Trascriptomic And Metabolic Profiling Of Lilium Infected With Botrytis Elliptica And Screening Of Key Genes For Resistant Diseases

Lilium is one of the most economically important genera of ornamental monocotyledons and is used worldwide as cut flowers,garden and potted plants.However,the ornamental value and yield of commercial lily are often limited by Botrytis cinerea.Lily is very sensitive to grey mold,which is exacerbated by high humidity and low temperatures.Screening and breeding of lily resistant varieties is the most economical and effective way to control gray mold.Therefore,revealing the defense mechanism of lily against this fungal pathogen will help to accelerate the selection process of lily resistance traits.The molecular and metabolic regulatory mechanisms of lily's defense response to grey mold infection have not been fully elucidated.In this study,transcriptome and metabolomics methods were used to determine the transcriptional and metabolic changes of Oriental Lily hybrid Sorbonne in response to the infection of B.elliptica.The time points of transcription and metabolome sampling and sequencing were 6h,24 h and 48 h after inoculation of Botrytis elliptica,which were in the early,middle and late stages of lily response to B.elliptica,respectively.Three biological replicates were taken from the control and treatment groups at each time point.The sequencing samples were numbered as CK?6 h,AI?6 h,CK?24 h,AI?24 h,CK?48 h,AI?48 h.Transcriptome sequencing resultsThe transcriptome results showed that the average sequencing amount of each sample of 18 RNA samples after the removal of low-quality sequencing data was 24.18G,the total sequencing amount was 435.16 G,and the error rate was 0.02%(97.4%for Q20,92.79%for Q30,and 49.87%for GC).A total of 430,835 transcripts were obtained after sequencing and splicing,with an average length of 701 bp,N50 length of 1213 bp and N90 length of 266 bp.The longest transcription in Trinity was named Unigenes,with 283,213 Unigenes and an average length of 534 bp,707 bp for N50 and 242 bp for N90.The results of NR database comparison showed that Asparagus(Asparagus officinalis 8.94%),oil palm(Elaeis Guineensis 8.72%),jujube(Phoenix Dactylifera7.39%),The grape(Vitis vinifera 3.63%)and the Cajanus cajan 3.45%were among the top five species in similarity to the lily.A total of 5295 differentially expressed genes were screened at three stages.At 6 h after infection,a total of 401 differentially expressed genes were detected,including 86up-regulated genes and 315 down-regulated genes.24 after infection,a total of 1887genes were differentially expressed,of which 1578 were up-regulated and 309 were down-regulated.Forty-eight hours after infection,a total of 4726 genes were differentially expressed,including 2772 up-regulated genes and 1954 down-regulated genes.KEGG enrichment analysis shows that the biosynthesis of secondary metabolites,flavonoids biosynthesis,biosynthesis of benzene propane class","plant hormone signal transduction","plant and pathogen interaction","MAPK signal pathway"pathways at the transcriptional level,such as grape lily of ellipse is a spore infect main reaction pathway.A total of 281 differential transcription factors were identified,among which the top three families were WRKY,TIFY and AP2/ERF,with 24,22 and 21 genes,respectively.WGCNA analysisWGCNA analysis identified key genes that played key roles in different stages of infection,revealing that JA(jasmonic acid),SA(salicylic acid),BR(brassinolactone)and Ca²⁺were key pathways in response to gray mold infection of Lily Sorbonne.The key genes in the early stage of infection were MYB30,AS1 and 2 LURP genes.PBL19,CRK2 and WAK genes were identified in the middle stage of infection.The key genes in the late stage of infection were WRKY22,WRKY30,three JAZ,DELLA,MAPK17/18and CML27.A total of 41 key genes were found to be up-regulated,including MYB61,WRKY75,MYC2,BRI1,2 PAL,HCT,2 CHS,F3'h,FLS,3 FLS2,RIN4,Pti5 and 7 PR proteins.Metabolome sequencing resultsThe metabolome results showed that a total of 524 kinds of metabolites were detected,and 115 kinds of differential accumulated metabolites.Phenolic acids,flavonoids,alkaloids,amino acids and their derivatives,and lipids were the main metabolites in all three stages of infection.The metabolites with the largest difference ratio were benzoyl salicylate,saint heliosol,tryptamine,3-(4-hydroxyphenyl)propionic acid,kaempferol,cryptochlorogenic acid,N-p-coumaryl sapropylamine,N'-ferulic sapropylamine,p-hydroxybenzoyl tartaric acid,syringa resin phenol-hexose,p-coumaryl malic acid,etc.In addition kaempferia galanga phenol-3-O-beta-D-glucose base(1->2)(6'-O-acetyl)-beta-D-galactose glucoside,mallow pigment3-O-(6'-acetyl glycosidase)-5-glucoside,nucleoside hesperidin-O-glycosidase cumulative differences,too.Venn diagram showed that there were 8 metabolites which were different in all three stages,namely,benzyl salicylate,saint heliosol,tryptamine,cetin,rivet,caffeic acid,dihydrokaempferol and N'-ferulic acid putrescine.Five of them belong to phenolic acid metabolic pathways,which are important secondary metabolites of plant resistance to pathogens.The pathways of phenylpropanoid biosynthesis,flavonoid biosynthesis and tryptophan metabolism were the main pathways of KEGG enrichment in Sorbonne leaves during all stages of Sorbonne leaf infection.Combined transcriptional and metabolome analysisCombined analysis of transcriptome and metabolome suggests that the biosynthetic pathways of phenylpropanoid and flavonoids in lily defense responses can be integrated into a transcriptional cascade and metabolic network,which together play a key role in the response of Lily Sorbonne to Botrytis elliptica.We mapped into this network all the differential genes and metabolites involved in the phenylpropanoid and flavonoid biosynthetic pathways,which were up-regulated or down-regulated at different stages of infection.Among the metabolites,phenylalanine,phenylethylamine,caffeic acid,cetin,chaletonin,naringin,cryptochlorogenic acid,saint heliosol,dihydrokaempferol,kaempferol and its derivatives,rivitin,coniferol,erucinaldehyde,erucinic acid and other flavonoids and phenylpropanoid-pathway metabolites were accumulated and activated.The expression levels of PAL,C4H,4CL,CCR,HCT,F3'h,C3'h,CSE,CAD,F5H,CHS,CHI,F3H,FLS,DFR and PGT1 genes were significantly different,among which PAL,HCT and CHS might play a more critical role.In vitro bacteriostatic experiments were conducted on 8 metabolites,and the results showed that except for 2 amino acids,the other 6 metabolites had strong bacteriostatic effects on 3 fungi taxa.The difference of bacteriostatic effects of 6 metabolites suggested that caffeic acid,kaneferol and tryptamine might be broad spectrum bacteriostatic metabolites.Naringin,Benson salicylate and coumarin may be the specific metabolites of Lily in response to B.elliptica.,which have strong pertinence.Cloning and bioinformatics analysis of WRKY family genesFive WRKY family genes were obtained by screening transcriptional database,among which Ls WRKY75 was the key gene analyzed by WGCNA.Gene cloning,protein sequence analysis,multi-gene alignment and phylogenetic tree analysis were carried out.The length of CDS region of Ls WRKY28 was 990bp;Ls WRKY31 is 1755bp in length.Ls WRKY40 is 963bp in length.Ls WRKY70 is 891bp in length.Ls WRKY75 is537bp in length.They were named Ls WRKY28,Ls WRKY31,Ls WRKY40,Ls WRKY70and Ls WRKY75,respectively,according to phylogenetic tree and multi-sequence alignment results.Ls WRKY70 is a C2HC WRKY family transcription factor,and the other four WRKY genes are ALL C2H2 WRKY family transcription factors.Ls WRKY70is a member of the second?family;Ls WRKY31 is a member of the?B subfamily.Ls WRKY40 is a member of the?A subfamily.Ls WRKY75 and Ls WRKY28are members of the?C subfamily.Expression pattern analysis of WRKY family genesOn this basis,the expression patterns of these five WRKY family genes were analyzed.The tissue specificity of Ls WRKY28 gene indicated that the expression level of Ls WRKY28 gene was the highest in roots and anthers of vegetative organs.The expression of Ls WRKY31 gene was highest in anther,but low in other organs.Ls WRKY40 gene was highly expressed in roots,stems,leaves and anthers,but almost not in other parts.Ls WRKY70 gene was expressed in almost all organs,with higher expression in stems and filaments and lower expression in anthers and petals.The expression of Ls WRKY75 gene was highest in roots,stems,leaves and anthers.The expression patterns of these five genes were different in the lily leaves infected by B.elliptica and Fusarium oxysporum.Ls WRKY28 gene was not only differentially expressed in the lily leaves infected by B.elliptica,but also could respond to Fusarium oxysporum infection,which might be a broad-spectrum response gene.The expression of Ls WRKY31 and Ls WRKY40 was significantly increased in the infection of B.elliptica,but not in fusarium oxysporum,which might be specific response to B.elliptica gene.The expressions of Ls WRKY70 and Ls WRKY75 were increased when induced by B.elliptica and decreased when induced by Fusarium oxysporum,which reflected the different functions and expression patterns of Ls WRKY70 and Ls WRKY75 in resisting the invasion of different pathogens,which could provide reference for further exploration of its functions.The expression patterns of these five genes were different after SA,JA and ET treatment.Ls WRKY28 gene is mainly regulated by jasmonic acid and ethylene,and responds to jasmonic acid more timely and lasts longer.Ls WRKY31 was affected by three hormones,but was more affected by jasmonic acid activation and ethylene inhibition.Ls WRKY40 was mainly induced by jasmonic acid and ethylene,but not by salicylic acid.Ls WRKY70 gene is activated by three hormone pathways,which may be more affected by salicylic acid.Ls WRKY75 gene is inhibited by salicylic acid and activated by jasmonic acid and ethylene induction,which may be more affected by jasmonic acid pathway.Transgenic overexpression of WRKY75 improved the resistance of tobacco to gray moldThe expression level and phenotype of Ls WRKY75 gene in transgenic tobacco showed that Ls WRK75-9 had the highest expression level,the most obvious resistance phenotype,and the largest difference between the diameter of disease spot and wild type,indicating that Ls WRKY75 overexpressed improved the resistance of tobacco,and Ls WRKY75 was a gene that positively regulated the resistance of lily to gray mold.This study provides a comprehensive perspective on the defense response and potential transcriptional regulatory network of Lily to B.elliptica,and thus provides candidate genes and metabolites for resistance breeding and metabolic engineering of Lily.