Control of Aflatoxin Production of Aspergillus flavus and Aspergillus parasiticus Using RNA Silencing Technology by Targeting aflD (nor-1) Gene

Abstract

Aspergillus flavus and Aspergillus parasiticus are important pathogens of cotton, corn, peanuts and other oil-seed crops, producing toxins both in the field and during storage. We have designed three siRNA sequences (Nor-Ia, Nor-Ib, Nor-Ic) to target the mRNA sequence of the aflD gene to examine the potential for using RNA silencing technology to control aflatoxin production. Thus, the effect of siRNAs targeting of two key genes in the aflatoxin biosynthetic pathway, aflD (structural) and aflR (regulatory gene) and on aflatoxin B1 (AFB1), and aflatoxin G1 (AFG1) production was examined. The study showed that Nor-Ib gave a significant decrease in aflD mRNA, aflR mRNA abundance, and AFB1 production (98, 97 and 97% when compared to the controls) in A. flavus NRRL3357, respectively. Reduction in aflD and aflR mRNA abundance and AFB1 production increased with concentration of siRNA tested. There was a significant inhibition in aflD and AFB1 production by A. flavus EGP9 and AFG1 production by A. parasiticus NRRL 13005. However, there was no significant decrease in AFG1 production by A. parasiticus SSWT 2999. Changes in AFB1 production in relation to mRNA levels of aflD showed a good correlation (R = 0.88; P = 0.00001); changes in aflR mRNA level in relation to mRNA level of aflD also showed good correlation (R = 0.82; P = 0.0001). The correlations between changes in aflR and aflD gene expression suggests a strong relationship between these structural and regulatory genes, and that aflD could be used as a target gene to develop efficient means for aflatoxin control using RNA silencing technology.

Natural antisense transcripts in fungi

“…Efficient RNA‐mediated gene silencing through the introduction of hairpin constructs has been reported for C. neoformans, M. oryzae , and S. commune (Liu et al ., 2002; Kadotani et al ., 2003; de Jong et al ., 2006). Synthetic siRNAs effectively silence gene expression in A. flavus (Abdel‐Hadi et al ., 2011).…”

Integrating toxin gene expression, growth and fumonisin B 1 and B 2 production by a strain of Fusarium verticillioides under different environmental factors

“…This could further help in developing a more targeted approach to design prevention strategies to control fumonisin biosynthesis in staple food commodities. For example, use of RNAi systems to interfere and reduce the function of the FUM1 or FUM21 gene may be an approach for the minimization of fumonisins in cereals [20]. This approach could be a powerful tool in examining the impact of climate change factors on toxin production [39].…”

Adenylate Cyclase AcyA Regulates Development, Aflatoxin Biosynthesis and Fungal Virulence in Aspergillus flavus

“…The recombinant plasmid, harboring acyA ORF with its native promoter and a pyrithiamine-resistance marker, was transformed into protoplasts of the Δ acyA mutant. Preparation of protoplasts and fungal transformation were performed as previously described (Cary et al, 2006; Abdel-Hadi et al, 2011; Yang et al, 2016). …”