Location: Food and Feed Safety ResearchTitle: Nuclear heterogeneity in conidial populations of Aspergillus flavus
|RUNA, FARHANA - North Carolina State University|
|CARBONE, IGNAZIO - North Carolina State University|
|PAYNE, GARY - North Carolina State University|
Submitted to: Fungal Genetics and Biology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 9/8/2015
Publication Date: 9/9/2015
Citation: Runa, F., Carbone, I., Bhatnagar, D., Payne, G.A. 2015. Nuclear heterogeneity in conidial populations of Aspergillus flavus. Fungal Genetics and Biology. 84:62-72. https://doi.org/10.1016/j.fgb.2015.09.003.
Interpretive Summary: Aspergillus flavus is a soil fungus that can be pathogenic on a diverse array of plant hosts and animals. It commonly infects and contaminates major seed crops such as maize, peanut and cottonseed with aflatoxin, which is highly toxic to humans and animals. In addition, A. flavus is the second leading cause for Invasive Aspergellosis (IA), which can be fatal in immunocompromised patients. Phenotypic and genotypic variations are common within strains of A. flavus, and studies have shown that genetic variation within A. flavus populations is correlated with the frequency and severity of aflatoxin contamination in crops. Several molecular genetic tools have been applied to better understand genetic diversity of A. flavus. To our knowledge, this is the first study to use fluorescently labeled nuclei to investigate the nuclear condition in A. flavus and its potential impact on fungal diversity and its ability to make aflatoxins.
Technical Abstract: Aspergillus flavus is a major producer of aflatoxin and an opportunistic pathogen for a wide range of hosts. Understanding genotypic and phenotypic variations within strains of A. flavus is important for controlling disease and reducing aflatoxin contamination. A. flavus is multinucleate and predominantly haploid (n) and homokaryotic. Although cryptic heterokaryosis may occur in nature, it is unclear how nuclei in A. flavus influence genetic heterogeneity and if nuclear condition plays a role in fungal ecology. A. flavus mainly reproduces asexually by producing conidia. In order to observe whether the nuclei in conidia are homokaryotic or heterokaryotic, we labeled nuclei of Aspergillus flavus using two different nuclear localized fluorescent reporters. The reporter constructs (pYH2A and pCH2B), encode histones HH2A and HH2B fused at the C terminus with either yellow (EYFP) or cyan (ECFP) fluorescent proteins, respectively. The constructs were transformed into the double auxotrophic strain AFC-1 (-pyrG, -argD) to generate a strain containing each reporter construct. Taking advantage of the nutritional requirement for each strain, we were able to generated fusants between FR36 (-argD) expressing yellow fluorescence, and FR46 (-pyr4) expressing cyan fluorescence. Conidia from the fusants between FR36 and FR46 showed three types of fluorescence: only EYFP, only ECFP or both EYFP+ECFP. Conidia containing nuclei expressing EYFP+ECFP were separated by Fluorescence–Activated Cell Sorting (FACS) and were found to contain both yellow and cyan fluorescent markers in the same nucleus. Further characterization of conidia having only one nucleus but expressing both EYFP+ECFP confirmed that they were diploid (2n). Our findings suggest that A. flavus maintains nuclear heterogeneity in conidial populations.