Silencing of Stress-Regulated miRNAs in Plants by Short Tandem Target Mimic (STTM) Approach

In plants, microRNAs (miRNAs) regulate more than hundred target genes comprising largely transcription factors that control growth and development as well as stress responses. However, the exact functions of miRNA families could not be deciphered because each miRNA family has multiple loci in the genome, thus are functionally redundant. Therefore, an ideal approach to study the function of a miRNA family is to silence the expression of all members simultaneously, which is a daunting task. However, this can be partly overcome by Target Mimic (TM) approach that can knockdown an entire miRNA family. STTM is a modification of TM approach and complements it. STTMs have been successfully used in monocots and dicots to block miRNA functions. miR159 has been shown to be differentially regulated by various abiotic stresses including ABA in various plant species. Here, we describe in detail the protocol for designing STTM construct to block miR159 functions in Arabidopsis, with the potential to apply this technique on a number of other stress-regulated miRNAs in plants.