Sp-miR396a-5p acts as a stress-responsive genes regulator by conferring tolerance to abiotic stresses and susceptibility to Phytophthora nicotianae infection in transgenic tobacco.

Overexpression of Sp-miR396a-5p in tobacco increased tolerance to salt, drought, cold stress and susceptibility to Phytophthora nicotianae infection. MicroRNA396 (miR396) is one of the conserved microRNA families in plants, and it targeted growth-regulating factors (GRFs) family. The GRF transcription factors are associated with growth and stress responses. However, the molecular mechanisms of miR396 responding to environmental stresses are elusive. The purpose of this study was to explore the function of tomato miR396a-5p (Sp-miR396a-5p) in Solanaceae responses to abiotic and biotic stresses. We showed that Sp-miR396a-5p transcript levels were up-regulated under salt and drought stresses and down-regulated after Phytophthora infestans (P. infestans) infection. Consistently, overexpression of Sp-miR396a-5p in tobacco enhanced its tolerance to salt, drought and cold stresses. Additionally, the expression of Sp-miR396a-5p was found to be down-regulated under pathogen-related biotic stress. Tobacco plants overexpressing Sp-miR396a-5p showed increased susceptibility to Phytophthora nicotianae (P. nicotianae) infection. Physiological analysis indicated that Sp-miR396a-5p overexpression enhanced osmoregulation and decreased production of reactive oxygen species (ROS). Furthermore, four Sp-miR396a-5p target genes, NtGRF1, NtGRF3, NtGRF7 and NtGRF8, were down-regulated in these plants. Our results suggested that Sp-miR396a-5p plays critical roles in both abiotic stresses through targeting NtGRF7-regulated expression of osmotic stress-responsive genes and pathogen infection via the regulatory networks of NtGRF1 and NtGRF3.