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Abstract
Maize pathogen Fusarium verticillioides is the causal agent of seedling blight disease, with production of the fumonisin B1 (FB1) mycotoxin being necessary for disease development. FB1 was previously observed to accumulate in the roots and leaves during systemic infection of the plant, but not in the leaves when watered into the soil. Here we detected FB1 in the roots and leaves during seedling blight infection by an aconidial F. verticillioides strain able to infect the mesocotyl but unable to colonize leaves, while wild type demonstrated colonization and FB1 accumulation in the same locations. Therefore, root colonization by F. verticillioides is necessary for accumulation of FB1 and leaf colonization is dispensable for FB1 mobility in planta. Since phytotoxic effects of fumonisin are known to include nitric oxide (NO) accumulation in planta, the biological and virulence effects of NO on F. verticillioides was evaluated. Specifically, we addressed the roles of flavohemoglobins FHB1 and FHB2 on NO detoxification by F. verticillioides. Microarray analysis revealed a significant induction of FHB2 (17-fold) transcription with exposure to NO (1.5 mM DETA NONOate, a NO donor) and a 2-fold increase in FHB1 transcription. In comparison, the highest induction at 246-fold was a dissimilatory nitrite reductase (NIR1), along with high induction of other denitrification genes, including a P450 nitric oxide reductase (NOR1). Deletion mutants (fhb1, fhb2, nor1, nir1, and fhb1/fhb2 double deletion) were generated and challenged on NO, nitrate, and nitrite media. fhb1, fhb2, and nor1 were restricted in growth with NO exposure, while fhb1/fhb2 showed no growth. Only 7% of fhb1/fhb2 conidia survived the NO exposure. All mutants grew on nitrate and nitrite media under normal atmospheric conditions. In contrast, fhb2, nir1, nor1, and fhb1/fhb2 were unable to grow on nitrite media under hypoxic conditions, thus linking these genes to denitrification. Also, fhb1/fhb2 had restricted growth on nitrite media under oxygenic conditions, demonstrating the importance of flavohemoglobins in nitrite metabolism. Maize seedling blight assays revealed fhb1/fhb2 to be more virulent than wild type, with double the production of FB1. Here we show denitrification is important in NO detoxification and link such detoxification to virulence and FB1 production.