AbstractTobacco brown spot caused by Alternaria fungal species is one of the most damaging diseases, and results in significant yield losses. However, little is known about the systematic response of tobacco to this fungal infection. To fill this knowledge gap, de novo assemblies of tobacco leaf transcriptomes were obtained in cultivars V2 and NC89 after the inoculation of either Alternaria longipes (AL) or Alternaria alternata (AA) at three different time points. We studied the gene expression profile of each cultivar-pathogen combination, and identified eight differentially expressed genes shared among all combinations. Gene ontology enrichment analysis of the differentially expressed genes revealed key components during the fungal infection, which included regulation of gene expression (GO:0010468), regulation of RNA metabolic process (GO:0051252), tetrapyrrole binding (GO:0046906), and external encapsulating structure (GO:0030312). Further analyses of the continuously upregulated/downregulated genes and the resistance genes demonstrated that the gene expression profile upon fungal infection was contingent on the specific cultivar and pathogen. In conclusion, this study provides a solid foundation for the investigation of plant-pathogen interaction, and is of great importance for disease prevention and molecular breeding.