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Endogenous microRNAs (miRNAs) play pivotal roles in plant development and responses to various biotic or abiotic stresses. Up to now, more than 500 maize miRNAs have been identified. However, functions of these identified miRNAs remained largely unknown due mainly to the lack of rapid and reliable tools. We previously reported a cucumber mosaic virus strain ZMBJ (ZMBJ-CMV)-based gene silencing vector for rapid and efficient gene function studies in maize lines with agronomical importance. Because ZMBJ-CMV induces very mild disease symptoms but strong gene silencing in maize, we decided to further modify this vector to suppress miRNA expressions in maize. The newly developed ZMBJ-CMV-2b N81 -STTM vector expresses a short tandem target mimic (STTM) containing two target-mimic sequences separated by a short spacer sequence. Our results showed that ZMBJ-CMV-2b N81 -STTM can be used to investigate miRNA function in  Nicotiana benthamiana  and maize seedlings. The ZMBJ-CMV-2b N81 -STTM-based downregulation of Nbe-miR165/166 or Nbe-miR159 induced specific and strong miRNA-sequestering phenotypes, and increased the expressions of their predicted target genes. For maize, the ZMBJ-CMV-2b N81 -STTM based downregulation of zma-miR167 or zma-miR482 caused a decrease of lateral roots growth and a plant stunting phenotypes, respectively. In both cases, the target genes of zma-miR167- or zma-miR482 were increased significantly. Thus, we consider ZMBJ-CMV based VbMS system as a useful tool for high-throughput investigations of miRNA functions in maize.

Analyses of MiRNA Functions in Maize Using a Newly Developed ZMBJ-CMV-2bN81-STTM Vector