Exploring host induced gene silencing of key biosynthetic pathway genes in management of aflatoxin accumulation in maize (zea mays) and groundnuts (arachis hypogaea l)

Abstract

Infestation of crops by mycotoxin-producing fungi is a major challenge in sub-Sahara Africa due to heavy economic losses and threats to human and animal health. Many crops are contaminated by fungi, particularly Aspergillus spp, that produce aflatoxins. Moreover, most studies on fungal contamination in plants have focused on maize due to its significance as a staple food in most parts of Africa. In Kenya, this has been driven by the outbreaks of aflatoxicosis in the eastern parts of the country, the worst in human history being in 2004 where 317 cases were reported with 125 deaths as a result of consumption of contaminated maize. Various pre and post-harvest control strategies have not effectively managed the aflatoxin menace in Africa therefore calling for a long term solution. This study aimed to develop transgenic maize and groundnut germplasm that accumulate low or no aflatoxins using host induced gene silencing approach. RNAi sequences targeting key enzymes and regulatory elements in the aflatoxin biosynthetic pathway were cloned into entry vector PCR8/TOPOTA and then introduced into monocot and dicot tailored binary vector pStargate and pHellsgate vectors respectively via the Gateway™ cloning technique. A regeneration protocol for ICGV-CG2 and CG2 groundnut varieties was first optimized using cotyledons for ease of regeneration of transgenics. Maize transformation was achieved by use of immature embryos co-cultivated with Agrobacetrium tumefaciens harboring the silencing constructs. Transgenic plants were confirmed via PCR using primers targeting respective selectable markers. Afterwards, transgenic maize were challenged using an aflatoxigenic Aspergillus flavus (MCKII) under controlled conditions in the glasshouse. Aflatoxin levels in transgenics and re-isolated fungi were first determined by fluorescence on neutral red desiccated coconut agar media and later quantified through enzyme-linked immunosorbent assay. Results showed reduced toxin levels in maize transformed with the constructs as compared to the wild type used as controls. StcJ tranagenic maize recorded the lowest aflatoxin levels of <1.75ppb followed by aflR and stcA at 10 and 12.4ppb respectively. Analysis of in vitro aflatoxigenecity of reisolated A. flavus also revealed higher levels of aflatoxins in fungal cultures reisolated from wild type and the MCKII culture in acamparison to transgenics. Aflatoxin levels of 117 and 70.3ppb were recorded in A. flavus cultures from wild type and the MCKII respectively. Those from maize transformed with stcJ, stcA and aflR recorded <1.75, 29.4 and 43.6 ppb respectively. Interestingly, the aflR construct was observed to have detrimental effects on both maize and groundnuts.These results indicate that transgenic maize synthesized double stranded RNAs that were later converted into complementary small interfering RNAs against the respective target aflatoxin pathway mRNA sequences. On colonization by toxigenic A. flavus, the complementary siRNA molecules trafficked via the plant-fungal cellular interface into the fungi where they chopped the respective target esquences into siRNAs of 21-28bp hence resulting in downregulation of respective gene expression. In conclusion, this study successfully downregulated aflatoxin biosynthesis and accumulation in maize. The transgenic maize generated could help in alleviation of the aflatoxin problem in SSA without any further investment by farmers except on seed acquisition