Location: Food and Feed Safety Research
2021 Annual Report
Objectives
Objective 1: Identify differentially expressed genes in resistant [R] and susceptible [S] corn lines that can serve as targets in controlling Aspergillus flavus and aflatoxin contamination.
Objective 2: Identify and characterize corn seed metabolites for enhancing resistance to aflatoxin contamination.
Objective 3: Develop and evaluate transgenic corn lines by over-expressing resistance-associated protein genes, or gene editing and silencing of Aspergillus flavus genes critical to growth and aflatoxin production.
Objective 4: Develop and evaluate different spectral-based imaging instruments for non-destructive detection of aflatoxin contamination. Advance and commercialize hyperspectral-based, rapid and non-invasive, imaging technology.
Approach
Aflatoxin (AF) contamination in food and feed crops such as corn, peanut, cottonseed, and tree nuts, caused by Aspergillus flavus, is a global concern that compromises food safety and marketability. Aflatoxins are potent carcinogens and their contamination in food are one of the major causes of liver cancer worldwide. The most efficient and practical approach to reduce pre-harvest AF contamination in corn is the development of resistant lines, the overall goal of this project plan. We will delineate the molecular basis of A. flavus resistance in corn seeds through “systems biology” approach that will involve a combination of transcriptomic, proteomic, and metabolomic analyses. Identification of novel regulatory genes and gene networks that play key roles in host plant resistance against the fungus will contribute to the development of robust markers for use in marker-assisted breeding and/or to the identification of candidate genes for editing. We will apply functional genomics to identify key metabolic pathways (polyamines, carotenoids, flavonoids) that contribute to resistance against A. flavus and AF production. Transgenic corn expressing antifungal proteins/peptides that strongly inhibit A. flavus growth will be generated. In addition, “host induced gene silencing” approach will be used to target fungal genes that are critical for growth, pathogenesis, and production of AFs. Finally, non-destructive, hyperspectral-based imaging systems for several platforms will be refined and commercialized to detect AF contamination in stored kernels. The knowledge and products generated from this research will be invaluable for the consumers, stakeholder groups, scientific community, and regulatory agencies to protect and preserve food safety in the United States and abroad.
Progress Report
This report documents progress for Project 6054-42000-027-00D, which started in April 2021. This project, under National Program 108 Food Safety, Component 1, Foodborne Contaminants, started only three months ago on 19 April 2021 and as such, ARS scientists in New Orleans, Louisiana report a limited progress report. The objective of this project is to develop aflatoxin resistant corn lines using omic (advanced molecular techniques such as genomics, proteomics, metabolomics and interactomics) technologies. To accomplish our objectives under Objective 1, scientists obtained genetically uniform lines of corn (called near isogenic lines or NILs) from ARS scientists in Mississippi State to avoid experimental error due to genetic variation. ARS scientists in New Orleans, Louisiana increased seed of the parental genotypes Mp313 (resistant) and Va35 (susceptible) and their crosses that harbor different genes within regions of the chromosomes that contribute to aflatoxin resistance. Seeds from these NILs were grown and self-pollinated in a temperature-controlled greenhouse. Experimental plants were grown soon after and the corn cobs were inoculated four weeks after pollination with A. flavus strain 3357 along with negative controls. Kernel samples were collected for RNA, microRNA sequencing, comparative proteomic analysis (Objective 1) and for various plant metabolites which may play a positive role in providing aflatoxin resistance - for example, carotenoids (pigmented phytonutrient chemicals), polyamines (nitrogenous amino groups ubiquitous in all living organisms which respond to stress and disease defense) and flavonoids (phytonutrient chemicals with antioxidant and antimicrobial traits) as indicated in Objective 2. ARS scientists in New Orleans, Louisiana objective is to use the same sample sets for analysis of gene expression as well as plant metabolites to get a comprehensive understanding of the resistance mechanism at the plant level. For this purpose, scientists recently acquired and installed a sophisticated, high resolution mass spectrometer (called Xevo G2 SX QTOF HRMS) to analyze primary and secondary metabolites produced by corn kernels in response to A. flavus infection. This sensitive instrument is being calibrated to detect metabolites in the low parts per billion (ppb) range, a large improvement over previous method.
Objective 3, ARS scientists have already made several gene constructs with potent synthetic antifungal peptide genes for corn transformation. This project is a collaboration with Genvor Company. ARS scientists in New Orleans, Louisiana are also continuing our efforts to generate corn lines which can selectively shutdown fungal genes upon infection to inhibit their growth or toxin production. Key fungal genes targeted will be derived from the results of the sister project 6054-41420-009-00D - “Aflatoxin control through identification of intrinsic and extrinsic factors governing the Aspergillus flavus-corn interaction”. Corn transformation experiments will be performed by ARS scientists on a limited scale, as most of the service providing laboratories are either closed or having operational difficulties. To control postharvest aflatoxin contamination of corn kernels, ARS scientists will be continuing our efforts to develop small, portable instruments to detect -contaminated corn kernels under Objective 4. ARS scientists are developing a tablet-based aflatoxin-detection device equipped with UV-LED light source. Contamination detection and sorting with the device has already been completed using field-grown corn in the United States. Continued efforts will seek to improve the performance of the tablet device and its portability for practical, real-world application, especially in small farms in developing countries. Improved accuracy and speed of detection will be analyzed through updated hardware and software.
Accomplishments
