Identification of wounding and topping responsive small RNAs in tobacco (Nicotiana tabacum)

To get insight into the role of small RNAs in damage-induced responses, we sequenced and analysed small RNA populations in roots and leaves from wounding or topping treated tobacco plants. In addition to confirmation of expression of 27 known miRNA families, we identified 59 novel tobacco-specific miRNA members of 38 families and a large number of loci generating phased 21- or 24-nt small RNAs (including ta-siRNAs). A number of miRNAs and phased small RNAs were found to be responsive to wounding or topping treatment. Targets of small RNAs were further surveyed by degradome sequencing.The expression changes of miRNAs and phased small RNAs responsive to wounding or topping and identification of defense related targets for these small RNAs suggest that the inducible defense response in tobacco might be controlled by pathways involving small RNAs.Small RNAs are a group of regulatory molecules that fall into two major classes, microRNAs (miRNAs) and short interfering RNAs (siRNAs). They play important roles in biological systems in eukaryotes by suppressing expression of target genes at the transcriptional and/or post-transcriptional level through specific base pairing with their targets [1]. In plants, siRNAs are further classified into trans-acting siRNAs (ta-siRNAs), natural antisense transcript-derived siRNAs (nat-siRNAs), and repeat-associated siRNAs (ra-siRNAs) [2]. In addition, a novel class of bacteria-induced 30- to 40-nt endogenous small RNAs, long siRNAs (lsiRNAs), was identified in Arabidopsis [3].As an important group of small RNAs, miRNA has attracted much attention. A number of studies have been performed to reveal the biogenesis of miRNAs and the mechanisms of miRNA-mediated gene regulation [4-6]. In plants, miRNA derives from primary miRNA transcript (pri-miRNA), which is transcribed by RNA polymerase II. After formation of a stem-loop secondary structure [7,8], pri-miRNA is cleaved twice by DICER-LIKE1 (DCL1), a RNase III enzyme [9]. The first cleavage