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

Abstract

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.

Gene discovery and gene function assignment in filamentous fungi

“…3). The bias does not correspond (21); N ϭ NUT1 (38). Published TI frequencies are 16%, 11%, 21%, 3%, and 22%, respectively.…”

“…Targeted integration (TI) for creating gene-specific mutations is very efficient and requires only 50-bp fragments of target gene homology on either side of a selectable marker (11,12). In contrast, many filamentous fungi have genome sizes in the range of [30][31][32][33][34][35][36][37][38][39][40] Mb and are estimated to contain at least 10,000 genes (13). Genome studies using expressed sequence tag analysis suggest that more than half of these genes lack homologues in S. cerevisiae (14).…”

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

“…The expression of selected genes involved in nitrogen metabolism is reduced in cells exposed to readily metabolizable nitrogen sources such as ammonia (Facklam & Marzluf, 1978;Sikora & Marzluf, 1982a, b). One transcriptional regulator, named NIT2 in N. crassa and AREA in A. nidulans, has been identified in several fungi (Fu & Marzluf, 1990b, c;Haas et al, 1995;Froeliger & Carpenter, 1996, Screen et al, 1998, and its binding to DNA is necessary for the expression of the genes involved in utilization of nitrogen sources (Scazzocchio, 2000). In the absence of ammonia, AREA binds to two closely spaced 59-GATA sequences in various gene promoters and activates their transcription .…”

Molecular mechanisms regulating plant infection in the rice blast fungus Magnaporthe oryzae

“…Ammonia is the preferred nitrogen source for M. oryzae. The NMR in M. oryzae occurs through the transcriptional activator Nut1, the M. oryzae AreA/Nit2 orthologue (Froeliger & Carpenter, 1996). The expression of a large number of genes encoding enzymes that are involved in the utilisation of various secondary nitrogen sources -nitrate, purines or amino acids-is subject to nitrogen metabolic repression and is positively regulated by Nut1.…”

“…The assimilation of nitrogen by M. oryzae from underground plant tissues is regulated by the global nitrogen regulator Nut1 (Froeliger & Carpenter, 1996). The Δnut1 mutant is non-pathogenic on roots but infects leaves as well as the wildtype strain .…”