Identification and Validation of Mirnas and their Targets that Regulate the Resistance Genes against Fusarium Wilt in Tomato

MicroRNAs (miRNAs) are a specialized group of small RNAs (sRNAs) that regulate gene expression in plants at both transcriptional and post-transcriptional levels. Numerous families of miRNA target genes are involved in regulating plant immunity. In this study, we studied the role of miRNAs in the defensive response against a fungal pathogen, Fusarium oxysporum f. sp. lycopersici, which causes wilt disease in tomatoes. Furthermore, the expression patterns of two novel miRNAs and their targets were validated by qRT-PCR. Moreover, two new miRNAs (miR30 and miR33) were further sequenced by Applied Biosystems, using gene-specific primers. The results showed that four miRNAs, two novel (miR30 and miR33), and two known miRNAs (miR46 and miR49) and their target genes were differentially expressed during the infection with the pathogen. On the other hand, two targets (P4) and (β-1,3-glucanase) showed an inverse correlation in expression with their corresponding (miR46), and (miR33, and miR49), respectively. Our results showed that tomato cv. Pusa Early Dwarf is moderately susceptible to the fungus because its resistance is not well-expressed enough to be attributed to miRNAs. Sequences analysis showed that miR30 and miR33 are highly conserved and are found in different plant species. We predicted the secondary structures of miR30 and miR33 by minimum free energy (MFE). The total free energy of miRNA30 and miR33 was -1.2 and -0.4 kcal/mol respectively, predicted by the Vienna RNA package program V.1.7. The result of this study could improve our comprehension of the role that miRNAs play in tomato resistance to F. oxysporum f. sp. lycopersici. In addition, it will provide novel gene sources to develop resistant breeds.