| Aspergillus is a genus consisting of several hundred mold species found in various climates worldwide. Species of Aspergillus are important medically and commercially. Some species are important in commercial microbial fermentations as example of Aspergillus niger and Aspergillus oryzae, other species can cause allergic disease and infection in humans and other animals such as Aspergillus fumigatus and Aspergillus clavatus. Aspergillus nidulans, which is considered as a premier model fungus for filamentous fungi especially for other Aspergilli due to its well-established genetic system for studying the process of growth, establishment and maintenance of polarity growth, and asexual sporulation. Moreover, it is hardly ever pathogenic, but is very closely related to other industrial and common pathogenic fungus; it was sequenced in part to better understand A. fumigatus pathogenicity.Calcium-mediated signaling mechanisms are widely employed in eukaryotes and are implicated in the regulation of diverse biological processes including growth development, cell morphology, exocytosis, cytoskeletal, differentiation, apoptosis, gene expression, and rearrangement. Consistently, calcium signaling pathway is critical for physiological function in yeast. Although the function of calcium signaling in Aspergillus nidulans and yeast are similar, there are also some differences. In comparsion, A.nidulans is more suitable for the study the hyphal polarity and conidiation which determined their virulence and pathogenicity. Therefore, my study will focus on this hot topic and have a further in-depth research.In fungi, at least two different carrier systems have been identified-a high-affinity calcium influx system (HACS) and a low-affinity calcium influx system (LACS). The HACS consists of at least two known subunits, CchA and MidA, which act as the major calcium entry route when calcium availability is low. Here, we have functionally characterized the yeast homologs, Cchl and Mid1, in A.nidulans using conditional and null deletion mutants.△cchA,△midA showed an aconidial phenotype in low-calcium concentration minimal medium. As expect, when down-regulated by the alcA promoter in the presence of glucose, both of cchA or midA conditional strains showed the similar and the consistent phenotypes with△cchA,△midA. More interestingly, the conidiation defect phenotype was induced in a density-dependent manner. To better understand these conidiation defects, the microcopy study was conducted. The results indicated that loss of either gene caused a sharp reduction in the number of conidiospores as well as abnormally shaped metulae and phialides, but these conidiation defects in mutants can, remarkably, be rescued either by extra-cellular calcium or by osmotic stress. CchA and MidA not only have unique and complex roles in regulating conidiation in low-calcium environments but also have functional benefits of hyphal polarity. Through microscopical observsation, we found that△cchA,△midA single or double mutants were significantly impaired in the formation of the single axis of hyphal polarity, resulting in the hypha branched early along with an abnormally wide and a bulbous growth pattern. This was in contrast to wild-type, which had organized, parallel, and defined hyphal filaments. In addition, we found that the mutant hyphae branched repeatedly to yield multiple polarity axes such that there was no single dominance; by contrast, wild-type had apical dominance as a result of suppression of secondary polarity axes in the general vicinity of a growing hyphal tip under the same culture conditions. Most interestingly,△cchA,△midA are more resistant to cell wall damaging agents than is the wild-type strain. As expected, the chitin content and β-1,3-glucan content in mutants also showed the statistically significant increased. Consistently, flow cytometry analysis labeled by β-1,3-glucan antibody revealed that the mutant strains had the differential rearrangement of surface carbohydrate epitopes compared with the wild type. Our results suggest that both CchA and MidA are critical for hyphal polarity establishment, conidial development, and cell wall components in low-calcium environmental conditions. Moreover, the fact that the phenotypic defects are not exacerbated by the presence of the double deletion, together with the yeast two-hybrid system assay, indicates that CchA and MidA may form a complex to function together.Based on the results described in previously published data, we know Calcineurin is probably a central key component in the calcium signaling pathway. Therefore, we want to know how Calcineurin, as an indicator of a high affinity calcium influx system, interact with CchA in the regulation of the developmental process of polarity morphogenesis in A.nidulans. Our results suggested that△cnaA/△cchA double mutant showed similar phenotypes to△cnaB/△cchA, both had more polarity defects than that of either single mutant in minimal medium. Moreover, adding extracellular salt stress (0.8M NaCl and0.6M KC1) induced more severe growth defects in AcnaA and△cnaB single mutants. In contrast, it was quite surprising that the extracellular salt stress was able to significantly improve the polarity growth defect of double mutants. Therefore, voltage-gated calcium channel homolog-CchA may act as a reverse way with Calcineurin in the regulation of polarity growth in response to extracellular salt stress, but act in parallel to determine cell polarity in A.nidulans under normal minimal medium.In conclusion, we have investigated the function of calcium signaling pathway with the example of Calcium Channel and Calcineurin in the regulation of cell polarized growth and morphogenesis in A. nidulans. We have demonstrated that the high affinity Ca2+channel, CchA, and its subunit MidA played important roles in regulating hyphal polarity, conidiation and cell wall components in low-calcium environment. Nevertheless, our findings in this paper enlighten a potentially viable and completely unexplored avenue to control conidiation in Aspergilli by the high affinity calcium channel. Moreover, elucidating the relationship of calcium channel and calcineurin signaling cascade during the response to salt stress will provide several new insights into the mechanisms of calcium signaling pathway. These findings will provide an excellent opportunity to increase the metabolic product and better understand the functional genes in human. Moreover, it has a great significance in finding novel drug therapies for fungal infections. |
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