| Cell polarity is a fundamental property of all cells and its importance is evident in a variety of cells. The growth pattern exhibited by filamentous fungi is a typical example of cell polarity. The well developed genetic system of Aspergillus nidulans(A. nidulans) makes it an excellent model system for studying the process of growth, establishment and maintenance of polarity.Ca2+-permeable, stretch-activated nonselective cation channel Midl, as a component of calcium-mediated signaling pathway, has been implicated in the regulation of diverse biological processes in eukaryotes. However, the role of Midl in different organisms was highly variable. So far little is known about the role of Midl homologues in Aspergillus nidulans, midA. In this study, we supposed that MidA, possibly as a stretch-activated calcium channel, may play an important role in A. nidulans.A conditional alcA(p)::CFP-midA strain HHA01 was successfully created by highly efficient gene homologous integration. It could be regulated by the carbon source: repression on glucose and induction on glycerol plus threonine. We found that on the repressing medium, YAG, HHA01 displayed a slower colony growth than that of wild-type. Furthermore, a microscopy study showed HHA01 on the YAG had a severe polarity maintenance defect accompanied by direction randomization of second or third even fourth germ tube formation. In addition, the conidia of HHA01 showed an abnormally extended isotropic swelling pattern upon germination. These results suggested that MidA was required for the normal hyphal polarity growth. Moreover, we demonstrated that HHA01 had a remarkable phenotype of a higher hyper-sensitivity to the low concentration of the extracellular [Ca2+] compared to the wild type. As expected, this phenotype could be partially rescued by increasing the concentration of Ca2+ in YAG media. This indicated that MidA may be especially important under lower [Ca2+]. Previous studies in other highly polarized cell types has strongly suggested that reactive oxygen species (ROS) generation at the hyphal tips plays a key role in enforcing apical dominance and the spatial regulation of polar growth. Under the repressing condition, the NBT staining in HHA01 was irregular and ROS no longer was accumulated at the hyphal tip only, suggesting the loss of the apical dominance of ROS maybe resulted in the polarity growth defect. TEM showed there was a defect in cell wall architecture in the depletion strain of MidA. In addition, the depletion strain displayed significantly decreased cell wall 1,3-β-D-glucan,while a compensatory increase in chitin content.Interestingly, when HHA01 growed on solid YAG, or was cultured in YAG liquid medium amended by the addition of 15% PEG,200 g/L sucrose or 1.25 M sorbitol, a recovery of hyphal polarity growth was observed. These results together suggested there was a cell wall defect in the MidA depletion strain.A deletion strain of midA by fusion PCR was also constructed. However, it didn't displayed the same severe polarity maintenance defect as HHA01 on YAG. Further study is still going on.In conclusion, MidA, as a putative stretch-activated calcium channel in Aspergillus nidulans, plays an important role in hyphal polarity establishment, maybe via regulating Ca2+ homeostasis, maintaining the ROS accumulation pattern and regulating the synthase of cell wall. |
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