Targeting Aflatoxin Resistance in Groundnut: Gene Discovery and CRISPRCas9-based Validation

Aflatoxin contamination, primarily caused by Aspergillus flavus, significantly threatens food safety and human health. Despite extensive research, the molecular and biochemical mechanisms underlying resistance remain only partially understood. Various strategies have been explored, including genetic analyses, cellular defense responses such as secondary cell wall reinforcement, biocontrol agents, and identifying resistance- and susceptibility-associated genes. While these studies have identified potential targets for precision breeding in groundnut, the complexity of resistance mechanisms during Aspergillus infection remains challenging. This study employed an integrative metabolomics and proteomics approach to investigate resistance mechanisms in two contrasting peanut genotypes: transgenic 4RNAi-expressing groundnut (resistant) and WT-ICGV 91114 (susceptible). We identified key regulatory events influencing enzyme activity, post-translational modifications, and gene expression during Aspergillus infection. Notably, proteomic changes preceded metabolite alterations, underscoring the significance of protein-level regulation in resistance. Integrating metabolomics and proteomics revealed metabolic and protein profiles linked to phenylpropanoid, flavonoid, and fatty acid biosynthesis, pathways crucial for suppressing aflatoxin production. Furthermore, we investigated putative susceptibility-associated genes, providing insights into potential targets for precise breeding interventions. To explore genetic modification strategies, we developed a proof-of-concept CRISPR/Cas9 system in groundnut via Agrobacterium-mediated transformation, achieving a transformation efficiency of 20% and an editing efficiency of 6% by targeting the phytoene desaturase (PDS) gene. Building on this system, we used CRISPR-Cas9 to manipulate susceptibility genes involved in host-pathogen interactions. This enabled us to study how groundnut recognizes fungal pathogens, triggers defense responses, and regulates metabolic pathways to counteract aflatoxin biosynthesis. Understanding these molecular interactions provides a foundation for breeding aflatoxin-resistant groundnut varieties. This comprehensive study advances knowledge of resistance mechanisms against Aspergillus infection and aflatoxin contamination, laying the groundwork for sustainable agricultural strategies to enhance food safety.