The fluffy gene of Neurospora crassa is required for asexual sporulation. It encodes an 88 kDa polypeptide containing a typical fungal Zn2Cys6 DNA binding motif. To identify the target genes on which FL may act, I sought to identify target sequences to which the FL protein binds. Several strategies were attempted to obtain purified FL protein. Purification was achieved by expressing the DNA binding domain of FL in Escherichia coli as a fusion with glutathione S-transferase followed by affinity purification using glutathione sepharose chromatography. DNA binding sites were selected by in vitro binding assays. Comparison of the sequences of selected clones suggested that FL binds to the motif 5??-CGG(N)9CCG-3??. A potential binding site was found in the promoter region of the eas (ccg-2) gene, which encodes a fungal hydrophobin. In vitro competitive binding assays revealed a preferred binding site for FL in the eas promoter, 5??-CGGAAGTTTCCTCCG-3??, which is located 1498 bp upstream of the eas translation initiation codon. In vivo experiments using a foreign DNA sequence tag confirmed that this sequence is a target site for FL regulation. Using Saccharomyces cerevisiae as an experimental system, I demonstrated that the C-terminal portion of FL functions in transcriptional activation. Microarray analysis was performed to study the role of fl in gene regulation on a large scale. mRNA levels in a fl mutant were compared to those in a strain overexpressing the fl gene. Experiments with cDNA microarray containing 13% of the total number of predicted N. crassa genes revealed 122 genes differentially expressed in response to overexpression of fl. Among these, eas displayed the greatest level of response. The cDNA microarray approach also revealed a number of genes that may be indirectly regulated by fl but may be involved in development. This information provides a foundation for further analysis of the role of fl in conidial development.

The orange bread mold Neurospora crassa is a useful model for the study of filamentous fungi. One of the asexual reproduction cycles in N. crassa, macroconidiation, can be induced by several environmental cues, including glucose starvation. The rco-3 gene is a regulator of sugar transport and macroconidiation in N. crassa and was proposed to encode a sugar sensor (Madi et al., 1997). To identify genes that are functionally related to RCO-3, three distinct suppressors of the sorbose resistance phenotype of rco-3 were isolated and characterized. The dgr-1 mutant phenotypically resembles rco-3 and may be part of the rco-3 signaling pathway. Epistatic relationship among rco-3, dgr-1 and the suppressors were carried out by analyzing rco-3; dgr-1 and sup; dgr-1 double mutants. These analyses indicate that rco-3 is epistatic to dgr-1. A cDNA microarray containing 1363 N. crassa genes was generated to examine the transcriptional response of wild type cells grown in the presence of glucose or starved for glucose for two hours. Comparing N. crassa profiling data with the published diauxic shift data from S. cerevisiae (DeRisi et al., 1997) revealed that S. cerevisiae and N. crassa share a similar, but not identical, transcriptional response pattern for genes belonging to central carbon metabolism. The microarray results indicate that N. crassa utilizes glucose through fermentation and respiration simultaneously in aerobic culture, a finding that is consistent with previous measurements of ethanol production and enzyme activities in N. crassa. The same microarray was used to examine the transcriptional response to glucose status in rco-3 and dgr-1 mutants. The two mutants display similar expression patterns for most of the genes on the microarray supporting a close functional relationship between them. In addition, I identified a high affinity glucose transport gene in N. crassa, whose transcription is under the control of glucose, rco-3 and dgr-1.

The fluffy gene of Neurospora crassa is required for asexual sporulation. It encodes an 88 kDa polypeptide containing a typical fungal Zn2Cys6 DNA binding motif. To identify the target genes on which FL may act, I sought to identify target sequences to which the FL protein binds. Several strategies were attempted to obtain purified FL protein. Purification was achieved by expressing the DNA binding domain of FL in Escherichia coli as a fusion with glutathione S-transferase followed by affinity purification using glutathione sepharose chromatography. DNA binding sites were selected by in vitro binding assays. Comparison of the sequences of selected clones suggested that FL binds to the motif 5??-CGG(N)9CCG-3??. A potential binding site was found in the promoter region of the eas (ccg-2) gene, which encodes a fungal hydrophobin. In vitro competitive binding assays revealed a preferred binding site for FL in the eas promoter, 5??-CGGAAGTTTCCTCCG-3??, which is located 1498 bp upstream of the eas translation initiation codon. In vivo experiments using a foreign DNA sequence tag confirmed that this sequence is a target site for FL regulation. Using Saccharomyces cerevisiae as an experimental system, I demonstrated that the C-terminal portion of FL functions in transcriptional activation. Microarray analysis was performed to study the role of fl in gene regulation on a large scale. mRNA levels in a fl mutant were compared to those in a strain overexpressing the fl gene. Experiments with cDNA microarray containing 13% of the total number of predicted N. crassa genes revealed 122 genes differentially expressed in response to overexpression of fl. Among these, eas displayed the greatest level of response. The cDNA microarray approach also revealed a number of genes that may be indirectly regulated by fl but may be involved in development. This information provides a foundation for further analysis of the role of fl in conidial development.

Conidiation is a common and critical asexual reproductive mode in fungi. The ascomycetes, the largest group in the kingdom Fungi undergo conidiation. The wide array of morphological difference in a conidiophore and conidial size, shape, and cellular organization demonstrates the importance of evolution in driving the morphological and functional diversity. An important unanswered question is how these conidiation processes evolve. We hypothesized that a conidiation regulatory pathway was present in the ancestral species, and became specialized in the extant species to lead to morphological and functional diversity. To address this hypothesis we assessed the conserved function of conidiation regulators in two distantly related ascomycetes, Aspergillus nidulans and Neurospora crassa. Using sequence similarity analysis, N. crassa orthologs were characterized to seven main conidiation regulatory genes in A. nidulans (fluG, flbC, flbD, abaA, wetA, medA, and stuA). Expression of the N. crassa orthologs complemented defective conidiation in the A. nidulans fluG, flbD, wetA, medA, and stuA mutants. In contrast, abaA and flbC and the N. crassa orthologs did not share conserved biochemical function. Taken in context of other recent studies of conidiation regulators, there are four distinct evolutionary patterns: (i) Non-homologous genes with analogous roles in conidiation (‘brlA’ and ‘fl’), (ii) Orthologs with retained biochemical function that lack analogous role in conidiation (‘fluG’, ‘flbD’, and ‘wetA’), (iii) Orthologs with retained biochemical function and analogous roles in conidiation (‘medA’ and ‘stuA’), and (iv) Orthologs with biochemical function not conserved but with analogous roles in conidiation (‘abaA’ and ‘flbC’). These studies set the stage for long-term studies of how evolution proceeded during the evolution of conidiation at different levels of phylogenetic diversity. An understanding of how evolutionary mechanisms shape the dynamics of developmental pathways will be significant for our understanding of fungal evolution of other novel adaptations such as pathogenesis.

Circadian clocks are internal timekeepers that allow organisms to anticipate and exploit predictable daily changes in their environment, aiding survival. Clock-driven rhythms, such as asexual spore development (conidiation) in Neurospora crassa, show temperature compensated periodicity that persists in constant conditions and can be reset by environmental time cues. This ability of circadian clocks to maintain a constant period and phase of behaviour over a range of temperatures is important, and whilst much of the machinery making up the circadian clock is known, the mechanism that underpins temperature compensation is not well understood. Further, it is unknown how the clock can control conidiation in the face of changing temperatures. To investigate possible mechanisms underlying temperature compensation, I first explored how compensation may arise within the central clock machinery using a comprehensive dynamic model of the Neurospora crassa circadian clock. This clock incorporates key components of the clock, and I introduced known temperature-sensitive component changes based on experimental observations. This analysis indicated that temperature-dependent changes in the binding of CK-1a to the FRQ-FRH complex may be pivotal in the temperature compensation mechanism. Previous work has highlighted the importance of the blue-light photoreceptor VIVID (VVD), as VVD knockout strains show a temperature-dependent delay in the phase of peak conidiation. Next I explored this potential role using a theoretical output model. By incorporating regulation of this pathway by VVD, I found that VVD may contribute to phase control by increasing expression of genes or proteins that peak early on in the output pathway. RNA-Seq experiments were carried out to assess the contribution of VVD to the overall transcriptomic profile of Neurospora. The analysis highlighted several key genes through which VVD may regulate the conidiation pathway, including the clock-controlled genes eas and ccg-9, which both show temperature- and strain-dependent changes in expression patterns over the time course of conidiation. In conclusion, VVD may indeed have an important role in the temperature compensation of output pathways, though further work is needed to assess the specific
contributions of genes highlighted by my RNA-Seq analysis to the compensatory mechanism.

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第21837号 / 農博第2350号 / 新制||農||1069(附属図書館) / 学位論文||H31||N5209(農学部図書室) / 京都大学大学院農学研究科地域環境科学専攻 / (主査)教授 田中 千尋, 教授 本田 与一, 准教授 刑部 正博 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DGAM

No description available.

Striatin family proteins contain a caveolin binding domain, a coiled-coil motif, a calmodulin binding domain, and a WD-repeat domain. Homologs of striatin protein have been However, our knowledge of the function and the molecular mechanism of fungal striatin homologs is limited. Based on the conserved sequences of functional domains, I hypothesized that the fungal striatin orthologs also act as scaffolding proteins that are functionally conserved among fungal species and involved in multiple types of development in the diverse kingdom Mycota. I used reverse genetic strategies to study the function of the Aspergillus nidulans striatin ortholog (strA) and the Colletotrichum graminicola striatin ortholog (str1). In assays of sexual development, the strA deletion strain (ΔstrA) produces fewer ascospores with smaller cleistothecia, while the str1 deletion strain (Δstr1) is defective in perithecia development. The ΔstrA phenotypes indicate that StrA is associated with ascosporogenesis in cleistothecia. Both ΔstrA and Δstr1 are reduced in radial growth and in conidia production. The Δstr1 strain is also altered in its spiral growth pattern and morphology of conidia and hyphopodia, but it produces appressoria similar to wild type. The pairing of nitrate non-utilizing mutants demonstrates that Str1 is required for hyphal fusion. In pathogenicity, Δstr1 is less virulent in maize anthracnose leaf blight and stalk rot. The phenotypes of Δstr1 are complemented by the Fusarium verticillioides striatin ortholog (fsr1), indicating that Fsr1 and Str1 are functionally conserved. Over-expression of StrA reveals its positive role in conidiation and the sexual production. StrA::eGFP localizes mainly to the endoplasmic reticulum. After comparing the results from these two species and other studied fungal species, I suggest that fungal striatins are involved in five types of development including hyphal growth, hyphal fusion, conidiation, sexual development, and virulence, and propose a model of fungal striatin protein interactions to account for these diverse phenotypes.

The Pezizomycotina, commonly known as the filamentous fungi, are a diverse group of organisms that have a major impact on human life. The filamentous fungi diverged from a common ancestor approximately 200 – 700 million years ago. Because of the diversity and the wealth of biological and genomic tools for the filamentous fungi it is possible to track the evolutionary history of genes and gene networks in these organisms. In this dissertation I focus on the evolution of two genes (lolC and lolD) in the LOL secondary metabolite gene cluster in Epichloë and Neotyphodium genera, the evolution of the MAP kinase-signaling cascade in the filamentous fungi, the regulation of the gene networks involved in asexual development in Neurospora crassa, and the identification of two genes in the N. crassa asexual development gene network, acon-2 and acon-3. I find that lolC and lolD originated as an ancient duplication in the ancestor of the filamentous fungi, which were later recruited in the LOL gene cluster in the fungal endophyte lineage. In the MAP kinase-signaling cascade, I find that the MAPK component is the most central gene in the gene network. I also find that the MAPK signaling cascade originated as three copies in the ancestor to eukaryotes, an arrangement that is maintained in filamentous fungi. My observations of gene expression profiling during N. crassa asexual development show tissue specific expression of genes. Both the vegetative mycelium and the aerial hyphae contribute to the formation of macroconidiophores. Also, with the help of genomic tools recently developed by researchers in the filamentous fungal community, I identified NCU00478 and NCU07617 as the genes with mutations responsible for two aconidial strains of N. crassa, acon-2 and acon-3 respectively.

A characteristic feature of species of Trichoderma is the production of concentric rings of conidia in response to alternating light-dark conditions. In response to a single burst of light, a single ring of conidia forms at what was the colony perimeter. On the basis of these observations, competency to photoconidiate has been proposed to be due to the age and metabolic rate of the hyphal cell. In this study, conidiation was investigated in five biocontrol isolates (T. hamatum, T. atroviride, T. asperellum, T. virens and T. harzianum) using both a morphological and molecular approach. All five isolates produced concentric conidial rings under alternating light-dark conditions on potato-dextrose agar (PDA), however, in response to a 15 min burst of blue light, only T. asperellum and T. virens produced a clearly, defined conidial ring which correlated with the colony margin at the time of light exposure. Both T. harzianum and T. hamatum photoconidiated in a disk-like fashion and T. atroviride produced a broken ring with a partially filled in appearance. On the basis of these results, it was postulated that competency to photoconidiate is a factor of the metabolic state of the hyphal cell rather than chronological age or metabolic rate. The influence of the source of nitrogen on photoconidiation was assessed on pH-buffered (pH 5.4) minimal medium (MM) amended with glutamine, urea or KNO₃. In the presence of glutamine or urea, T. asperellum and T. harzianum conidiated in a disk, whereas, when KNO₃ was the sole nitrogen source, a ring of conidia was produced. Further, in the presence of increasing amounts of glutamine, the clearly defined photoconidial ring produced on PDA by T. asperellum became disk-like. These results clearly demonstrated that primary nitrogen promotes photoconidiation in these isolates and strongly suggests that competency of a hyphal cell to conidiate in response to light is dependent on the nitrogen catabolite repression state of the cell. The experiments were repeated for all five isolates on unbuffered MM. Differences were apparent between the buffered and unbuffered experiments for T. atroviride. No photoconidiation was observed in T. atroviride on buffered medium whereas on unbuffered medium, rings of conidia were produced on both primary and secondary nitrogen. These results show that photoconidiation in T. atroviride is influenced by the buffering capacity of the medium. Conidiation in response to light by T. hamatum and T. virens was absent in all nitrogen experiments, regardless of the nitrogen source and buffering capacity, whereas both isolates conidiated in response to light on PDA. These results imply that either both sources of nitrogen are required for photoconidiation, or a factor essential for conidiation in these two isolates was absent in the minimal medium. Mycelial injury was also investigated in five biocontrol isolates of Trichoderma. On PDA, all isolates except T. hamatum conidiated in response to injury. On nitrogen amended MM, conidiation in response to injury was again observed in all isolates except for T. hamatum. In T. atroviride, injury-induced conidiation was observed on all medium combinations except the pH-buffered MM amended with glutamine or urea and T. virens conidiated in response to injury on primary nitrogen only, regardless of the buffering capacity. These results have revealed conidiation in response to injury to be differentially regulated between isolates/species of Trichoderma. On unbuffered MM amended with glutamine or urea, conidiation in response to injury occurred at the colony perimeter only in T. atroviride. It was hypothesised that the restriction of conidiation to the perimeter may be due to changes in the pH of the agar. The experiment was repeated and the pH values of the agar under the growing colony measured at the time of light induction (48 h) or injury (72 h). The areas under the hyphal fronts were acidified to below the starting value of the medium (pH 5.4) and the centres of the plates were alkalinised. The region of acidification at the time of stimuli correlated with the production of conidia, which implicates a role for crossregulation of conidiation by the ambient pH. The influence of the ambient pH on injury-induced conidiation was investigated in T. hamatum and T. atroviride on MM amended with glutamine and PDA, pH-buffered from pH 2.8 to 5.6. Thickening of the hyphae around the injury site was observed at the lowest pH values on MM in both T. atroviride and T. hamatum, however no conidia were produced, whereas both Trichoderma species conidiated on pH-buffered PDA in a strictly low pH-dependent fashion. This is the first observation of injury-induced conidiation in T. hamatum. The influence of the ambient pH on photoconidiation was assessed in T. hamatum, T. atroviride and T. harzianum using both buffered and unbuffered PDA from pH 2.8 to 5.2. On buffered PDA, no conidiation in response to light was observed above pH 3.2 in T. hamatum, above 4.0 in T. atroviride and above 4.4 in T. harzianum, whereas on unbuffered PDA it occurred at all pH values tested. It was postulated that conidiation at pH values above 4.4 on unbuffered PDA was due to acidification of the agar. The pH values of the agar under the growing colony were measured at the time of light exposure and in contrast to the MM with glutamine experiments, alkalisation of the agar had occurred in both T. atroviride and T. hamatum. No change in medium pH was recorded under the growing T. harzianum colony. These results indicate that low pH-dependence of photoconidiation is directly related to the buffering capacity of the medium. Recent studies have linked regulation of conidiation in T. harzianum to Pac1, the PacC orthologue. In fungi, PacC regulates gene expression in response to the ambient pH. In these studies pH-dependent photoconidiation occurred only on buffered PDA and on unbuffered PDA conidiation occurred at significantly higher ambient pH levels. It is proposed that the influence of ambient pH on conidiation in the isolates used in this study is not due to direct Pac1 regulation. The T. harzianum isolate used in this study produced profuse amounts of the yellow anthraquinone pachybasin. Production of this secondary metabolite was strictly pH-dependent, irrespective of the buffering capacity of the medium. Studies in T. harzianum have linked Pac1 regulation to production of an antifungal α-pyrone. pH-dependence on both buffered and unbuffered media strongly suggests that pachybasin production may also be under the control of Pac1. Photoconidiation studies on broth-soaked filter paper, revealed rhythmic conidiation in the pachybasin producing T. harzianum isolate. Diffuse rings of conidia were produced in dark-grown cultures and, in cultures exposed to light for 15 min at 48 h, the rings were clearly defined. These results show that conidiation is under the control of an endogenous rhythm in T. harzianum and represent the first report of circadian conidiation in a wild-type Trichoderma. A Free-Running Rhythm (FRR) assay was used to investigate rhythmic gene expression in T. atroviride IMI206040 and a mutant derivative, in which the wc-2 orthologue, blr-2, was disrupted. Over a 3 d period, expression of gpd, which encodes the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase, oscillated with a period of about 48 h. In the Δblr-2 mutant, the gpd rhythm was absent. These results revealed that in T. atroviride, gpd expression is under the control of an endogenous clock and that clock-regulated expression of gpd is associated with a functional BLR complex. Using degenerate primers, a portion of frq, which encodes the N. crassa clock oscillator FREQUENCY, was isolated from T. atroviride and used to probe the FRR assay northern blots. No frq expression was detected at any time point, which suggests that the circadian clock in Trichoderma does not involve FREQUENCY. In a concurrent study, orthologues of rco-1 (rcoT) were isolated and sequenced from T. atroviride and T. hamatum using a combination of degenerate, inverse and specific PCR. RcoT is an orthologue of the yeast global co-repressor Tup1 and in the filamentous fungi, RcoT orthologues have been demonstrated to negatively regulate conidiation. Genomic analysis of all available rcoT orthologues revealed the conservation of erg3, a major ergosterol biosynthesis gene, upstream from rcoT in ascomycetous filamentous fungi, but not in the ascomycetous yeast or in the basidiomycetes. These studies have significantly contributed to our understanding of the regulatory factors controlling conidiation in Trichoderma and have multiple implications for Trichoderma biocontrol; most notable the promotion of conidiation by primary nitrogen and low pH. Incubation conditions can be altered to suit the nitrogen and pH preferences of a biocontrol strain in order to promote cost effective conidial production, however this is not easily achieved in the soil, where the biocontrol strain must perform in a highly buffered environment optimised for plant growth. Successful use of Trichoderma biocontrol strains may involve the screening and targeting of strains to the appropriate pH conditions or the selection of new strains on the basis of capacity to perform under a given range of conditions.