Resistance to aflatoxin accumulation in maize mediated by host-induced silencing of the Aspergillus flavus alkaline protease (alk) gene

Authors: OMOLEHIN, OLANIKE - LSU Agcenter; RARUANG, YENJIT - LSU Agcenter; HU, DONGFANG - LSU Agcenter; HAN, ZHU-QIANG - Guangxi Academy Of Agricultural Sciences; Wei, Qijian - Mei Mei; WANG, KAN - Iowa State University; Rajasekaran, Kanniah - Rajah; Cary, Jeffrey; CHEN, ZHI-YUAN - LSU Agcenter

Submitted to: The Journal of Fungi
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 10/22/2021
Publication Date: 10/26/2021
Citation: Omolehin, O., Raruang, Y., Hu, D., Han, Z.-Q., Wei, Q., Wang, K., Rajasekaran, K., Cary, J.W., Chen, Z.-Y. 2021. Resistance to aflatoxin accumulation in maize mediated by host-induced silencing of the Aspergillus flavus alkaline protease (alk) gene. The Journal of Fungi. 7(11):904. https://doi.org/10.3390/jof7110904.
DOI: https://doi.org/10.3390/jof7110904

Interpretive Summary: Aflatoxin contamination of maize and other food supplies results in hundreds of millions of dollars in crop loss annually, and globally imposes a severe health risk to exposed populations. The consumption of tainted crops can lead to liver cancer, stunted growth in children, and eventually death. Currently no single method of remediation has proven fully successful, thus the development of pre-harvest technologies is vital in reducing the effects of this toxigenic fungus. Our research demonstrated it is possible to reduce aflatoxin accumulation and Aspergillus flavus growth in maize using RNAi technology. RNAi molecules are small fragments of RNA that are generated in plants, animals and other eukaryotes. Once produced, these molecules can then recognize and bind to specific genes that are also being produced by the invading fungus. The binding of this RNAi molecule results in degradation of the target gene. We have exploited this natural system by generating transgenic maize plants that produce small RNAi molecules designed to target a key fungal enzyme, alkaline protease (alk), involved in the colonization of maize kernels and degrade it resulting in significant reduction of fungal growth and aflatoxin levels. Further, since this method that generates the small RNAi molecules does not generate a transgenic protein in crops thus reducing the public concerns regarding GMO. These findings are useful to corn breeders, biotechnologists, and corn food industry for reducing aflatoxin-related toxicity and illness in animals and humans.

Technical Abstract: Aspergillus flavus is a fungal pathogen that infects maize and produces aflatoxins. Host-Induced Gene Silencing (HIGS) has been shown to reduce host infection by various fungal pathogens. Here, the A. flavus alkaline protease (alk) gene was targeted for silencing through HIGS. An RNAi vector carrying a portion of the alk gene was incorporated into the B104 maize zygotic embryo. Four out of eight transformation events containing the alk gene, Alk-3, Alk-4, Alk-7 and Alk-9, were self-pollinated to T4/T6 generations. At T3, the Alk-transgenic lines showed up to 87% reduction in aflatoxin accumulation under laboratory conditions. T4 transgenic Alk-3 and Alk-7 lines, and T5 and T6 Alk-4 and Alk-9 showed an average of 84% reduction in aflatoxin accumulation compared to their null controls under field inoculations (P<0.05). F1 hybrids of three elite maize inbred lines and the transgenic lines also showed significant improvement in aflatoxin resistance (P<0.006 to P<0.045). Reduced A. flavus growth and levels of ß-tubulin DNA was observed in transgenic kernels during in-vitro inoculation. Alk-4 transgenic leaf and immature kernel tissues also contained about 1000-fold higher levels of alk-specific small RNAs compared to null controls, indicating that the enhanced aflatoxin resistance in the transgenic maize kernels is due to suppression of A. flavus infection through HIGS of alk gene.