Functional Identification And Application Of MicroRNAs In Tomato

MicroRNA is an important regulator of plant development and plant-enviroment interaction. MicroRNA can be modified to target new sites, termed artificial microRNA. Artificial microRNA is an important tool in genetic study and plant biotechnology.Expression of artificial microRNAs (amiRNAs) can confer virus resistance in plants. CMV is one of most infective viruses and causes huge lost in tomato industry. We made two amiRNA constructs; one targeting the coding sequence shared by the 2a and 2b RNA and the other one targeting the highly conserved 3’untranslated region (UTR) of Cucumber mosaic virus (CMV). The transgenic tomato plants displayed effective resistance to CMV infection. The resistance is stable even facing the mixed infection of a nontarget virus, Tobacco mosaic virus (TMV) or Tomato yellow leaf curl virus (TYLCV). The grafting assays indicate that the amiRNAs can not transmit through the vascular system over long distances and act in a cell-autonomous manner. When grafted onto a CMV-infected rootstock, the transgenic scions stay intact for a long time under a constantly high concentration of virus invasion from the rootstock. This indicates that amiRNAs can protect the plant under high viral pressure. Northern blotting showed amiR-2a/b transgenic tomato lines accumulated less CMV RNAs compared to that expressing amiR-3’UTR. This suggests that amiRNAs targeting the coding region can induce a more effective RNA degradation than targeting of the untranslated region. Our work gives a good sample of how to use amiRNAs as an effective approach to engineering viral resistance in the tomato and possibly in other crops, and also adds some new knowledge to amiRNA-induced antiviral resistance and plant-virus interaction in miRNA level.Plant miRNA regulates multiple developmental and physiological processes, including drought responses. Drought is one of the most important factors restricting agricultural development and food security. We found that the accumulation of Sly-miR169 in tomato (Solanum lycopersicum) w as induced by drought stress. Consequently, Sly-miR169 targets, namely, three nuclear factor Y subunit genes (S1NF-YA1/2/3) and one multidrug resistance-associated protein gene (S1MRP1), were significantly down-regulated by drought stress. Constitutive over-expression of a miR169 family member, Sly-miR169c, in tomato plant can efficiently down-regulate the transcripts of the target genes in leaf and stem tissues. Compared with non-transgenic plants, transgenic plants over-expressing Sly-miR169c displayed reduced stomatal opening, decreased transpiration rate, lowered leaf water loss, and enhanced drought tolerance. Transgenic plants showed more sensitive to NaCl treatment. Since MRP protein involves in cellular ion transport, suggesting that sly-miR169 controls stomatal opening by regulating the ion transport switch in stomatal guard cell. Phylogenetic analysis revealed that MRP1 was an ancient target of miR169 in Magnoliophyta plants, but the microRNA recognition element (MRE) rapidly evolved to escape miR169 regulations. Our study is the first to provide evidence that the evolution of miRNA regulatory networks significantly contributes to plant environmental adaptation. Among these miRNA regulatory networks, Sly-miR169c negatively regulates stomatal movement in plant drought responses.Plant microRNAs (miRNAs) are vital components of the translation control system that regulates plant development and reproduction. In this study, the biological function of sly-miR156 was investigated by over-expression in tomato plants. Transgenic tomato plants exhibited a drastically altered phenotype, with reduced height, smaller but more numerous leaves, smaller fruit and patricianly phenocopied sft mutant in Inflorescence. The putative targets of sly-miR156 including six SBP-box transcription factor genes were identified by data base search and their expression patterns were determined in 35S-miR156a and wild type tomato plants. The target genes and SFT (a tomato FT ortholog) was significantly down-regulated in sly-miR156 over-expressing plants.