Isolation of a nitrogen response regulator gene (nrr1) from Metarhizium anisopliae.

Attempts to improve the effectiveness of entomopathogenic fungi as biological control agents require a clear understanding of the pathogenicity determinants at both the biochemical and molecular level. Proteases play a key role in entomopathogenicity, allowing the fungus to penetrate the insect cuticle and rapidly invade the host. The most extensively studied of these protease activities, PR1A and PR2, are both subject to nitrogen derepression. The Metarhizium anisopliae nrr1 (nitrogen response regulator 1) gene was identified using a PCR-based strategy; it encodes a putative DNA-binding protein with a single zinc finger motif defined by the C-X2-C-X17-C-X2-C sequence. M. anisopliae NRR1 shows a significant sequence similarity to Neurospora crassa NIT2. Sequence analysis identified the presence of two introns, suggesting a greater degree of similarity to N. crassa nit2 than to the areA-like genes that have been identified. However, functional equivalence of nrr1 to areA was demonstrated, by co-transformation and complementation of an A. nidulans areA loss-of-function mutant (areA18 argB2 pabaA1 inoB2) with the M. anisopliae nrr1 gene. The areA-/nrr1+ Aspergillus transformants were able to grow on media with nitrate and glutamate as the sole nitrogen source, whereas the areA- strain is unable to grow under these conditions. The possible relevance of nitrogen regulation to pathogenicity is discussed.