Potential Roles of WRKY Transcription Factors in Resistance to \u3ci\u3eAspergillus flavus\u3c/i\u3e Colonization of Immature Maize Kernels

The infection and colonization of maize (Zea mays L.) by the necrotrophic fungal pathogen Aspergillus flavus results in contamination of kernel tissues with carcinogenic mycotoxins known as aflatoxins, resulting in severe economic losses as well as negative effects on human and animal health. Resistance to A. flavus is mediated by both inducible and constitutively expressed defense proteins; however the mechanism regulating the expression of these defenses is poorly understood. This study examined the potential roles of six maize WRKY transcription factors, ZmWRKY19, ZmWRKY21, ZmWRKY53, ZmWRKY53.1, ZmWRKY67, and ZmWRKY68, in regulating defense responses against A. flavus. The responses of these WRKY transcription factors to A. flavus inoculation were examined over a time course in the immature kernel tissues of two maize lines, B73 (susceptible) and TZAR101 (resistant) using real-time quantitative PCR. Three defense genes, Nonexpressor of Pathogenesis-Related Protein 1 (ZmNPR1), Pathogenesis-Related Protein 1 (ZmPR-1), and Ethylene Responsive Factor 1 (ZmERF1), were also examined in order to determine whether salicylic acid, jasmonic acid, or ethylene-mediated defense mechanisms were induced in response to A. flavus inoculation. The genes ZmWRKY19, ZmWRKY53, and ZmWRKY67 were found to be induced by inoculation and constitutively expressed at higher levels in the resistant maize line. Both the putative ZmWRKY19 and ZmWRKY53 are homologs of Arabidopsis WRKY53 and WRKY33, respectively, with ZmWRKY53 also being homologous to rice and wheat WRKY53. These genes may function in promoting antioxidant enzymes to sequester reactive oxygen species (ROS) during pathogen infection or abiotic stress. ZmWRKY67 is homologous to AtWRKY50 and may function in the suppression of jasmonic acid-regulated defenses in the resistant maize line. The expression of ZmNPR1 was also induced by inoculation in the resistant variety without concurrent induction of ZmPR-1, possibly due to the observed induction of ZmERF1 and, therefore, ethylene-based defenses. These findings indicate that resistant maize lines may possess elevated oxidative stress tolerance potentially conferring resistance to programmed cell death as part of the hypersensitive response induced by ROS during necrotrophic pathogen infections. Future studies of these WRKY transcription factors are necessary to better understand their regulation and involvement in resistance to A. flavus infection and aflatoxin production