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

Location: Food and Feed Safety Research

2019 Annual Report

Objectives
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.

Approach
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, continue to identify several native proteins in corn kernels with enhanced resistance to infection by the aflatoxin producing fungus, Aspergillus (A.) flavus. 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, ARS researchers produced six corn lines named TZAR 101-106 that 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 conducted as a part of Southeast Regional Aflatoxin Test (SERAT2) in multiple locations (Starkville, Mississippi; College Station, Texas; Lubbock, Texas; and Carbondale, Illinois). TZAR lines and test-crosses performed well against aflatoxin contamination. Meanwhile, detailed analyses of the proteins (proteomic analysis) contributing to resistance to toxin-producing fungi in TZAR lines and the test-crosses 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 by polyamines (PAs), which are ubiquitous nitrogenous molecules that control growth and development of plants under biotic (pest) and abiotic (drought, salt) stress. First, inactivation of a key fungal gene, spermidine synthase (Spds), was demonstrated to reduce fungal growth, pathogenicity, and aflatoxin production in corn kernels. In addition, analysis of maize genotypes susceptible or resistant to Aspergillus flavus identified a key gene (S-adenosylmethionine decarboxylase or SAMDC) that regulates the production of higher PAs, possibly contributing to fungal resistance. The fungal and plant genes identified from this work provide potential targets for improvement of maize resistance to fungal colonization and aflatoxin production. 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 the expression of genes during the corn-A. flavus interaction. Comparative analysis will be made of A. flavus-infected kernels of the aflatoxin and drought resistant hybrid TZAR 102, released by ARS, along with an aflatoxin-resistant line (MI82) and a susceptible control (Va35). This will allow us to delineate the molecular genetic differences that might explain the enhanced resistance to A. flavus. Big data analysis of ribonucleic acid (RNA) sequencing is on-going with Louisiana State University, Baton Rouge, Louisiana, to identify key genes at the mRNA (transmit genetic information from DNA to make amino acid sequence of the protein) and microRNA (miRNA - small non-coding RNA molecule containing about 22 nucleotides that functions in RNA silencing and regulation of gene expression) levels. An interactome analysis which analyzes the whole set of molecular interactions in a particular cell based on the RNA-Seq data of the A. flavus-corn interaction is also being performed to enable the 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. Using a similar approach ARS researchers 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. Transgenic cotton lines expressing this gene are being regenerated and will be analyzed for molecular and phenotypic resistance to fungus. Under Objective 3, Agricultural Research Service (ARS) researchers in New Orleans, Louisiana, made significant progress in the genetic engineering of corn for resistance to Aspergillus (A.) flavus and aflatoxin production. ARS researchers analyzed transgenic maize lines expressing double-stranded ribonucleic acid (RNA) molecules from maize seed that target two specific A. flavus genes, either alone or in combination. These two target genes, nsdC and veA, which are required by A. flavus for production of aflatoxin and fungal survival structures, sclerotia, along with numerous other toxic secondary metabolites. An in vitro maize seed infection assay with A. flavus showed up to 75% reduction of fungal growth in the infected ribonucleic acid interference (RNAi) seeds targeting nsdC and veA as compared to the seeds from a control plant. A significant 86% reduction of aflatoxins was observed along with 80% reduction of another important fungal secondary metabolite called cyclopiazonic acid (CPA). ARS researchers in New Orleans, Louisiana, have also generated more corn lines in which up to four fungal toxin biosynthetic genes or global regulators (aflR, aflS, aflM, aflC, veA, nsdC) have been targeted for silencing. These corn lines will be analyzed further for their effectiveness in controlling fungal growth and toxin production. 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 (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 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 toxigenic and atoxigenic Aspergillus flavus strains. A spectral signature to detect aflatoxin-contaminated corn has been developed and licensed. A hyperspectral camera system based on pushbroom and rotational scan for whole corn ear surface imaging and contamination mapping has been developed and tested. A joint effort between ARS researchers, Mississippi State University and a collaborator in Martin, Tennessee, was established to develop a novel method to improve aflatoxin detection utilizing accuracy of multispectral imaging technology and to develop commercial, rapid, screening equipment for aflatoxin-contaminated corn. For this effort, a prototype dual-camera based multispectral imaging system for the purpose of rapidly screening aflatoxin contamination has been designed, developed, and evaluated. Funds were also secured from the National Science Foundation (2017-2018) for a project on “Novel method to improve aflatoxin detection accuracy of multispectral imaging technology.” The dual-camera system has been tested with field-inoculated corn as well as commercial samples from three states, Arkansas, Mississippi, and Tennessee, in 2018. With support from the United States Agency for International Development, ARS researchers and collaborators developed a low-cost portable technology to detect aflatoxin contamination in corn kernels for use in developing countries. The current outcome is a tablet-based sorting device equipped with UV-LED light source. The research team has secured funds from LaunchTN in Nashville, Tennessee, and is actively seeking support from other sources to implement field tests in developing countries.

Accomplishments
1. Host-induced gene silencing of Aspergillus (A.) flavus genes to reduce infection and aflatoxin production in maize kernels. Maize is an important food and feed crop and it is highly susceptible to Aspergillus (A.) flavus infection and aflatoxin (AF, a carcinogen) contamination. ARS researchers in New Orleans, Louisiana, have produced transgenic maize plants, which can withstand fungal infection and toxin production through ribonucleic acid (RNA) interference-mediated host-induced gene silencing (HIGS; a mechanism in the plant that produces small RNA molecules that can target specific genes for silencing). The HIGS process targets key A. flavus genes to reduce fungal virulence, growth and/or AF biosynthesis in susceptible maize crop and it has proven to be a promising and consumer-friendly approach to control dangerous levels of carcinogenic AF. The genes that were targeted for silencing include the veA and nsdC genes, both global regulators required for normal A. flavus development and AF production. Fungal growth was monitored in transgenic kernels using a green fluorescent protein (GFP)-expressing A. flavus and AF measurement was carried out by UPLC (Ultra High Pressure Liquid Chromatography). Results from transgenic kernel infection studies demonstrated significant reductions in fungal growth, invasion, and AF production in transgenic maize ribonucleic acid interference lines (85-90% reduction compared to controls). A significant reduction by up to 82% was also observed in another toxic secondary metabolite called cyclopiazonic acid (CPA).

2. Transgenic maize kernels expressing a synthetic peptide are resistant to aflatoxin contamination. Maize is an important food and feed crop and it is highly susceptible to Aspergillus flavus infection and aflatoxin (a carcinogen) contamination. ARS researchers in New Orleans, Louisiana, have determined that a synthetic peptide (small protein) called AGM 182, modeled after an antimicrobial peptide from horseshoe crab, provides significant control of Aspergillus flavus growth and toxin production. ARS researchers also determined this peptide is not toxic to animals and human beings. Genetically altered corn kernels expressing the AGM 182 gene demonstrated a significant reduction in fungal growth and aflatoxin contamination (76-98% reduction in third generation kernels). The development of corn containing the peptide was a collaborative effort between ARS researchers and a CRADA partner that provided the synthetic peptide, along with a scientist at University of Arkansas, Fayetteville, Arkansas. Transgenic lines will be advanced to at least two more progenies and multiplied in the greenhouse for evaluation under field conditions.

3. Maize genotypes resistant to aflatoxin contamination have higher levels of polyamines than susceptible lines. Maize, an important food and feed crop, is highly susceptible to Aspergillus flavus infection. Upon infection, the fungus produces carcinogenic aflatoxins and numerous other toxic secondary metabolites that adversely affect crop value and human health worldwide. The role of polyamines (PAs), which are ubiquitous nitrogenous molecules that act as global regulators of growth and development fortifying plant productivity under ambient or stress conditions, in Aspergillus (A.) flavus resistance and aflatoxin production in resistant and susceptible maize lines was determined. In collaboration betweem ARS researchers in New Orleans, Louisiana, scientists from USDA Forest Service and University of New Hampshire, Durham, New Hampshire, analysis of PA content in both resistant (TZAR102 and MI82) and susceptible (SC212) corn kernels was conducted. The analysis indicated that resistant varieties showed higher expression of PA biosynthetic genes upon A. flavus infection compared to the susceptible control kernels. The resistant lines accumulated higher amounts of PAs such as spermidine (Spd), spermine (Spm), and specific antimicrobial PA conjugates, showed altered amino acids content, and had lower levels of fungal load and aflatoxin contamination. In a parallel study with A. flavus, inactivation of a key PA gene, spermidine synthase (Spds), was demonstrated to reduce fungal growth, pathogenicity, and aflatoxin production in corn kernels. Combined together, these results provide a valid means of controlling A. flavus growth and aflatoxin production in corn through engineering of PA biosynthesis, either by over expression in the plant or by silencing the fungal gene by host-induced gene silencing.

Review Publications
Tao, F., Yao, H., Hruska, Z., Liu, Y., Rajasekaran, K., Bhatnagar, D. 2019. Use of visible-near-infrared (Vis/NIR) spectroscopy to detect aflatoxin B1 on peanut kernels. Applied Spectroscopy. 73(4):415-423. https://doi.org/10.1177/0003702819829725.
Rajasekaran, K., Sayler, R.J., Majumdar, R., Sickler, C.M., Cary, J.W. 2019. Inhibition of Aspergillus flavus growth and aflatoxin production in transgenic maize expresing the a-amylase inhibitor from Lablab purpureus L. Journal of Visualized Experiments. 144:e59169. https://doi.org/10.3791/59169.
Sengupta, S., Rajasekaran, K., Baisakh, N. 2018. Natural and targeted isovariants of the rice actin depolymerizing factor 2 can alter its functional and regulatory binding properties. Biochemical and Biophysical Research Communications. 503:1516-1523. https://doi.org/10.1016/j.bbrc.2018.07.073.
Moore, J., Rajasekaran, K., Cary, J.W., Chlan, C. 2018. Mode of action of the antimicrobial peptide D4E1 on Aspergillus flavus. International Journal of Peptide Research and Therapeutics. 25(3):1135-1145. https://doi.org/10.1007/s10989-018-9762-1.
Majumdar, R., Minocha, R., Lebar, M.D., Rajasekaran, K., Long, S., Carter-Wientjes, C.H., Minocha, S., Cary, J.W. 2019. Contribution of maize polyamine and amino acid metabolism toward resistance against Aspergillus flavus infection and aflatoxin production. Frontiers in Plant Science. 10:692. https://doi.org/10.3389/fpls.2019.00692.
Han, D., Yao, H., Hruska, Z., Kincaid, R., Rajasekaran, K., Bhatnagar, D. 2019. Development of high-speed dual-camera system for batch screening of aflatoxin contamination of corn using multispectral fluorescence imaging. Transactions of the ASABE. 62(2):381-391. https://doi.org/10.13031/trans.13125.
Hruska, Z., Yao, H., Kincaid, R., Brown, R.L., Bhatnagar, D., Cleveland, T.E. 2017. Temporal effects on internal fluorescence emissions associated with aflatoxin contamination from corn kernel cross-sections inoculated with toxigenic and atoxigenic Aspergillus flavus. Frontiers in Microbiology. 8:1718. https://doi.org/10.3389/fmicb.2017.01718.
Xing, F., Yao, H., Liu, Y., Dai, X., Brown, R.L., Bhatnagar, D. 2017. Recent developments and applications of hyperspectral imaging for rapid detection of mycotoxins and mycotoxigenic fungi in food products. Critical Reviews in Food Science and Nutrition. 59(1):173-180. https://doi.org/10.1080/10408398.2017.1363709.
Tao, F., Yao, H., Zhu, F., Hruska, Z., Liu, Y., Rajasekaran, K., Bhatnagar, D. 2019. A rapid and nondestructive method for simultaneous determination of aflatoxigenic fungus and aflatoxin contamination on corn kernels. Journal of Agricultural and Food Chemistry. 67:5230-5239. https://doi.org/10.1021/acs.jafc.9b01044.