Altered secretion patterns and cell wall organization caused by loss of PodB function in the filamentous fungus Aspergillus nidulans
Karthik BoppidiLiliane Fraga Costa RibeiroSirasa IambamrungSidney M. NelsonYan WangMichelle MomanyElizabeth RichardsonStephen LincolnRanjan SrivastavaSteven D. HarrisMark R. Marten
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Filamentous fungi are widely used in the production of a variety of industrially relevant enzymes and proteins as they have the unique ability to secrete tremendous amounts of proteins. However, the secretory pathways in filamentous fungi are not completely understood. Here, we investigated the role of a mutation in the POlarity Defective (podB) gene on growth, protein secretion, and cell wall organization in Aspergillus nidulans using a temperature sensitive (Ts) mutant. At restrictive temperature, the mutation resulted in lack of biomass accumulation, but led to a significant increase in specific protein productivity. Proteomic analysis of the secretome showed that the relative abundance of 584 (out of 747 identified) proteins was altered due to the mutation. Of these, 517 were secreted at higher levels. Other phenotypic differences observed in the mutant include up-regulation of unfolded protein response (UPR), deformation of Golgi apparatus and uneven cell wall thickness. Furthermore, proteomic analysis of cell wall components in the mutant revealed the presence of intracellular proteins in higher abundance accompanied by lower levels of most cell wall proteins. Taken together, results from this study suggest the importance of PodB as a target when engineering fungal strains for enhanced secretion of valuable biomolecules.Keywords:
Aspergillus nidulans
Secretory protein
Filamentous fungus
Fungal protein
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Department of Pharmaceutical Engineering, Woosuk University, Wanju, 565-701, Republic of Korea (Received December 13, 2010. Accepted December 21, 2010)ABSTRACT: A homothallic filamentous fungus Aspergillus nidulans has been used as the a model organism forstudying growth and development for eukaryotic system. Various studies about specific transcription factors havebeen performed for elucidating the molecular mechanisms of growth, asexual and sexual developmental processes.Among them, the fkhE gene (AN2025.3) is located in chromosome VII and contains an ORF encoding 718 aminoacid polypeptide intervening with two short introns. The cDNA sequencing revealed that at least four types of alter-native splicing events were occurred when the fkhE gene was transcribed. The putative FkhE polypeptide containsa conserved forkhead domain and a bipartite nuclear localization signal at it's N-terminus and C-terminus, respec-tively. Deletion of fkhE resulted in impaired conidiophore formation in a solid medium. However, the sexual devel-opmental process or cleistothecia formation was normal. Furthermore, fkhE deletion mutant produced conidiophoresand conidia under the submerged culture, indicating that the fkhE gene is involved in asexual developmental processsimilar to the fkhF gene.KEYWORDS :
Aspergillus nidulans
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Filamentous fungi are unique organisms-rivaled only by actinomycetes and plants-in producing a wide range of natural products called secondary metabolites. These compounds are very diverse in structure and perform functions that are not always known. However, most secondary metabolites are produced after the fungus has completed its initial growth phase and is beginning a stage of development represented by the formation of spores. In this review, we describe secondary metabolites produced by fungi that act as sporogenic factors to influence fungal development, are required for spore viability, or are produced at a time in the life cycle that coincides with development. We describe environmental and genetic factors that can influence the production of secondary metabolites. In the case of the filamentous fungus Aspergillus nidulans, we review the only described work that genetically links the sporulation of this fungus to the production of the mycotoxin sterigmatocystin through a shared G-protein signaling pathway.
Aspergillus nidulans
Sterigmatocystin
Secondary metabolism
Filamentous fungus
Aspergillus versicolor
Secondary metabolite
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Filamentous fungi are widely used in the production of a variety of industrially relevant enzymes and proteins as they have the unique ability to secrete tremendous amounts of proteins. However, the secretory pathways in filamentous fungi are not completely understood. Here, we investigated the role of a mutation in the POlarity Defective (podB) gene on growth, protein secretion, and cell wall organization in Aspergillus nidulans using a temperature sensitive (Ts) mutant. At restrictive temperature, the mutation resulted in lack of biomass accumulation, but led to a significant increase in specific protein productivity. Proteomic analysis of the secretome showed that the relative abundance of 584 (out of 747 identified) proteins was altered due to the mutation. Of these, 517 were secreted at higher levels. Other phenotypic differences observed in the mutant include up-regulation of unfolded protein response (UPR), deformation of Golgi apparatus and uneven cell wall thickness. Furthermore, proteomic analysis of cell wall components in the mutant revealed the presence of intracellular proteins in higher abundance accompanied by lower levels of most cell wall proteins. Taken together, results from this study suggest the importance of PodB as a target when engineering fungal strains for enhanced secretion of valuable biomolecules.
Aspergillus nidulans
Secretory protein
Filamentous fungus
Fungal protein
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Aspergillus nidulans
Filamentous fungus
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Aspergillus nidulans
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Keywords:selection arena, progeny choice, Aspergillus nidulans , fungus, spores, ascospore, conidiospore, asexual, sexual, fruiting body, cleistothecium, zygote, dikaryon, self-sterility, self-fertility, mutation accumulation, fitness, modular organism, auxotrophic.Chapter 1: IntroductionThe selection arena hypothesis states that overproduction of zygotes -a widespread phenomenon in animals and plants- could be explained as part of a quality control mechanism: An enlarged array of zygotes is created of which only a genetically superior subset will fully develop; zygotes with a low future fitness fail, while zygotes with a high future fitness thrive. In this way, parental energy for reproduction is invested in the most promising zygotes. This hypothesis further assumes that 1) zygotes are cheap to produce, 2) parental time, energy and/or risk are invested in the zygotes, 3) offspring vary in fitness, and 4) this fitness difference can be identified. In this introducing chapter, we review several examples which strongly suggest the operation of selection arenas in nature. Yet, although the idea of the selection arena is appealing and plausible, it has been difficult to actually demonstrate a mechanism of selection against low quality zygotes in favour of the production of higher quality ones.This thesis examines the selection arena hypothesis in the filamentous ascomycete fungus Aspergilus nidulans.This fungus may overproduce dikaryotic fruit initials, called dikaryons, like animals and plants may overproduce zygotes. Then, progeny choice might involve selection on which of these dikaryons will thrive to produce thousands of zygotes. These zygotes each produce eight sexual spores which together fill up one fruiting body, namedcleistothecium.In this introducing chapter we explain that A. nidulans is highly suited to study the selection arena hypothesis as it fulfils the first three of the four conditions of the selection arena hypothesis. Moreover, the predicted result of the arena can be tested in this fungus, namely that dikaryotic fruit initials with a low future fitness fail, while dikaryotic fruit initials with a high future fitness thrive.Chapter 2: The male and female role in fruiting body formationOnly if a dikaryon passes the selection arena, it will eventually develop into a ripe fruiting body. However, the initiation of a fruiting body and especially - in the case of outcrossing - the roles of the nuclei and mitochondria from the two parental strains in its formation have remained unclear. In chapter 2 we resolved these roles by analysing the genetic constituents of these fruiting bodies (called cleistothecia) from crosses between vegetatively compatible parents and from crosses between vegetatively incompatible parents. In compatible parents, nuclear genomes and cytoplasm usually mix in the vegetative hyphae prior to the formation of the sexual stage, after which any cleistothecial composition is possible. In incompatible parents, the maternal strain contributes the nuclei for the cleistothecial wall and one nucleus as well as mitochondria for the ascospore origin. The paternal strain donates one nucleus for the ascospore origin. Only in crosses between vegetatively incompatible partners, it is possible to assign a female and male role to the parental strains. Our results confirm that the vegetative heterokaryotic stage is not a prerequisite for cleistothecium formation.Chapter 3-5: Testing the selection arena in Aspergillus nidulansIn these chapters we tested the selection arena hypothesis in A. nidulans with emphasis on the predicted outcome, namely that dikaryons with a low future fitness would fail, while dikaryons with a high future fitness would thrive. We reformulated this into a testable prediction for this mycelial fungus: Nuclei with deleterious mutations will not be transmitted via the sexual route, or at least less easily than via the asexual route.In chapter 3,we analysed 2 mitochondrial and 15 auxotrophic mutations for consequences on sexual and asexual reproduction. We found that many of these mutations confer sexual self-sterility as pleiotropic effect under conditions of normal asexual spore production:fruiting bodies were very tiny and contained hardly any ascospores. This confirms an important prediction of the selection arena, namely that dikaryons carrying a (slightly) deleterious mutation are not able to proliferate and produce sexual spores.So, no reproductive energy is invested in sexual spores carrying deleterious mutations, while asexual spore production is unaffected. Note that asexual and sexual spores would have carried the same genotype. Although self-sterility has been previously reported for a few mutations, these results have never been discussed as a manifestation of a selection arena.In chapter 4 we tested the selection arena theory using a different approach. We exploited the coexistence of asexual and sexual reproduction in A. nidulans , especially in cases of sexual self-fertilisation, where offspring from the sexual and asexual pathway have the same genotype. The selection arena hypothesis predicts that nuclei with deleterious mutations will not be transmitted via the sexual route, or at least less easily than via the asexual route. This impliesthen that the sexual spores - produced by selfing - will have a higher average fitness than the asexual spores with the same genotype. To investigate this, we started a mutation accumulation experiment with 40 asexual and 40 sexual selfing lines, all derived from one single ancestral strain of A. nidulans . In these lines, mutations were allowed to accumulate during 40 generations by single-spore transfer of respectively asexual and sexual spores. After 40 generations of mutation accumulation, the fitness of each line was estimated relative to that of the founder. Fitness was estimated by measuring the colony diameter grown in a fixed time period (RCD = relative colony diameter). The results show that mutations accumulate in both groups, but, in accordance with the selection arena hypothesis, with a significantly lower'fitness impact' in the sexual lines than in the asexual lines. We argue that genetic recombination can be excluded as an explanation for this result.In chapter 5 we estimated the fitness of the mutation accumulation lines using a different measure, namely asexual spore production under competitive conditions ( W ij ). Such competition is harsh and provides a condition that likely amplifies potential differences in fitness between mutation accumulation lines and the ancestor. However, we did not find such a result . Instead, we found that the two fitness measures RCD (relative colony diameter) and W ij (asexual spore production under competitive conditions) were uncorrelated. This appeared to be due to an 'ultra-sexual' phenotype in more than half of the sexual lines: these lines produced many sexual spores at the expense of asexual spores. The 'ultra-sexual' phenotype must have been caused by the experimental history in the mutation accumulation procedure and made the fitness estimate W ij based on asexual spore production problematic. We discuss difficulties in fitness estimation in modular organism in general, especially when they produce progeny (spores) via both a sexual and an asexual pathway. Chapter 6: Summary and discussionIn this chapter we summarize the findings of this thesis. Furthermore, we speculate about the actual mechanism of the selection arena. The generality of the selection arena mechanism is discussed as well as directions for future research.
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Dikaryon
Filamentous fungus
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Aspergillus nidulans
Filamentous fungus
Glucanase
Aspergillus oryzae
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Abstract The filamentous fungus Aspergillus nidulans has proved to be an excellent experimental system for the identification, isolation, and analysis of genes that are essential lo cell-cycle progression and regulation. Studies in A. nidulans have led to the discovery of several key elements of cell-cycle progression and regulation that were not first identified in other experimental systems (1). It is a homothallic ascomycetous fungus that has both a sexual and an asexual life cycle (Figure 1). The classical genetics and molecular genetics of A. nidulans have been previously described (2) and are, in many respects, as advanced as the yeast systems. his the purpose of this chapter to describe some of the techniques that can be utilized to study the cell cycle in A. nidulans.
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We have studied compensatory evolution in a fludioxonil resistant mutant of the filamentous fungus Aspergillus nidulans. In an evolution experiment lasting for 27 weeks (about 3000 cell cycles) 35 parallel strains of this mutant evolved in three different environmental conditions. Our results show a severe cost of resistance (56%) in the absence of fludioxonil and in all conditions the mutant strain was able to restore fitness without loss of the resistance. In several cases, the evolved strain reached a higher fitness than the original sensitive ancestor. Fitness compensation occurred in one, two or three discrete steps. Genetic analysis of crosses between different evolved strains and between evolved and ancestral strains revealed interaction between compensatory mutations and provided information on the number of loci involved in fitness compensation. In addition, we discuss the opportunities for the experimental study of evolutionary processes provided by the filamentous fungus A. nidulans.
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Fludioxonil
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