ABSTRACTAmerican elm (Ulmus americana), highly prized for its ornamental value, has suffered two successive outbreaks of Dutch elm disease (DED) caused by ascomycete fungi belonging to the genus Ophiostoma. To identify the genes linked to the pathogenicity of different species and lineages of Ophiostoma, we inoculated 2-year-old U. americana saplings with six strains representing three species of DED fungi, and one strain of the saprotroph Ophiostoma quercus. Differential expression analyses were performed following RNA sequencing of fungal transcripts recovered at 3- and 10-days post-infection. Based on a total of 8,640 Ophiostoma genes, we observed a difference in fungal gene expression depending on the strain inoculated and the time of incubation in host tissue. Some genes overexpressed in the more virulent strains of Ophiostoma encode hydrolases that possibly act synergistically. A mutant of Ophiostoma novo-ulmi in which the gene encoding the ogf1 transcription factor had been deleted did not produce transcripts for the gene encoding the hydrophobin cerato-ulmin and was less virulent. Weighted gene correlation network analyses identified several candidate pathogenicity genes distributed among 13 modules of interconnected genes.IMPORTANCEOphiostoma is a genus of cosmopolitan fungi that belongs to the family Ophiostomataceae and includes the pathogens responsible for two devastating pandemics of Dutch elm disease (DED). As the mechanisms of action of DED agents remain unclear, we carried out the first comparative transcriptomic study including representative strains of the three Ophiostoma species causing DED, along with the phylogenetically close saprotrophic species Ophiostoma quercus. Statistical analyses of the fungal transcriptomes recovered at 3 and 10 days following infection of Ulmus americana saplings highlighted several candidate genes associated with virulence and host-pathogen interactions wherein each strain showed a distinct transcriptome. The results of this research underscore the importance of ...