Combined Transcriptome And Metabolome Analysis Initially Reveals The Molecular Mechanism Of Powdery Mildew Resistance In Tomato

Tomato powdery mildew（PM）is a fungal disease caused mainly by the Oidium neolycopersici（O.neolycopersici）,which seriously affects its production and yield.Mining for tomato PM resistance genes and breeding tomato PM resistant varieties are the most economical and effective control measures.At present,research on tomato PM resistance in China is still relatively weak,and there are few reports on the use of combined transcriptome and metabolome analysis to find candidate genes,metabolites and metabolic pathways for tomato PM resistance.In response to this situation,the PM-resistant material,’63187’,and the high-susceptible tomato material,’Moneymaker（MM）’,were used as experimental materials for the combined analysis of transcriptome and widely targeted metabolome on tomato leaves at 0 hour post inoculation（hpi）,12 hpi,and 48 hpi.The main results of the study were as follows:1.After screening 18 tomato germplasm resources with varying resistance in the field,an indoor precision evaluation was conducted during the seedling stage to determine resistance to PM.Combining the field preliminary screening and the indoor precision evaluation at the seedling stage,the phenotypes of the high-susceptible material ’MM’ and the resistant material ’63187’ were observed after 25 days of inoculation with O.neolycopersici.It was found that ’63187’ was more resistant to PM,,and its growth was better than ’MM’.The white spore spots on the surface of the ’MM’ leaf almost covered the entire leaf,while the’63187’ only had sporadic white spore spots.The above two materials were used as test materials for transcriptome and metabolome determination and analysis.2.The transcriptome of different resistant tomato leaves inoculated with O.neolycopersici was determined and analyzed.A total of 9855 differentially expressed genes（DEGs）were identified.The 276 genes were common in the four comparison groups,0 hpi of the disease-resistant materials_vs₁2 hpi of disease-resistant materials（R0_vs_R12）,0 hpi of the disease-resistant materials_vs₄8 hpi of disease-resistant materials（R0_vs_R48）,0 hpi of the high-susceptible materials_vs₁2 hpi of high-susceptible materials（S0_vs_S12）,and 0 hpi of the high-susceptible materials_vs₄8 hpi of high-susceptible materials（S0_vs_S48）.The selected 790,266,1364,and 739 DEGs were unique to the 4 comparison groups.GO classification and GO enrichment analysis of common and unique DEGs showed no significant difference in the number of GO terms,but some differences in the enriched terms.The results of the KEGG enrichment analysis showed that the common DEGs were significantly enriched in pathways such as photosynthetic-antennal proteins,amino acid biosynthesis and metabolic pathways,while the specific DEGs were significantly enriched in different pathways.A total of 276 DEGs were analyzed by K-means clustering analysis,and it was found that they were divided into eight classes in ’63187’ and ten classes in ’MM’.A total of 2031 new genes were identified,including 802 differentially expressed new genes,with significantly enriched metabolic pathways beta-Alanine metabolism,valine,leucine and isoleucine degradation,and carbon metabolism,et al.Further analysis of the WRKY family genes revealed that the expression of four transcription factors increased in ’63187’after inoculation with O.neolycopersici,without significant changes in ’MM’,while the expression of one transcription factor increased in ’MM’,without significant changes in ’63187’.Through WGCNA analysis,19401 genes were classified into 15 modules,and based on KEGG enrichment analysis,the pathways significantly enriched in the cyan and greenyellow modules were plant-pathogen interactions.The reliability of transcriptome data was verified by qRT-PCR.3.The widely targeted metabolomics of different resistant tomato leaves inoculated with O.neolycopersici was determined and analyzed.A total of 1058 metabolites were detected,which were divided into 10 categories,and 46 differentially accumulated metabolites（DAMs）were common.The 27,84,52,and 76 DAMs identified were unique to the four comparison groups,R0_vs_R12,R0_vs_R48,S0_vs_S12,and S0_vs_S48,respectively.The KEGG enrichment analysis of common and unique DAMs in four comparative groups showed that the pathways significantly enriched in common DAMs were the pentose phosphate pathway and alpha-linolenic acid metabolism.The pathways significantly enriched for unique DAMs varied.Screening the top 10 metabolites with the highest absolute values of log2Fold Change in each comparative group revealed that the accumulation of terpenoids and flavonoids may have a positive response to tomato inoculation with O.neolycopersici.K-means clustering analysis showed that 46 common DAMs were divided into nine classes in ’63187’and’MM’.4.Combined analysis of transcriptome and extensively targeted metabolome data of different resistant tomato leaves inoculated with O.neolycopersici revealed that candidate genes were negatively correlated with lysoPE16:3 and positively correlated with other candidate metabolites.Phenylpropanoid biosynthesis and flavonoid biosynthesis pathways were both enriched in ’63187’ and ’MM’.In ’63187’,six metabolites involved in this pathway were downregulated,and four genes were highly expressed.While in ’MM’,three metabolites were upregulated,and four metabolites were downregulated,and ten genes with high expression.These metabolites and genes above might be candidates for PM resistance or susceptibility for subsequent studies.The biosynthesis of unsaturated fatty acids was a pathway in which the specific DEGs and DAMs of PM resistant tomato leaves were significantly enriched at 48 hpi.There were two DEGs and four DAMs involved in this pathway.This study aims to identify candidate genes,metabolites,and metabolic pathways related to PM in tomatoes through the mining of DEGs,DAMs,and regulatory metabolic pathways,initially analyze the molecular mechanisms of tomato resistance to and susceptibility to PM,providing a theoretical basis for cultivating tomato varieties resistant to PM.