In order to identify Nicotiana megalosiphon genes for novel inhibitors of microbial serine proteinase involved in resistance to the oomycete Phytophthora parasitica var. nicotianae, SuperSAGE technology combined with next-generation sequencing were used to generate libraries in order to identify transcripts that are differentially up-regulated. We identified a N. megalosiphon inhibitor of microbial serine proteinase gene (NmIMSP) rapidly induced during the interaction. Silencing of NmIMSP gene was found to compromise the resistance of N. megalosiphon to P. parasitica var. nicotianae. Herewith, the transient expression of NmIMSP gene in a susceptible genotype conferred high level of resistance to P. parasitica var. nicotianae and Peronospora hyoscyami f. sp. tabacina. This is the first evidence of this inhibitor of microbial serine proteinase as a positive regulator of defense response against oomycete. (C) 2013 Elsevier Ltd. All rights reserved.