| Calcium-mediated signaling pathways are widely employed in eukaryotes and are implicated in the regulation of diverse biological processes.In Saccharomyces cerevisiae,at least two different calcium uptake systems have been identified—the high-affinity calcium influx system(HACS)and the low-affinity calcium influx system(LACS).Compared to the HACS,little is known about the LACS in fungi.Up to date,Figl is the only characterized member of the LACS in fungi.In S.cerevisiae,fig1 is up-regulated in response to mating pheromone and loss of fig1 results in incomplete fusion between the tips of mating shmoos.Therefore,the name figl(mating factor-induced gene1)is initially used to describe this gene.In this study,FigA,a homolog of the LACS member Fig1 fromS.cerevisiae,was functionally characterized in the filamentous fungus Aspergillus nidulans.Aspergillus species are among the most abundant fungi worldwide.The model filamentous fungus Aspergillus nidulans develops both sexual and asexual spores through complicated regulated mechanisms.Our previous studies reported the HACS components CchA/MidA play unique and complex roles in regulating conidiation,hyphal polarity and cell wall components in low-calcium environment.Compared to the HACS,the function of the LACS during fungal development is barely known.In this study,The roles of figA during the life cycle of A.nidulans,especially for hyphal growth,asuxual and sexual development were investigated by studying calcium homeostasis,gene expression,and protein localization.To study the function of figA in A.nidulans,a figA knockout strain and a conditional strain,were constructed.Our results showed that loss of figA resulted in retardant hyphal growth and sharp reduction in conidia production.Our results clearly indicate that with increasing figA expression,hypal growth and conidiation could be enhanced accordingly,suggesting that figA indeed plays important roles in hypal growth and asexual development.Most importantly,FigA was essential for the homothallic mating(self-fertilization)process,the ?figA mutants did not produce either any visible cleistothecia or aggregated Hulle cells when induced for sexual development.FigA was also required for the heterothallic mating(outcross)in the absence of HACS-cchA/midA.Interestingly,in figA deletion mutant,adding extracellular Ca2+ was able to rescue the hyphal growth defects but could not restore asexual and sexual reproduction.In addition,we found riboB2 mutants in A.nidulans confer self-sterile and thus are unable to form hybrid cleistothecia by outcross when both of parent strains harboring riboB2 auxotrophic marker under the standard protocol.To assess the role of riboflavin during the different developmental stages of A.nidulans,the limited concentrations of riboflavin were monitored.We found the addition of riboflavin could restore self-sterile defects and outcross defects in riboB2 mutants.We then test if high concentration of riboflavin could rescue the self-sterile defects and outcross defects in figA deletion mutant.And we found in figA deletion mutant,adding high concentration of riboflavin could not restore sexual reproduction,suggesting that figA indeed plays important roles in sexual development.Furthermore,the quantitative PCR results revealed that figA deletion sharply decreased the expression of brlA and nsdD,which are known as the key regulators during asexual and sexual development,respectively.In addition,the green fluorescent protein(GFP)tag at the C-terminus of FigA(FigA::GFP)showed that FigA localized to the center of septum in the mature hyphal cell and to the location between vesicle-metulae,and between the junctions of metulae-phialide in conidiophores.Thus,our findings suggest that FigA localized to location betwwen the junctions of cell-cell,and play multiple roles during hyphal growth,asexual and sexual development,via being a member of calcium uptake system in A.nidulans and enable communication between cell-to-cell and change gene expression of transcription factor... |
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