps to better understand pathways in detoxification gene expression and can be helpful to design next generation insect control measures by interfering with these pathways and detoxification gene expression.

Keywords:

Cap’n’collar isoform C (CncC), detoxification, resistance, plant adaptation, gene regulation