1a.Objectives (from AD-416):
Characterize the role of maize kernel proteins and corresponding genes in resistance to aflatoxin contamination and aflatoxin-producing fungi. Identify and characterize resistance-associated proteins/genes in soybean that may enhance our understanding of aflatoxin-resistance and that may be exploited through enhancement of homologous genes in maize. Determine the most useful maize resistance markers for breeders to use in transferring resistance to commercially useful germplasm.
1b.Approach (from AD-416):
Characterize resistance-associated proteins (RAP's), e.g., antifungal, stress
responsive, or interfering with toxin signaling pathways, in developing and mature maize kernels identified at SRRC through proteomic comparisons of resistant and susceptible maize inbreds, some with very similar genetic backgrounds. Identify, using proteome analyses, resistance-associated proteins/genes in soybean with efficacy against A. flavus and aflatoxin production. Soybean is an aflatoxin resistant species, therefore, the possibility exists that resistance factors may be identified that could be exploited through enhancement of homologous genes in maize. Characterize function of maize and soybean proteins/genes through molecular and physiological laboratory and greenhouse investigations. Determine role of proteins/genes in aflatoxin-resistance using RNAi gene silencing technology.
Five aflatoxin resistant maize inbred (resulting from cross between genetically related parents) lines obtained from the International Institute of Tropical Agriculture-Southern Regional Research Center (IITA-SRRC) collaborative breeding program were evaluated in 2012 (previously evaluated in 2010 and 2011) for aflatoxin-resistance under Louisiana field conditions. Two of the six lines were found to have high levels of aflatoxin resistance, comparable to resistant checks used in the study. These results were similar to those obtained in field evaluations in 2010 and 2011. A novel antifungal glucanase gene previously identified by this lab had been partially characterized, including comparing the differences in expression pattern, tissue specificity of this gene in response to biotic and abiotic stresses. A manuscript summarizing the characterization work has been revised after additional experiments (suggested by a refereed journal) were conducted. These include antifungal assays using transgenic Arabidopsis (an experimental laboratory plant) expressing the corn glucanase gene, as well as studies to determine whether this glucanase gene is regulated through the salicylic acid signal transduction pathway (a cascade of biochemical reactions possibly leading to resistance). Three WRKY (protein that binds to deoxyribonucleic acid and controls information flow) transcription factor genes that show differential expression between resistant and susceptible corn lines after Aspergillus (A.) flavus inoculation have been identified. Also, data suggests that plant hormones are involved in regulating gene expression in response to A. flavus infection. Promoter analysis (promoter is a region of deoxyribonucleic acid of a gene that initiates expression of that gene) of two PR10 genes has been initiated in order to gain insight on how the expression of host resistance related genes is regulated. Five different promoter deletions of the corn PR10 gene have been created and transformed into Arabidopsis for testing gene expression levels. A manuscript summarizing the results on the maize PR10.1 promoter analysis is being prepared. In this study, we have identified a small region (0.6 kb) of PR10.1 promoter that can drive high level of target gus gene (a tagged gene that can be monitored) expression in Arabidopsis. This can be used in the future to enhance host resistance associated genes in corn. Experiments to comparatively investigate the proteomes (entire protein complement of an organism) of the five resistant IITA-SRRC maize lines are being planned to determine the similarity of the components involved in aflatoxin resistance of the five lines.