Location: Food and Feed Safety Research
2017 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
Progress was made on all four objectives. The primary objective of this project is to identify native and foreign proteins or peptides (very small proteins) that provide resistance to the host crop (primarily corn and cotton) against fungal invasion and/or aflatoxin (compounds that are toxic and carcinogenic to humans and animals) contamination, and to test the efficacy of these genes by classical or molecular breeding of cotton and corn.
Under Objective 1, Agricultural Research Service (ARS) scientists in New Orleans, Louisiana, made significant progress in identifying 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. After several field tests in the U.S. these varieties demonstrated aflatoxin-resistance comparable to known resistant lines. In collaboration with the International Institute of Tropical Agriculture, Nigeria, six corn lines (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. Using these resistant lines, several drought-tolerant lines were developed and they are being field/lab-tested in Nigeria/Southern Regional Research Center (SRRC).
Under Objective 2, ARS scientists in New Orleans, Louisiana, have initiated experiments on a genome-wide transcriptome (the sum of all the actively expressed genes of a corn plant) analysis of the corn-A. flavus interaction. In collaboration with The J. Craig Venter Institute (JCVI) we will be using 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. We will be comparing A. flavus-infected kernels of TZAR 102 (a hybrid corn line derived from aflatoxin resistant germplasm developed in the U.S. and an African line that exhibits superior resistance to drought and ear rot), released by ARS-SRRC, 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 observed in the TZAR 102 line. Corn seed has been infected with the A. flavus strain and we have collected the infected seed at 4 different time points (0, 8, 72 and 168 hours). The infected seed has been processed to stabilize the RNA needed for RNA-Seq analysis and the samples have been shipped to JCVI for sequencing. Data generated from RNA sequencing will be analyzed in-house. An interactome (the whole set of molecular interactions in a particular cell) analysis based on the RNA-Seq data will 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. In cotton, we identified twenty-eight genes from infected cotton boll pericarp (outer coat) and seeds whose expression differed significantly compared to non-infected bolls. The Spot11 catalase gene showed up-regulation in inoculated locules (a segment of a cotton boll) and it is currently being evaluated in transgenic cotton. Recently, following a genome-wide transcriptome profiling that identified differentially expressed cotton genes in response to infection with both toxigenic and atoxigenic strains of A. flavus, a comparative transcriptome analysis was also performed that identified genes that were significantly differentially expressed in corn and peanut in response to A. flavus. We identified 732 unique genes with only 26 genes common across all three crops that were considered candidate A. flavus resistance genes which could be used to improve resistance to aflatoxin resistance.
Under Objective 3, we made significant progress in genetic engineering of corn and cotton to introduce genes for resistance to A. flavus growth and aflatoxin production.
(a) Transgenic corn kernels expressing a synthetic peptide 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). In a similar experiment, reduction in fungal growth and toxin production was also observed from sixth generation corn kernels expressing a gene from another plant, hyacinth beans, that encodes a novel antifungal seed protein. This antifungal protein inhibits a key enzyme (a-amylase) that is necessary for the fungus to grow and infect seed.
(b) 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 A. 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 believed to be involved in disease resistance. Similar RNAi-based approaches were carried out to silence fungal genes as well that are critical for the fungus to grow, infect and produce toxins. Our efforts to silence fungal genes such as fungal a-amylase (essential for fungal growth), VeA and NsdC [required by A. flavus for the production of aflatoxin and sclerotia (fungal survival structures)] have yielded aflatoxin resistance in transgenic kernels.
(c) Unlike in corn, no resistant germplasm (seed stock) to A. flavus has been identified in cotton so far. ARS scientists in New Orleans, Louisiana, in collaboration with scientists at the University of Louisiana-Lafayette studied a wide variety of cotton germplasm to try and identify lines that demonstrate natural resistance to A. flavus infection and aflatoxin accumulation. Representative lines from three different species: Gossypium (G.) arboreum (old world cotton), G. barbadense (Pima cotton), and G. hirsutum (upland cotton) were screened with a green fluorescent protein (GFP) expressing A. flavus strain to assess any innate resistance to the fungus. Some cotton varieties of G. arboreum were the most resistant and commercially cultivated upland cotton varieties were moderately susceptible to A. flavus infection.
(d) Substantial progress was also made on the correlation between cottonseed with rich reserves of lipids (a type of fat molecule that can serve as a nutrient source for fungi) and A. flavus infection and aflatoxin accumulation. Lipid accumulation in developing cottonseed was well-correlated with the ability of an aflatoxin-producing fungal strain to grow and produce aflatoxins compared to no toxin production by a non-toxin producing fungal strain. This study is useful to understand factors controlling A. flavus infection and aflatoxin production in developing cottonseed.
(e) Transgenic cotton lines expressing a synthetic peptide designated D4E1 demonstrated antifungal effects against A. flavus under greenhouse conditions or seedling pathogens under field conditions. They were field-tested in collaboration with University of Arizona for resistance to aflatoxin contamination. Recent results from the small field experiment were inconclusive due to lack of A. flavus infection and aflatoxin contamination.
Toxin detection procedures in corn and other crops are time consuming, destructive, inconsistent, and costly. Therefore, under Objective 4, ARS scientists in New Orleans, Louisiana, in collaboration with Geosystems Research Institute (GRI) of Mississippi State University (MSU) based at the Stennis Space Center, Mississippi, developed a non-invasive hyperspectral imaging technique (collecting and processing information from across the light-spectrum) to detect and quantify aflatoxins in corn kernels. Hyperspectral instruments have already demonstrated the ability to differentiate toxigenic and atoxigenic A. flavus strains. A spectral signature to detect aflatoxin contaminated corn has been developed (U.S. Patent #8,563,934). A prototype multi-spectral imaging system for use in the field, as well as at grain inspection facilities has been designed. The patent has been licensed to Secure Food Solutions, Inc. (SFS), a Tennesse-based company, towards developing aflatoxin screening and removal technologies for automated grain handling systems in the U.S. and international markets. For this purpose, a Cooperative Research and Development Agreement was established on “Specialized high-resolution imaging system for rapid batch screening of aflatoxin in corn.” With funding from Gates Foundation and United States Agency for International Development we developed a portable technology to detect aflatoxin contamination in single corn cobs for farmers in the developing countries. We are also in the process of developing “AflaGoggles" 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 SFS, MSU, and USDA on “Novel method to improve aflatoxin detection accuracy of multispectral imaging technology” to develop commercial, rapid, screening equipment for aflatoxin contaminated corn.
Accomplishments
1. Development of aflatoxin and fumonisin-tolerant corn lines. Agricultural Research Service (ARS) scientists in New Orleans, Louisiana, in collaboration with the International Institute of Tropical Agriculture, Nigeria developed six corn varieties (TZAR101-106) with resistance to contamination by the aflatoxin (compounds that are toxic and carcinogenic to humans and animals) producing fungus, Aspergillus flavus. In a recent field trial these six lines also demonstrated resistance to another fungus, Fusarium, responsible for producing a toxin called fumonisin. Using these lines several drought tolerant lines were also developed that will be used by growers in African countries to reduce the incidence of aflatoxin and fumonisin contamination in corn.
2. Aflatoxin-resistance conferred by the expression of a synthetic peptide (very small protein). Agricultural Research Service (ARS) scientists in New Orleans, Louisiana, in collaboration with scientists at Tuskegee University and University of Arkansas developed transgenic corn lines expressing a synthetic antifungal peptide AGM 182 (modeled after an antimicrobial peptide from horseshoe crab). Transgenic corn kernels expressing AGM 182 demonstrated a significant reduction in fungal growth and aflatoxin contamination (76-98% reduction in third generation kernels). Transgenic corn lines with resistance to aflatoxin contamination will serve as a valuable germplasm for breeding new hybrids.
3. Demonstration of the role of a native corn kernel protein in aflatoxin resistance. The contribution of a native corn kernel protein, Pathogenesis–Related maize seed protein (PRms) to Aspergillus (A.) flavus resistance was validated by Agricultural Research Service (ARS) scientists in New Orleans, Louisiana. Using ribonucleic acid interference (RNAi, a technology that enables specific genes to be targeted for down-regulation or silencing) -mediated silencing, the PRms gene was made ineffective in transgenic corn lines and the kernels were evaluated for A. flavus growth and aflatoxin contamination. Down-regulation of PRms in transgenic kernels resulted in a 250-350% increase in A. 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 believed to be involved in disease resistance in corn. Increased expression of PRms or breeding for it will provide resistance to aflatoxin contamination in transgenic kernels.
Review Publications
Rajasekaran, K., Ford, G., Sethumadhavan, K., Carter Wientjes, C., Bland, J., Cao, H., Bhatnagar, D. 2016. Aspergillus flavus growth and aflatoxin production as influenced by total lipid content during growth and development of cottonseed. Journal of Crop Improvement. 31(1):91-99. https://doi.org/10.1080/15427528.2016.1263811.
Majumdar, R., Rajasekaran, K., Cary, J.W. 2017. RNA interference (RNAi) as a potential tool for control of mycotoxin contamination in crop plants: concepts and considerations. Frontiers in Plant Science. 8:200. https://doi.org/10.3389/fpls.2017.00200.
Anderson, D.M., Rajasekaran, K. 2016. The global importance of transgenic cotton. In: Ramawat, K.G., Ahuja, M.R., editors. Fiber Plants: Biology, Biotechnology and Applications, Volume 13. Switzerland: Springer International Publishing. p. 17-33. https://doi.org/10.1007/978-3-319-44570-0_2.
Brown, R.L., Williams, W.P., Windham, G.L., Menkir, A., Chen, Z.-Y. 2016. Evaluation of African-bred maize germplasm lines for resistance to aflatoxin accumulation. Agronomy. 6(2):24. doi:10.3390/agronomy6020024.
Srour, A.Y., Fakhoury, A.M., Brown, R.L. 2016. Targeting mycotoxin biosynthesis pathway genes. In: Moretti, A., Susca, A., editors. Mycotoxigenic Fungi: Methods and Protocols, Methods in Molecular Biology. New York, NY: Springer. 1542:159-171.
Sakhanokho, H.F., Rajasekaran, K. 2016. Cotton regeneration in vitro. In: Ramawat, K.G., Ahuja, M.R., editors. Fiber Plants: Biology, Biotechnology and Applications. Gewerbestrasse, Switzerland: Springer International Publishing AG. p. 87-110.
Rajasekaran, K., Majumdar, R., Sickler, C., Wei, Q., Cary, J.W., Bhatnagar, D. 2017. Fidelity of a simple Liberty leaf-painting assay to validate transgenic maize plants expressing the selectable marker gene, bar. Journal of Crop Improvement. 31(4):628-636. https://doi.org/10.1080/15427528.2017.1327913.
