Characterisation of tomato short RNAs involved in fruit development

Most of the RNA molecules in cells are involved in protein production (ribosomal, transfer or messenger RNAs), however there are RNA molecules with other functions. A very recently discovered class of non-coding RNAs have been called microRNAs (miRNAs) because they are very short (19-24 nucleotides). These miRNAs can recognise specific mRNAs because they have partially complementary sequences to them. As a result of this interaction between miRNAs and mRNAs, the expression of the targeted mRNAs is significantly reduced. Most plant miRNAs regulate the production of transcription factors required for normal development of plants. Many miRNAs have been identified in arabidopsis and rice and several miRNAs found in one species were absent from the other suggesting that there are miRNAs which are present only in a certain group of plants. We hypothesized that miRNAs could regulate the development of different plant characteristics that are missing from arabidopsis and rice. If this was true, novel miRNAs regulating valuable plant traits could be discovered in other species. We tested this hypothesis on fleshy fruit development because of its agronomic importance. Tomato is the model plant for fleshy fruit development and ripening; therefore we sequenced more than half a million short RNAs from tomato fruit and leaf. We have demonstrated that conserved miRNAs are present in fruit and that one of them targeted a known transcription factor required for fruit ripening. We also identified four novel miRNAs, which are absent from arabidopsis and other plant genomes and showed that one of them targeted a CTR family (genes that are involved in fruit ripening) member. In addition we identified novel candidates of another class of short RNA that are produced through a different biogenesis pathway than miRNAs. These are called ta-siRNAs and are produced in a phased manner from precursor non-coding RNAs. Our preliminary work demonstrated that the process of fleshy fruit development is regulated by miRNAs and we would like to establish the biological role of the two miRNAs we showed to target ripening genes through transgenic tomato plants that produce more miRNAs or target genes that are resistant to miRNA targeting. We also propose to validate and characterise the ta-siRNA candidates. Further deep sequencing work will establish the expression profile of short RNAs during fruit development and ripening and also identify short RNAs that are regulated by known transcription factors involved in fruit development. Finally, we developed a novel technique to generate a library of mRNAs that are cleaved by short RNAs and we will apply this protocol for tomato fruit tissue. This tool will help target prediction and will have a big impact for crop research. Most crop species' genome is not known and this tool will enable researchers to investigate short RNA targets in these species. Understanding this layer of regulation in fruit development and ripening may lead to the improvement of key characteristics of fleshy fruits.

MicroRNAs (miRNAs) are small RNAs, 19-24 nucleotides in length that regulate gene expression by targeting mRNAs. Although many plant miRNAs are phylogenetically conserved, several rice miRNAs are absent from the arabidopsis genome and a few arabidopsis miRNAs are not present in the rice genome. This observation suggests that non-conserved miRNAs could play a role in the development of plant traits, which are missing from arabidopsis and rice. To test whether sRNAs play a role in fleshy fruit development we have sequenced more than half a million sRNAs from tomato fruit. We have demonstrated that one of the conserved miRNAs targeted CNR, a gene required for fruit ripening. We also identified four novel tomato-specific miRNAs and showed that one of them targeted splice variants of CTR4. In addition we identified novel candidates of another class of sRNAs. These are the ta-siRNAs, which are produced in a phased manner from precursors. Our work demonstrated that the process of fleshy fruit development is regulated by miRNAs and we would like to establish the biological role of the two miRNAs we showed to target ripening genes through transgenic tomato plants that over-express miRNAs or target genes that are resistant to miRNAs. We also propose to validate ta-siRNAs using mutant tomato lines that are defective in this pathway and characterise these novel ta-siRNAs. Further high throughput sequencing will establish sRNA expression profile during fruit development and ripening. We will also identify sRNAs that are regulated by known transcription factors involved in fruit development. Finally, we developed a novel technique to generate a library of mRNAs that are cleaved by sRNAs and we will apply this protocol for tomato fruit tissue. It will be used to predict targets of sRNAs identified in the different stages of fruit development. This protocol will be useful to investigate sRNAs in other crop species, since it does not require any genome sequence information.