关键词: 转录组, 代谢组, 玉米茎腐病, 层出镰孢, 苯丙氨酸代谢

Abstract:

【Objective】Stalk rot is a prevalent disease of maize (Zea mays) that severely affects maize yield and quality worldwide. The diseases are caused by several types of fungi and bacteria that are part of the complex of microorganisms. Among which, the ascomycete fungus Fusarium proliferatum has become one of the most aggressive causal agents of maize diseases in China in recent years. The study aims to provide a comprehensive analysis of multi-omics of maize stalks following F. proliferatum inoculation and valuable insights into the molecular basis of the response, including functional annotation and enrichment analyses of the major pathways enriched for differentially expressed genes (DEGs) and differentially expressed metabolites (DEMs) of maize conditioning F. proliferatum infection, and to lay theoretical foundation for maize breeding and disease management.【Method】Maize inbred lines ZC17 (ZhengC17, resistant) and CH72 (Chang72, susceptible), with different levels of resistance to F. proliferatum were used for sample collection. Seedlings at the nine-leaf stage with uniform performance were selected for artificial inoculation. Maize plants were inoculated by punching a hole in the stem at the second or third internode above the soil line using a sterile micropipette tip, followed by injection of 50 μL of freshly prepared F. proliferatum inoculum. A similar number of plants were inoculated with PDB as a mock treatment. The wounds were sealed with vaseline after inoculation. The upper and lower stem segments immediately adjacent to the inoculation segments were sampled at 7 dpi (days post inoculation), all individual samples were used for further transcriptome and nontargeted-metabolomics analysis. The inoculated internodes of the individual plants were split and symptoms were observed for evaluating of SRS_A (stalk rot score on average) and DSI (disease severity index). Multiple bioinformatics tools were used to conduct in-depth analysis of the transcriptome sequencing data and metabolomics data, and the DEGs were verified by real-time fluorescence quantitative PCR (RT-qPCR).【Result】Phenotypic and physiological characteristics indicated that the inoculated group of samples from the resistant inbred line ZC17 showed significantly lower lesion areas and symptoms than those of the sensitive inbred line CH72 after inoculation with F. proliferatum. The SRS_A and DSI of the ZC17 and CH72 stalks were consistent with the symptom phenotypes: the susceptible inbred CH72 had approximately 2.48-fold more SRSAs than the resistant inbred ZC17 line, and its DSI increased by 35.36% compared to that of ZC17. The results of the principal component analysis (PCA) showed a high reproducibility of the samples within the group. In PC1, ZC17 and CH72 were separated from each other. In PC2, FP group and MK group were separated from each other. The DEGs in the two comparison pairs (ZC17_FP vs. ZC17_MK, CH72_FP vs. CH72_MK) were analyzed. More DEGs were found in CH72 than those of ZC17 post inoculation, whereas nearly 50% of the DEGs share the same trend of expression between the two comparison pairs. Functional annotation and enrichment analysis found that DEGs and DEMs were enriched in pathways such as biosynthesis of plant secondary metabolites, phenylalanine metabolism, biosynthesis of plant hormones, and plant-pathogen interactions. Integrated analysis of transcriptomics and metabolomics data revealed that phenylalanine, tyrosine, and tryptophan biosynthesis and phenylalanine metabolism were significantly enriched in both the transcriptomic and metabolomic data for CH72 and ZC17. This result suggested that these pathways play a key role in the maize response to F. proliferatum. In addition, transcriptional factors of bHLH, MYB, NAC, and WRKY families were significantly activated after fungal inoculation, demonstrating the important role of transcription factors in the maize response to F. proliferatum infestation. To further confirm the reliability of the sequencing data, 11 genes were randomly selected for qPCR validation, which showed that the trends of the RNA-Seq and qPCR results were consistent in both CH72 and ZC17, Spearman rank correlation analysis also showed high concordance between the RNA-Seq and qPCR results (r=0.75, P=7.5e-05).【Conclusion】Phenylalanine metabolism-related pathways are crucial in maize response to F. proliferatum stalk rot. Key enzymes such as C4H, PAL, ADT, GOT and significantly up-regulated metabolites such as 2-coumaric acid, 3-hydroxycinnamic acid, indole, phenylalanine, tryptophan and tyrosine play an important role in plant disease resistance. The potential disease resistance-related transcription factors, genes and metabolites excavated in this study can provide an important basis for further analysis of the molecular response mechanism of maize to F. proliferatum stalk rot.

Key words: transcriptome, metabolome, maize stalk rot, Fusarium proliferatum, phenylalanine metabolism