Isolation and characterization from pathogenic fungi of genes encoding ammonium permeases and their roles in dimorphism

Abstract

SummaryNutrient sensing plays important roles in fungal development in general, and specifically in critical aspects of pathogenicity and virulence, for both animal and plant pathogens. Dimorphic pathogens such as the phytopathogenic smut fungi, Ustilago maydis and Microbotryum violaceum , must switch from a yeast-like to a filamentous form in order to cause disease. Two genes encoding methylammonium permeases (MEPs) were identified from each of these latter fungi and all the encoded proteins were most similar to Mep2p, the high-affinity permease from Saccharomyces cerevisiae that plays a direct role in pseudohyphal or filamentous growth for that organism. This is the first report of MEPs from pathogenic fungi. The two genes from U. maydis and one of the genes from M. violaceum were expressed in diploid S. cerevisiae mutants deleted for all three mep genes ( mep1mep2mep3 ). Each of the heterologous genes could complement the severe growth defect of the S. cerevisiae mutant on low ammonium. Moreover, the U. maydis ump2 gene, initially detected as an upregulated gene in budding cells, was also able to complement the pseudohyphal defect characteristic of the mutant yeast. This gene is thus one of few heterologous MEP genes capable of efficiently restoring pseudohyphal growth in yeast. For U. maydis , disruption of ump2 eliminated the filamentous phenotype of haploid cells on low ammonium, while ump1 disruption only slightly reduced methylamine uptake. The most significant drop in methylamine uptake was seen for the ump2 and the ump1ump2 double mutants. Moreover, when grown in liquid medium, the ump1ump2 double mutant aggregated and sedimented. Also, the importance of a putative site for phosphorylation by protein kinase A was investigated in both Mep2p and Ump2p via site-directed mutagenesis of the respective genes. A mutation predicted to prevent phosphorylation of either protein, still allowed each to provide growth on low ammonium, but eliminated their abilities to provide pseudohyphal growth for the S. cerevisiae triple mutant. These findings allow us to present a model of how ammonium transporters play a role in regulating dimorphic growth in fungi.

Ustilago maydis phosphodiesterases play a role in the dimorphic switch and in pathogenicity

“…maydis cells were grown at 25 uC in YPS and/or SLAD (synthetic low-ammonium medium; contains 50 mM ammonium as ammonium sulfate) as described previously (Smith et al, 2003). Solid media were made with 2 % agar, and 1 % activated charcoal was added to enhance contrast of mating assays (Gold et al, 1997).…”

“…Wild-type U. maydis cells produce filamentous cells when starved for ammonium (Smith et al, 2003). When grown on SLAD low-ammonium agar, wild-type strain 2/9 produced colonies with extensive filamentation, while umpde2 deletion strains showed reduced filamentation.…”

Fungal sensing of host environment

“…Fungal adaptation mechanisms include regulation of metabolism, stress response, virulence factors and toxins, sexual reproduction and modulation of the cell wall. (Smith et al, 2003) and is important in a host environment because the availability of nitrogen regulates pathogenicity and filamentation. Normally, easily available sources of nitrogen such as ammonium are utilized by fungi.…”

Sulphur and nitrogen regulation of the protease-encoding ACP1 gene in the fungus Botrytis cinerea: correlation with a phospholipase D activity

“…Fungi sense the nutritional status of their environment via multiple mechanisms that first relay the information about particular nutrients and then trigger the appropriate cellular response (Gagiano et al, 2002;Eckert-Boulet et al, 2004;Smith et al, 2003). Three essential nutrients, namely carbon, nitrogen and sulphur, are present in the environment as readily usable sources or as complex molecules from which they can be enzymically extracted by fungi.…”