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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

2018 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. Under Objective 1, Agricultural Research Service (ARS) researchers in New Orleans, Louisiana, previously identified several native proteins in corn kernels that resist infection by the fungus, Aspergillus (A.) flavus that produces aflatoxins. Such resistance-associated proteins and their corresponding genes have been identified in corn lines and these genes have been transferred to commercial varieties by classical breeding. In collaboration with the International Institute of Tropical Agriculture, Nigeria, six corn lines named TZAR 101-106 were developed and they showed resistance not only to aflatoxin-producing fungi but also to a Fusarium fungus that produces another toxin called fumonisin. Limited field evaluations of these six corn lines and test-crosses with domestic lines have been planned and will be conducted in the coming years. Meanwhile, detailed analysis of the proteins (proteomic analysis) contributing to the resistance to the toxin-producing fungi is being conducted at the Southern Regional Research Center (SRRC). Under Objective 2, Agricultural Research Service (ARS) researchers in New Orleans, Louisiana, conducted experiments on a genome-wide transcriptome (the sum of all the actively expressed genes of a corn plant) analysis of the corn-Aspergillus (A.) flavus interaction. In collaboration with the J. Craig Venter Institute, La Jolla, California, ARS researchers used the modern ribonucleic acid-sequencing (RNA-Seq; a means of determining levels of activity of individual genes in both the fungus and corn) technique to study expression of genes during the corn-A. flavus interaction. Comparative analysis will be made of A. flavus-infected kernels of hybrid TZAR 102, resistant to aflatoxin contamination and drought, released by ARS, along with a resistant line (MI 82) and a susceptible check (Va35) to delineate the molecular genetic differences that might explain the enhanced resistance to A. flavus. Data generated from RNA sequencing is currently being analyzed in-house. An interactome (the whole set of molecular interactions in a particular cell) analysis based on the RNA-Seq data will also be performed enabling identification of key global regulators of A. flavus growth and aflatoxin biosynthesis as well as developmental and virulence (ability to cause infection) factors that can serve as targets for intervention strategies. This analysis will shed light specifically on the mechanisms of fungal pathogenesis and corn resistance. A similar genome-wide transcriptome profiling study has already been completed in cotton where ARS researchers identified several key genes that were turned on during infection of A. flavus in cottonseed. Recently, a comparative transcriptome analysis was also performed in collaboration with scientists at Louisiana State University, Baton Rouge, Louisiana, that identified common genes that were significantly differentially expressed in cotton, corn, and peanut in response to A. flavus. ARS researchers and collaborators identified 26 genes common across all three crops that were considered candidate A. flavus resistant genes, which could be used to improve resistance to aflatoxin production in susceptible crops. Under Objective 3, Agricultural Research Service (ARS) researchers in New Orleans, Louisiana, made significant progress in genetic engineering of corn and cotton for resistance to Aspergillus (A.) flavus and aflatoxin production. a) Transgenic corn kernels expressing a synthetic peptide (small protein) gene AGM 182 (modeled after an antimicrobial peptide from horseshoe crab) demonstrated a significant reduction in fungal growth and aflatoxin contamination (76-98% reduction in third generation kernels). ARS researchers obtained the synthetic peptide from Nexion, Inc. and a scientist at University of Arkansas, Fayetteville, Arkansas, collaborated on transformation of corn. b) In another experiment in collaboration with scientists at the University of Arkansas in Fayetteville, Arkansas, reduction in fungal growth and toxin production was also observed from sixth generation transgenic corn kernels expressing a gene from another plant, hyacinth beans, that encodes a novel antifungal seed protein. This antifungal protein inhibits a key enzyme (alpha-amylase) that is necessary for the fungus to grow and infect seed. Significantly, reduced fungal growth and toxin production (up to 88%) was observed with transgenic corn kernels expressing this natural protein. c) Significant progress has also been achieved in experiments to understand the contribution of native corn kernel proteins to A. flavus resistance. One such protein is called Pathogenesis–Related maize seed protein (PRms). To understand its role in fungal resistance, the gene was first silenced using a ribonucleic acid interference (RNAi, a technology that enables specific genes to be targeted for down-regulation or silencing) approach in transgenic corn lines. Down-regulation of PRms in transgenic kernels resulted in a ~250-350% increase in Aspergillus flavus growth accompanied by a 4.5 to 7.5-fold higher accumulation of aflatoxins than the control plants, confirming its central role in fungal resistance. This particular gene also affected the functioning of other genes associated with disease resistance. d) Other RNAi-based approaches were carried out to silence fungal genes as well that are critical for the fungus to grow, infect and produce toxins. One such experiment to target and silence a fungal gene that codes for alpha-amylase (essential for fungal growth) resulted in development of transgenic corn lines capable of resisting Aspergillus flavus growth and aflatoxin production. The transgenic corn plants not only reduced the fungal amylase gene expression resulting in decreased fungal growth but also significantly reduced aflatoxin production by 98% in kernels. e) ARS researchers analyzed transgenic maize lines expressing RNAi in maize seed only that target two specific Aspergillus flavus genes- nsdC and veA which are required by A. flavus for production of aflatoxin and sclerotia [fungal survival structures], and numerous other toxic secondary metabolites. In vitro maize seed infection assay with A. flavus showed up to 75% reduction of fungal growth in the infected RNAi seeds as compared to the seeds from control. A significant reduction was observed of aflatoxins (up to 86%) as well as another important fungal secondary metabolite called cyclopiazonic acid (CPA) (33-82%). f) Regeneration of transgenic cotton lines expressing selected resistant genes, for example, spot11 catalase, identified through comparative transcriptomic analysis (see Objective 2) is underway. g) Transgenic cotton lines expressing a synthetic peptide named D4E1 demonstrated antifungal effects against Aspergillus flavus under greenhouse conditions or seedling pathogens under field conditions. To ascertain the mode of action of the D4E1, experiments were conducted in collaboration with University of Louisiana, Lafayette, Louisiana, using a specialized dye (Sytox Green) to track movement of peptide molecules in fungal cells. ARS researchers demonstrated that the antimicrobial activity of D4E1 is due to membrane permeabilization and accumulation of reactive oxygen species (ROS) that induce cell death. In addition to developing crops resistant to aflatoxin contamination, ARS researchers in New Orleans, Louisiana, in collaboration with Geosystems Research Institute of Mississippi State University in Mississippi State, Mississippi, based at the Stennis Space Center, Hancock county, Mississippi, developed a non-invasive, inexpensive and rapid hyperspectral imaging technique (collecting and processing information from across the light-spectrum) to detect and quantify aflatoxins in corn kernels under Objective 4. Hyperspectral instruments have already demonstrated the ability to differentiate toxigenic and atoxigenic Aspergillus flavus strains. A spectral signature to detect aflatoxin-contaminated corn has been developed and licensed. A prototype dual-camera based multispectral imaging system for rapid detection has been designed, developed, and evaluated for screening and removal technologies for automated grain handling systems in the U.S. and international markets. With funding from Gates Foundation and United States Agency for International Development (USAID) ARS researchers and collaborators developed a low cost portable technology to detect aflatoxin contamination in corn kernels for use in the developing countries. With additional funding from LaunchTN in Nashville, Tennessee, ARS researchers are in the process of assessing and refining a large version of an inspection device based on our “AflaGoggles" concept for screening aflatoxin contamination in maize - a quick and simple tool for detecting aflatoxin in corn especially in African countries. Funding from the National Science Foundation has also enabled a joint effort among Mississippi State University at Mississippi State, Mississippi, Secure Food Solutions, Inc. in Martin, Tennessee, and ARS researchers on a novel method to improve aflatoxin detection utilizing accuracy of multispectral imaging technology to develop commercial, rapid, screening equipment for aflatoxin-contaminated corn.

1. Host-induced gene silencing of fungal alpha-amylase to reduce infection and toxin production. Preharvest contamination of corn kernels with dangerous levels of aflatoxin not only reduces the value of the crop but also poses a health hazard to humans and livestock. Transgenic maize plants were generated by Agricultural Research Service (ARS) researchers in New Orleans, Louisiana, with an RNAi (ribonucleic acid interference) construct to silence the fungal alpha-amylase gene, which is essential for its growth and infection. As a result, fungal growth and aflatoxin production were significantly reduced (up to 98%). This technology, made possible by the short-lived RNA, does not require expression of a foreign protein so food produced from resistant lines of transgenic maize should be more acceptable to regulatory agencies and consumers. Corn plants carrying this gene will serve as an excellent parent material to transfer the resistant trait to other commercial varieties.

2. Transfer of a natural antifungal protein from another plant to maize for controlling aflatoxin contamination. Preharvest contamination of corn kernels with dangerous levels of aflatoxin not only reduces the value of the crop but also poses a health hazard to humans and livestock. It has been established before that fungal alpha-amylase is essential for its growth and infection. Transgenic maize plants were regenerated expressing an antifungal protein from hyacinth beans that inhibits alpha-amylase in Aspergillus flavus. This research was conducted by Agricultural Research Service (ARS) researchers in New Orleans, Louisiana, in collaboration with University of Arkansas, Fayetteville, Arkansas. Kernels from sixth generation transgenic maize plants and their progenies were screened for their ability to withstand fungal infection and toxin production. Significant reduction was obtained in fungal growth and aflatoxin production (63-88%). Corn plants carrying this gene will serve as an excellent parent material to transfer the resistant trait to other commercial varieties.

3. Detection of aflatoxin contamination using a dual-camera based multispectral imaging system. Current screening methods for aflatoxin contamination in food and feed products is wrought with detection and sampling problems and the analysis by chemical methods is a time-consuming process. A prototype dual-camera based multispectral imaging system for the purpose of rapidly screening aflatoxin contamination has been designed, developed, and evaluated. This was achieved by Agricultural Research Service (ARS) researchers in New Orleans, Louisiana. This technology enables rapid and non-destructive spectral-based aflatoxin contamination detection and removal in corn to provide toxin-free food supply.

Review Publications
Mehanathan, M., Bedre, R., Mangu, V., Rajasekaran, K., Bhatnagar, D., Baisakh, N. 2018. Identification of candidate resistance genes of cotton against Aspergillus flavus infection using a comparative transcriptomics approach. Physiology and Molecular Biology of Plants. 24(3):513–519.
Sharma, K.K., Pothana, A., Prasad, K., Shah, D., Kaur, J., Bhatnagar, D., Chen, Z.-Y., Raruang, Y., Cary, J.W., Rajasekaran, K., Sudini, H.K., Bhatnagar-Mathur, P. 2018. Peanuts that keep aflatoxin at bay: a threshold that matters. Plant Biotechnology Journal. 16:1024-1033.
Majumdar, R., Rajasekaran, K., Sickler, C.M., Lebar, M.D., Musungu, B.M., Fakhoury, A.M., Payne, G.A., Geisler, M., Carter-Wientjes, C.H., Wei, Q., Bhatnagar, D., Cary, J.W. 2017. The pathogenesis-related maize seed (PRms) gene plays a role in resistance to Aspergillus flavus infection and aflatoxin contamination. Frontiers in Plant Science. 8:1758.
Tao, F., Yao, H., Hruska, Z., Burger, L.W., Rajasekaran, K., Bhatnagar, D. 2018. Recent development of optical methods in rapid and non-destructive detection of aflatoxin and fungal contamination in agricultural products. Trends in Analytical Chemistry. 100:65-81.
Rajasekaran, K., Sayler, R.J., Sickler, C.M., Majumdar, R., Jaynes, J.M., Cary, J.W. 2018. Control of Aspergillus flavus growth and aflatoxin production in transgenic maize kernels expressing a tachyplesin-derived synthetic peptide, AGM182. Plant Science. 270:150-156.
Bhatnagar, D., Rajasekaran, K., Gilbert, M.K., Cary, J.W., Magan, N. 2018. Advances in molecular and genomic research to safeguard food and feed supply from aflatoxin contamination. World Mycotoxin Journal. 11(1):47-72.
Gilbert, M.K., Majumdar, R., Rajasekaran, K., Chen, Z.-Y., Wei, Q., Sickler, C.M., Lebar, M.D., Cary, J.W., Frame, B.R., Wang, K. 2018. RNA interference-based silencing of the alpha-amylase (amy1) gene in Aspergillus flavus decreases fungal growth and aflatoxin production in maize kernels. Planta. 247:1465–1473.
Moore, J., Rajasekaran, K., Cary, J.W., Chlan, C. 2017. Identification of resistance to Aspergillus flavus infection in cotton germplasm. Journal of Crop Improvement. 31(5):727-741.
Majumdar, R., Lebar, M.D., Mack, B.M., Minocha, R., Minocha, S., Carter-Wientjes, C.H., Sickler, C.M., Rajasekaran, K., Cary, J.W. 2018. The Aspergillus flavus spermidine synthase (spds) gene, is required for normal development, aflatoxin production, and pathogenesis during infection of maize kernels. Frontiers in Plant Science. 9:317.