Multiplexed host-induced gene silencing of Aspergillus flavus genes confers aflatoxin resistance in groundnut

Authors: PRASAD, KALYANI - International Crops Research Institute For Semi-Arid Tropics (ICRISAT) - India; YOGENDRA, KALENAHALLI - International Crops Research Institute For Semi-Arid Tropics (ICRISAT) - India; SANIVARAPU, HEMALATHA - International Crops Research Institute For Semi-Arid Tropics (ICRISAT) - India; Rajasekaran, Kanniah - Rajah; Cary, Jeffrey; SHARMA, KIRAN - The Energy And Resources Institute (TERI); BHATNAGAR-MATHUR, POOJA - International Maize & Wheat Improvement Center (CIMMYT)

Submitted to: Toxins
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 4/23/2023
Publication Date: 5/5/2023
Citation: Prasad, K., Yogendra, K., Sanivarapu, H., Rajasekaran, K., Cary, J.W., Sharma, K.K., Bhatnagar-Mathur, P. 2023. Multiplexed host-induced gene silencing of Aspergillus flavus genes confers aflatoxin resistance in groundnut. Toxins. 15:319. https://doi.org/10.3390/toxins15050319.
DOI: https://doi.org/10.3390/toxins15050319

Interpretive Summary: Aflatoxin contamination of one of the most edible crops around the world - peanuts otherwise called groundnuts, pose major health hazards for vulnerable populations especially in Sub-Saharan Africa and South Asia. First, we analyzed defense-related protein profile in resistant peanut lines with objective of identifying and boosting the natural resistance first with simultaneous suppression of key genes responsible for aflatoxin production. Both the processes provided an effective combination strategy for controlling fungal infection and aflatoxin contamination of peanuts. In addition to development of safe peanut crop, free of dangerous aflatoxins, the results provided in this manuscript will also be useful to other scientists for identification of key molecular targets for improvement of peanuts through biotechnology.

Technical Abstract: Aflatoxin contamination in groundnuts is a serious constraint for food safety and human health. In this study, we show that host-induced gene silencing (HIGS) is a promising method to eliminate aflatoxin contamination in transgenic groundnut. We transformed groundnut with targeted RNAi silencing of fungal aflM (ver1), essential for sporulation and aflatoxin production. Fungal biomass and aflatoxin production (<20 ppb) were significantly reduced in RNAi transgenic groundnut lines compared to WT control lines after A. flavus infection. Further, comparative proteomic analysis was performed in two contrasting groundnut genotypes, variety ICGV 91114 (susceptible) and its near-isogenic resistant HIGS lines to better understand the molecular processes of resistance to aflatoxin accumulation. Fungal differentiation and pathogenicity proteins including calmodulin, transcriptional activator-hacA, kynurenine 3-monooxygenase 2, veA, velC, abaB, and a number of aflatoxin pathway biosynthetic enzymes were downregulated in A. flavus infecting the transgenic HIGS lines. Additionally, several host resistance-associated proteins linked with fatty acid metabolism were strongly induced in the resistant HIGS lines including phosphatidylinositol phosphate kinase, lysophosphatidic acyltransferase-5, palmitoyl-monogalactosyldiacylglycerol '-7 desaturase, ceramide kinase-related protein, sphingolipid '-8 desaturase, and phospholipase-D. This study demonstrates the efficacy of RNAi-based HIGS technology in generating groundnut lines with enhanced resistance to A. flavus growth and aflatoxin contamination. Comparative proteomic analysis was also used as a means to identify groundnut metabolites that may play a significant role in resistance to A. flavus infection and aflatoxin contamination. Combined, this knowledge can be used to develop transgenic or marker-assisted groundnut breeding programs with the goal of providing a more safe and secure food supply.