|BALDWIN, THOMAS - Former ARS Employee|
|Glenn, Anthony - Tony|
Submitted to: Inoculum
Publication Type: Abstract Only
Publication Acceptance Date: 3/31/2014
Publication Date: 6/8/2014
Citation: Baldwin, T., Glenn, A.E. 2014. Detoxification of nitric oxide by Fusarium verticillioides is linked to denitrification. Inoculum. 65(3):28.
Technical Abstract: Nitric oxide (NO) is a potent cellular signaling molecule and a byproduct of nitrogen metabolism. High concentrations of NO are a form of nitrosative stress, and to alleviate this stress, organisms utilize flavohemoglobins to convert NO into nontoxic nitrate ions. We have investigated the capacity of Fusarium verticillioides, a maize pathogen well-known for its production of fumonisin mycotoxins, to detoxify NO using two flavohemoglobins, FHB1 and FHB2. Microarray analysis revealed a 17-fold induction of FHB2 transcription and a 2-fold induction of FHB1 when the fungus was exposed to exogenous NO (1.5 mM of NO-donor DETA NONOate). Surprisingly, the highest induction was a 246-fold increase in transcription of the dissimilatory nitrite reductase (NIR1) from the denitrification pathway. This ecologically important nitrogen dissimilatory pathway is generally associated with metabolism under hypoxic conditions. The pathway includes a unique nitrate reductase (NAR1) and a P450 nitric oxide reductase (NOR1) that are also induced by NO exposure, 16 and 22-fold respectively. Deletion mutants nir1, nor1, and fhb2 were unable to grow on nitrite media under hypoxic conditions, thus linking FHB2 to denitrification. Under normal, oxygenated conditions, deletion of either flavohemoglobin increased F. verticillioides sensitivity to exogenous NO, with complete inhibition of growth when both genes were deleted (fhb1/fhb2). Maize seedling blight assays demonstrated increased virulence and fumonisin production by the fhb1/fhb2 deletion mutant, indicating loss of NO detoxification may also impact nitrogen regulated secondary metabolism. Further studies will address this observation. Phylogenetic evaluation of the denitrification pathway proteins suggested the capacity for dissimilatory nitrogen metabolism may be restricted to only a narrow range of genera, and the NIR1, NOR1, and the FHB1 and FHB2 flavohemoglobin genes appear to be derived from bacteria via horizontal gene transfer. In contrast the dissimilatory nitrate reductase NAR1 arose from duplication and divergence of an assimilatory nitrate reductase. Interestingly we were unable to detect a NAR1 ortholog in any other fungi besides F. verticillioides and F. oxysporum. These species have uniquely evolved NAR1, which may confer enhanced denitrification activity over other fungi. Overall this work extends our understanding of both NO detoxification and the denitrification of nitrate and nitrite in fungi.