Studies On The Function Of AaMR1 And AaSNF1 Genes Of Alternaria Alternata Tangerine Pathotype

Alternaria brown spot(ABS)is a fungal disease caused by Alternaria alternata pathotype tangerine,which seriously harms the tender shoots,young leaves and fruits of citrus,and it brings serious problems to the production of some mandarins and tangerines.Along with the completion of the whole genome sequencing of A.alternata,the growth/development and pathogenicity related genes of A.alternata have been gradually discovered,providing a new platform for understanding the molecular mechanism of A.alternata at the genomic level.Transcription factors have been shown to be involved in regulating the growth/development of a variety of plant pathogenic fungi,response to external stress,pathogenicity,drug tolerance and secondary metabolite synthesis.In this study,the function of AaMR1 and AaSNF1 was characterized using gene knock-out and complementation strategy.This research is based on transcriptome sequencing to study the regulation mechanism of transcription factor AaMR1 on the growth and pathogenesis of citrus brown spot pathogen,and the transcriptional expression of citrus in response to citrus brown spot pathogen infection.The main results are as follows:1.AaMR1 and AaSNF1 genes knockout mutants and complementary strains were obtained.Split-marker strategy was used to construct AaMR1 and AaSNF1 gene knockout box,and PEG(Polyethylene glycol)-mediated protoplast transformation was carried out to gain gene knock-out mutants.The mutants were picked out after screening and verification by hygromycin,PCR(Polymerase chain reaction)and Southern blot.The efficient homologous recombination mechanism in Saccharomyces cerevisiae was used to construct the complementation plasmid.The complementation plasmid was transformed into protoplasts of mutants.The complementary strains were obtained from medium supplemented with antibiotic Bleomycin and examined by PCR.2.The AaMR1 gene was involved in the regulation of spore formation,virulence and melanin synthesis of A.alternata tangerine pathotype.Deletion of AaMR1 gene resulted in the loss of colony and conidia melanin of of A.alternata tangerine pathotype,conidia became shorter,sporulation decreased significantly,and pathogenicity increased,but did not affect the growth of the strain,osmotic stress and cell wall function.Real-time quantitative PCR(qPCR)was used to analyze the expression of AaMR1 gene,melanin synthesis,and toxin synthesis-related genes of wild-type and mutants at different stages of infection with mandarin orange.The results showed that the mutant melanin synthesis gene expression was significantly down-regulated,the AaMR1 gene expression was down-regulated,and the toxin synthesis gene ACTTS3 was significantly up-regulated,indicating that the AaMR1 gene negatively regulates the pathogenicity of brown spot citrus.It is speculated that the up-regulation of ACTTS3 toxin gene expression may be one of the reasons leading to enhanced pathogenicity of AaMR1 gene deletion mutants.3.The AaSNF1 gene was involved in the growth,spore production,carbon source utilization,cell wall integrity,and pathogenicity of A.alternata tangerine pathotype.Deletion of AaSNF1 affects the mycelial growth,spore production,carbon source utilization,cell wall function,and pathogenicity of A.alternata tangerine pathotype.The mutants spores became shorter,the spore production decreased significantly,the spore germination rate was significantly slowed,and the growth rate on different media decreased.The growth rate was inhibited to varying degrees on MM media with different single carbon sources,especially on polygalacturonic acid,sucrose,and ethanol media.The mutants' pathogenicity was significantly reduced.4.The AaMR1 gene played an important role in regulating the growth and development,spore production,melanin synthesis,toxin production,and carbohydrate enzyme production of A.alternata tangerine pathotype.The transcriptional expression profiles of wild type(WT)strain Z7 and AaMR1 knockout mutant ?Aamr1-91 of citrus brown spot pathogen were obtained by transcriptome sequencing,and 19 differentially expressed genes(DEGs)were screened for qRT-PCR verification.Differential factors related to the pathogenicity of brown spot were obtained from the differential genes of WT strain Z7 in vegetative growth stage and plant infection stage.The results showed that MAPK signal,G protein signal and two-component signal transduction pathway were detected in the process of infecting plants,which regulated the reproductive development,stress resistance and other life processes of fungi.In the process of infection,there were a large number of genes up-regulated in the C metabolic pathway,among which hydrolase gene families that degraded plant cell wall were significantly differentially expressed.In addition,there were secondary metabolic gene clusters producing toxins,protective enzymes of antioxidant system,secretory proteins that produce effectors,transporters regulating material and energy balance and a large number of transcriptional factors are highly expressed in the infection process,indicating that brown spot infecting plants is a complex process dependent on a variety of pathways.A total of 370 DEGs were obtained by comparing the difference between transcription factor AaMR1 knockout and WT strains under vegetative growth conditions,among which 337 genes were up-regulated and 33 genes were down-regulated in?Aamr1-91.Gene Ontology(GO)functional enrichment analysis of DEGs showed that these DEGs were involved in corresponding stimulation,signal transduction,cell membrane,nucleic acid binding and antioxidant activity in vegetative growth stage.The expression of melanin synthesis related SCD1 gene was significantly down-regulated by AaMR1 mutation,which was consistent with the morphological decrease of melanin in the mutant strain.DEGs related to the growth,development and pathogenicity of mutant strain ?Aamr1-91 and WT strain Z7 were analyzed by KEGG enrichment analysis.The results showed that the up-regulated genes were enriched in MAPK signaling pathway,tryptophan metabolism,amino acid sugar and nucleic acid sugar metabolism.Among them,AaMR1 mutation resulted in up-regulation of gene expression in MAPK signaling pathway,and the expression of hydrolase gene in mutant strain was significantly higher than that in WT.Amr1 also negatively regulated the expression of some antioxidant genes.The effects of AaMR1 on the transcriptional regulation of citrus brown spot pathogen were analyzed by GO enrichment analysis by comparing the differences between WT and mutants in the process of infection.Here we mainly focuse on the transcriptional regulation of other transcription factors that interact with AaMR1 transcription factors.GO enrichment analysis showed that the transcription factors annotated by nucleic acid binding were down-regulated by AaMR1 deletion,so AaMR1 positively regulated the expression of transcription factors during plant inoculation.On the contrary,among the DEGs between mutants and WT at vegetative growth stage,except for the down-regulation of gene expression caused by AaMR1 self-mutation,the expression of other transcription factors in WT was higher than that in mutants.Therefore,AaMR1 negatively regulates the expression of transcription factors in vegetative growth stage,which indicates that the regulation mode of AaMR1 in vegetative growth and plant infection is opposite.Through phenotype,it can be seen that AaMR1 positively regulates melanin formation in vegetative growth stage.During the infection stage,the pathogenicity was negatively regulated,and it was speculated that the function of AaMR1 was related to the regulation of transcription factors.5.The up-regulated DEGs induced by A.alternata tangerine pathotype in tangerine were mainly enriched in biological stress-related gene categories such as pathogen recognition,signal transduction,active oxygen elimination,transcriptional regulation,secondary metabolic reactions,disease-related proteins,etc.The AaMR1 gene affected the defense mechanisms mediated by secondary metabolites,hormones,and resistance genes / proteins/ transcription factors in tangerine.The transcriptional group expression profiles of citrus susceptible materials before and after infection by WT strain Z7 and ?Aamr1-91 mutant strain were obtained by transcriptome sequencing,and 19 DEGs were screened for q RT(Reverse transcriptional)-PCR verification.The results showed that tangerine had strong response to WT and mutant of brown spot,and a large number of DEGs related to stress were produced after 28 hours of inoculation,which were analyzed by GO,KEGG enrichment and Map Man software.In the process of citrus brown spot stress,tangerine basic metabolism was seriously damaged,the process of C metabolism changed significantly,and photosynthesis was seriously blocked,resulting in insufficient material and energy supply.The hormone signal dominated by ET/JA(Ethylene/jasmine acid)was induced to activate downstream gene expression,and the changes related to the synthesis of secondary metabolites represented by terpenoids and polyphenols significantly produced antibacterial substances.A large number of transcription factors involved in stress resistance,such as WRKY,ERF,MYB and NAC,were activated by signal transduction,and PTI and ETI response genes such as receptor kinase and NBS resistance protein were also expressed under the regulation of transcription factors.At the same time,the expression of resistance gene PR and antioxidant protective enzyme system related gene POD were also induced by brown spot,which indicated that citrus brown spot infection had a great effect on the physiological state of tangerine plants.By comparing the difference between WT and?Aamr1-91 mutant strains in tangerine,it was found that the gene expression of JA synthesis pathway in ?Aamr1-91 infected plants was significantly lower than that in wild-type strains through the hormone signaling pathway annotated by Map Man.The results showed that the JA synthesis and signal transduction during the infection of mutant strains were weaker than those of WT infection,and the KEGG metabolic pathway showed that phenylpropanes were found in tangerine during mutant infection.Expression of some genes related to the synthesis of secondary metabolites such as flavonoids and anthocyanins were lower than those of wild strains,and ascorbic acid expression in the pathway of ascorbic acid metabolism was relatively lower than that of wild-type strain.Among the DEGs,24 transcription factors were also identified.These transcription factors were mainly down-regulated,and 16 receptor kinase proteins were also down-regulated.Due to the aggravation of tangerine infection by AaMR1 knockout,the disease of tangerine was more serious than that of WT infected.Transcriptional expression results showed that the expression of genes involved in hormone synthesis pathway,secondary metabolite synthesis pathway and important resistance related genes and proteins mediated by tangerine defense were affected to various degrees.Defense-related genes were mainly down-regulated.Therefore,compared with the tangerine resistance induced by WT infection,the mutant strain inhibited the expression of receptor kinase or JA signal and affected the downstream resistance gene expression,resulting in relatively weak tangerine resistance.The results were also consistent with the results that AaMR1 mainly negatively regulated the virulence of the strain.