NUT1, a major nitrogen regulatory gene in Magnaporthe grisea, is dispensable for pathogenicity

NUT1, a gene homologous to the major nitrogen regulatory genes nit-2 of Neurospora crassa and areA of Aspergillus nidulans, was isolated from the rice blast fungus, Magnaporthe grisea. NUT1 encodes a protein of 956 amino acid residues and, like nit-2 and areA, has a single putative zinc finger DNA-binding domain. Functional equivalence of NUT1 to areA was demonstrated by introducing the NUT1 gene by DNA-mediated transformation into an areA loss-of-function mutant of A. nidulans. The introduced NUT1 gene fully complemented the areA null mutation, restoring to the mutant the ability to utilize a variety of nitrogen sources. In addition, the sensitivity of Aspergillus NUT1 transformants to ammonium repression of extracellular protease activity was comparable to that of wild-type A. nidulans. Thus, NUT1 and areA encode functionally equivalent gene products that activate expression of nitrogen-regulated genes. A one-step disruption strategy was used to generate nut1- mutants of M. grisea by transforming a rice-infecting strain with a disruption vector in which a gene for hygromycin B phosphotransferase (Hyg) replaced the zinc-finger DNA-binding motif of NUT1. Of 31 hygromycin B (hyg-B)-resistant transformants shown by Southern hybridization to contain a disrupted NUT1 gene (nut1 : : Hyg), 26 resulted from single-copy replacement events at the NUT1 locus. Although nut1- transformants of M. grisea failed to grown on a variety of nitrogen sources, glutamate, proline and alanine could still be utilized. This contrasts with A. nidulans where disruption of the zinc-finger region of areA prevents utilization of nitrogen sources other than ammonium and glutamine. The role of NUT1 and regulation of nitrogen metabolism in the disease process was evaluated by pathogenicity assays. The infection efficiency of nut1- transformants on susceptible rice plants was similar to that of the parental strain, although lesions were reduced in size. These studies demonstrate that the M. grisea NUT1 gene activates expression of nitrogen-regulated genes but is dispensable for pathogenicity.