Mechanism Of Aspergillus Niger CbsA And CysA In Antioxidation And Pathogen-plant Interaction

Aspergillus niger is a common strain in Aspergillus fungi.It is an important industrial fermentation strain and a pathogenic fungus that causes mildew in food and fruits.When a fungal pathogen infects a plant,the plant host cell initiates a defense mechanism such as the outbreak of reactive oxygen species to limit the growth of the pathogen,and the pathogen activates the body's antioxidant enzymes and antioxidants to ensure that it is not immune to plant immunity.The mechanism of antioxidant enzymes and antioxidants in the pathogen-plant interaction has always been a topic of concern to scholars.cbsA?An05g00160?and cysA?An12g09880?are genes respectively encoding cystathionine-?-synthase and cysteine synthetase in Aspergillus niger,which are involved in the metabolism of Cys and H₂S,and Cys and H₂S are important antioxidants.But the function in antioxidant and bacterial interaction is still unclear.In this paper,Aspergillus niger was used as experimental material,and the Aspergillus niger cbsA and cysA knockout vectors were constructed by using the principle of homologous recombination.The Aspergillus niger protoplasts were transformed with PEG,and the mutant strains?cbsA and?cysA were identified by genomic PCR and qRT-PCR.The following work was carried out using the mutant strain?cbsA and A.niger MA70.15?wild type?as materials.First,under normal culture conditions,the cbsA gene deletion did not affect the growth of A.niger and grew on GMM?Lack of Amino Acid?medium,the colonies of?cbsA growth were found to be small,indicating that cbsA is an amino acid-dependent enzyme.By detecting the content of cysteine and GSH in A.niger,the content of cysteine and GSH in?cbsA was increased by 0.28times and 0.94 times compared with WT,respectively.In addition,the formation of hydrogen sulfide was detected by BiGGY agar chromogenic medium.The WT colonies were found to be darker in color than the?cbsA colonies,indicating that the loss of the cbsA gene reduced the release of H₂S in A.niger.Under the stress condition of 0.5mM CdCl₂,the plaque diameter of WT was significantly smaller than?cbsA.On the fourth day of culture,the colony diameter of WT was even less than half of?cbsA,indicating that cbsA gene is a negative in oxidative stress under cadmium stress.The regulatory factor,however,was not significantly different between the?cbsA mutant and the wild type under the conditions of menadione,manganese sulfate,and fluconazole treatment.In the spore germination experiment,the spore germination rate of the control strain was only 45.1%,while the spore germination rate of cbsA reached71.3%,further indicating that the loss of cbsA gene enhanced the ability of A.niger to resist Cd stress.The spore suspension of WT and?cbsA was inoculated on the pear fruit.The deletion of cbsA gene caused the infective diameter of pear fruit to be larger than that of wild type,which enhanced the pathogenicity of A.niger to pear,indicating that cbsA is a negative regulator of A.niger infecting fruit.The contents of?O2^–,H₂O₂and MDA were detected in the WT and?cbsA inoculated fruit tissues.It was found that the content of?O2^–,H₂O₂ and MDA in fruit tissues was increased after?cbsA infested pear fruits compared with WT.A stronger ROS response was shown,indicating that cbsA deletion induces a stronger response process in pear fruit.The deletion of cbsA can increase the content of Cys and decrease the release of H₂S.Therefore,we speculate that cbsA has the function of catalyzing the production of H₂S by Cys in A.niger.To verify the hypothesis,we successfully constructed the prokaryotic expression vector of A.niger cbsA.And found CS and CSX1 with the highest homology to cbsA in tomato as a reference.In the study of protease properties,we found that cbsA,CS and CSX1 can catalyze the production of H₂S by Cys,and the catalytic ability of cbsA after induction is increased by 1.4 times.Cysteine synthetase is the rate-limiting enzyme for the synthesis of cysteine and cysteine plays an important role in stress resistance.Whether cysA is involved in the anti-reverse response and bacterial interaction mechanism of A.niger is not clear.A.niger has six genes encoding cysteine synthetase,all of which contain a PALP domain characterized by a conserved pyridoxal-phosphate-dependent enzyme.Growth in GMM medium revealed that the?cysA mutant showed smaller colonies,indicating that cysA is also an amino acid dependent enzyme.Under the abiotic stress of CdCl₂,AlCl₃,menadione,ZnSO₄,Pb?NO3?₂,MnCl₂,NaCl and H₂O₂,no significant difference was found in the colony diameters of WT and?cysA,indicating that cysA may not participate in the stress response under stress condition of A.niger.In the experiments of infecting pears and apples,there was no significant difference in the diameter of lesions between WT and?cysA,so it was concluded that the loss of cysA gene did not affect the ability of A.niger to infect pears and apples.The activity of WT and?cysA cysteine synthetase and cysteine content were detected.The cysA gene deletion had no significant effect on the cysteine synthase activity and cysteine content of A.niger,This indicates that cysA is not a key gene involved in the synthesis of cysteine,which may be due to redundancy of gene function.qRT-PCR showed that the expression levels of An14g00960,An15g00160 and An02g10750 gene which encoding cysteine synthase in the?cysA mutant were increased by 1.5 times,0.3 times and 0.75 times,respectively,compared with WT.Therefore,we hypothesized that the up-regulated expression of other genes can compensate for the loss of the cysA gene,which means that there may be negative feedback regulation of cysteine synthase gene expression in A.niger.Summary:The deletion of the gene cbsA leads to the accumulation of cysteine content in A.niger,which increases the ability to resist cadmium stress and the ability to infect pear fruit.The enzymatic properties of prokaryotic expression showed that cbsA can catalyze the production of H₂S by Cys.After the cysA gene was deleted,it did not affect the anti-oxidation and infectivity of A.niger.The results of qRT-PCR showed that the expression levels of the genes encoding cysteine synthetase An14g00960,An15g00160 and An02g10750 were up-regulated in A.niger,so it was speculated that there may be negative feedback regulation of cysteine synthase gene expression in A.niger.