Suppression subtractive hybridization analyses identify genes central to phosphate sensing functions in Neurospora crassa

GRAS DE; SILVEIRA HC; MARTINEZ-ROSSI NM; ROSSI A

Águas de Lindóia

Encuentro; XXXV Reunião Anual da Sociedade Brasileira de Bioquímica e Biologia Molecular; 2006

Sociedade Brasileira de Bioquímica e Biologia Molecular

Microorganisms have evolved complex signal transduction networks that enable them to make optimal use of the nutrient sources available. Inorganic phosphate (Pi), an essential nutrient for all organisms, is required for the biosynthesis of nucleic acids, phospholipids and cellular metabolites, as well as for the energy transduction and metabolic signaling responses. The phosphorus acquisition system in N. crassa includes four regulatory genes: nuc-2, preg, pgov, and nuc-1. Under limiting Pi, NUC-2 inhibits the functioning of the complex PREG-PGOV through the interaction of ankyrin-like repeats, allowing the activation of the wide domain transcription regulator NUC-1. In order to identify candidate genes involved in Pi sensing, we employed suppression subtractive hybridization (SSH) between RNA isolated from the 74A and nuc-2A mutant strains of N. crassa after growth under limiting Pi. Following SSH, expression of the clones was examined using macro-arrays dot-blot and virtual Northern blots. Sixty-six cDNA clones differentially expressed in the nuc-2A mutant strain were selected for sequencing. Twenty-five of them had significant homology to fungal sequences from N. crassa databases. Functional grouping, performed according to the N. crassa protein sequence reference set from MIPS, revealed that 63% corresponded to unclassified proteins and 37% corresponded to putative proteins involved in diverse cells process. Further examination of eight clones by virtual Northern blots reveled that six clones were significantly up regulated in the nuc-2 mutant strain, including two MAP kinases (predicted MAP kinase, accession no. NCU 02393.2, and mitogen-activated protein kinase sty1, accession no. NCU07024.2). The fact that these genes were strongly expressed in the nuc-2 mutant indicates that a Pi rich environment induces their expression in the wild type strain. Thus, we propose that these genes are involved in posttranscriptional modifications of NUC-1 to repress the expression of the phosphorus acquisition genes. Our results provide the first insight into the expression of genes under the action of the nuc-2 gene in N. crassa.