A breakthrough against aflatoxin

Efforts to alleviate aflatoxin from food/feed has taken a multi-prong research approach for decades from both the commercial and academic communities. Efforts include using non-toxin producing Aspergillus strains to out compete naturally-occurring toxin-producing strains, breeding for either fungal or toxin resistance in susceptible crops and the use of chelating agents to bind the toxin and render it no longer biologically available. Despite these combined efforts, it is currently estimated that millions of tonnes of various crops, maize being chief among them, are lost every year due to harvested crops containing above regulatory limits of aflatoxin.

Alleviating aflatoxin in maize

Maize is widely consumed worldwide and is very susceptible to aflatoxin contamination with current estimates of nearly five billion people, predominantly in developing countries, being chronically exposed to aflatoxin through consumption of contaminated crops. However, an RNAi suppression biotechnology strategy to alleviate aflatoxin in developing maize kernels has been successfully used. This novel approach takes advantage of two recent scientific findings: one, that all eukaryotic cells contain gene suppression machinery involving a protein called dicer and this proteins’ use of a template molecule consisting of small interfering RNA (siRNA); and two, that these siRNA molecules are capable of passing between a plant host and its contaminating pathogen. When siRNA molecules with a specific sequence are introduced and expressed by an organism, it initiates the degradation of endogenous transcripts containing homologous sequences to the introduced siRNA molecule. The end result is the silencing or suppression of a targeted gene within an organism by the introduction of a designed siRNA molecule. If the RNA transcript of a gene is suppressed in this manner, the transcript is not available to be translated into a protein, and with the protein absent it is not capable of performing its enzymatic activity, and in turn, in this example, the targeted enzyme is part of a biosynthetic toxin pathway, no toxin would be produced. If the gene to be silenced is within the siRNA expressing organism itself, the technology is referred to as RNAi technology. This has proven to be a useful technique in studying gene function in various organisms. If the siRNA molecule is expressed in one organism, the host cell for example and in this instance a maize plant, yet the targeted gene of suppression lies within a contaminating pathogen, the RNAi suppression transcends species boundaries and is referred to as host-induced gene silencing (HIGS).

Specific gene targeted

In research, HIGS was used to silence the production of aflatoxin in Aspergillus-contaminated maize kernels by expressing an RNAi cassette that would target a biosynthetic gene in the fungal aflatoxin pathway. The gene chosen to target for suppression by RNAi technology was the Aspergillus polyketide synthase gene (denoted aflC). This gene was chosen as a target because it encodes for a protein that is necessary to produce a biochemical precursor to all four aflatoxin compounds so the suppression of this enzymatic step should result in no toxin production. Additionally, the polyketide synthase encoding gene is large in size so selecting it to target for suppression enhanced the probability of using regions of the gene in the RNAi cassette that were specific to the Aspergillus gene but did not display significant homology to any of the genes expressed in the maize host organism, or any likely downstream consumers, such as humans, pigs and cattle. Specifically, three regions were chosen, each approximately 200 bp in length, of the Aspergillus polyketide synthase to construct an RNAi suppression cassette to be expressed in the edible portion of maize kernels.

A bioinformatics analysis of the chosen three regions of the Aspergillus aflC gene did not display homology in the maize, human, pig or cattle genomes. Three regions of the targeted gene were chosen so that the gene would be completely suppressed, as opposed to be merely truncated and the encoding enzyme having some activity, and to lessen the likelihood that contaminating Aspergillus would be able to readily evolve resistance as resistance gain would consist of mutating the three targeted areas within the alfC gene simultaneously in the pathogen. Transgenic maize plants were produced and through molecular analysis shown to be expressing both the herbicide resistance selectable marker Bar gene and the inserted RNAiaflC gene cassette. The highest RNAiaflC expressing transgenic maize events were self-pollinated until plants that were breeding true for the introduced RNAiaflC cassette were achieved. Kernels from these expressing plants were inoculated while still developing on the cob at 10 days post pollination with a known and equal amount of an aflatoxin-producing Aspergillus fungal strain. The infections were allowed to continue for 30 days throughout kernel development. At the end of the infection point, the kernels surrounding each infection site were harvested and combined from each plant to determine toxin load. Each cob was infected 3-4 times with each infection site contained 6-8 surrounding kernels. Toxin was detected in all nontransgenic control maize kernels with values ranging from 1,000-220,000 ppb while no toxin was detected in any of the transgenic kernels (Figure 1).

Figure 1 – Aflatoxin-producing Aspergillus infection challenge assay of RNAi suppression transgenic maize cobbs.

A quantitative reverse transcription polymerase chain reaction was then performed with RNA extracted from contaminated maize tissue and showed that the targeted polyketide synthase gene (aflC) was not expressing in any of the fungal tissue interacting with the transgenic kernels but was expressing in the nontransgenic control kernels while another fungal gene used as an expression control, chitin synthase, was expressed at comparable levels in both transgenic and nontransgenic contaminated kernels. Together these findings indicate that HIGS can be used to alleviate aflatoxin contamination in developing maize kernels and as such can aid in the efforts to eliminate this toxin from global food/feed.

Exploring unintended gene suppression

RNAi and HIGS technologies rely heavily on target sequence specificity to suppress the desired gene to be silenced. A potential disadvantage of this technology is the introduced siRNA molecule might have sequence homology to other genes rather than just the desired targeted gene and, in the case of HIGS, perhaps share enough sequence similarly to genes in interacting organisms to cause unintended gene suppression. Such undesired gene suppression events are often referred to as ‘unintended phenotypes’ or ‘off-targets’ in the biotechnology community. As this research involved the expression of a siRNA molecule into maize kernels, it was investigated if any endogenous maize RNA transcripts had been unintentionally suppressed as a result of the introduced RNAiaflC cassette. Total RNA was extracted from two nontransgenic control plants and three RNAiaflC expressing transgenic events. Through a novel pairwise comparison, all RNA transcript samples to one another were compared, with particular attention to the six comparisons that involved a transgenic event to a nontransgenic control sample. Although each pairwise comparison of a transgenic/nontransgenic yielded 70-100 significant transcript differences, when the six transgenic/nontransgenic comparisons were analysed there was no single transcript seen to be consistently significantly different in the transgenic samples compared to the nontransgenic controls. This indicates that when two samples (transgenics/nontransgenics) are compared there will be small transcript differences as the plants are responding to their own micro-environments and their slightly different stages of development. Yet, to determine if the expressed introduced RNAi cassette caused differences in transcripts in maize kernels, a six pairwise comparison analysis showed no consistently different transcripts in the transgenic kernels. If the expression of the introduced RNAiaflC cassette was causing any off-targets by having homology to endogenous maize genes then it was
expected that the RNA transcript analysis should show gene expression consistently altered in the three expressing lines compared to the two nontransgenic controls. This shows that if the sequence used to construct an RNAi suppression cassette is specific to the targeted desired gene to be suppressed than no off-targets or unintended phenotypes should occur. In demonstrating no significant transcript differences in the RNAiaflC transgenic kernels to the controls, this is the first step towards showing substantial equivalence, in essence a demonstration that the biotechnology event mirrors the nontransgenic counterpart in all aspects except the introduced trait, which is a regulatory mandate when moving a biotechnology trait towards commercialisation.

This research has shown that HIGS is both a promising and exciting technology to use for the alleviation of aflatoxin in maize during pre-harvest that can be extended to other aflatoxin-susceptible crops and used to suppress mycotoxins produced from other contaminating fungus. The suppression of a mycotoxin biosynthetic pathway by HIGS is a powerful tool to be used to improve human health as well as contribute substantially to global food security and sustainability.

Author: Monica Schmidt, School Of Plant Sciences, University Of Arizona