The Function Of Transient Receptor Potential Channel Proteins YvcA, TrpA, TrpR In Aspergillus Nidulans In Response To Stress And Regulation Of Cytoplasmic Calcium Homeostasis

Calcium signal plays an important role in many cellular processes,and it is crucial for cells to maintain the homeostasis of free calcium.In order to keep optimal cytoplasmic calcium concentration,many calcium-related genes are involved to this regulation,including Ca2+ pumps,Ca2+ exchangers,Ca2+ channels,enzymes and so on.Transient receptor potential(TRP)superfamily is one class of ion channels,and most of them are Ca2+ permeable.In mammals,flies and worms,TRP superfamily members predominantly function in "sensory physiology",like vision,taste,olfaction,touch,and hearing.In fungi,one TRP protein called Yvc1 in Saccharomyces cerevisiae and Candida albicans has been reported functioning in cell growth and affecting.Ca2+homeostasis under several stresses.Besides,Yvc1 in C.albicans and its ortholog in Aspergillus fumigatus both influence the virulence of these two opportunistic pathogens.However,AfYvcA seems not involved in stress responses and cytoplasmic Ca2+regulation.According to the information of TRP protein in yeast,we found four trp genes in Aspergillus nidulans via alignment,followed as yvcA,trpA,trpR and pkd2A.Among these four genes,yvcA and trpA are both related to yvcl in S.cerevisiae,while trpR and pkd2A are on the aliment of trp1322 and pkd2 in Schizosaccharomyces pombe,respectively.The bioinformatic analysis showed that the four genes above shared some features of TRP superfamily,like owning transmembrane regions at least for the amount of six,sharing identity in some transmembrane regions and possessing TRP domain.Finally,we chose yvcA,trpA and trpR to further study on the basis of bioinformatic analysis.In consistent with the rusults in A.fumigatus,yvcA showed unconspicuous effects on cell growth and cytoplasmic Ca2+fluctuation caused by extracellular stimuli,and another S.c.yvc1-related gene,trpA,appeared like yvcA.Besides,yvcA and trpA seemed not compensated for each other in function.In addition,neither yvcA nor trpA cooperated with other calcium-related genes in regulating cell growth under several conditions.Surprisingly,trpR worked like a trp gene mostly among the three trp genes in Aspergillus nidulans.The results showed that trpR worked in heat sensitivity,because ?trpR appeared more sensitive in rather high temperature compared with wild type.And the addition of Ca2+ in medium could recover the defect of ?trpR under 37 ?.Meanwhile,?trpR could also be sensitive to a low-Ca2+ environment.And the peak value of cytoplasmic Ca2+ concentration in ?trpR responded to excellular Ca2+showed higher than that in wild type.These results suggested that the function of trpR might be related to free Ca2+ regulation And in other stimuli tests,it showed that ?trpR was sensitive to cell wall stresses,including Congo Red and caspofingin.Therefore,trpR might be also involved in cell wall integrity.As to the connection between trpR and the members in calcium signal network,the reults showed that trpR cooperated with midA and cchA which coded the high-affinity calcium uptake system on conidiation.Besides,knocking out trpR on the background of ?cchA could recover the lowered Ca2+ peak value caused by the deletion of cchA.And the sensitivity to CR of?trpR?cchA and ?trpR?midA were between that of the parental strains.pmrA coded a Ca2+ channel on the membrane of Golgi apparatus and the double deletion of trpR and pmrA caused a severe conidial defects,which could not be restored completely on the addition of Ca2+.Gene clxA coded Calnexin on endoplasmic reticulum membrane,and the double mutant ?trpR ?clx A also showed a less conidiation than single deletions,but excellular Ca2+ could recover the conidial defect of the double mutant to a certain degree.Gene cnaA coded the catalytic subunit of Calcineurin,and ?trpR?cnaA appeared as ?cnaA in phenotype.However,the conidiation of ?trpR?cnaA could not be restored by excellular Ca2+ unlike the situation in ?cnaA.Furthermore,?trpR?clxA,?trpR?pmrA,?trpR?cnaA are all more sensitive to CR than the single deletions.In conclusion,trpR involved in several stress responses,including high temperature,cell wall stress and low-Ca2+ environment.And trpR regulated the conidiation and cell wall stress response together with the components in calcium signalling pathway.Besides,trpR took a opposite role with cchA in cytoplasmic Ca2+regulation responded to high concentration of Ca2+.