Functional Characterization Of The APSES Transcription Factor Pcg2 In The Rice Blast Fungus

APSES (Asmlp Phdlp, Sok2p, Efg1p, and StuAp) proteins are a class of transcription regulators specific to fungi. Fungal APSES proteins play important roles in regulating developmental processes. However, the regulation mechanisms of these members, especially in plant pathogenic fungi are little known. In this study, we characterize a novel APSES transcription factor Pcg2 in Magnaporthe oryzae. The APCG2 mutants show defects in hyphal growth, conidiogenesis, germination, appressorium formation, penetration peg formation, invasive hyphae formation and is reduced in virulence toward host rice and barley. Further, the molecular function of Pcg2 is fully characterized by means of molecular techniques in the rice blast fungus Magnaporthe oryzae.Pcg2 is able to substitute the function of Swi4 or Mbpl in Saccharomyces cerevisiae, and is the homolog of both Mbpl and Swi4, which are key regulators in cell cycle process. Transformants with the fusion PCG2::GFP restore the defects of △PCG2 deletion mutant, and the fusion protein Pcg2-GFP is localized into nuclei. The APSES domain is a DNA binding domain specific to fungal transcription regulators. Therefore Pcg2-GST fusion protein is expressed and purified, and in vitro it binds both MCB (ACGCGNT, ACGCGT) motifs and SCB (CACGAAA) motifs. Fusion with the DNA binding domain of GAL4, we prove Pcg2 has transcriptional activation activity with two independent transcription activation domains. So, Pcg2 is a novel APSES transcription factor that binds both MCB and SCB box and the APSES domain, ANK domains, C-terminus are required for Pcg2 function.To uncover the genes regulated by Pcg2, microarray based gene expression analysis is used to compare gene expression patterns in the △PCG2 mutant and the wild type strain. Microarray analysis reveals that 417 genes are activated two-fold in the △PCG2 mutant, in which 138 genes contain MCB or SCB box in the promoter region. Most function known product of the 138 genes take part in DNA replication and repair, which indicates Pcg2 functions as transcription repressor and plays important roles in regulate DNA replication and repair related genes. In the Pcg2-repressed genes, a novel gene with one MCB-box in its promoter region, encodes a zinc finger protein, and contains a BTB/POZ domain (named MoBTBl) captures our attention. △foBTBl mutants show no difference with the wild-type strain. However, we delete the MCB box in the promoter region, and PCG2 is up-regulated and shows slower hyphal growth in the transformants. In addition, MoBTBl and PCG2 double mutants obviously grow faster than the APCG2 sole mutant. Date suggest MoBTBl function downstream of PCG2, and acts as an important repressor of hyphal growth. One important function of Pcg2 is repressing the expression of MoBTBl in order to regulate hyphal growth.On the other hand, microarray analysis shows 271 genes are suppressed two-fold in the △PCG2 mutant, in which 50 genes contain MCB or SCB box in the promoter region. In the Pcg2-activated genes,20 genes are unique to Magnaporthe oryzae. In which, a novel gene, named MoUNQl (Magnaporthe oryzae unique gene 1) alters its expression level over 10-fold. Knocking out of MoUNQ1 results in both slower hyphal growth and reduction in virulence. Moreover, in vitro Pcg2 binds the MCB box in the MoUNQl promoter region. Therefore, the author speculated another important function of Pcg2 is activating the expression level of MoUNQl to regulate hyphal growth and virulence.To open up the molecular mechanism of how Pcg2 function as both transcription activator and repressor, three tandem flags are fusion to the N-terminus of Pcg2, and the fusion construct is able to restore the defects of APCG2 mutant. Western analysis shows in M.oryzae, except for the full length Pcg2715, a truncated form about Pcg2～5CO occurs. Interestingly, the Pcg2-500 form has no transcriptional activation activity, suggesting the full length Pcg2715 form may act as transcriptional activator and the truncated form Pcg2-500 may function as transcriptional repressor to regulate the expression level of different genes. This conclusion has to be confirmed by Q-PCR detection. In addition, the author is driving on separating Pcg2 complex by pull-down technique.Overall, this research fully characterizes mechanism of the Pcg2, not only identifys the binding cis-elements of Pcg2, but discovers two downstream targets regulated directly by Pcg2. As well, this research firstly discoverys the APSES member protein occuring two forms in vivo, and each form may functions as activator or repressor regulated the expression level of different genes. Therefore, deeply research on mechanism of the APSES transcription factor of Pcg2 will provide clues for dissecting the function of other TFs in human, animals, plants and other fungi.