Stem trichome small RNA-seq analysis of wild tomato relatives (Solanum section Lycopersicon)
Insects are gaining resistance to commercially available pesticides and effective insecticides (e.g. neonicotinoids) are currently being banned.Consequently, there is a real threat that the vegetable production will be affected. Wild tomato species have the ability to defend themselves by producing natural defence compounds that have a toxic or repellent effect on insects. These compounds are produced and stored in glandular trichomes on the plant stem and leaf surface. Extensive breeding in protected environments led to the loss of these defence compounds in cultivated tomatoes. It was previously shown that the introduction of the terpene 7-epizingiberene biosynthetic pathway from a wild tomato in trichomes of cultivated tomato results in an enhanced resistance to insects. However, the regulatory factors that govern the production of these defence compounds seem also essential for successful incorporation of 'wild resistance' into breeding material. This study aims to discover metabolic defence mechanisms present in the trichomes of wild tomato species that can be re-introduced into cultivated tomato with a focus on posttranscriptional regulation of biosynthetic pathways. Small RNA were sequenced to identify post-transcriptional regulations.  RNA isolation Total RNA from stem trichomes (n = 1) were isolated using concentrated TRIzol reagent (Life Technologies). Total RNA was isolated using the E.Z.N.A.® MicroElute RNA Clean Up Kit (Omega Bio-Tek). Briefly, TRIzol Reagent (Life Technologies) and chloroform was added according to the manufacturer's instructions. After centrifugation, the RNA-containing aqueous phase was collected, mixed with 1.5 volume of 100% ethanol and applied to a MicroElute spin column (Omega Bio-Tek). The column was washed according to the manufacturers's instructions: once with RWT buffer (Qiagen), once with RPE washing buffer (Qiagen) and finally with 80% ethanol. The RNA concentration was measured on a NanoDrop ND-2000 (Thermo Scientific) and RNA integrity was examined using the 2200 TapeStation System with Agilent RNA ScreenTapes (Agilent Technologies). Total RNA was spiked with ERCCs spike-in mix 1 (Life Technologies) as well as a synthetic spike-in set for Size Range Quality Control (SRQC) together with an External Reference for Data Normalization (ERDN; Locati et al., 2015).  The total RNA was divided in a large and a small fraction. The large RNA fraction was bound to a mirVana™ spin column (mirVana™ miRNA Isolation Kit, Life Technologies) according to the manufacturer's instructions. Small RNAs (<200 nts) were purified from the flow-through by adding ethanol to a final concentration of 65% (v/v) and bound to an E.Z.N.A.® MicroElute spin column. The column was washed once with RWT buffer, once with RPE buffer and once with 80% ethanol (Qiagen). The concentration and integrity of small RNA was examined as described above. Library preparation Bar-coded small RNA libraries were generated according to the manufacturer's protocols using the Ion Total RNA-Seq Kit v2 and the Ion Xpress™ RNA-Seq bar-coding kit (Life Technologies). The size distribution and yield of the bar-coded libraries were assessed using the 2200 TapeStation System with Agilent D1K ScreenTapes (Agilent Technologies). Sequencing templates were prepared on the Ion Chef™ System using the Ion PI Hi-Q Chef Kit (Life Technologies). Sequencing was performed on an Ion Proton™ System using Ion PI v3 chips (Life Technologies) according to the manufacturer's instructions.