Molecular function of LAMMER kinase in Aspergillus nidulans development

국문 초록 (Abstract)

진핵 세포 생물의 LAMMER kinase는 다양한 세포 생리적 과정에서 중요한 역할을 한다. 하지만, 사상성 진균에서 그 기능이 아직 밝혀지지 않았기 때문에, 대표적인 사상성 진균인 Aspergillus nidulans를 이용하여 LAMMER kinase의 역할을 규명하고자 하였다.
A. nidulans LAMMER kinase를 암호화한 유전자인 lkhA ORF는 2,253bp로, 6개의 exon과 5개의 intron으로 구성되어 있었다. LkhA은 667개의 아미노산으로 이루어진 분자량이 약 74.8 kDa 의 단백질이다. 아미노산 서열 분석과 계통분석을 통하여 LkhA가 LAMMER kinase 그룹에 속함을 확인하였다. lkhA 전사체 분석 결과A. nidulans의 생활사 동안 지속적으로 발현되었는데, 특히 무성 및 유성분화 시기의 특정 시간에서 발현이 증가하였다. 또한, 적어도 2개 이상의 isoform이 영양생장 시기에 발현되었다.
lkhA 결손 돌연변이는 생존에 필수적이지는 않으나, 생장의 감소, 배지의 색소 침착등의 비정상적인 표현형을 보였디. 이 돌연변이주는 germination동안 bipolar 및 multipolar germling들이 증가하는 것으로 보아 LkhA는 polarity의 유지 및 germ tube emergency의 양상에 영향을 미친다. 고체 배지에서 결손 돌연변이주의 균사체 생장이 감소하는데 반해, 액체 배지에서는 지속적인 배양시 자가분해 현상이 지연되었다. 또한 균사의 분지는 증가하고, 격벽의 간격이 일정치 않거나 짧아지고, 두꺼워졌으며, 핵의 분포와 수가 비정상적이었다. 이러한 결과들은 lkhA가 세포 주기에 따른 핵의 분포와 격벽 형성 및 격벽 간격 조정에 관여하는 조절 기작에 영향을 줌을 나타낸다.
무성분화 동안 형성되는 생식기관인 conidiophore는 lkhA 결손시 primary sterigmata의 수가 감소하고, vesicle로부터 secondary conidiophore의 형성, sterigmata의 길이 변화 및 stalk 내의 septa가 형성되었다. 또한, sterigmata의 대칭성 결손으로 인해 conidia의 생성율도 감소하였다. Northern blotting을 통해 asexual development의 시작에 관여하는 upstream developmental regulator들의 전사체량을 분석한 결과 변화를 보이지는 않았다. 하지만, brlAβ 와 brlAα 및 abaA의 전사체량은 매우 감소하였다. 이러한 결과들은 lkhA가 brlA 의 전사 조절과정을 통해 conidiophore development에 관여함을 시사한다.
lkhA 결손 돌연변이주는 nimXcdc2AF돌연변이주와 유사한 conidiophore형태 결함을 보이기 때문에 lkhA 와 nimX 유전자를 이용하여 nimXcdc2AF돌연변이주와 lkhA 결손돌연변이를 대상으로 suppression test를 하였고, NimX의 Northern과 Western 분석을 하였다. 이 결과, lkhA는 세포 주기 조절자인 NimX 조절을 통해 영양생장 및 conidiophore 형태에 영향을 주는 것으로 결론지었다.
lkhA결손 돌연변이주의 cleistothecia는 야생형에 비해 크기가 작고, 아주 적은 수의 ascospore를 가지고 있었다. Transmission electron microscopy (TEM) 관찰 결과에 의하면 lkhA 결손돌연변이주의 cleistothecia는 ascogenous cell들로 보이는 비정형화된 세포들과 적은 수의 asci로 채워져 있었다. Electron-dense한 물질인 cleistin은 야생형에서는 가장 바깥 쪽의 peridial-layer들에 축적되어있는 반면, 돌연변이주에는 cleistothecia의 안쪽 부분으로 퍼져있음을 확인하였다. 이 결과는 lkhA는 peridial layer 및 ascogenous cell들의 구성을 포함한 cleistothecia의 성숙과정에 필요함을 나타내었다. Cleistothecia의 성숙에 관여하는csnD의 전사체량은 매우 낮았고, 이 유전자의 과발현은 cleistothecial outer later의 분화 및 성숙은 회복시켰으나, 크기 및 ascospore의 생성에는 영향이 나타나지 않았다. 이 결과는 LkhA가 csnD 전사조절뿐만 아니라 다른 유성분화 관련 유전자들의 조절을 통해 cleistothecia의 성숙과정에 관여함을 시사한다. 또 다른 분화 관련 유전자 중 ppoA의 발현에도 관여함으로써 ascosporogenesis에도 영향을 주는 것으로 확인하였다.
lkhA 결손 돌연변이주는 야생형에 비해 H？lle cell이 이른 시기에 나타났다. H？lle cell에 특이적인 α-1,3-glucanase를 암호화한 mutA유전자의 발현을 무성분화 후기 시기에 확인한 결과, lkhA 결손 돌연변이주에서만 발현되었다. 이러한 결과는 LkhA가 A. nidulans에서 분화 관련 유전자 발현의 시간적 조절 기작에 영향을 줌을 확인할 수 있었다. 더욱이 stuA transcript의 profile이 변한 것으로 보아 LkhA는 stuA 발현을 통해 생식기관의 분화 동안 시간 조절 기작을 조정하는 것으로 추정된다.
본 연구를 통해 LkhA가 생존에 필수적이지는 않으나, germ tube 발생, germling의 방향성 축의 형성, 세포 주기와 관련된 격벽 형성 및 생식 기관의 형성에 관여함을 밝혔다. LkhA는 무성 분화 동안의 BrlA, StuA, NimX와 유성분화 동안의 CsnD 및 PpoA의 공조적인 효과를 통해 A. nidulans 분화 과정에 관여하는 것으로 보인다. 이 결과들은 A. nidulans의 분화과정에 대한 분자적 기작에 새로운 시각을 제공할 것이다.

다국어 초록 (Multilingual Abstract)

LAMMER kinase plays pivotal roles in various physiological processes in eukaryotes; however, its function in filamentous fungi is not known. To understand function of the Aspergillus nidulans LAMMER kinase, LkhA, molecular studies were performed.
The ORF of A. nidulans LAMMER kinase gene, lkhA, which was 2,253bp in length, was divided into six exons and 5 introns. The predicted LkhA protein had 667 amino acids and an estimated mass of 74.8 kDa. Amino acid sequence and phylogenetic analysis indicated that LkhA belongs to the family of LAMMER kinases. Northern blot analysis showed continuous lkhA expression throughout the A. nidulans life cycle, but the level of expression was relatively high at certain time points during asexual and sexual development. At least two isoforms of the lkhA transcript were detected during vegetative growth,
The lkhAΔ strain was viable, but showed abnormalities in growth such as reduced radial growth and deposition of reddish-yellow pigments in the agar culture medium. The proportion of bipolar or multipolar germlings was increased in the lkhA deletion mutants, suggesting that the pattern of germ tube emergence and the maintenance of polarity axis were affected by LkhA. The lkhAΔ strain showed significant reduction of mycelia production on complete agar medium, but delay of disintegration (autolysis) of the mycelia by the lkhA deletion was suggested in liquid culture. The lkhAΔ strain showed hyper-branching of the hyphae, uneven and shorter septum interval than that in the wild type, and thickening of the hyphae with frequent septation. Most interestingly, the lkhAΔ strain revealed numerical and morphological abnormalities in its nuclei. These results indicate that lkhA affects regulatory mechanisms that are associated with the formation of the septum and its intervals and to the distribution of nuclei in accordance with the cell cycle.
The lkhAΔ strain displayed a reduced number of primary sterigmatae, the emergency of secondary conidiophores from its vesicle, irregularity in the length of sterigmata (non-separated), and multiple septa in its stalk. The lkhAΔ strain also showed a lack of symmetry in sterigmata attributable to the absence of or just one phialide budding from a metula, resulting in decrease of conidia production. In Northern blot analysis, the expression of upstream developmental activators, flbB, flbC, and flbD, showed no dramatic changes during the vegetative stage following by lkhA deletion. After the initiation of asexual development, however, the level of brlAβ and brlAα expression was dramatically decreased in the lkhA deletion mutants. The level of abaA expression was also reduced significantly in the lkhA deletion mutants after asexual development. These results suggest that the lkhA may play a role in conidiophore development through transcriptional modulation of brlA expression.
Since morphogenetic defects in conidiophores caused by lkhA deletion were similar to those of nimXcdc2AF mutants, suppression test of the nimXcdc2AF mutant and the lkhA-deletion strain with lkhA and nimX alleles, and Northern and Western analysis of NimX was performed. The results show that the lkhA deletion may have deleterious effects on vegetative growth and conidiophore morphogenesis based on changes in the cell division regulator NimX.
The cleistothecia from the lkhAΔ strain were smaller than the wild type and immature. It showed more fragile shells filled with very few ascospores. Results from transmission electron microscopy (TEM) revealed that the lkhAΔ strains were mostly filled with amorphous aggregates that resembled ascogenous cells and contained very few asci. The electron-dense cleistins were not restricted to the outer-most peridial layers, but infiltrated into the inner parts of the cleistothecia of the lkhAΔ strain. These results indicate that lkhA is required for the completion of cleistothecial development, including organization of the peridial layers and the ascogenous cells, which collectively comprise the process of ascosporogenesis. On the basis of the immaturity of cleistothcia, the level of csnD transcription was lower, particularly during the early sexual stage (S24) of the lkhAΔ strain. Failures in development and maturation of the cleistothecial outer layers, such as toughness and pigmentation, were reversed by the over-expression of the csnD; however, no reversals were observed in terms of size of cleistothecia and the production of ascospores. These results indicate that LkhA affects the maturation of cleistothecia from primordia not only by modulating csnD transcription but also by modulating other sexual gene(s). A decrease in ppoA transcription was observed at the vegetative stage (V12), whereas an upregulation of transcription was observed during sexual development in the wild type, suggesting that lkhA influences ascosporogenesis through the transcriptional regulation of ppoA.
The lkhAΔ strain cultured under normoxia showed an earlier appearance of H？lle cells. In the lkhAΔ strain, expression of mutA, encoding H？lle cell-specific α-1,3-glucanase, was detected 48 h after asexual induction, whereas this was not observed in the wild type. The early appearance of sexual organs even under optimal conditions for asexual development in the lkhAΔ strain indicates that LkhA affects the temporal regulatory mechanism through the expression of development-specific genes in A. nidulans. In the lkhA deletion mutants, the expression levels of both stuAα and stuAβ were significantly decreased at 48 h during late asexual development, as well as at 48 h during sexual development compared to that in the wild type. These data suggest that LkhA modulates the temporal regulatory circuit for the differentiation of reproductive organs through stuA expression.
In summary, this study presents several pieces of evidence, supporting the connection that LkhA is not essential for viability, but is involved in germ-tube development, maintenance of axis polarity in germlings, septation in conjunction with cell division cycle, and development of reproductive organs, such as conidiophores and cleistothecia, during the life cycle of the filamentous fungus, A. nidulans. These pleiotropic effects of LkhA in A. nidulans differentiation are mainly attributed to the sequential and concerted actions of BrlA, StuA, and NimX during asexual development and of CsnD and PpoA during sexual development. The results presented here will provide new insights into the molecular mechanisms underlying development in A. nidulans.

목차 (Table of Contents)

• ABSTRACT* 1
• I. Introduction 10
• 1. LAMMER kinase 10
• 1. 1. Characteristics of LAMMER kinase family 10
• 1.2. Biological functions of members in LAMMER kinase family 10
• 1.3. Mechanism for modulation of LAMMER kinase 14
• 2. Aspergillus nidulans 16
• 2.1. Characteristics as model organism 16
• 2.2. Germination 16
• 2.3. Development 17
• 2.3.3. Transcription factors in asexual development 20
• 2.3.3.1. Upstream developmental activators (UDA) 20
• 2.3.3.2. BrlA and AbaA in central regulatory pathway 20
• 2.3.3.3. StuA as developmental modifier 22
• 2.3.4. Development of sexual organs 23
• 2.4. Relationship between development and cell cycle in A. nidulans 28
• II. Materials and methods 30
• 1. Strains and media 30
• 1.1. Strains, media, and culture conditions 30
• 1.2. Media and culture condition 30
• 2. Transformation and DNA manipulation in E. coli 37
• 3. Genetic and molecular techniques of A. nidulans 37
• 3.1. Genetic techniques 37
• 3.2. Molecular techniques 42
• 4. Thin layer chromatography 46
• 5. Microscopy 46
• 5.1. Staining 47
• 5.2. Scanning electron microscopy 47
• 5.3. Transmission electron microscopy 48
• III. Results 49
• 1. Identification of LAMMER kinase in A. nidulans (LkhA) 49
• 2. Transcription profile of lkhA during life cycle 53
• 3. Disruption and reversion of lkhA 55
• 4. Germination 57
• 5. Vegetative growth 59
• 6. Asexual development 65
• 6.1. Morphogenesis of asexual reproductive organ 65
• 6.2. Regulation of transcription factors related with conidiation 69
• 7. Relationship between LkhA and cell cycle regulator, NimXcdc2 71
• 8. Sexual development 75
• 8.1. Morphogenesis of sexual reproductive organ 75
• 8.2. Ultrastructure of mature cleistothecia 75
• 8.3. Expression of the genes related with cleistothecia maturation and ascosporogenesis 76
• 9. Temporal regulation during development 83
• 9.1. Abnormal development caused by lkhA deletion 83
• 9.2. Modulation in transcriptional expression of stuA by LkhA 84
• 10. Alternative splicing of lkhA 86
• 11. Response to various stresses 91
• 11.1. Oxidative stress 91
• 11.2. Cell wall damage, osmotic stress, and antifungal agents 91
• IV. Discussion 94
• V. References 106
• VI. Abstract in korean 118
• 감사의 글 122