JAST
2012 March;3(1):81-84.
Published online 2012 February 10.
doi:http://dx.doi.org/10.5355/JAST.2012.81
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| Copyright ⓒ 2010 Journal of Analytical Science & Technology |
| The roles of phosducin-like protein in sexual development of Aspergillus nidulans |
| Sei-Jin Lee1*, Dong-Min Han2, Keon-Sang Chae3, Dae-Hyuk Kim3, Tae-Boong Uhm4, Kwang-Yeop Jahng4* |
1Korea Basic Science Institute, Jeonju Center
2Division of Life Science, Wonkwang University, Iksan 570-749, Republic of Korea
3Department of Molecular Biology, Chonbuk National University, Chonju, 561-756, Republic of Korea
4Department of Biological Sciences, Chonbuk National University, Chonju, 561-756, Republic of Korea |
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Corresponding Author:
Sei-Jin Lee ,Tel: +82-63-270-4516, Fax: +82-63-270-4308, Email: lsj@kbsi.re.kr |
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ABSTRACT |
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| To better understand the function of phnA encoding a Phosducin-like protein in Aspergillus nidulans, we made the phnA deletion mutant. Compared with wild type, the deletion of phnA resulted in increase in the diameter and total volume of cleistothecia as well as increase in the number of ascospore even though it did not affect to the number of hülle cell. From these results, we suggest that PhnA did not affect the number. |
| Keywords: Aspergillus nidulans, Sexual development, phnA |
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Introduction |
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| Aspergillus nidulans, the filamentous fungus ascomycetes, has three distinguishing developmental cycles as sexual, asexual and parasexual (vegetative) cycle [1]. The parasexual cycle involves the formation of undifferentiated cells (hyphae as constitute the mycelium). Hyphae are grown by apical extension and specialized for the acquisition of nutrients from the environment. Under appropriate conditions, some hyphae cannot grow normally but begins asexual cycle. During asexual cycle, several morphological changes occur to form multicellular conidiophores including uninucleated conidia (asexual spores). During sexual cycle, cleistothecia (sexual organ) covered with hülle cells are developed by mycelial aggregation. According to asexual cycle, several genes (stuA, brlA etc.) have been genetically characterized and interactions between them have been investigated [1]. Otherwise, the sexual development process during sexual cycle has not been well elucidated. Recently, we reported that the MAP kinses, mpkB was required for some post-karyogamy process as well as at the hyphal anastomosis stage to accomplish sexual development successfully in A. nidulans [2]. Kim et al. reported that the veA gene activates sexual development in A. nidulans [3]. However, the deletion of phosducin-like protein, phnA in A. nidulans with abnormal veA gene expression resulted in defective fruiting body formation during sexual cycle [4]. Therefore, we tried to investigate the role of phnA, per se, in A. nidulans during sexual development. |
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Materials and Methods |
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Isolation of the phnA gene
The phnA was isolated by PCR using the A. nidulans FGSC A4 genomic DNA as a template and the atb1phnAo and atb2phnA as primers. The DNA polymerase was used the PrimeSTAR (Takara).
Disruption of the phnA gene
The phnA deleted mutant was made by DJ-PCR procedure [5]. The phnA ORF was replaced by the pyrG of A. fumigatus. The 3rd round PCR product was used for template to amplify the ΔphnA::pyrG cassette with primers FRphnA550 and REphnA543.
Cleistothecium formation rate
Conidia of wild types and phnA mutants were spread in solid MM. To facilitate sexual development, the plates were sealed with parafilm for 24 hr after inoculation and all strains were incubated at 37 ℃. The number of cleistothecia was counted in microscopic field and the values reported represent the number of cleistothecia in 1 cm2of plates.
To investigate the diameter of cleistothecium, conidia (1ⅹ105) of wild type and mutants were spread in solid CM plates and incubated for 5 days. 30 different cleistothecia of wild type were compared to these of phnA deletion mutant. Measurements were done in triplicate.
Count the number of hülle cell and ascospore
Conidia of wild type and mutants were spread in solid MM and CM plates. After spreading, every day for 7 days, 0.8 cm agar blocks were vortexed in 200 μl of 0.08% Triton X-100 solution included 0.2 g beads (0.5 mm zirconia/silica beads, catalog No. 11079105z, BioSpec Products, Inc.) for 2 min and the resulting suspension was counted with hemacytometer.
To investigate the diameter of cleistothecium, conidia of wild type and phnA deletion mutant were spread in solid CM plates and incubated for 5 days. 10 different cleistothecia of wild type and phnA deletion mutant were broken in distilled water. The resulting suspension was counted with hemacytometer. Measurements were done in triplicate.
Growth and development
To investigate growth rate, radial colony extension and cell density of mycelia were calculated. After 2 μl of conidial suspension (1ⅹ106 conidia/ml) of wild type and phnA mutant strains were point inoculated onto supplemented minimal medium plates, these were incubated for 3, 4, or 5 days. |
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Results and Discussion |
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A. nidulans FLC2A21-RT (wild type, pabaA1, pyrG89,yA2;mpkB+;veA+;pyrG+) was used as host strain for integration of manipulated genes. To investigate the function of phnA, the phnA deletion mutant FLC2A21-DP (pabaA1,pyrG89,yA2;mpkB+; ΔphnA::pyrG;veA+) was constructed by the double-joint PCR procedure [5]. The gene replacement region was confirmed by PCR, and southern blot analysis (data not shown). After inoculating for 5 days, we illustrated the typical cleistothecia of wild type and the phnA deletion mutant (Figure 1). Phenotypes of mutants were observed in sexual induced condition. To induce the sexual development, 105 conidia of designated strains were grown in minimal media (MM) with casamino acid (0.1 %) to induce sexual development. MM plates containing casamino acid were tightly sealed, incubated for 24 h and unsealed. After unsealing, each strain was incubated at 37 °C for 5 days. As shown in Table 1, the number of cleistothecia per cm2 from the phnA deletion mutant was less 6 times than that of wild type. Otherwise, the diameter of cleistothecia from the phnA deletion mutant was longer 2 times than that of wild type. We calculated volume of cleistothecia from number and diameter assuming that the cleistothecia were complete spheres. Therefore, the calculated total volume of cleistothecia (mm3) from the phnA deletion mutant was bigger 10 times than that of wild type. Even if the number of hulle cell was not changed by the phnA deletion, the number of ascospore from phnA deletion mutant was more 5 times than that of wild type.
Phosducin is a member of a family of phosducin-like protein (PhLP) found in eukaryotes. Phylogeny of 33 PhLP from metazoan, plants and lower eukaryotes identified three distinct groups named PhLP I-III [6]. PhLP I has been shown to be essential for assembly of G protein signaling. PhLP II and PhLP III have no role in G protein signaling, but they appear to assist in the folding of proteins essential in regulating cell cycle progression as well as actin and tublin [7]. There are three PhLP homologues, phnA, phnB, and phnC in A. nidulans. phnA is included to PhLP I that bind Gβγ subunits with high affinity [4]. phnB has similarity to PhLP I and PhLP II. phnC has similarity to PhLP III. The phnA deletion mutant without normal veA expression did not make the cleistothecium in A. nidulans [4]. They showed that the A. nidulans phnA gene, encoding PhLP, is required for G protein signaling for vegetative growth, regulation of development, and production of secondary metabolites. The phnA deletion mutant was found to be severely impaired in sexual reproduction suggesting the PhnA is needed for G protein signaling for cleistothecium development. As described above, they used A. nidulans without normal veA expression. In A. nidulans, veA null mutants had never formed sexual structures, even under conditions where sexual development preferentially occurs in wild types [3]. Cells over-expressing veA formed larger numbers of sexual structures with a much reduced number of conidial heads than a control strain (a veA1 mutant), even under conditions where wild type strains form little sexual structure but form conidial heads very well. Therefore, the authors suggested that the veA gene is a positive regulator in sexual development in A. nidulans. In this study, we showed that the deletion of phnA in A. nidulans with normal veA expression resulted in increase in the diameter and total volume of cleistothecia as well as increase in the number of ascospore. In conclusion, phnA appeared to be involved in formation of normal cleistothecium in A. nidulans. |
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Acknowledgement |
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| This study was supported by research funds of Chonbuk national University in 2010 and KBSI, Jeonju Center. |
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FIGURES |
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Fig.1
Phenotypes of wild type (FLC2A21-RT) and phnA deletion mutants (FLC2A21-DP). The typical cleistothecia from wild type and phnA deletion mutant were taken from a plate using the needle. |
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TABLES |
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Table.1
The sexual organs in wild type and phnA mutant strain. |
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REFERENCE |
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| 1. |
Adams, T. H.; Wieser, J. K.; Yu, J. H. Asexual sporulation in Aspergillus niulans. Microbiol. Mol. Biol. Rev. 1998, 62, 35?54. |
| 2. |
Jun, S. C.; Lee; S. J.; Park, H. J.; Kang, J. Y.; Leem, Y. E.; Yang, T. H.; Chang, M. H.; Kim, J. M.; Jang, S. H.; Kim, H. G.; Han, D. M.; Chae, K. S.; Jahng, K. Y. The MpkB MAP Kinase Plays a Role in Post-karyogamy Processes as well as in Hyphal Anastomosis during Sexual Development in Aspergillus nidulans. J. Microbiol. 2011, 49, 418-430. |
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| 3. |
Kim, H.; Han, K.; Kim, K.; Han, D.; Jahng, K.; Chae, K. The veA gene activates sexual development in Aspergillus nidulans. Fungal Genet. Biol. 2002, 37, 72-80. |
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Seo, J. A. and Yu, J. H. The phosducin-like protein PhnA is required for Gβγ-mediated signaling for vegetative growth, developmental control, and toxin biosynthesis in Aspergillus nidulans. Eukaryot. Cell. 2006, 5, 400-410. |
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Yu, J. H.; Hamari, Z.; Han, K. H.; Seo, J. A.; Reyes-Dominguez, Y.; Scazzocchio, C. Double-joint PCR: a PCR-based molecular tool for gene manipulations in filamentous fungi. Fungal. Genet. Biol. 2004, 41, 973-981. |
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Blaauw, M.; Knol, J. C.; Kortholt, A.; Roelofs, J.; Ruchira; Postma, M.; Visser, A. J.; van Haastert, P. J. Phosducin-like proteins in Dictyostelium discoideum: implications for the phosducin family of proteins. EMBO J. 2003, 22, 5047-5057. |
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| 7. |
Willardson, B. M.; Howlett, A. C. Function of phosducin-like proteins in G protein signaling and chaperone-assisted protein folding. Cell Signal 2007, 19, 2417-2427. |
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