mrflbA, encoding a putative FlbA, is involved in aerial hyphal development and secondary metabolite production in Monascus ruber M-7
Highlights
► RGS named MrflbA sharing high similarity with FlbA was cloned from Monascus ruber. ► Loss of mrflbA led to changes in morphology and reduction of pigment and mycotoxin. ► Loss of mrflbA elevated the transcript level of G protein α subunit. ► Loss of mrflbA reduced the transcription of genes for pigment and mycotoxin synthesis.
Introduction
Regulators of G protein signalling (RGSs) are a group of proteins which govern the intensity and duration of G protein signalling by interacting with an activated GTP-G protein α subunit (Gα) (Gold et al., 1997, Ross and Wilkie, 2000, Seo et al., 2005). To date, six RGS protein families (orthologues of RgsA, RgsB, RgsC, RgsD, fluffy low brlA (FlbA), and Gprk) from Aspergillus spp. have been identified (Han et al., 2004, Lafon et al., 2006). FlbA in Aspergillus nidulans was the first identified RGS protein in filamentous fungi and the functions of which have been elucidated in several fungi over the last two decades. For instance, a flbA deletion strain in A. nidulans formed the fluffy autolytic colony that lacked sexual/asexual sporulation and sterigmatocystin (ST) production (Lee and Adams, 1994, Yu et al., 1996, Hicks et al., 1997); the phenotypes of AfflbA (Aspergillus fumigatus flbA) and AorflbA (Aspergillus oryzae flbA) mutants were almost identical to that of the flbA mutant in A. nidulans (Mah and Yu, 2006, Ogawa et al., 2010); cag8 (conidiation-associated gene, orthologous to flbA) in Metarhizium anisopliae, was involved in the modulation of conidiation, virulence, and hydrophobin synthesis (Fang et al. 2007); rgs1 in Magnaporthe grisea regulated pathogenesis, asexual growth, and thigmotropism (Liu et al. 2007), indicating that RGSs play pivotal roles in the regulation of developmental and secondary metabolic processes including vegetative growth, sporulation, mycotoxin/pigment production, and pathogenicity in filamentous fungi.
Substantial basic knowledge concerning the functions of the components in G protein signalling pathway has been obtained mainly from model filamentous fungi and pathogenic fungi. However, little is known about the biological significance of the components in industrial fungi. Monascus spp. are the producers of red yeast rice (RYR), also called Hongqu, which have been widely used as food or food additives, folk medicine, as well as fermentation starters to brew rice wine and vinegar for many centuries in oriental countries (Lin et al., 2008, Li et al., 2010). Nowadays, a variety of bioactive metabolites, such as monacolins, gamma aminobutyric acid, pigment, and antioxidant (dimerumic acid) et al., have been identified and utilized world-wide (Aniya et al., 2000, Su et al., 2003); however, the isolation of citrinin, a kind of mycotoxin which might cause kidney and liver damage in humans, aroused controversy over the safety of Monascus spp. (Blanc et al. 1995). Therefore, genes associated with these secondary metabolites synthesis attract extensive attention from researchers. In recent years, several genes involved in the biosynthesis of citrinin, monacolin K, and pigments (Shimizu et al., 2005, Shimizu et al., 2007, Chen et al., 2008, Chen et al., 2010, Li et al., 2010) have been cloned and analyzed, which made an important step forward in understanding the secondary metabolism in Monascus spp.
In this report, we cloned an RGS protein gene, mrflbA (Monascus ruber flbA) from M. ruber, which had significant homology to flbA (Lee & Adams 1994). To investigate the role of mrflbA in the process of development and metabolism in M. ruber, we constructed the mrflbA deletion mutant for observing the phenotypes, detecting the production of pigments and citrinin, and analyzing the variation of transcription level for Gα subunit mga1 (accession number: FJ640858, Li et al. 2010) of the mutant. To our knowledge, this is the first report describing RGS protein coding genes in Monascus spp., enhancing our understanding of the function of G protein signalling pathways in development and secondary metabolism in M. ruber.
Section snippets
Fungal strains and growth conditions
The Monascus ruber M-7 (wild-type strain) and the mrflbA deletion mutant were maintained on potato dextrose agar (PDA) slants at 4 °C. G25N (25 % glycerol nitrate agar) medium was used to prepare spore suspensions because of the low production of spores on PDA medium. For analysis of the production of pigment and citrinin, strains were grown in PDB (potato dextrose broth) medium. Every 50 mL of the culture medium was inoculated with 1.5 × 106 spores and cultivated at 28 °C with continuous shaking at
Isolation and sequence analysis of the mrflbA
Based on the partial DNA sequence isolated from Monascus mutant MZ805 (Shao et al. 2009), a DNA fragment of 6281 bp in length was generated by assembling the 5′ and 3′ flanking sequences (Fig 1A and B) amplified via SON-PCR. The sequence prediction (http://linuxl.softberry.com/berry.phtml) revealed that it consisted of a 2419 bp length of ORF and three introns (Fig 1C), which had significant homology to flbA (Lee & Adams 1994), and was called mrflbA. Sequence comparison of mrflbA cDNA to genomic
Discussion
In fungi, G protein signalling pathways play an important role in regulating growth and development, stress response, virulence, and biosynthesis of secondary metabolites (Gao and Nuss, 1996, Segers and Nuss, 2003, Seo et al., 2005). The negative roles of RGS proteins in the regulation of Gα subunit signalling in several filamentous fungi have been studied (Yu et al., 1996, Hicks et al., 1997, Han et al., 2004, Liu et al., 2007). The characterization of the MrflbA described here indicates the
Acknowledgements
We appreciate Dr Youxiang Zhou from Food Quality Inspection and Testing Center of Agricultural Ministry of China in Hubei providing technical assistance in citrinin analysis. This study was financially supported by Doctoral Fund of Ministry of Education of China (RFDP No. 200805041022) and National High Technology Research and Development Program of the People’s Republic of China (863 Program: 2006AA10Z1A3 and 2008AA10Z416).
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