Function Analysis Of Thioredoxin And Glutaredoxin Systems, Histone Acetyltransferases And Deacetylases In Alternaria Alternata

Citrus brown spot is a fungal disease that seriously harms citrus tender leaves,shoots,and fruits.It often causes symptoms such as defoliation,fruit-drop,and shoot blight,which pose a serious threat to the citrus industry.Current research shows that decline in the ability to resist oxidative stress will cause the pathogenicity of the Alternaria alternata to be severely reduced or even completely lost.In cells,the thioredoxin and glutaredoxin systems play an important role in the removal of reactive oxygen species(ROS).In many plant pathogens,the loss of histone acetyltransferases(such as Gcn5)or deacetylases(such as Hos2)often causes a significant decrease in the antioxidant capacity and pathogenicity of the corresponding pathogens.However,the research results of genes related to the thioredoxin and glutaredoxin systems in A.alternata are rarely published,and the functional analysis of histone acetyltransferase and deacetylase family genes is even less in this pathogen.Therefore,this study used gene knockout method to systematically knock out genes related to the thioredoxin and glutaredoxin systems,histone acetyltransferases and deacetylases in the A.alternata.The main research results are as follows:1.Functional analysis of thioredoxin and glutaredoxin systemsThioredoxin peroxidase(Tsa1)and thioredoxin reductase(Trr1),glutathione peroxidase(Gpx3)and glutathione reductase(Glr1)are peroxidase or reductase that regulate the redox homeostasis of thioredoxin and glutathione,while glutamyl-cysteine synthetase(Gsh1)and glutathione synthetase(Gsh2)are the two enzymes necessary for the synthesis of glutathione.In Saccharomyces cerevisiae,the knockout of Tsa1,Trr1,Gpx3,Glr1,Gsh1,and Gsh2 caused their ability to remove external oxidative stress to be impaired.These six genes are highly conserved among pathogens such as Magnaporthe grisea and Fusarium graminearum.The transcription levels of Tsa1,Trr1,Gpx3,Glr1,Gsh1 and Gsh2 were all up-regulated in A.alternata treated by oxidants.Trr1 and Glr1 are important genes in the vegetative growth and sporulation process of A.alternata.The deletion of Trr1 or Glr1 will lead to serious damage to the pathogen's growth,development,and sporulation ability.The addition of exogenous glutathione to the medium can restore the vegetative growth and sporulation ability of Trr1 and Glr1 deletion mutants.The knockout mutants of Tsa1,Trr1 and Glr1 were severely inhibited under oxidative stress,and their pathogenicity was significantly reduced or lost.The absence of Gsh1 and Gsh2 caused the vegetative growth of the A.alternata to be severely inhibited,and the antioxidant capacity was severely reduced.In addition,Trr1 and Glr1 help A.alternata adapt to salt stress caused by NaCl.Glr1 is also an important gene for A.alternata to adapt to osmotic stress,mancozeb and boscalid fungicides.Trr1 is involved in the resistance reaction of boscalid and chlorothalonil.Tsa1 did not respond to the above three fungicides.qRT-PCR analysis showed that Tsa1 and Trr1 transcription were regulated by oxidative stress regulators Ap1,Hog1 and Skn7.The results showed that genes related to the thioredoxin and glutaredoxin systems are essential for adapting A.alternata to ROS,and have an important impact on the growth and pathogenicity of the pathogen.2.Functional analysis of histone acetyltransferases and deacetylasesHistone acetylation is one of the main regulation methods of epigenetics,and plays an important role in regulating chromosome disaggregation,gene transcription and protein function.In this study,a total of 7 histone acetyltransferases(including Gcn5,RTT109,Elp3,Sas3,Sas2,Nat3,and Esa1)and 6 histone deacetylases(Hos2,Rpd3,Hda1,Hos3,Hst2 and Sir2)were identified in A.alternata.These 13 genes are highly conserved among different fungi.Chromosome location analysis showed that these 13 genes were all located in the essential chromosome of the A.alternata.Through gene knockout experiment,a total of 12 gene knockout mutants of histone acetyltransferases and deacetylases were obtained.Compared with the wild type,the gene deletion mutants of 6 histone acetyltransferases and 2 histone deacetylases were significantly inhibited in vegetative growth.The pathogenicity test results showed that the pathogenicity of Gcn5,Sas3 and Nat3 deletion mutants was completely lost,the pathogenicity of RTT109,Elp3,Hos2 and Rpd3 deletion mutants decreased significantly.The pathogenicity of Sas2,Hda1,Hos3,Hst2 and Sir2 deletion mutants did not change significantly.Microscopic observations revealed that Sas3,RTT109,Nat3,Hos2,and Rpd3 deletion mutants had significantly reduced spore production.Sas2,Elp3,Hda1,Hos3,Hst2,and Sir2 did not play a significant role in the conidiation of A.alternata.The sporulation ability of the Gcn5 deletion mutant was completely lost,and the mycelial cells were shortened,stubby and swelling.The results of various stress experiments indicated that Gcn5,Sas3 and Hos2 play an important role in adapting A.alternata to oxidative stress,cell wall interfering agents,osmotic stress and starvation stress.In addition,the absence of Gcn5,Sas3 and Hos2 also led to defects in the ability of the A.alternata to utilize carbon sources.Transcriptome analysis results showed that Gcn5 and Hos2 can regulate the transcription of genes related to energy production,carbon source metabolism,DNA damage repair,antioxidant,cellular autophagy,ubiquitin degradation,and protein processing on the endoplasmic reticulum membrane.In addition,Gcn5,Hos2,and RTT109 deletion mutants are significantly more sensitive to genome toxic agents such as hydroxyurea,methyl methanesulfonate or camptothecin.Genome resequencing data analysis indicated that Gcn5 plays an important in repairing DNA damage caused by ultraviolet radiation.This study revealed that histone acetyltransferases and deacetylases have important regulatory effects on the vegetative growth,conidiation,pathogenicity,stress resistance and carbon source utilization of citrus brown spot pathogen.Our studies indicate that thioredoxin and glutaredoxin systems,and histone acetylation are crucial to the vegetative growth,development,multiple stress resistance and fungicide tolerance of A.alternata.The results expand our understanding of the regulation mechanism between the antioxidant systems,histone acetylation and pathogenicity of A.alternata,which providing a basis for further research on molecular biology.