Production of the 14 kDa trypsin inhibitor protein is important for maize resistance against Aspergillus flavus infection/aflatoxin accumulation

Abstract

Maize (Zea mays L.) is one of the major crops susceptible to Aspergillus flavus Link ex. Fries infection and subsequent aflatoxin contamination. Previous studies found the production of an antifungal 14 kDa trypsin inhibitor (TI) was associated with maize aflatoxin resistance. To further investigate whether the TI plays any direct role in resistance, a TI gene silencing vector was constructed and transformed into maize. Mature kernels were produced from 66 transgenic lines representing 18 independent events. A final total of twelve lines representing four independent events were confirmed positive for transformation, five of which showed significant reduction (63 to 88%) in TI transcript abundance in seedling leaf tissue and seven of which showed significant TI protein reduction (39-85%) in mature kernels. Six of the seven silenced transgenic lines supported higher levels of aflatoxin production compared to negative controls. To further confirm the role of TI in field resistance to aflatoxin accumulation, DNA sequence polymorphisms from within the gene or linked simple sequence repeats were tested in four quantitative trait loci (QTL) mapping populations for QTL effect, and three QTL with log of the odds scores of 11, 4.5, and 3.0 and possibly caused by the TI protein encoding gene were found. Sequence polymorphisms were also tested for association to aflatoxin levels in an association mapping panel, and three single nucleotide polymorphisms were found associated with aflatoxin accumulation (P<0.01). The data from both RNAi and genetic mapping studies demonstrated that production of the TI in maize is important for its resistance to A. flavus infection and/or aflatoxin production.