**8. RNA interference Strategy and its mechanisms**

RNA interference (RNAi) refers to post-transcriptional gene silencing mediated by either degradation or translation arrest. This mechanism was first discovered in plants where transgene and viral RNAs guide DNA methylation [74, 34, 52]. The process is a naturally occurring biological process that is highly conserved among multicellular organisms includ‐ ing plants. The process is mediated by small interfering RNAs (siRNAs) that are produced from long dsRNA of exogenous or endogenous origin by an endonuclease (an enzyme) called a dicer. The resulting siRNAs are about 21-24 nucleotides long with 2 nucleotide sin‐ gle stranded 3' end overhangs on each strand. The siRNAs are then incorporated into a nu‐ clease complex called the RNA-induced silencing complex (RISC), which then targets and cleaves mRNA that is complementary to the siRNA [86].

In plants, RNAi plays a role in cellular defense, protecting the cell from inappropriate ex‐ pression of repetitive sequences, transposable elements and viral infections [43]. RNAi has proved to have ability to regulate the expression of genes involved in a variety of cell proc‐ esses such as proliferation, apoptosis and differentiation [2]. Moreover, it is thought to play a role in protecting the genome against damage caused by transposons [44]. More recently, these findings have been extended by the observations that siRNA-directed DNA methyla‐ tion in plants is linked to histone modification [89]. In fission yeast, hetero-chromatin forma‐ tion at centromere boundaries is associated with siRNAs [72].

aquaporins that aid in loosening of the host plant cell wall during parasite infection [63]. This ability to tap into native pathways has yielded crucial breakthrough in parasitic plant

A New Approach in Aflatoxin Management in Africa: Targeting Aflatoxin/Sterigmatocystin Biosynthesis in Aspergillus

Species by RNA Silencing Technique http://dx.doi.org/10.5772/51440 49

**10. Prospects of applying RNAi in management of Aspergillus species**

Early genetic studies have identifiedaflatoxin biosynthesis to becontrolled by a cluster of aflatoxin and sterigmatocystin gene in an ~70kb region [85, 13, 69, 76]. A critical analysis of the pathway indicates that their exists three enzymes that catalyse the two rate-limiting steps in aflatoxinbiosynthesis. Two enzymes stcJ and stcKcatalyse the first step that involves the conversion of Acetate and Malonyl-CoA into Hexanoyl-CoA. A further critical examina‐ tion of the pathway identifies another enzyme stcA which catalyses the conversion of Hexa‐ noyl-CoA to Norsolorinic acid. To addstrength to this observation, Brown et al.,[1996] reported that Aspergillus species with mutations in the stcJ and stcK grew normally but could not produce aflatoxin and sterigmatocystin. In the same study addition of Hexanoic acid to growth media restored aflatoxin and sterigmatocystin production. Recent studies have reported the trafficking of molecular cues between hosts and parasites including fungi. Among the molecules are small interfering RNA SiRNA. This targeted downregulation of gene expression bySiRNA has been used to engineer crops against virueses, nematodes and parasitic plants in cross species version. The key steps for this strategy to succeed are;i) iden‐ tifying a key gene to a process, ii) cloning the target sequence of the gene from the parasite, iii) making an RNAi construct with the target sequence of the parasite in sense and antisense direction separated by an intron so as to allow formation of primary small intereferingRNAs (SiRNA) in host (maize), iv) transforming the host (maize) with the construct tailored for RNAi. In this case the rate-limiting steps in aflatoxin biosynthesis are known to be catalysed bystcJ, stcK and stcA [85,]. The strategy is therefore be to make an RNAi construct contain‐ ing either combined partial or full sequences of the stcA, stcK and stcJ in sense and antisense orientation and transform it into maize. Upon colonization with aflatoxigenic fungi in the field, the primary SiRNA molecules will then cross from transgenic maize into Aspergil‐ lussppfungi through the haustoria connection at infection. The siRNAs will then cleave the stcJ, stcK and stcA mRNAs into 20 to 28bp long double molecules hence downregulating or inhibiting aflatoxin and sterigmatocystin biosynthesis.The transformed aflatoxigenic species in the field will be unable to synthesizeaflatoxins both in field and storage. RNAi will suc‐ ceed in this case because it can be both local (cell–cell) and systemic (spread through the vas‐ cular system), hence all parts of the transgenic plant shall remain armed against aflatoxin biosynthesis. The stcJ, stcK and stcA do not exist in maize hence their silencing will not af‐

management [23].

fect the crop.

**and aflatoxins in grains**

Application of RNAi in crop improvement has been derived from targeted degradation of gene products with significant homology to the introduced sequence. Successful utilization of RNAi-based resistance effects rely on; (i) identification of a target gene (ii) dsRNA deliv‐ ery, which includes in plantaexpression of dsRNA and (iii) delivery of sufficient amounts of intact dsRNA. RNAi can therefore be an important tool for crop improvement given that the RNAi signal can be both local (cell-cell) and systemic (spread through vascular system) [8, 71]. RNAi mediated silencing for agricultural traits offers the advantage of transmission across many cells and application in multigene family silencing.
