Two Tales Of Alternaria Alternata:comparative Genomics And Function Of BZIP Transcription Factor

Alternaria brown spot(ABS)is an important citrus disease caused by Alternaria alternata tangerine pathotype,which pose a serious threat to young leaves,shoots and fruits of fruit trees.It has been reported that A.alternata tangerine pathotype can secrete a host selective toxins(HSTs),which kill host cell before invasion.In the meantime,there is a large number of endogenous A.alternata in citrus,which is not pathogenic to citrus leaves,and its biological significance is still unclear.In addition,methionine is essential for fungi to maintain normal life activities.S-adenosylmethionine,which is converted by methionine,is the main methyl donor in biomethylation,involving methyl transfer,transsulfurization and aminopropylation.In this paper,78 Alternaria strains isolated from citrus were genome-wide sequenced and compared with the genome of other Alternaria species.We identified all bZIP transcription factors in the genome of A.alternata Z7,knocked out 13 genes encoding bZIP transcription factor using PEG-mediated protoplast knockout technique and analyzed the phenotypes of bZIP gene disrupted mutants.We focused on the phenotype and transcriptome analysis of the bZIP22(AaMetR)mutant,predicting the regulation of the methionine metabolism pathway by AaMetR.Several target gene regulated by AaMetR was knocked out and verified to further elucidate the mechanism by which AaMetR regulates in the synthesis of methionine pathway.The research in this paper is mainly in the following aspects:1.Comparative genomic analysis of Alternaria spp.from citrusThe genome of Alternaria species isolated from citrus was 33036879 bp～34687595 bp,with 111～443 scaffolds,and 50.87%～51.31%GC content.The genome of Alternaria species isolated from citrus encodes 12159～12585 genes,130～142 tRNA genes,11～15 rRNA genes,and 25～37 secondary metabolite gene clusters.The secondary metabolite gene clusters mainly include non-ribosomal peptide synthase(nrps),polyketide synthase Ⅰ(tlpks),polyketide synthase Ⅱ(t2pks),polyketide synthase Ⅲ(t3pks),terpenes,lanthipeptide,fatty acid.The number of nrps and tlpks in pathogenic strains is significantly more than that of non-pathogenic strains,which may be related to the pathogenicity of Alternaria.Since pathogenic strains have a conditionally dispensable chromosome(CDC)carrying multiple copies of ACT toxin gene cluster,the number of coding genes and genome size is significantly more than that of non-pathogenic strain.The genome of Z7 was assembled to chromosome level,containing 10 essential chromosomes(ECs)and one conditionally dispensable chromosome(CDC).Multi-gene phylogenetic analysis and genome-wide comparison revealed that pathogenic strains isolated from citrus were clustered on three different clades,suggesting that Alternaria species causing citrus brown spot may be including at least three completely different Alternaria species:A.alternata,A.gaisen and A.tangelonis.In addition,most of A.alternata isolates isolated from citrus,regardless of multi-gene phylogenetic analysis and ANI,are similar to A.turkisafria BMP 3436 and A.turkisafria EGS 44-159,EGS 44-110,but are different from A.tangelonis EV-MIL-2S,A.tangelonis BMP 2327,A.citriarbusti BMP2343,suggesting that A.alternata and A.turkisafria may be synonymous.Analysis of carbohydrate enzymes(CAZymes)showed that Alteranariar spp.isolated from citrus contains 290～299 glycoside hydrolases(GHs),110～116 glycosyltransferases(GTs),24～26 polysaccharide lyases(PLs),168～181 carbohydrate esterases(CEs),152～160 coenzymes(AAs)and 67～76 carbohydrate-binding modular enzymes(CBM).Since the types and number of these carbohydrate enzymes are not significantly different between pathogenic strains and non-pathogenic strains of Alternaria species,indicating that carbohydrate enzymes are not associated with pathogenicity of A.alternata.2.Function analysis of bZIP transcription factorWe used bioinformatics methods to predict all bZIP transcription factors in the genome of A.alternata,knocking out 13 bZIP transcription factor encoding genes by protoplast transformation and gene homologous recombination,and studying its biological functions.bZIPJ5,bZIP16,bZIP18,bZIP20,bZIP23(AaAP1),bZIP25 and bZIP26 disrupted mutants grow normally on PDA,CM,V8 and MM media.The vegetative growth of the bZIP14 disrupted mutant on MM medium was significantly different from wild-type.The growth rate of bZIP17(AaAtf1)and bZIP24 disrupted mutant was slowely than wide-type.the aerial mycelia of bZIP19(AaHacl)disrupted mutant was significantly decreased PDA,CM,V8 and MM media.bZIP21(AaMetR)disrupted mutant was unable to grow on V8 and MM medium and showed pale colony on PDA medium without producing melanin.Pathogenicity test showed that the virulence of AaAP1 disrupted mutant decreased,while the pathogenicity of AaMetR disrupted mutant completely lost.The light microscopical observation showed that AaMetR disrupted mutant was unable to produce conidia on PDA medium.Real-time PCR analysis showed that the expression of AaMetR gene was significantly up-regulated under oxidative stress and metal ion stress.All phenotypes of AaMetR disrupted mutants could be restored to wild-type on PDA,V8 and MM medium supplemented with exogenous methionine,indicating AaMetR disrupted mutant is a typical methionine auxotrophic type.The oxidative stress sensitivity test revealed that the AaAPl and AaMetR disrupted mutants were extremely sensitive to oxidative stress,and could not grow on PDA plates containing slight of hydrogen peroxide.These results indicate that bZIP14,AaAtfl,AalHacl,bZIP22 and bZIP24 play an important role in the growth of A.alternata.AaMetR plays a key role in the vegetative growth,pathogenicity,sporulation,melanin formation,antioxidant stress and aerial hyphal formation of A.alternata.3.AaMetR affects several genes in the methionine metabolic pathwayThe transcriptome sequencing results of wild-type and AaMetR disrupted mutant with thresholds of log2ratio≥1.5 and FDR:≤0.001 showed that the number of differentially expressed genes(DEGs)was up to 2042,of which 956 genes were up-regulated and 1086 genes were down-regulated in the AaMetR disrupted mutant.The DEGs were significantly enriched in 29 GO categories,such as catalytic activity,binding,structural molecule activity and transporter activity.KEGG pathway enrichment analysis showed that a total of 798 DEGs were mapped to the KEGG pathway,mainly in aminoacyl-tRNA biosynthesis,cysteine and methionine metabolism,proteasome,valine,leucine and isoleucine biosynthesis.Transcriptome data showed that 13 DEGs were mapped in the cysteine and methionine metabolic pathways.Interestingly,12 of the DEGs were up-regulated in the AaMetR disrupted mutant,indicating that AaMetR acts as a negative regulator in the methionine synthesis pathway.Five genes predicted by the transcriptome,including AaMetB,AaMetC,AaMetE,AaMsrC,and AaMetX knocked out to further investigate the mechanism regulated by AaMetR.Morphological analysis on PDA,V8,MM,CM media showed that the phenotype of AaMetE,AaMsrC disrupted mutants are similar to wild-type,and the AaMetB,AaMetC and AaMetX disrupted mutants showed methionine auxotrophy in PDA medium.AaMetB,AaMetC and AaMetX disrupted mutants grew poorly on V8 medium was poor,and were unalbe to grow on the MM medium.The mutant phenotype could be completely restored by exogenous methionine,and partially restored by exogenous homocysteine.Combined with KEGG PATHWAY analysis,we found that AaMetC is a crucial gene in the fungal methionine biosynthesis pathway A,AaMetC catalyzes the synthesis of homocysteine by cystathionine;AaMetB and AaMetX are crucial genes in the methionine biosynthesis pathway B,AaMetB catalyzing the synthesis of homocysteine or cystathionine by O-acetyl homoserine,AaMetX catalyzing the synthesis of O-acetyl homoserine by homocysteine.Oxidative stress sensitivity test showed that AaMetB,AaMetC and AaMetX mutants were not sensitive to oxidative stress.These results indicate that MetR acts as a crucial regulator that regulates the methionine synthesis pathway A and the methionine synthesis B and may have other unknown regulatory mechanisms to regulate the stress response to hydrogen peroxide.