Location: Food and Feed Safety Research2012 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:
In a comparison between two toxin producing fungi, Aspergillus (A.) flavus and Fusarium (F.) verticillioides, we showed that the two fungi differ somewhat in their colonization of maize seeds. Field grown maize was harvested at 4, 12, 24, 48, and 72 hours post inoculation (hpi). In the first 48 hpi, colonization was restricted to aleurone and endosperm tissues (part of a seed) that received the inoculum (fungal material to cause infection). By 72 hpi, both fungi were detected in the aleurone, endosperm, and embryo-endosperm interface/in seeds. F. verticillioides more extensively colonized the kernel than A. flavus. To determine if known maize defense genes are expressed in tissues colonized by the fungi, ribonucleic acid (RNA) in situ hybridization was used to assess transcript (conversion from DNA to RNA) accumulation of PRms (Pathogenesis related protein, maize seeds) and UGT (UDP-glucosyltransferases) during infection. PRms was differentially expressed in the aleurone and scutellum (three different seed compartiments and tissues) of greenhouse and field grown seeds infected with each fungal species. UGT was expressed in the aleurone, endosperm, and scutellum of the field-grown seeds, but was only expressed in the scutellum of the greenhouse-grown seeds infected each fungus. With both fungi, PRms and UGT were expressed in advance of visible colonization. Current studies are directed at profiling gene transcription in maize tissue at these time points. 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 DNA into specific cell structures) fused at the C terminus (part of protein) with either the yellow (EYFP) or cyan (ECFP) fluorescent proteins. These constructs were transformed into A. flavus strain AFC to generate two strains, (AFC-pyr-arg+ECFP) expressing cyan fluorescence that requires the compound uracil and another strain (AFC–pyr+arg-EYFP) expressing yellow fluorescence that requires arginine (an amino acid) to grow. Further, fusants between the two strains were obtained through the technique of polyethylene mediated cell fusion and selection on minimum medium, which favored the growth of the fusants over that of either of the parent strains. One fusant selected for further study was heterokayotic (cells with different types of nuclei) with nuclei expressing either EYFP or ECFP or both EYFP+ECFP in mycelia and conidia (reproductive structures of the fungi). Conidia (spores of the fungus) containing nuclei expressing EYFP+ECFP were separated and found to contain both yellow and cyan fluorescent proteins in the same nucleus. Current research is focused on determining whether nuclei expressing EYFP+ECFP are maintained predominantly as heterokaryons or as diploids (having two sets of chromosomes).