Downregulation of transcription factor aflR in Aspergillus flavus confers reduction to aflatoxin accumulation in transgenic maize with alteration of host plant architecture

Abstract

We report success of host-induced gene silencing in downregulation of aflatoxin biosynthesis in Aspergillus flavus infecting maize transformed with a hairpin construct targeting transcription factor aflR. Infestation of crops by aflatoxin-producing fungi results in economic losses as well as negative human and animal health effects. Currently, the control strategies against aflatoxin accumulation are not effective to the small holder farming systems in Africa and this has led to widespread aflatoxin exposure especially in rural populations of sub-Saharan Africa that rely on maize as a staple food crop. A recent strategy called host-induced gene silencing holds great potential for developing aflatoxin-resistant plant germplasm for the African context where farmers are unable to make further investments other than access to the germplasm. We transformed maize with a hairpin construct targeting the aflatoxin biosynthesis transcription factor aflR. The developed transgenic maize were challenged with an aflatoxigenic Aspergillus flavus strain from Eastern Kenya, a region endemic to aflatoxin outbreaks. Our results indicated that aflR was downregulated in A. flavus colonizing transgenic maize. Further, maize kernels from transgenic plants accumulated significantly lower levels of aflatoxins (14-fold) than those from wild type plants. Interestingly, we observed that our silencing cassette caused stunting and reduced kernel placement in the transgenic maize. This could have been due to "off-target" silencing of unintended genes in transformed plants by aflR siRNAs. Overall, this work indicates that host-induced gene silencing has potential in developing aflatoxin-resistant germplasm.

Aflatoxin-free transgenic maize using host-induced gene silencing

“…Previous research efforts to use RNAi to silence toxin production resulted in a mere 5- to 10-fold reduction and impaired the growth of transgenic plants, presumably because the RNAi cassette targeted a fungal transcription factor gene that may have had other silencing effects ( 23 ). Before embarking on this project, a bioinformatics analysis determined that the Aspergillus polyketide synthase gene does not have any notable DNA sequence homology with the maize genome.…”

Characterization of small RNA populations in non-transgenic and aflatoxin-reducing-transformed peanut

“…Research on plant-induced RNAi silencing of single fungal genes has shown reduction of aflatoxin levels in the range of 60 and 97% [17,23,38]. Masanga et al [23] demonstrated a 14-fold reduction in aflatoxin accumulation in transformed maize, expressing a hairpin construct targeting aflR.…”

A review of agricultural aflatoxin management strategies and emerging innovations in sub-Saharan Africa

“…A follow-up study on this prediction by a research group in Kenya led to the transformation of aflR hairpin constructs into a susceptible tropical maize line resulting in a significant (14-fold) reduction in aflatoxin levels. While these results were commendable, the transgenic maize had an altered plant phenotype possibly due to HIGS mistargeting by aflRsi RNAs [55]. Consequently, this study creates a platform from which the effects of silencing other aflatoxin biosynthetic genes on aflatoxin levels could be evaluated.…”