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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). F 1  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.

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