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ARS Home » Southeast Area » New Orleans, Louisiana » Southern Regional Research Center » Food and Feed Safety Research » Research » Research Project #430862

Research Project: Use of Classical and Molecular Technologies for Developing Aflatoxin Resistance in Crops

Location: Food and Feed Safety Research

2020 Annual Report

Objective 1. Develop aflatoxin-resistant corn with enhanced resistance traits against other mycotoxins and drought tolerance. Identify gene regulatory factors, networks and pathways related to resistance-associated proteins (RAPs). These data are then transferred to others to assist in selection by marker-assisted breeding. Objective 2. Identify resistance associated protein (RAPs) genes from corn and cotton using transcriptomic analyses of the Aspergillus flavus-host plant interaction and evaluate for control of fungal growth and aflatoxin contamination. Objective 3. Develop and evaluate transgenic corn and cotton containing over-expressed identified RAP genes (Objectives 1 and 2) or with RNA interference (RNAi)-based silencing of Aspergillus flavus genes critical to growth and aflatoxin production. Objective 4. Advance and license the rapid, non-destructive hyperspectral imaging technology; develop and evaluate instruments suitable for different user platforms.

Aflatoxin contamination in crops such as corn, cottonseed, peanut, and tree nuts caused by Aspergillus (A.) flavus is a worldwide food safety problem. Aflatoxins are potent carcinogens and cause enormous economic losses from destruction of contaminated crops. Utilizing resistant germplasm against A. flavus growth and aflatoxin contamination is the most practical solution for pre-harvest control, the overall goal of this project plan. To this end, we plan to elucidate the complex, multi-genic resistance mechanisms in corn identified in resistant genotypes bred through a collaborative program. We will understand the molecular basis of seed-based resistance through transcriptomic analysis of the corn-A. flavus interaction allowing identification of genes and networks correlated with resistance for use in marker-assisted breeding. RNA interference technology will be used to a) determine the roles and contribution of selected corn genes to overall resistance; and b) to target genes critical to A. flavus growth and toxin production to generate corn varieties with enhanced resistance. Resistance genes identified from transcriptomic analysis of the A. flavus-cottonseed interaction, along with identified corn resistance genes will be over-expressed in cotton to achieve enhanced resistance. Finally, instrumentation for non-destructive, hyperspectral imaging detection will be refined and modified to address practical applications suitable for different user-specified platforms. The proposed research will result in development of cotton and corn germplasm with enhanced resistance to A. flavus growth and aflatoxin contamination. Information and material generated from this research will benefit the scientific community, stakeholder groups, food and feed safety regulatory agencies and consumers, both nationally and internationally.

Progress Report
Substantial progress has been made in all four objectives of the project, all of which fall under National Program 108 Food Safety, Component 1, Foodborne Contaminants. Objective 1, ARS researchers in New Orleans, Louisiana, have identified several native proteins in corn kernels linked with enhanced resistance to infection by the aflatoxin (AF, a carcinogen)-producing fungus, Aspergillus (A.) flavus. Genes encoding such resistant-proteins have been transferred into commercial varieties by classical breeding. In collaboration with the International Institute of Tropical Agriculture, Nigeria, ARS reseachers at New Orleans, Louisiana, produced six inbred lines (with less genetic variation) that were deposited into the corn germplasm or repository and made available to other researchers. The combining ability of exotic corn lines resistant to AF accumulation was examined as a means to generate new inbred lines for cultivation in the United States with enhanced resistance to AF contamination. Two productive corn varieties with resistance to AF contamination were released as “Sammaz45” in Nigeria and “Syn2-YF2” in Cameroon. Several elite hybrids formed from AF resistant inbred lines are currently in advanced stage of testing for possible cultivation and they are available to all breeders from the ARS corn germplasm. Meanwhile, detailed analyses of the proteins contributing to resistance to toxin-producing A. flavus in these inbreds and hybrids are being conducted at the Southern Regional Research Center (SRRC), New Orleans, Louisiana. ARS researchers in New Orleans, Louisiana, have also demonstrated the critical roles in fungal growth and toxin production of polyamines (PAs), ubiquitous nitrogenous molecules that control growth and development of plants under biotic (pest) and abiotic (environmental) stress. ARS scientists in New Orleans, Louisiana, have initiated a novel project based on engineering for PA content by introducing a native key PA biosynthetic gene (S-adenosylmethionine decarboxylase or SAMDC) into corn kernels to protect from AF contamination (Objective 3). Under Objective 2, ARS researchers in New Orleans, Louisiana, in collaboration with the J. Craig Venter Institute, La Jolla, California, used modern RNA-Sequencing (RNA-seq, a means of determining levels of activity of individual genes in both the fungus and corn) technology to study the expression of genes during the corn-A. flavus interaction. Comparative analysis indicated several novel gene candidates and biological processes related to host resistance to fungal infection and AF contamination. The genes and gene pathways of interest identified from the RNA-seq study include tetraspanins, acetylornithine deacetylase, macrophage migratory inhibitory factor, and genes in the flavonoid and flavone biosynthetic pathway and protein detoxification. Gene co-expression analysis identified at least two gene network modules that were highly preserved in resistant lines compared to susceptible corn line, indicating their role(s) in resistance mechanisms. For example, genes for jasmonic acid signaling, nitrate assimilation, and production of glucoside and long chain fatty acid were highly enriched in these modules. Non-coding small RNAs (ncRNAs, do not encode for proteins) present in corn lines were also sequenced and analyzed in collaboration with researchers at Louisiana State University, Baton Rouge, Louisiana to identify microRNAs (miRNAs, a class of ncRNAs that can control the expression of specific genes) with potential roles in A. flavus resistance. Differences in miRNAs were observed between resistant and susceptible corn lines. Expression analysis of the candidate target genes of such miRNAs and potential novel miRNAs are being carried out. These data will be compared with the RNA-seq data (presented above) to identify the miRNA-mRNA modules demonstrating roles in resistance to A. flavus infection in corn. Analysis of A. flavus small RNA (smRNA) sequence data by ARS researchers in New Orleans, Louisiana, indicated the presence of unconventionally sized (approx. 15 base pairs, normally 21-24 base pairs) smRNA molecules. These inhibitory smRNAs were generated by three potential Dicer-like proteins (Dicer is an enzyme which facilitates the activation of the RNA-induced silencing complex or RISC, which is essential for targeted gene silencing). A series of A. flavus Dicer mutant strains have been generated where one, two, or all three of these putative Dicer-like genes were knocked out in an effort to determine which of these genes is responsible for production of these unusually small RNAs. Objective 3, ARS researchers in New Orleans, Louisiana, continued to make significant progress in the genetic engineering of corn for resistance to A. flavus and AF contamination. Transgenic corn lines expressing double-stranded RNA molecules in corn seed were analyzed that specifically target fungal toxin biosynthetic genes for silencing. Recently, corn plants with the capability to silence a key enzyme involved in the AF biosynthetic pathway (aflM) were field tested for three years and were found to provide significant resistance to natural AF contamination (up to 95% reduction). ARS researchers in New Orleans, Louisiana, are also generating more corn lines in which up to four fungal genes required for AF production (for example, aflR, aflS, aflM, aflC, veA, nsdC) have been targeted for silencing. These transgenic corn lines will be analyzed further for their effectiveness in controlling fungal growth and toxin production. New experiments to produce transgenic corn lines expressing improved synthetic peptides have also been initiated under a cooperative agreement. Similarly, ARS researchers New Orleans, Louisiana, in collaboration with Louisiana State University, Baton Rouge, Louisiana, identified a key gene that is overexpressed in cotton boll pericarp (the outer wall) called spot11 catalase from RNA-seq analyses (Objective 2). Limited number of transgenic cotton lines expressing this gene have been regenerated for further molecular analyses and resistance to fungal infection and AF contamination. In addition to developing crops resistant to AF contamination, ARS researchers in New Orleans, Louisiana, in collaboration with Geosystems Research Institute of Mississippi State University (MSU) in Mississippi State, Mississippi, based at the Stennis Space Center, Hancock county, Mississippi, developed a non-invasive, inexpensive and rapid hyperspectral imaging technique (that employs a large part of the electromagnetic spectrum, especially that includes those parts of the spectrum invisible to the eye) that collects and processes information from across the light-spectrum. This imaging technique detects and quantifies aflatoxins in corn kernels under Objective 4. Hyperspectral instruments have already demonstrated the ability to differentiate AF and non-AF A. flavus strains. A spectral signature to detect AF-contaminated corn has been developed and licensed. A joint effort between ARS researchers, Mississippi State University and a collaborator in Martin, Tennessee, was established to develop a novel method to improve AF detection utilizing the accuracy of multispectral imaging technology and to develop commercial, rapid, screening equipment for AF-contaminated corn. With support from the United States Agency for International Development, ARS researchers and collaborators upgraded the initial AflaGoggles concept into a low-cost portable technology to detect AF contamination in corn kernels for use in developing countries. The current outcome is a portable tablet-based detection device equipped with UV-LED light source. The device employs an Android App developed in-house for image acquisition, detection, and marks the contaminated kernels so the user can manually sort them out. Experiments have been implemented using inoculated corn kernels, which mimics natural field AF contamination. The research team also continues to seek support from other sources to implement field tests in developing countries where AF contamination of crops is endemic.

1. Classic breeding results in aflatoxin resistant corn varieties. Aflatoxin (a carcinogen) contamination in a major food crop worldwide, corn, caused by Aspergillus (A.) flavus is not only a food safety problem impacting human and animal health but also it results in huge economic losses to farmers and stakeholders due to contaminated produce being unsuitable for market. In order to develop strategies to mitigate aflatoxin contamination of corn and other crops, it is important to develop corn lines resistant to A. flavus infection and aflatoxin contamination in edible kernels. Resistant proteins and their corresponding genes have been identified in corn lines by ARS researchers in New Orleans, Louisiana, and these genes have been transferred to commercial varieties by classical breeding and they are available to U.S. breeders. In collaboration with the International Institute of Tropical Agriculture, Nigeria, additional hybrids were developed which showed resistance to aflatoxin contamination. Two productive corn varieties with resistance to aflatoxin contamination were released as “Sammaz45” in Nigeria and “Syn2-YF2” in Cameroon for cultivation. Additional hybrids for other countries where aflatoxin contamination is endemic are also being developed. Aflatoxin-resistant corn lines improve food safety and security worldwide while reducing economic losses borne by farmers.

2. Host-induced gene silencing of Aspergillus (A.) flavus genes reduces infection and aflatoxin production in corn kernels. Corn is an important food and feed crop and it is highly susceptible to A. flavus infection and aflatoxin (a carcinogen) contamination. Transgenic corn plants were generated by ARS researchers in New Orleans, Louisiana, in collaboration with scientists at Louisiana State University, Baton Rouge, Louisiana, to selectively silence one of the key aflatoxin pathway genes called aflM using a host-induced gene silencing approach. The transgenic lines with the capability of silencing fungal aflM were field tested for three years. Aflatoxin production was significantly reduced (up to 95%) under natural infection and even under artificial infection the aflatoxin levels were reduced by 77%. This technology, made possible by the short-lived silencing RNA molecules, does not require expression of a foreign protein in the plant so food produced from resistant transgenic lines of corn should be more acceptable to regulatory agencies and consumers. Corn plants carrying this gene will also serve as an excellent parent material to transfer the resistance trait to other commercial varieties. Aflatoxin-resistant corn lines improve food safety and security while minimizing economic losses borne by farmers.

Review Publications
Tao, F., Yao, H., Hruska, Z., Liu, Y., Rajasekaran, K., Bhatnagar, D. 2019. Detection of aflatoxin B1 on corn kernel surfaces using visible-near infrared spectroscopy. Journal of Near Infrared Spectroscopy. 28(2):59-69.
O'Neill, M.J., Chan, K., Jaynes, J.M., Knotts, Z., Xu, X., Abisoye-Ogunniyan, A., Guerin, T., Schlomer, J., Li, D., Cary, J.W., Rajasekaran, K., Yates, C., Kozlov, S., Andresson, T., Rudloff, U. 2020. LC-MS/MS assay coupled with carboxylic acid magnetic bead affinity capture to quantitatively measure cationic host defense peptides (HDPs) in complex matrices with application to preclinical pharmacokinetic studies. Journal of Pharmaceutical and Biomedical Analysis. 181:113093.
Raruang, Y., Omolehin, O., Hu, D., Wei, Q., Han, Z.-Q., Rajasekaran, K., Cary, J.W., Wang, K., Chen, Z.-Y. 2020. Host induced gene silencing targeting Aspergillus flavus aflM reduced aflatoxin contamination in transgenic maize under field conditions. Frontiers in Microbiology. 11:754.
Sengupta, S., Rajasekaran, K., Baisakh, N. 2019. Basic characterization of plant actin depolymerizing factors: a simplified, streamlined guide. Protocol Exchange.
Hruska, Z., Yao, H., Kincaid, R., Tao, F., Brown, R.L., Cleveland, T.E., Rajasekaran, K., Bhatnagar, D. 2020. Spectral-based screening approach evaluating two specific maize lines with divergent resistance to invasion by aflatoxigenic fungi. Frontiers in Microbiology. 10:3152.
Meseka, S., Williams, W.P., Warburton, M.L., Brown, R.L., Ortega, A., Bandyopadhyay, R., Menkir, A. 2018. Heterotic affinity and combining ability of exotic maize inbred lines for resistance to aflatoxin accumulation. Euphytica. 214:184.
Musungu, B.M., Bhatnagar, D., Payne, G.A., O'Brian, G., Quiniou, S., Geisler, M., Fakhoury, A. 2020. Use of dual RNA-seq for systems biology analysis of zea mays and Aspergillus flavus interaction. Frontiers in Microbiology. 11:853.