ISOLATION OF A NITROGEN RESPONSE REGULATOR GENE (NRR1) FROM METARHIZIUM-ANISOPLIAE

Attempts to improve the effectiveness of entomopathogenic fungi as bio logical control agents require a clear understanding of the pathogenic ity determinants at both the biochemical and molecular level. Protease s play a key role in entomopathogenicity, allowing the fungus to penet rate the insect cuticle and rapidly invade the host. The most extensiv ely studied of these protease activities, PRIA and PR2, are both subje ct to nitrogen derepression. The Metarhizium anisopliae nrrl ((nitroge n 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-X-2-C-X-17-C-X-2-C sequence. M. anisopliae NRR1 shows a significant sequence similarity to Neurospora crassa NIT2. Se quence analysis identified the presence of two introns, suggesting a g reater degree of similarity to N. crassa nit2 than to the ar ed-like g enes that have been identified. However, functional equivalence of nrr 1 to areA was demonstrated, by co-transformation and complementation o f an A. nidulans areA loss-of-function mutant (areA18 argB2 pabaA1 ino B2) with the M. anisopliae nrrl 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 gro w under these conditions. The possible relevance of nitrogen regulatio n to pathogenicity is discussed. (C) 1998 Elsevier Science B.V. All ri ghts reserved.