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.

The characterization and compare expression profiling of the miRNA transcriptome lay a solid foundation for unraveling the complex miRNA-mediated regulatory network in tomato resistance mechanisms against LB. MicroRNAs (miRNAs) are a class of small endogenous non-coding RNAs with 20-24 nt. They have been identified in many plants with their diverse regulatory roles in biotic stresses. The knowledge, that miRNAs regulate late blight (LB), caused by Phytophthora infestans, is rather limited. In this study, we used miRNA-Seq to investigate the miRNA expression difference between the tomatoes treated with and without P. infestans. A total of 42,714,516 raw reads were generated from two small RNA libraries by high-throughput sequencing. Finally, 207 known miRNAs and 67 new miRNAs were obtained. The differential expression profile of miRNAs in tomato was further analyzed with twofold change (P value ≤0.01). A total of 70 miRNAs were manifested to change significantly in samples treated with P. infestans, including 50 down-regulated miRNAs and 20 up-regulated miRNAs. Moreover, a total of 73 target genes were acquired for 28 differentially expressed miRNAs by psRNATarget analysis. By enrichment pathway analysis of target genes, plant-pathogen interaction was the most highly relevant pathway which played an important role in disease defense. In addition, 30 miRNAs were selected for qRT-PCR to validate their expression patterns. The expression patterns for targets of miR6027, miR5300, miR476b, miR159a, miR164a and miRn13 were selectively examined, and the results showed that there was a negative correlation on the expression patterns between miRNAs and their targets. The targets have previously been reported to be related with plant immune and involved in plant-pathogen interaction pathway in this study, suggesting these miRNAs might act as regulators in process of tomato resistance against P. infestans. These discoveries will provide us useful information to explain tomato resistance mechanisms against LB.

Being one kind of approximately 22nt long small RNA, miRNA has shown its roles in host-pathogen interaction, providing one possible way for pathogen infection. Though Phytophthora infestans is a major pathogen that causes devastating late blight of potato, tomato and so on, so far there have not been any systematic researches on miRNAs and even pathogenic miRNAs in P. infestans. Here, for the first time we comprehensively predicted and identified pathogenic miRNAs that may exist in P. infestans. First, a total of 128 putative miRNAs belonging to 66 miRNA family were identified by bioinformatic approaches. Then, 33 vital pathogenic miRNAs were screened by constructing miRNA-miRNA relationship networks. Finally, four potential pathogenic miRNAs were chosen for detection, two of which are chosen for validation. The expression quantity of pi-miR466 and pi-miR1918 changed dramatically during incubation of tomato leaves, implying that they are potential pathogenic miRNAs.

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