Targeting the Aspergillus flavus p2c gene through host-induced gene silencing reduces A. flavus infection and aflatoxin contamination in transgenic maize

Authors: RARUANG, YENJIT - Louisiana State University Agcenter; OMOLEHIN, OLANIKE - Louisiana State University Agcenter; HU, DONGFANG - Louisiana State University Agcenter; Wei, Qijian - Mei Mei; PROMYOU, SURASSAWADEE - Kasetsart University; PAREKATTIL, LIDIYA - Louisiana State University Agcenter; Rajasekaran, Kanniah - Rajah; Cary, Jeffrey; WANG, KAN - Iowa State University; CHEN, ZHI-YUAN - Louisiana State University Agcenter

Submitted to: Frontiers in Plant Science
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 4/11/2023
Publication Date: 5/9/2023
Citation: Raruang, Y., Omolehin, O., Hu, D., Wei, Q., Promyou, S., Parekattil, L.J., Rajasekaran, K., Cary, J.W., Wang, K., Chen, Z.-Y. 2023. Targeting the Aspergillus flavus p2c gene through host-induced gene silencing reduces A. flavus infection and aflatoxin contamination in transgenic maize. Frontiers in Plant Science. 14:1150086. https://doi.org/10.3389/fpls.2023.1150086.
DOI: https://doi.org/10.3389/fpls.2023.1150086

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, p2C (pectinase or polygalacturonase), 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, biotechnologist, and corn food industry for reducing aflatoxin-related toxicity and illness in animals and humans.

Technical Abstract: Maize is susceptible to Aspergillus flavus infection, and subsequent contamination with aflatoxin. Currently, biocontrol and developing resistant cultivars have met with limited success. In the present study, host-induced gene silencing (HIGS) was employed to mitigate aflatoxin contamination in maize by suppressing the expression of the A. flavus p2c gene encoding a polygalacturonase, a key enzyme involved in the colonization of maize kernels by A. flavus. An RNAi vector containing a portion of the p2c gene was constructed and introduced into B104 immature embryos through Agrobacterium transformation. Twenty-eight transgenic plants were produced from fifteen independent transformation events. Thirteen of the fifteen transformation events were confirmed by PCR to have the p2c gene region. The T2 generation kernels containing the p2c silencing transgene from six events out of eleven examined had less aflatoxin than those without the transgene. Homozygous T3 transgenic kernels from four events also produced significantly less aflatoxins (P=0.02) than kernels from the null or B104 controls under field inoculation conditions. Six elite inbred lines were crossed with pollens from two of the events and the F1 kernels from the resulting crosses also supported significantly less aflatoxins (P=0.02) than those from the crosses with pollens from non-transgenic plants. In addition, significantly higher levels of p2c gene-specific small RNAs were detected in the transgenic leaf (T0 and T3) and kernel tissue (T4). Significantly less fungal growth (27~40 fold) was detected in homozygous transgenic maize kernels than in the null control kernels 10 days after fungal inoculation in the field, indicating that the reduced aflatoxin production in the homozygous transgenic kernels is likely due to suppression of p2c expression through HIGS, which results in reduced fungal growth.