Discipline: Ecology Environmental and Earth Sciences
Subcategory: Plant Research
Zandria Chambers - Fort Valley State University
Co-Author(s): Gold Scott, University of Georgia, Athens, GA
Fusarium verticillioides is a pathogen predominantly found worldwide in association with maize plants and kernels. The fungus produces and secretes harmful secondary metabolites (mycotoxins), most notably fumonisin B1 (FB1), which affects a variety of animals and potentially humans. FB₁ causes equine leukoencephalomalacia (ELEM) in horses and porcine pulmonary edema (PPE) in pigs and was shown to be carcinogenic in laboratory animals. It is also correlated with neural tube birth defects in humans. Further, FB1 is also a phytotoxin to maize, causing leaf necrosis and reduced root growth. Fumonisins are similar in structure to free sphingoid bases sphinganine and sphingosine, which are used in the synthesis of ceramide. In turn ceramide is modified to form sphingolipids that are essential for proper membrane structure, localization of membrane-bound proteins, cell-to-cell recognition, signal transduction pathways, and other cellular functions. The structural similarity of FB1 to sphinganine and sphingosine is the basis of its toxicity and mechanism of action since it is an inhibitor of ceramide synthase, the enzyme needed to produce ceramide. Inhibition of ceramide synthase results in depleted sphingolipids. Given that FB1 inhibits ceramide synthase in a wide range of eukaryotic organisms, we are curious to understand how F. verticillioides is resistant to its own toxin. Perhaps it has an FB1-insensitive ceramide synthase, or maybe it possesses a mechanism to detoxify any FB1 inside of its own cells. We are examining this latter possibility. A bacterium, Sphingopyxis sp., has been found to encode a carboxylesterase (fumD) that is able to detoxify FB1 by cleaving off its two tricarballylic acid side chains. BLASTP of fumD against F. verticillioides identified a putative homolog encoded by FVEG_04320 (E value 6e-66). Interestingly, when this encoded protein is used for BLASTP against the entire NR database at NCBI, the only other fungi possessing orthologs are a few Fusarium species, and these have significant similarity to proteins from a diversity of bacteria. Thus, we propose that Fusarium acquired this gene from bacteria via horizontal gene transfer (HGT). FVEG_04320 was targeted for gene deletion in F. verticillioides using the OSCAR protocol, or One Step Construction of Agrobacterium Recombination-ready-plasmids. The process for creating this deletion construct is described. Once a verified mutant is obtained, it will be compared to wild type to determine if FVEG_04320 is needed by F. verticillioides for its insensitivity to FB1 (standards).
References: Menke, J., et al., 2013. Cellular development associated with induced secondary metabolism in the filamentous fungus Fusarium graminearum. PLoS Pathogens. 8(5):e63077. Kistler, H.C., Rep, M. 2013. Structural dynamics of Fusarium genomes. In: Fusarium: genomics, molecular and cellular biology. Brown, D.W. and Proctor, R.H., Editors. Norwich, United Kingdom. Caister Academic Press. p. 46-60.
Funder Acknowledgement(s): This work was supported in part by a grant from the NSF funded Fungal Genomics & Computational Biology REU Program at the University of Georgia, Genetics Department, Athens, GA; NSF HBCU-UP/Targeted Infusion #1332503 awarded to Sarwan Dhir, Fort Valley State University.
Faculty Advisor: Sarwan Dhir, firstname.lastname@example.org
Role: Design the experiments, collect the data, take the pictures, prepare the presentation.