Location: Food and Feed Safety Research2013 Annual Report
1a. Objectives (from AD-416):
Facilitate marker assisted corn breeding efforts as well as development of transgenic cotton to enhance resistance to aflatoxin contamination. Identify genes encoding resistance associated proteins (RAPs), e.g., antifungal, stress responsive, or interfering with toxin signaling pathways, in developing and mature corn kernels through transcriptional profiling of Aspergillus flavus infected corn seed. Determine if hyperdiversity in aflatoxigenicity is driven by variation in nuclear composition.
1b. Approach (from AD-416):
Aspergillus flavus microarrays containing the 5,200 expressed gene sequences will be analyzed for identification of specific genes involved in plant-fungal interactions, as well as those expressed under conditions conducive to aflatoxin production in the fungus. Histological examination of fungal colonization within the seed will be used to identify seed tissues that are most impacted by Aspergillus flavus during the infection process. Nuclear diversity in Aspergillus flavus and Aspergillus parasiticus that are either aflatoxigenic or nontoxigenic will be examined by using 1) qPCR of parents and offspring derived from sexual crosses and 2) differential labeling of nuclei in several homokaryons derived by sorting conidia with a single nucleus and setting up parasexual and sexual crosses to monitor nuclear inheritance. Offspring will be grown serially in culture to track changes in nuclear ratios over several generations of asexual growth and will also be inoculated on corn to determine if crops are selecting for specific toxin (nuclear) traits. The possibility of supernumerary/dispensable chromosomes influencing toxicity will also be explored using protoplasting and karyotyping methods.
3. Progress Report:
To better understand the dynamics of the interaction between corn seeds and Aspergillus (A.) flavus, its interaction is being compared with that of the fungus Fusarium verticillioides, which is another toxin-producing fungus that is thought to have a different type of interaction with corn seeds than A. flavus, while co-existing with this fungus on corn seed. Field grown maize was harvested at 4, 12, 24, 48, and 72 hours post inoculation (hpi) and gene transcription monitored by ribonucleic acid (RNA)-seq technology. Bioinformatic analysis (analysis using computer tools for gaining biological information) of these data is not yet complete, but the emerging picture is that both fungi elicit the expression of a core set of host genes. Each fungus also changes the expression of a set of genes unique to that species. Analysis for corn genes differentially expressed at three different time points show that all three of the plant’s natural defense signaling pathways are induced in response to these fungi. Preliminary data also indicate that these two fungi affect the abscisic acid, auxin, and gilbberillic acid (plant hormone) signaling pathways, which are also known to modulate the salicylic acid, jasmonate, and ethylene (all hormone signals) defense signaling pathways. Based on gene expression analysis, infection by each fungus causes a change in the plant’s carbon metabolism by shifting from the synthesis of starch to the breakdown of starch. This is more pronounced in the A. flavus/corn seed interaction than in the Fusarium verticillioides/corn seed interaction. Gene expression analysis of putative resistance genes is being coupled with RNA in situ hybridization (joining of two complementary strands of deoxyribonucleic acid) and histology (microscopic anatomy of cells and tissues) to determine the timing and location of gene expression in the host. The tools are in hand to identify, quantify, localize and functionally analyze putative corn resistance genes to these two fungi. Results to date show early recognition of the fungi by the host, tissue specific colonization by the fungus, and tissue specific defense responses by the host. Data analysis by new bioinformatics tools will allow the construction of gene networks and lead to the identification of new putative resistance genes. In studies designed to understand the nuclear condition of A. flavus we are using two nuclear localized fluorescent reporter constructs (vehicles to transport genes) that encode histones HH2A or HH2B (proteins that package deoxyribonucleic acid (DNA) into specific cell structures) fused at the C-terminus (part of protein) with either the yellow (EYFP) or cyan (ECFP) fluorescent proteins (allow the visualization of either yellow or cyan-fluorescing nuclei by microscopy). Results suggest that the number of nuclei in the conidial (fungal spores) population of Aspergillus flavus is not homogenous and that different types of nuclei can be selectively packaged into conidia during the process of conidiation in A. flavus. The available genetic markers, the ability to fuse genetically different fungal strains, and the ability to rapidly sort nuclei by fluorescence assisted cell sorting (FACS) provides a very powerful system to better understand the biology and ecology of A. flavus.