Resistance to Aflatoxin Accumulation in Maize Mediated by Host-Induced Silencing of the Aspergillus flavus Alkaline Protease (alk) Gene.

Olanike Omolehin,Zhu-Qiang Han,Zhi-Yuan Chen,Jeffrey W Cary,Kanniah Rajasekaran,Qijian Wei,Dongfang Hu,Yenjit Raruang,Kan Wang

doi:10.3390/jof7110904

Abstract

Aspergillus flavus is a fungal pathogen that infects maize and produces aflatoxins. Host-Induced Gene Silencing (HIGS) has been shown to reduce host infection by various fungal pathogens. Here, the A. flavus alkaline protease (alk) gene was targeted for silencing through HIGS. An RNAi vector carrying a portion of the alk gene was incorporated into the B104 maize genome. Four out of eight transformation events containing the alk gene, Alk-3, Alk-4, Alk-7 and Alk-9, were self-pollinated to T4/T6 generations. At T3, the Alk-transgenic lines showed up to 87% reduction in aflatoxin accumulation under laboratory conditions. T4 transgenic Alk-3 and Alk-7 lines, and T5 and T6 Alk-4 and Alk-9 showed an average of 84% reduction in aflatoxin accumulation compared to their null controls under field inoculations (p < 0.05). F1 hybrids of three elite maize inbred lines and the transgenic lines also showed significant improvement in aflatoxin resistance (p < 0.006 to p < 0.045). Reduced A. flavus growth and levels of fungal ß-tubulin DNA were observed in transgenic kernels during in vitro inoculation. Alk-4 transgenic leaf and immature kernel tissues also contained about 1000-fold higher levels of alk-specific small RNAs compared to null controls, indicating that the enhanced aflatoxin resistance in the transgenic maize kernels is due to suppression of A. flavus infection through HIGS of alk gene.

Highlights

Aspergillus flavus is an opportunistic plant pathogen that resides in the soil and has a global distribution
Major economically important crops including maize, peanut and cotton are highly susceptible to both A. flavus infection and subsequent contamination with carcinogenic secondary metabolites known as aflatoxins [3,4,5]
After inserting the alk and 30 arm and the intron containing chloramphenicol selection marker (CmR) into pBS-d35S-attR4- attR4 through LR recombination, the resulting construct was verified through digestions with EcoR V and EcoR I/Kpn I restriction enzymes, and the resulting fragment sizes were in agreement with expected sizes

Summary

Introduction

Aspergillus flavus is an opportunistic plant pathogen that resides in the soil and has a global distribution It reproduces predominately asexually through the release of a large number of conidia under natural conditions [1,2]. Major economically important crops including maize, peanut and cotton are highly susceptible to both A. flavus infection and subsequent contamination with carcinogenic secondary metabolites known as aflatoxins [3,4,5]. The annual economic loss of maize due to aflatoxin contamination was estimated to be in the hundreds of millions of dollars and has been predicted to reach as high as USD 1.68 billion due to global climate change [6], making aflatoxin contamination a serious economic threat to world trade of aflatoxin-susceptible crops. The options available to manage aflatoxin contamination in maize include biological control through field application of atoxigenic A. flavus strains, such as NRRL30797, AF36 and NRRL21882, which are capable of reducing aflatoxin accumulation up to 94.8% in maize by competitively displacing native A. flavus strains that produce aflatoxin [11,12,13]

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