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Sesame (Sesamum indicum L.; Pedaliaceae) is a diploid (2n = 26) dicotyledonous and one of the oldest oil seed crop grown widely in tropical and subtropical areas for its edible oil and food products. Beside of large land covered for cultivation of sesame there is a wide demand–supply gap as its production is constrained by various biotic and abiotic stresses which leads to less productivity. Biotic stresses such as diseases, insects, and pests affect sesame crops adversely causing substantial yield losses. Among the major diseases, charcoal rot caused by Macrophomina phaseolina affects severely at the all stages of the crop growth. In India, this disease incidence was recorded up to 50%. Keeping this constrain in mind present study was undertaken to understand the molecular responses of the host cell against a fungal infection and the putative role of regulatory players. In present study, two different genotypes GT-10 [resistant(R)] and RT-373 [susceptible(S)] were selected to perform transcriptome and four genotypes GT-10, Rama (resistant) and RT-373, AT-306 (susceptible) for biochemical characterization. Total 8 samples were collected at post flowering stage at four different post inoculation stages. Root samples of resistant and susceptible genotypes were collected at 0 hpi (hours post inoculation), 24 hpi, 48 hpi, 72 hpi. The mRNA was isolated from all the collected samples and was sequenced in IonS5 next generation sequencer. After completions of sequencing run the total raw sequence generated were assessed through FastQC quality control tool in which all 8 (eight) samples having good quality sequence were selected for further analysis. Trimming of raw reads yielded a total of 8443423, 8381018, 10143124, 11552102 reads in R1, R2, R3 and R4 respectively and 8713438, 8149237, 11265254, 9281491reads yielded in S1, S2, S3 and S4 respectively. This HQ data was mapped on M. phaseolina genome to remove possible contamination of fungus genome. Unmapped data was extracted and mapped on sesame genome which was downloaded from NCBI. Clear reads were used to carry out differential gene expression analysis. Differential expression analysis of both the genotypes yielded top 10 up and down regulated transcripts expressed at each PIS which were selected for the Gene Ontology enrichment analysis. GO terms like response to auxin (GO:0009733), integral component of membrane (GO:0016021), lipid metabolic process (GO:0006629) were found to be significantly over-represented in sequences having higher expression in GT-10. Moreover, glutathione transferase activity (GO:0004364), oxidoreductase activity (GO:0016491), alpha,alpha-trehalose-phosphate synthase (UDP-forming) activity (GO:0003825), oxidoreductase activity (GO:0016491), integral component of membrane (GO:0016021), beta-fructofuranosidase activity ii (GO:0004564), malate transport(GO:0015743) were found in up regulated sequence in GT-10 over the RT-373. In pathway enrichment analysis, common KEGG pathways like starch and sucrose metabolism and phenyl propanoid biosynthesis were enriched in up-regulated transcripts, however their fold expression was different in both the genotypes. Fatty acid degradation, Arachidonic acid metabolism, Glycerophospholipid metabolism, Tryptophan metabolism, Glyoxylate and dicarboxylate metabolism pathways were enriched in down regulated transcripts in susceptible genotype. Inositol phosphate metabolism, Ascorbate and aldarate metabolism and zeatin biosynthesis were enriched in up regulated transcripts of susceptible genotypes. The DEGs obtained from Deseq software were validated by qRT PCR. The expression patterns of the randomly selected DEGs in to RT-qPCR were in agreement with those obtained by the RNA-Seq, suggesting that the RNA-seq data reflected the real expression patterns of the sesame genes in the compatible interaction. The leaves and roots were analyzed at the interval of 24 hours up to 72 hours after pathogen inoculation. The β-1,3 glucanase and chitinase activities were increased up to 48 hpi thereafter decreased at 72 hpi. The activation of chitinase was more rapid and higher in plants of resistant genotypes than in susceptible genotypes. The results of phenol determination using LC-MS/MS reveals that in both the resistant genotypes, Rama and GT-10, vanillic acid, chlorogenic acid and salicylic acid were found to be higher in diseased condition which indicates these phenolics might play important role in defense mechanism. While in susceptible genotype (RT-373) syringic acid, catechin, coumaric acid, salicylic acid, ferulic acid and cinnamic acid were high in post inoculation stages. In AT-306 vanllic acid, catechin, gallic acid and cinnamic acid were increased during interaction with pathogen. In phytohormone profiling, the levels of IAA, Zeatin, ABA, ACC, JA and SA in the plant extracts were successfully quantified in MRM mode, but this was not possible for GA because of the low level of this phytohormone in plant tissues. At 48 hpi the level of IAA increased in resistant genotypes. The concentration of JA was found higher at 24 hpi in leaves and roots of both susceptible genotypes. No significant change in JA concentration was found in both resistant genotypes among all PIS. The level of SA increased at 48 hpi in all genotypes however its concentration varied among the all. The level of ACC increased in all genotypes after fungus inoculation in both the tissues. Zeatin slightly increased in leaves and root of GT-10 at 48 hpi. It drastically increased in root of Rama at 72 hpi.